[International Review of Cell and Molecular Biology] International Review of Cell and Molecular...

C H A P T E R F I V E

In

IS

DV

ternati

SN 1

epartmeterin

Genetics of Meiosisand Recombination in Mice

Ewelina Bolcun-Filas and John C. Schimenti

Contents

1. O

onal

937

enary

verview of Meiosis

Review of Cell and Molecular Biology, Volume 298 # 2012

-6448, http://dx.doi.org/10.1016/B978-0-12-394309-5.00005-5 All rig

t of Biomedical Sciences and Center for Vertebrate Genomics, Cornell UniversityMedicine, Ithaca, New York, USA

Else

hts

, C

180

2. Id
entification of Mouse Meiosis Genes 182
2.1.
F orward genetic screens 182
2.2.
R everse genetics: Yeast orthologs 183
2.3.
G ene expression analyses 184
2.4.
O ther methods: Proteomics 185
3. E
ntry into Meiosis: Male Versus Female 186
4. P
rophase I 187
4.1.
T ransposon and repetitive element silencing 187
4.2.
C hromosome structure: Cohesins, telomeres, nuclear
envelope attachment
188
4.3.
H omolog recognition and alignment 191
4.4.
S ynapsis and SC 192
4.5.
In itiation of recombination (DSB induction: Hot spots) 194
4.6.
R ecombination (DSB repair: NCO vs. CO) 196
4.7.
C hromosome segregation: Chiasmata resolution and removal
of abnormal recombination intermediates
199
4.8.
X Y pairing and silencing 200
4.9.
C heckpoint control 201
4
.10. S mall RNAs 203
4
.11. C ell cycle regulation and exit from prophase I 205
4
.12. P rotein modification during prophase I: Phosphorylation,
SUMOylation, ubiquitination, methylation, acetylation, etc.
206
4
.13. C oncluding remarks 208
Refe
rences 214
Abstract

Meiosis is one of the most critical developmental processes in sexually repro-

ducing organisms. One round of DNA replication followed by two rounds of cell

vier Inc.

reserved.

ollege of

179

http://dx.doi.org/10.1016/B978-0-12-394309-5.00005-5

180 Ewelina Bolcun-Filas and John C. Schimenti

divisions results in generation of haploid gametes (sperm and eggs in mam-

mals). Meiotic failure typically leads to infertility in mammals. In the process of

meiotic recombination, maternal and paternal genomes are shuffled, creating

new allelic combinations and thus genetic variety. However, in order to achieve

this, meiotic cells must self-inflict DNA damage in the form of programmed

double-strand breaks (DSBs). Complex processes evolved to ensure proper DSB

repair, and to do so in a way that favors interhomolog reciprocal recombination

and crossovers. The hallmark of meiosis, a structurally conserved proteina-

ceous structure called the synaptonemal complex, is found only in meiotic

cells. Conversely, meiotic homologous recombination is an adaptation of the

mitotic DNA repair process but involving specialized proteins. In this chapter,

we summarize current developments in mammalian meiosis enabled by geneti-

cally modified mice.

Key Words: Meiosis, Cell divisions, Meiotic recombination, Meiotic DNA

repair, Double-strand breaks, Meiotic mutants, Synaptonemal complex.

� 2012 Elsevier Inc.

1. Overview of Meiosis

The word “meiosis” comes from the Greek meioun, meaning “tolessen.” This is a fitting adjective for a process in which number of chromo-somes per cell is reduced by half. This reduction is achieved by a singleround of DNA replication followed by two rounds of chromosome segre-gation in the germ cells of sexually reproducing organisms. Meiosis mostlikely evolved from mitosis but acquired new critical steps: pairing andsynapsis of homologous chromosomes, recombination between nonsisterchromatids, suppression of sister chromatid separation during the first mei-otic prophase, and bypassing DNA replication between the two meioticdivisions (Wilkins and Holliday, 2009).

Following premeiotic DNA replication, germ cells enter an extendedmeiotic prophase I that is divided into cytologically discernable substagesbased on the behavior of chromosomes and the proteinaceous scaffoldknown as the synaptonemal complex (SC) (Fig. 5.1). During the earlieststage, leptonema, a protein-rich backbone forms between sister chromatidsknown as the axial element (AE) that will keep them together until thesecond meiotic division. During this stage, chromosomes are relativelydecondensed and long. AEs start as short stretches that become increasinglylonger as cells progress through leptonema to next stage of zygonema.Throughout zygonema, homologous chromosomes pair as two AEs apposeand are then tethered together by a zipper-like structure called the centralelement (CE). At this point, AEs become the lateral elements (LEs) of the

Interphase Leptonema Zygonema Pachynema

RAD51/DMC1

HORMAD1

HORMAD2

SYCP2/3

Cohesin core

Chromatin loop

SYCP1

SYCE1–3 /TEX12

MSH4/MSH5 MLH1/MLH3

Diplonema Metaphase I

Chromosomes condensechiasma visible

Chromosomesdesynapse

Synapsis is completedcrossover occurs

Chromosomesbegin to pair

DSBs fromchromosomes

condenseDNA synthesis

Figure 5.1 Schematic representation of the events occurring between homologouschromosomes during prophase of the first meiotic division. Substages of prophase I andrelative progression of synapsis and recombination are depicted with spatiotemporaldistribution of proteins involved in the synaptonemal complex formation andrecombination.

Genetics of Meiosis and Recombination 181

SC. Formation of tripartite SC along the entire length of chromosome axes(synapsis) is the hallmark of the next substage—pachynema. At the end of thepachytene stage, homologs are fully synapsed and chromosomes are shortand condensed. Toward the end of prophase I, the SC starts to disassemble,marking entry to diplonema. However, homologs remain physicallyconnected by chiasmata. Chiasmata are formed during a process of homol-ogous/meiotic recombination that runs in synchrony with chromosomesynapsis. Recombination is initiated early in prophase I by DNA double-strand breaks (DSBs). During recombination, these breaks are repaired bycrossover (CO) or noncrossover (NCO) repair pathways, the former result-ing in chiasmata. Chiasmata are essential for correct alignment and segrega-tion of homologous chromosomes during metaphase I. Chromosomes thatfail to establish COs/chiasma frequently fail to disjoin properly leading toaneuploidy. The first meiotic division is reductional where maternal andpaternal chromosomes are segregated to daughter cells. In the secondmeiotic division, sister chromatids are separated, culminating in the genera-tion of haploid gametes. The principles of meiosis are shared between twosexes of heterogametic organisms such as mouse or human, but timing andregulation are sexually dimorphic as discussed later.


2. Identification of Mouse Meiosis Genes

Meiosis is a highly specialized process that evolved in the ancestors ofsexually reproducing organisms. The basic principles of chromosomebehavior and recombination are relatively conserved from single-celledyeast to multicellular organisms such as worms, flies and mammals. How-ever, the underlying molecular mechanisms and regulation have distinc-tions, making it often difficult to extrapolate between different organisms.This confounds the understanding of mammalian meiosis, which occurs inthe context of complex gametogenesis processes. Fortunately, the orthologsof core proteins involved in conserved meiotic processes or structuresusually possess identical or similar functions. However, there are manyother instances where critical meiotic genes in one taxon have no clearorthologs in another. The complexity of mammalian meiotic transcriptomeshows we still have a lot to discover and offers a springboard for identifyingfunctional homologs or mammalian-specific meiotic players. In the follow-ing sections, multiple strategies for identifying the genes that play importantroles in mammalian meiosis are outlined.

2.1. Forward genetic screens

Although we have extensive knowledge about simpler meiotic systems, it isnot sufficient to explain the complexity of mammalian meiosis. Forwardgenetic screens proved to be a powerful tool to link genes to meioticphenotypes in other model organisms and were successfully used in mam-mals to study other phenotypes. Forward genetic screening in the mouseinvolves induction of random mutations, identification of a desired mutantphenotype, and finally mapping and isolation of the causal allele. Meioticdefects usually cause infertility; thus, the nature of the phenotype compli-cates the screening process and subsequent mapping of the potential under-lying mutation. Furthermore, it is only feasible with recessive mutations.Nevertheless, a large-scale genome-wide ENU mutagenesis screen forinfertility alleles has been undertaken by the Reproductive Genomicsgroup at The Jackson Laboratory. In a heroic effort, over 17,000 micewere fertility tested, and as a result, 44 mutant lines were selected and 42mapped. Interestingly, the majority of mutant lines (32) had an impact onmale fertility, while only 3 on female and 7 on both sexes (http://repro-ductivegenomics.jax.org/). Among meiotic genes identified in this screenare Mei1—novel protein critical for formation of programmed meioticDSBs (Libby et al., 2002, 2003), Recmei8—point mutation in alreadyknown cohesin (Bannister et al., 2004), Ccnb1ip1mei4—novel proteinrequired for generation of COs (Ward et al., 2007), Mybl1repro9—point

http://reproductivegenomics.jax.org/




mutation in known transcription factor required for meiotic progression(Bolcun-Filas et al., 2011), Eif4g3—a novel translation initiation factorcritical for meiotic exit (Sun et al., 2010), Spata22—a novel vertebrate-specific gene of unknown function that is required for meiotic progression(La Salle et al., 2011), and Marf1—another novel gene that is requiredspecifically for female meiosis and appears to have roles in RNA degradationand suppression of retrotransposon expression (Su et al., 2012). Althoughthis approach successfully identified new alleles causing infertility due tomeiotic defects, it has its limitations: it is laborious, time consuming, andexpensive. It also necessitates generation and analysis of large numbers ofmice for mapping purposes. However, as whole-genome sequencing costsdecrease, genetic mapping may become dispensible (Arnold et al., 2011)and make forward genetic screens more effective and efficient.

2.2. Reverse genetics: Yeast orthologs

Model organisms such as yeast have been instrumental in understandingmammalian meiosis. Saccharomyces cerevisiae, the single-celled yeast withpowerful genetic tools, allowed phenotype-oriented genetic screens thatidentified genes and epistatic groups involved in meiotic recombination.Many of the meiotic processes are highly conserved across diverse eukar-yotes and allowed identification of orthologs in mouse mainly based onsequence homology and conserved motifs in their functional domains. Thisway, many of the core meiotic proteins were identified and their inferredfunction was shown to be conserved in knockout mouse models. Theinitiator of meiotic recombination, the Spo11 topoisomerase, was shownfirst in yeast to be responsible for generation of DSBs. Spo11 orthologs werealso identified in Caenorhabditis elegans and Drosophila, and thus Spo11 was agood candidate for a role in mammalian meiosis. Indeed, mouse Spo11 wasidentified based on sequence homology (Metzler-Guillemain and de Massy,2000; Romanienko and Camerini-Otero, 1999) and confirmed to haveconserved function in mice (Baudat et al., 2000; Romanienko andCamerini-Otero, 2000). Two RecA homologs, Rad51 and Dmc1, shownto catalyze pairing and strand exchange between homologous DNA strandsin yeast were identified in mouse based on sequence homology in the RecAdomain (Habu et al., 1996; Matsuda et al., 1996; Morita et al., 1993;Shinohara et al., 1993). Both genes were then targeted in mouse to investi-gate their role in mammalian meiosis. Rad51 is a ubiquitously expressedgene, and not surprisingly, null mutants displayed early embryonic lethalityprecluding meiotic phenotype analysis (Tsuzuki et al., 1996). In contrast,Dmc1 is specific to meiosis and mutants were viable and showed meioticphenotypes similar to those in yeast (Pittman et al., 1998; Yoshida et al.,1998). However, not all yeast meiotic proteins are evolutionarily conservedand sequence homology-based searches in mammals were unable to identify


orthologs in higher eukaryotes. Recently, a combination of phylogenomichomology searches coupled with multiple sequence alignments and second-ary protein structure prediction analyses identified orthologs of Mei4 andRec114 in mouse (Kumar et al., 2010). Targeted inactivation of Mei4 inmice confirmed functional conservation even though protein sequenceswere highly divergent. Both yeast and mouse MEI4 interact with REC114and are critical for generation of DSBs. There are also cases in which theclosest mouse orthologs of yeast meiotic proteins do not have the identicalpredicted functions as in the case of ATM (yeast Tel1) or TRIP13 (yeastPch2) (Elson et al., 1996; Li and Schimenti, 2007; Roig et al., 2010; Xu et al.,1996). Finally, proteins that play mostly structural roles have no obviousorthologs such as Zip1, and their apparent functional homologs were identi-fied using other approaches.

2.3. Gene expression analyses

Another method that proved to be successful in identification of potentialcandidates for meiotic roles is gene expression analysis. The first wave ofmale spermatogenesis that occurs during first 3–4 weeks after birth is rela-tively synchronous, with cell cohorts entering subsequent stages in a coor-dinated and timely manner. In particular, meiotic prophase I is wellcharacterized. Specific prophase I stages are correlated with prepubertalage (in days postpartum, dpp; Bellve et al., 1977; Goetz et al., 1984).Therefore, on a particular dpp, the mouse testis will be enriched for a substagepopulation of spermatogenic cells. For example, at 10–11 dpp, most of thecells are in leptonema; at 15–16 dpp in pachynema; and at 17–18 dpp indiplonema. However, late-stage testis would also contain earlier stages.Highly enriched populations of the various spermatogenic cell types can beobtained using gradient sedimentation methods like STA-PUT (Meistrichet al., 1973) or flow cytometric sorting (Mays-Hoopes et al., 1995). Analysisof gene expression profiles in enriched spermatogenic populations from testesof mouse, rat, and human revealed characteristic expression patterns (Chalmelet al., 2007; Pang et al., 2003, 2006; Rossi et al., 2004; Schultz et al., 2003;Sha et al., 2002; Shima et al., 2004; Wu et al., 2004). Transcripts can beclustered into groups based on their first appearance, peak and level ofexpression. Thus, transcripts detected after 10 dpp and peaking around15 dpp most likely encode proteins functioning during meiosis.

Studies of mutants with meiotic failure (Dazl�/�, Spo11�/�) provide analternative to pinpoint transcripts that are expressed during prophase I(Maratou et al., 2004; Smirnova et al., 2006). The advantage of using testesarrested at early stages of prophase I (leptotene/zygotene) is that they do notcontain later stages of meiosis (pachytene/diplotene) as compared to thesame age wild-type testis. Therefore, transcripts that are elevated afterpostnatal day 10 but are downregulated in mutant testis represent the


potential meiotic genes. Using this approach, the long list of testis-expressedgenes can be narrowed down to the most promising potential candidatesplaying a role in meiotic processes. Examples that were identified using thismethod and later confirmed to have meiotic function include the threesynaptonemal complex central element proteins SYCE1–3 (Bolcun-Filaset al., 2007, 2009; Schramm et al., 2011). The sheer quantity of testistranscriptome profiling data is overwhelming; however, some of the expres-sion data have been consolidated in the GermOnline database (Lardenoiset al., 2010; www.germonline.org/) focused on germ cell development inmice. This database is easily searchable and allows extraction of meiosis-specific transcripts. The most recent version also includes yeast data.Once the promising candidate have been chosen based on expressionpatterns or homology to known proteins/functional domains, the finalproof for its meiotic function comes from mice mutant lacking the func-tional protein. In recent years, generation of knockout animals has becomeeasier with many programs generating targeted mutations by means of genetrapping or targeted knockouts (Skarnes et al., 2011; http://www.knock-outmouse.org/).

2.4. Other methods: Proteomics

Proteomics is a new frontier in biology that can complement genomicsand transcriptomics approaches. The development and availability of massspectrometry (MS) combined with co-immunoprecipitation (co-IP) andaffinity purification techniques bring additional clues/confirmations topotential meiotic proteins identified by methods described above. His-torically, the three major nonconserved components of the mammaliansynaptonemal complex SYCP1, SYCP2, and SYCP3 were identified in aprotein-based approach (Heyting et al., 1987; Meuwissen et al., 1992;Offenberg et al., 1998) and later genetically proven to be essential for SCassembly (de Vries et al., 2005; Yang et al., 2006; Yuan et al., 2000). Inbrief, SCs were isolated from rat spermatocytes and used to raise mono-clonal and polyclonal antibodies. Selected antibodies were then utilizedto identify corresponding cDNA clones from expression libraries(Heyting et al., 1989). Now, over two decades later, protein identifica-tion can be done using MS with far lower quantities of protein. Manyproteins involved in meiotic processes do not act alone; they formcomplexes such as recombination nodules or substructures of the SC(axial, lateral, and central elements). Recently, a new component of themeiotic cohesin complex RAD21L was identified using co-IP/MS(Ishiguro et al., 2011). Interestingly, the same protein was identified inanother study based on the sequence similarity to the RAD21 cohesin(Herran et al., 2011).

http://www.germonline.org/

http://www.knockoutmouse.org/




3. Entry into Meiosis: Male Versus Female

Entry into meiosis is sexually dimorphic in mammals. Meiosis isinitiated during a brief window of fetal development in females and inearly postnatal life in males. These differences are regulated by intrinsicand extrinsic factors (Lin et al., 2008). The sexual fate of primordial germcells (PGCs) depends on the gonadal environment. Bipotential PGCs thatarrive at the female primitive gonad (prospective ovary) receive cues toinitiate meiotic entry and become oocytes. On the other hand, PGCsreaching the male gonad (prospective testis) receive other cues, preventingthem from initiating meiosis and directing them to the male differentiationprogram (Adams and McLaren, 2002). Those cues represent extrinsic fac-tors that revolve mostly around retinoic acid (RA) signaling. RA is synthe-sized in the mesonephroi of the developing embryos of both sexes (Bowleset al., 2006) and can activate genes with retinoic acid-responsive elements(RARE) such as Stra8 (stimulated by retinoic acid 8). Genetic analysis hasshown that Stra8 is critical to initiate meiotic entry in both female and malegerm cells; however, the timing of its activation is sexually dimorphic(Anderson et al., 2008; Baltus et al., 2006). Fetal gonads of both sexes areexposed to RA from the mesonephros, but only female gonads induce Stra8expression and initiate meiosis. Male gonads do not respond to RA due tothe expression of the retinoid-degrading enzyme, CYP26b1 (a member ofthe cytochrome P450 family) in fetal Sertoli cells, thus preventing inductionof Stra8 and onset of meiosis. Cyp26b1-deficient male germ cells entermeiosis precociously at the same embryonic time point as do normal femalegerm cells (Bowles et al., 2006). Stra8-deficient male mice also fail to initiatemeiosis, suggesting that despite the different timings of meiotic entry, theunderlying mechanisms are most likely the same and involve STRA8 andRA (Anderson et al., 2008). However, because retinoids are also found inother tissues, the final outcome of RA induction depends on the meioticcompetence of germ cells. A meiosis-permissive environment is implemen-ted by intrinsic factors such as RNA-binding protein DAZL.Dazl-deficientembryonic germ cells do not respond to the RA cues, fail to activate Stra8expression (Lin et al., 2008), and remain in an undifferentiated state.Gill et al. (2011) proposed a term for DAZL action as “licensing of gameto-genesis”—a gateway to sex-specific gametogenesis programs.

Once meiosis in the female embryo is initiated, oocytes progress throughthe first meiotic prophase and arrest neonatally at the diplotene/diakinesisstage in which they remain until they are recruited for resumption ofmeiosis following sexual maturation. In the male gonad, germ cells remainarrested at G0/G1 until they initiate proliferation postnatally for spermato-gonial stem cell pool expansion. The maintenance of fetal male germ cell


arrest is dependent on Nanos2. NANOS2 suppresses meiosis in male germcells (Suzuki and Saga, 2008), and in the absence of NANOS2, malegerm cells precociously enter meiosis in the fetal testis and are eliminatedby apoptosis. Nanos2 is not expressed in female fetal germ cells and isdownregulated in male germ cells prior to meiotic entry. Once meiosis isinitiated in the male, consecutive waves of spermatogenesis ensue through-out the life of the male. Following meiotic divisions, haploid spermatidsundergo specialized morphological differentiation known as spermiogenesis.

4. Prophase I

4.1. Transposon and repetitive element silencing

Transposable elements (TEs) can have beneficial as well as detrimental effecton the evolution of genomes. New integrations can disrupt a gene, andrecombination between nonallelic TEs results in genomic rearrangementssuch as deletions, duplications, or inversions (Goodier and Kazazian, 2008).Transposons and transposon-derived repetitive elements constitute 3–5% ofyeast, 12% of worm, and 15–22% of fly genomes, but in mice and human,these elements make up almost half of their genomes (40% and 45%,respectively) (Biemont and Vieira, 2006). This abundance of potentiallyharmful elements requires a restraining system to prevent them from unre-strained movement and expansion. Despite the quantity of TEs in thegenome, only a small fraction (estimated 0.05%) remains potentially mobile(Mills et al., 2007). TEs are kept in check in two ways: epigenetic silencingby DNA methylation and piRNA-induced transcript degradation. piRNAswere also suggested to play a role in de novo DNA methylation of TEs(Kuramochi-Miyagawa et al., 2008). Protection from jumping elementsis even more important in the germ line to protect genomic integrity ofnew individuals and the species. Therefore, DNA methyltransferases(DNMT1, DNMT3L, DNMT3A) with their accessory proteins (LSH)and proteins involved in piRNA biogenesis (MILI, MIWI, MIWI2,MAEL, MOV10L1, TDRD1, TDRD9, GASZ) are expressed in thegerm line (Kuramochi-Miyagawa et al., 2001; La Salle et al., 2004;Zamudio and Bourc’his, 2010). Retrotransposon derepression during mei-otic prophase I was observed in mutants defective in DNA methylation(de novo and maintenance) and piRNA pathways. Interestingly, thesemutants also displayed defects in meiotic progression. These defects aremore prominent in males, probably reflecting different timing and regula-tion of meiotic events with respect to transposon silencing pathways(Zamudio and Bourc’his, 2010). Germ cells of both sexes undergo globalDNA demethylation soon after colonizing fetal gonads, which results intransient/partial derepression of transposon silencing (Lees-Murdock et al.,


2003). In females, widespread DNA demethylation directly precedes mei-otic initiation and de novo DNA methylation occurs postmeiotically ingrowing oocytes. On the other hand, in the male germ line de novo DNAmethylation is established in quiescent fetal germ cells and is maintained insubsequent generations of germ cells long before they enter meiotic division(Hajkova et al., 2002). Almost all of the mutations resulting in TEs dere-pression cause male infertility (Mili, Mael, Mov10L1, and Tex19.1) (Aravinet al., 2007; Frost et al., 2010; Ollinger et al., 2008; Soper et al., 2008;Zheng et al., 2010). Only in the case of Lsh/Hells, both sexes showeddefective meiotic progression (De La Fuente et al., 2006; Zeng et al., 2011).

How exactly transposable and repetitive elements affect meiosis is notknown, but there are a few possibilities. Active transposition generatesDNA DSBs. During meiosis, DSBs are formed naturally and are requiredfor recombination, homologous chromosome synapsis, and ultimate com-pletion of meiosis (see below). Therefore, additional breaks, particularly innonunique repetitive sequences, could interfere with these processes.Indeed, synapsis defects are characteristic of the aforementioned mutants.Analysis of Spo11�/� Mael�/� spermatocytes confirmed the presence ofSpo11-independent DSB caused by reactivation of TEs in Mael mutants(Soper et al., 2008).

4.2. Chromosome structure: Cohesins, telomeres, nuclearenvelope attachment

During meiosis, accurate chromosome segregation depends on tightlycoordinated control of sister chromatid cohesion (SCC) with chromosomesynapsis and SC assembly/disassembly (described later). The chromatin ofmeiotic chromosomes is arranged into a series of loops originating from themeiotic chromosome axis, which is composed of a cohesin core and the SC.Mitotic and meiotic cohesin complexes are composed of four core subunits:two SMC (structural maintenance of chromosomes) and two non-SMCproteins (a-kleisin and stromalin/SA) (Nasmyth and Haering, 2009). Insomatic cells, an SMC1a/SMC3 heterodimer forms a ring-like structureclosed by interaction with RAD21 kleisin (from the Greek word kleisimofor closure), while the stromalin SA1/SA2 subunit interacts with the kleisinto maintain the ring-like arrangement embracing sister chromatids. Meio-sis-specific paralogs of cohesin subunits exist in germ cells in addition tocanonical subunits, suggesting coexistence of more than one cohesin com-plex in meiotic cells. The meiosis-specific cohesin subunits SMC1b, REC8,RAD21L, and STAG3 together with the mitotic SMC1a, SMC3, andRAD21 can form distinct cohesin complexes as shown by immunoprecipi-tation experiments (Ishiguro et al., 2011; Fig. 5.2A). At least three differentcomplexes appear to contain one of the kleisin subunits REC8, RAD21L,or RAD21. Their different spatiotemporal expression patterns suggest

Somatic and meiotic

A

B

SA1/SA2 STAG3 STAG3 STAG3

REC8 RAD21L

MEIOTIC

AE/LESYCP1

SYCE1

SYCE2

SYCE3

TEX12

SYCP2/3

Cohesincore

Chromatinloops

AE/LE

TF

TF

CE CR

SM

C1a

SM

C1a

/b

SM

C1a

/b

SM

C3

SM

C3

SM

C3

SM

C1a

/b

SM

C3

RAD21RAD21

Figure 5.2 (A) Schematic summary of putative cohesin complexes and their subunitsfound in meiotic cells. (B) Model for the synaptonemal complex (SC) assembly. SYCP1homodimers form unstable N-terminal self-associations and require SYCE1/3 complexfor stabilization and initiation of synapsis. Propagation of the SC and formation of thecentral element (CE) requires interaction with SYCE2/TEX12 complex (adapted fromBolcun-Filas et al., 2007).


distinctive roles in SCC in prophase I and possibly beyond. Most of ourknowledge about the role of cohesins comes from genetic studies. Mousemutants lacking the meiotic SMC protein SMC1b are infertile; malemeiosis arrests at early/mid pachytene due to synapsis and recombinationdefects and oocytes progress to dictyate with fewer COs, resulting in severeaneuploidies (Revenkova et al., 2004). AEs of mutant chromosomes wereshortened by 50% and accompanied by increased DNA loop size, revealinga role for SMC1b in chromatin loop organization along the AE. Additionalanalysis of mouse oocytes deficient for Sycp3 showed longer AEs with moreand smaller loops, while in oocytes doubly deficient for Smc1b and Sycp3,the average loop size was increased compared to Sycp3 single mutants or


wild type. These results implicate SMC1b in determination of DNA loopsize and illustrate important interplay between the cohesin cores and SC AEcomponents in establishing meiotic chromosome structure (Novak et al.,2008). It was previously postulated by Zickler and Kleckner (1999) that SClength and DNA loop size are reciprocally correlated; importantly, SClength/loop size ratios in these mouse mutants support this idea.

SMC1b was also implicated in protecting meiotic telomere integrity(Adelfalk et al., 2009). In the absence of SMC1b, telomeres display a widerange of abnormalities: telomeres are shortened, fail to attach to the nuclearmembrane (NE), and are often found broken off of the chromosome.SMC1a and b localize to the SC until diplonema, but only SMC1b remainsat centromeres until metaphase II, signifying its sole involvement in SCCand chromosome segregation during meiosis. SMC1b forms a complexwith meiotic-specific kleisin REC8. Two mutant mouse models implicateREC8 as an important factor for restricting synapsis between homologouschromosomes (Bannister et al., 2004; Xu et al., 2005). Rec8-deficient miceshowed unexpected phenotype compared to other model organisms(Molnar et al., 1995; Watanabe and Nurse, 1999). In contrast to yeast,murine REC8 is dispensable for AE formation and synapsis and in itsabsence, SC forms between sister chromatids instead of homologous chro-mosomes. However, this abnormal synapsis results in recombination defectsand CO failure and thus sterility of both sexes. As expected, REC8 shows alocalization pattern similar to SMC1b (Lee et al., 2003), indicating its role inSCC. However, REC8 is not the only meiosis-specific kleisin. Recently, anew vertebrate-specific meiotic kleisin, RAD21L, was identified that bearsclosest similarity to mitotic RAD21 (Herran et al., 2011; Ishiguro et al.,2011). Interestingly, one of the studies shows that REC8 and RAD21Lcontaining cohesin complexes show symmetrical and mutually exclusivelocalization patterns along the AE of unsynapsed chromosomes (Ishiguroet al., 2011). The authors postulate that this “barcode-like” patterningof AEs could aid in homology establishment prior to recombination-dependent DNA associations. RAD21L appears first in leptotene sperma-tocytes associated with newly forming AE, peaks at pachynema, and beginsto disappear from late pachynema onward but remains associated withcentromeres at metaphase I. However, RAD21L does not localize tocentromeres in metaphase I oocytes, unlike SMC1b and REC8, suggestingsexually dimorphic requirements for cohesin complexes. Although STAG3function in meiosis has not yet been confirmed by means of null mutants, itscolocalization and interactions with other cohesin subunits suggest its role inchromatid cohesion (Kouznetsova et al., 2005; Prieto et al., 2001).

Telomeres play a crucial role in chromosome and genome stability.During prophase I, telomeres are attached to the nuclear envelope (NE).During meiotic prophase I, telomeres display dynamic movements, espe-cially at the leptotene/zygotene transition when they transiently cluster


together within a limited area forming the so-called meiotic “bouquet”which is thought to facilitate homologous chromosome interactions andpairing. Telomere movements during zygonema have been implicated inremoving chromosome interlocks that happen during synapsis (Zickler andKleckner, 1998). It has been shown that in various meiotic mutants defec-tive in DSB repair and synapsis, meiotic telomere dynamics is affected(Adelfalk et al., 2009; Liebe et al., 2004, 2006; Scherthan, 2003). In micedeficient for ATM, SPO11, gH2AX, SYCP3, or MLH1, the length of thebouquet stage was significantly extended, indicating that exit from this stageis mediated by processes monitoring DSB repair progression, and suggestinga tight connection between meiotic events and telomere dynamics. The firstgenetic evidence showing the importance of telomere attachment andmovement in mammalian meiosis came from SUN1-deficient mice (Dinget al., 2007). Sun1 mutant males and females were infertile due to DNArepair and synapsis defects. Without SUN1, telomeres fail to attach to the NEand lose their mobility, confirming an important role of these phenomena inhomologous pairing and synapsis. In other model organisms, meiotic telo-meres attach to the NE by interaction with SUN–KASH domain proteinsthat reside in the inner nuclear membrane. However, the mammalian coun-terparts have not been identified (Hiraoka and Dernburg, 2009). Notably,Rap1, protein essential for telomere attachment in Schizosaccharomyces pombe,is dispensable for mammalian meiosis (Scherthan et al., 2011). Mammaliantelomere behavior remains poorly understood and awaits identification andfunctional analysis of other components of the NE telomere-tetheringcomplex.

4.3. Homolog recognition and alignment

Chromosome numbers vary significantly in different organisms. Fruit flieshave only 4 pairs of chromosomes, whereas worms, yeast, mice, humans,and dogs have 6, 16, 20, 23, and 39, respectively. Especially in thoseorganisms with more chromosomes, the ability of homologs to find oneanother, pair, and remain together during the first meiotic division is criticalto avoid chromosome mis-segregation and resulting aneuploidy.

In most organisms including mice, stable homolog alignment and chro-mosome synapsis require DNA DSBs. In the absence of SPO11, a topo-isomerase responsible for generating DSBs, homologous chromosomes failto pair, and synapsis can occur between nonhomologs. Telomere-drivennuclear rearrangements that occur during the “bouquet” stage (when telo-meres are clustered on the NE resembling the stems of a floral bouquet)greatly increase the likelihood of homolog encounters. DSB-dependenthomology searching and formation of DNA joint molecules stabilize thealignment between homologs. It has been postulated that telomere-driventransient contacts between chromosomes serve as homology-testing


mechanism (Bass et al., 2000; Scherthan et al., 1998). The requirement forstable DNA interactions between chromosomes to maintain homologousalignment has been observed in various meiotic mutants. In mice deficientfor structural components of the SC (SYCP1, SYCE1–3, TEX12), homo-logs maintain alignment despite lack of the SC, presumably due to stableDNA joint molecules. Mutants defective in DNA repair such as Dmc1 orMsh4 fail to engage/maintain DNA-mediated interactions with the homo-log and thus fail to sustain homolog pairing. Genetic analysis of Hormad1mutants (Daniel et al., 2011) showed that sufficient numbers of single-stranded ends at DSBs are required for full homologous synapsis. Theauthors proposed that among other functions, this HORMA domain pro-tein ensures sufficient steady-state numbers of ssDNA ends and thus facil-itates homology searching.

4.4. Synapsis and SC

The SC was first observed independently by Moses and Fawcett in 1956(Fawcett, 1956; Moses, 1956). The fully formed SC is a zipper-like tripartitestructure that is so protein rich that it can be observed on spread prepara-tions under light-phase microscopy (Goodpasture and Bloom, 1975; Moses,1977). Before the use of antibodies, the SC was typically visualized andanalyzed using silver staining (Dresser and Moses, 1979; Goodpasture andBloom, 1975). Since then, many proteins have been described that buildthis crucial biological structure. Genetic analysis of mutant mice lacking anyof the SC components shows that this conserved structure is crucial forgeneration of gametes. However, phenotypes are often sexually dimorphic(Table 5.A1), mostly for the AE/LE components, reflecting differences inthe regulation and stringency of male and female meiosis (Fraune et al.,2012; Yang and Wang, 2009).

The mature SC is composed of two parallel AEs called LEs in thecontext of the SC. The AE/LE coalign with cohesin cores and areconnected by transverse filaments (TFs) with one end anchored in the LEand the other in the CE (Fig. 5.2B). The SC structure is fairly conservedfrom yeast to human; however, the same cannot be said about its compo-nents. There is no obvious protein sequence similarity between mouse andyeast/worm SC components. What seems to have been under evolutionarypressure is the domain organization and three-dimensional structure(Bogdanov et al., 2007). SC proteins often possess coiled-coil domainsable to self-polymerize and form homo/heteropolymers as observed inheterologous overexpression systems (Costa et al., 2005; Ollinger et al.,2005). From immunolocalization studies, co-IP experiments and geneticanalyses, major structural components, and interactions between them havebeen described (Yang and Wang, 2009). Additionally, there are manyproteins known to be transiently associated with the SC. The two major


components of the AE/LE are SYCP2 and SYCP3 (SC protein). Mousemutants deficient for either protein revealed that their localization to the SCaxial cores is interdependent (Yang et al., 2006; Yuan et al., 2000). TFs, themajor building blocks of the central region (CR), consist of SYCP1. TheSYCP1 molecule contains a long coiled-coil segment with two globulardomains at the ends that form a parallel dimer. The length of the coiled-coildomain was shown to be the determining factor of the SC CR width(Ollinger et al., 2005). SYCP2 (but not SYCP3) serves as a linker betweenAE and the TF (Liu et al., 1996; Tarsounas et al., 1997). SYCP2 interactswith SYCP3 within the axial core and with the C-terminal ends of theSYCP1 dimer embedding TFs within the LE. The N-terminal domain ofthe SYCP1 dimer forms homotypic interaction with the N-terminus of thedimer anchored in the opposite LE, connecting parallel cores in a zipper-like fashion, creating CE where the TFs overlap. Although SYCP2 has TF-binding ability, in the absence of SYCP2 and SYCP3, short stretches ofsynapsis can be found in males and almost normal levels in females (whichare fertile), suggesting that SYCP1 can directly bind to DNA (Dobson et al.,1994) or other SC protein. So far, only SYCP1 has been shown to consti-tute TFs in the mouse. Genetic analysis of Sycp1�/� mice confirmed thecritical role for SYCP1 in the SC assembly (de Vries et al., 2005). Both maleand female mutant mice fail to assemble SC but retain the ability tohomologously pair and align chromosomes. Synapsis failure results inrecombination block and meiotic arrest.

The CE can be observed by electron microscopy as an electron denseribbon-like structure in the middle of the SC. Four mammalian-specific CEcomponents have been identified. All of them have been shown to berequired for the zipper-like assembly of the CR (Fig. 5.2B). SYCE1–3can form homodimers and interact with each other (Costa et al., 2005;Schramm et al., 2011). SYCE1 and SYCE2 bind to the N-terminus ofSYCP1, presumably stabilizing the interaction within CE. SYCE3 binds toSYCE1/2, while TEX12 interacts only with SYCE2; both are onlyindirectly associated with the TF. Interestingly, presence of all four CEcomponents is required for proper assembly of TFs between two LEs(Bolcun-Filas et al., 2007, 2009; Hamer et al., 2008b; Schramm et al.,2011). For detailed review of the mammalian SC, see Yang and Wang(2009) and Fraune et al. (2012). The mature SC keeps homologs physicallylinked and acts as a scaffold for meiotic recombination but prior to synapsis,AEs seem to regulate chromosome compaction. In the absence of SYCP3/2 chromosome, cores are elongated and chromatin loops are smaller. Addi-tionally, in contrast to wild type, Sycp3�/� mutants are able to incorporateforeign transgenes into the DNA loop array, suggesting loss of chromatinattachment specificity (Kolas et al., 2004). In the absence of SYCP2/3,chromatin loops might not be attached correctly, causing release or mis-alignment of repetitive sequences, usually associated with the AE, thus


affecting pairing, synapsis, and recombination. Indeed, it has been shown bychromatin immunoprecipitation that SYCP3 associates with repetitivesequences, named LEARS—lateral elements-associated repetitivesequences (Hernandez-Hernandez et al., 2008, 2009).

In addition to the canonical components, there are other proteins thatare associated with the SC. FKBP6 (FK506-binding protein 6) is requiredfor completion/maintenance of homologous synapsis in males. FKBP6weakly associates with the chromosome cores prior to synapsis and stronglycolocalizes with the SYCP1 on synapsed chromosomes, even in the absenceof SYCP3; however, its exact function remains unknown (Crackower et al.,2003; Noguchi et al., 2008). Mouse orthologs of conserved HORMAdomain-containing proteins, HORMAD1 and HORMAD2, have beenidentified (Chen et al., 2005; Fukuda et al., 2010; Pangas et al., 2004;Wojtasz et al., 2009). Both HORMAD proteins associate with unsynapsedchromosome cores, prior to and after synapsis, and do not overlap withSYCP1. Genetic analysis revealed that HORMAD displacement fromchromosome axes depends on the presence of TRIP13 (Wojtasz et al.,2009). In the Hormad1-deficient mouse, homologous chromosomes fail topair and synapse causing recombination defects, confirming a conservedrole for mammalian HORMA domain proteins in coordinating progressionof chromosome synapsis with meiotic recombination (Daniel et al., 2011).

4.5. Initiation of recombination (DSB induction: Hot spots)

Meiotic recombination is a fundamental process for many reasons. First, itgenerates genetic diversity; allelic recombination creates new alleles andunique allelic combinations that are subjected to natural selection. Second,it results in physical links between homologous chromosomes that ensureproper chromosome segregation during the first meiotic division and correctploidy of the future gametes. Meiotic recombination requires programmedDNA DSBs for initiation. DSBs are generated by SPO11 topoisomerase(Keeney et al., 1997). Spo11-null animals lack DSBs and thus do not initiatemeiotic recombination (Baudat et al., 2000; Romanienko and Camerini-Otero, 2000). Recent findings shed more light on the regulation of SPO11activity. SPO11 has two isoforms a and b displaying different expressionpatterns in males, suggesting different roles during male meiosis (Bellani et al.,2010; Kauppi et al., 2011). Indeed, generation of mice containing onlySpo11b confirmed its major role in global DSB formation and suggestedthat the SPO11a isoform has additional function in ensuring proper pairing ofsex chromosomes (Kauppi et al., 2011). Both SPO11 isoforms are capable ofgenerating DSBs and are expressed during the whole prophase I; therefore,SPO11 activity seemed to be regulated at the posttranscriptional level. Arecent study from Scott Keeney’s group implicates ATM kinase in regulatingSPO11’s DSB-generating activity (Lange et al., 2011). Increased levels of


DSBs observed in Atm�/� spermatocytes combined with the fact that Spo11heterozygosity can partially rescue early meiotic arrest in Atm-null mice(Barchi et al., 2008; Bellani et al., 2005), prompted authors to suggest thatATM kinase acts via a negative feedback loop to regulate SPO11 activity andthe number of DSBs. SPO11-induced DSBs trigger the DNA damageresponse and activate the ATM checkpoint kinase. ATM activation resultsin a signaling cascade, probably phosphorylating SPO11 or its accessoryproteins, and this in turn leads to inhibition of further DSB formation(Lange et al., 2011).

In addition to SPO11, there are other proteins that have been geneticallyshown to be critical for generation of DSBs. Most of them have been foundin yeast, but their mammalian orthologs have not been identified yet ortheir role is not conserved (Keeney, 2008; Maleki et al., 2007). Theexception seems to be the two recently identified SPO11 accessory proteinsMEI4 and REC114 (Kumar et al., 2010).Mei4-null mice display Spo11-likephenotype; both males and females are DSB deficient and fail to initiaterecombination. REC114’s role has not yet been established and awaitsgeneration of mutant mice. The exact biochemical function of MEI4/REC114 is not known, but parallels can be drawn from yeast. It has beenrecently proposed that S. cerevisiae Mei4/Rec114 proteins play a role intethering future DSB sites to the chromosome axis (Panizza et al., 2011).The only other protein in mice demonstrated to play a role in DSBgeneration is MEI1 (meiosis defective 1). It was identified in forwardgenetic chemical mutagenesis screen for genes involved in mammalianfertility, and so far, its only nonmammalian functional ortholog wasidentified in Arabidopsis thaliana (AtPRD1) (De Muyt et al., 2007; Libbyet al., 2002). Although MEI1’s biochemical function remains unknown(AtPRD1 interacts with AtSPO11), functional analysis of mutants produc-ing a truncated form of MEI1 clearly shows its essential role in DSBgeneration. Mei1�/� oocytes and spermatocytes lack DSBs as shown bythe absence of DNA damage markers (gH2AX and RAD51) and pheno-copying Spo11-null animals. For a recent detailed review of recombinationinitiation see Kumar and De Massy, 2011.

Analysis of recombination events in various model organisms revealedthat DSBs are nonrandomly distributed throughout the genome. Theytend to cluster in discrete genomic regions called hot spots and are under-represented in other regions called coldspots. Recombination hot spots inmice and humans were first identified by mapping CO events in pedigrees,populations or sperm. It has been observed in mouse that the landscape ofrecombination hot spots differs between strains and sexes (Baudat and deMassy, 2007). The strain differences were exploited to genetically map andidentify the first trans-acting hot spot-regulating locus on chromosome 17 inmouse (Grey et al., 2009; Parvanov et al., 2009). The mapped locuscontained Prdm9 (Parvanov et al., 2010). Interestingly, Prdm9 has been


previously shown to be critical for meiotic progression (Hayashi et al.,2005). Moreover, Prdm9 has been implicated in cross-hybrid sterility(Mihola et al., 2009). Prdm9 encodes H3K4 histone methyltransferase.It also contains a tandem array of 12 DNA-binding zinc fingers whichdetermine sequence specificity of the PRDM9 DNA-binding motif(Baudat et al., 2010). Analyses of hot spot features revealed strong correla-tion with sites of H3K4 trimethylation (Buard et al., 2009), providing anadditional link between PRDM9 and regulation of hot spots. Even moreconvincing is the finding of Smagulova et al. (2011) that the zinc finger-binding motif enriched at hot spot sites matches the predicted PRDM9-binding sequence of the same mouse strain. However, H3K4me3 on itsown is not sufficient to explain recombination site specification. It seemsthat multiple histone modifications contribute to the regulation of hot spotuse (Buard et al., 2009; Grey et al., 2009). Emerging data suggest multiplelevels of regulation. First, on the DNA level, specific motifs could bringcertain proteins to the potential hot spot site, such as methyltransferases ortranscription factors that in turn would alter chromatin structure. On thesecond level of regulation, changes in chromatin state such as nucleosomeoccupancy (Getun et al., 2010) or histone modifications (Buard et al., 2009)would further refine hot spot activity. Finally, it seems that the highest degreeof regulation takes place at the chromosome organization level. The differ-ences in male and female recombination rates may be attributable to thelength of the SC and DNA loop size, which in females are longer and smaller,respectively, translating to higher recombination frequencies. A similar phe-nomenon was observed in the pseudoautosomal region (PAR) of X and Ychromosomes. Considering the genomic size of the PAR region, it is packedinto much longer axis than expected with shorter chromatin loops (Kauppiet al., 2011) and shows clustering of overlapping hot spots (Smagulova et al.,2011). Therefore, higher order chromatin structure executed by cohesins andaxial core proteins, known to influence DNA loop size, can also affect DSBdistribution. Currently, the finest analysis of mouse DSB distributions isdescribed in Smagulova et al. (2011).

4.6. Recombination (DSB repair: NCO vs. CO)

The estimated 200–300 programmed meiotic DSBs create high levels ofDNA damage, which if left unrepaired is lethal to the cell. In contrast tomitotic DSB repair, which occurs mostly using the nonhomologous end-joining pathway, meiotic breaks are repaired by homologous recombination(HR) (Andersen and Sekelsky, 2010). This pathway is not only less errorprone but also capable of generating interhomolog (IH) connections (CO),so crucial for chromosome segregation. However, only a subset of DSBsresults in formation of COs; the majority of DNA breaks are resolved asNCO products (gene conversions). Both CO and NCO repair pathways


start with resection of the 50-end of the break, creating a 30-single-strandedoverhang able to pair invade a homologous repair template. The choice ofthe template is crucial for the outcome of HR, as only the use of nonsisterchromatid can result in CO. However, the sister chromatid is in closerproximity. To avoid its use as a template, a mechanism evolved to preventHR between sisters and/or to promote recombination between nonsisters.Evidence from yeast suggests that NCO products emerge by synthesis-dependent strand annealing and they precede the appearance of COs(Terasawa et al., 2007). On the other hand, COs are formed in independentpathways and involve double-Holliday junction (dHJ) intermediates. Theexistence of independent NCO and CO pathways has been also describedin the mouse (Guillon et al., 2005). DSB repair via HR is a fundamentalprocess for genomic stability which is reflected in a high level of conserva-tion between organisms as well as mitotic and meiotic processes (Andersenand Sekelsky, 2010). Here, we will focus on some recent developments inregulation of meiotic recombination in mouse.

DSBs generated in early prophase are resected and form single-strandedDNA to which RAD51 and DMC1 recombinases are recruited, formingearly recombination nodules. RAD51/DMC1-coated nucleofilaments pro-mote homology search and strand exchange, to form joint molecule inter-mediates, and are then displaced from synapsing chromosomes asrecombination intermediates are processed. DMC1 is known to be requiredfor proficient DSB repair (Pittman et al., 1998; Yoshida et al., 1998). TheTex15-null mutant, which fails to recruit DMC1 and RAD51 to DSB sites,fails in DSB repair and synapsis. However, unlike Dmc1�/� mutants, Tex15deficiency is sexually dimorphic; males are infertile due to meiotic arrest,while females show normal meiotic progression and fertility (Yang et al.,2008a). This sexually dimorphic requirement for TEX15 in RAD51/DMC1 recruitment is perplexing and would suggest different regulationsof recombination between sexes. As homologs synapse, RAD51/DMC1are being replaced and recombination nodules transiently associate withRPA and MSH4/MSH5 before they acquire late recombination nodulecomponents MLH1/MLH3 (Plug et al., 1998). Mismatch repair proteins ofthe MutS (MSH4/5) and MutL (MLH1/3) families are essential for meioticrecombination (de Vries et al., 1999; Edelmann et al., 1996; Kneitz et al.,2000; Lipkin et al., 2002).

One enigmatic story concerns TEX11 (ZIP4H), an ortholog of yeastZip4 protein required for synapsis and CO distribution (Tsubouchi et al.,2006). However, two studies describing genetic analysis of Tex11-null miceindicate that mammalian TEX11/ZIP4H is generally dispensable for synap-sis (although one of the mutants shows individual asynaptic chromosomes).It is required for timely DSB repair and normal numbers of COs. In theabsence of TEX11/ZIP4H, DMC1-containing recombination nodulespersist longer and coexist on the same SC with the MLH1 containing late


nodules, suggesting delayed or defective repair (Adelman and Petrini,2008). Additionally, numbers of COs were significantly decreased resultingin meiocytes with achiasmate chromosomes (Adelman and Petrini, 2008;Yang et al., 2008b). One of the groups developed specific antibodiesshowing TEX11/ZIP4H focal localization to synapsed regions in zygoteneand disappearing by late pachytene. Colocalization analysis showed thatTEX11/ZIP4H mostly colocalizes with RPA and less so with MSH4, butnot with DMC1 or MLH1, suggesting it could be a new component of“transition nodules” (Plug et al., 1998). Although each study identifieddifferent interacting partners, the exact function of TEX11/ZIP4H remainsunknown. Yang et al. identified, and confirmed by Co-IP, SYCP2 as aninteracting partner and suggested TEX11/ZIP4H involvement in synapsis,it needs to be mentioned that only this group described synapsis defects(completely asynapsed pairs next to fully synapsed chromosomes that con-stitute the majority). Adelman et al. have not described synaptic defects intheir mutant. Using yeast two-hybrid experiments, they have identifiedinteraction with NBS1, a component of Mre11 complex involved inmany aspects of DSB repair (Stracker et al., 2004). Although this interactionpresents attractive link between TEX11/ZIP4H and recombination, it hasnot been confirmed in vivo.

Another interesting meiotic recombination protein identified recently isthe vertebrate-specific SPATA22, found to be required for meiotic progres-sion (La Salle et al., 2011). In SPATA22 deficient mice DSBs were formed atnormal levels as judged by recruitment of RAD51 but failed to be repaired.Chromosome synapsis was severely affected; only short stretches of SYCP1were observed in males and only rare fully synapsed single chromosomes infemales. Apart from a few BRCT-binding motifs and SQ/TQ sites forATM/ATR kinases, there is no further clue to SPATA22 function.

The choice of template for DSB repair is essential for generation of IHconnections. The regulation of partner choice in meiosis has been extensivelyinvestigated in yeast due to available methods allowing distinction betweenintersister (IS) and IH repair intermediates. The repair template choice, or IHto IS bias, is thought to be established early, prior to, or at the time of strandinvasion. It ismediated byRAD51 andmeiosis-specific DMC1 recombinases,with the latter executing the IH and the former performing primarily ISrecombination in concert with RAD54. Emerging data from budding yeastsuggest that there exists a barrier to sister chromatid repair (BSCR) and itinvolves impeding Rad51 ability to perform IS recombination. This isachieved in two ways: (1) axial protein Red1 binds to Rad51 preventingRad51/Rad54 association; (2) phosphorylation of Hop1 (HORMA domainprotein) in response to DSBs by Mec1/Tel1 (ATR/ATM) activates Mek1kinasewhich in turn phosphorylatesRad54, reducing its affinity toRad51 andpossibly other targets involved in BSCR (Callender andHollingsworth, 2010;Wu et al., 2010). The Mek1-mediated BSCR seems to be independent of


Rec8 cohesion (Callender and Hollingsworth, 2010). However, it has beenalso suggested that the IH:IS bias is implemented at the level of chromosomeorganization by differential release of DSB ends. In this scenario, Rec8promotes IS bias, keeping sister chromatids in close proximity and this bias islocally counteracted by Hop1/Mek1 pathway (Kim et al., 2010). Anotherstudy suggests that Mek1-mediated BSCR could be, in fact, a “kineticimpediment” slowing down the processing of IS recombination to allowcompletion of slower IH repair. Regardless of what can be concluded fromyeast experiments, it is difficult to extrapolate to mouse due to lack of clearorthologs (Red1, Mek1) or difference in mutant phenotypes (Rec8). Theputative Hop1 orthologs HORMAD1 and HORMAD2 were identified inmammals as components of unsynapsed AEs (Chen et al., 2005; Fukuda et al.,2010; Wojtasz et al., 2009). HORMAD1-deficient mice are characterized bydefective synapsis and persistent DSBs, resulting in meiotic arrest in males.Females progress through meiosis but are infertile due to aneuploidies causedby lower CO numbers (Shin et al., 2010). HORMAD1 was implicated in achromosome synapsis “checkpoint” because Hormad1 nullizygosity rescuedsurvival of Spo11�/� oocytes (Daniel et al., 2011). Interestingly,Hormad1�/�

mutant ovaries, despite synapsis defects and persistent DSBs, show increasednumbers of oocytes compared to wild type. One explanation is thatHORMAD1 deficiency allows repair by sister chromatids, thereby suggestinga conserved role in IS:IH bias (Shin et al., 2010). It is possible thatHORMAD2 plays a redundant role.

Genetic studies of multiple mutants in our laboratory suggest that mam-malian AEs themselves, or their components SYCP2/SYCP3, influencepartner choice in favor of the IH recombination (Li et al., 2011). Meioticmutants such asDmc1�/�, Trip13�/� and others show abnormal synapsis andpersistent DNA damage causing checkpoint-mediated oocyte death aroundbirth, and complete elimination by 3 weeks of age. We observed that in theabsence of certain AE components,Dmc1�/� and Trip13�/� mutant oocytescan survive the initial elimination, and that this “rescue” is abolished in theabsence of RAD54. This, and the finding that surviving oocytes have reducedmarkers of DSBs compared to those with intact AEs, suggests that eliminationof SYCP3/2 (or functional AE) allows DSB repair using IS recombination.Lack of suitable methodologies to assay IS versus IH recombination con-founds investigations into mammalian partner choice regulation.

4.7. Chromosome segregation: Chiasmata resolution andremoval of abnormal recombination intermediates

The preference of IH over IS recombination for DSB repair allows genera-tion of joint molecules between maternal and paternal chromosomes. Ifboth ends of DSBs are engaged in interaction with the same nonsisterchromatid, they form dHJs that can be resolved into COs. The identity of


the meiotic dHJ resolvase in mouse is still unclear. It seems that differentorganisms use different pathways to generate COs (Schwartz and Heyer,2011). In mouse, the MLH1/MLH3-dependent pathway is responsible forthe majority of CO events (Guillon et al., 2005). Most fission yeasts and athird of budding yeast COs occur in the Mus81-dependent pathway(Schwartz and Heyer, 2011). A recent study suggests that this pathwayalso exists in mammals (Holloway et al., 2008). One can imagine thatwithout some regulation, the two ends of the DSB could invade twodifferent chromatids forming multichromatid joint molecules (mcJMs),which could cause aberrant COs. Indeed, large JMs involving three oreven four chromatids have been detected in yeast and shown to be removedby the action of Sgs1 (ortholog of Bloom helicase, BLM) (Oh et al., 2007).A similar role for mammalian BLM was recently described in a conditionalmouse mutant (Holloway et al., 2010). In the absence of BLM, aberrantmcJMs accumulate in mutant cells, also involving nonhomologous chro-matids. Additionally, cells that progress to metaphase I exhibit increasednumbers of chiasmata on single chromosomes and chiasmata-like structuresbetween chromosomes. Thus, mammalian BLM is required to resolvemcJM and prevent nonhomologous synapsis and aberrant COs.

4.8. XY pairing and silencing

In heterogametic organisms, pairing of sex chromosomes with limitedhomology presents an additional challenge for the meiotic machinery. Inthe mouse, X and Y chromosomes are of different sizes and share only a smallregion of homology, called the Pseudoautosomal Region (PAR). Properpairing and generation of COs/chiasmata ensure correct segregation of sexchromosomes. When compared to autosomes, the size of the PAR availablefor pairing is minute. Kauppi et al. (2011) recently described a mechanism bywhich meiotic recombination machinery ensures pairing of sex chromo-somes. They have observed that (1) pairing of XY occurs later than auto-somes, (2) additional DSBs on the XY pair form late in zygonema and aregenerated by SPO11a isoform, (3) those late DSBs appear concurrent withsex body (see below) which may facilitate or maintain XY pairing, and (4) thePAR AE length/loop size ratio suggests a higher frequency of DSBs (dis-cussed in Section 4.5). Thus, to ensure pairing, sex chromosomes receiveadditional late DSBs that appear to serve two purposes: one is to locallyactivate DDR machinery that results in formation of the sex body aroundthe XY pair, and the other is to facilitate homology searching and stableinteractions via joint molecules. In the absence of SPO11a, the majority ofmeiocytes contain unsynapsed X and Y chromosomes (Kauppi et al., 2011).

During pachynema and diplonema, sex chromosomes are sequestered to aspecialized chromatin domain called sex body. This chromatin domain isenriched in heterochromatinmarks and suggested to transcriptionally inactivate


sex chromosomes (meiotic sex chromosomes inactivation—MSCI) (Heard andTurner, 2011; Turner, 2007). Meiotic mutants defective in chromosomesynapsis or recombination display MSCI failure and arrest at prophase I(Burgoyne et al., 2009). It was speculated that expression of sex-linked geneswould be detrimental to male meiosis (Kelly and Aramayo, 2007;Wu and Xu,2003). Indeed, it has been shown that in XYYmales, the two Y chromosomessynapse and evade MSCI causing meiotic arrest. This suggests that transcrip-tional inactivation of the Y chromosome during pachynema is essential formeiotic progression. Direct evidence comes from the observation that expres-sion of Y-linked transgenes from an autosome causes the same meiotic arrest asin XYY or mutants with defective MSCI (Royo et al., 2010). The authorsfurther suggested that the X also contains pachytene-lethal genes.

The mechanism of sex body formation is not yet fully understood, butrecent studies suggest an important role for MDC1 (mediator of DNAdamage 1). MDC1 is a gH2AX-binding partner and was previously shownto function in somatic DNA damage responses (Goldberg et al., 2003; Louet al., 2003; Stewart et al., 2003). Upon entry into pachynema, BRCA1,TOPBP1, and ATR coat unsynapsed AEs of the sex chromosomes, followedby spreading of ATR and TOPBP1 into the X and Y chromatin domainscoinciding with phosphorylation of H2AX (gH2AX) (Turner, 2007). In theabsence of MDC1, the recruitment of BRCA1, TOPBP1, and ATR isunaffected as they are DDR dependent. However, TOPBP1, ATR, andgH2AX failed to spread across XY chromatin, and thus typical heterochro-matic sex bodies do not form. X and Y genes became derepressed due toMSCI failure. Additionally, other marks usually acquired by the sex body inpachynema were absent or mislocalized (SUMO1, FK2, ubi-H2A, XMR,H2K27me3) (Ichijima et al., 2011). The authors propose that MSCI consistsof two steps. One is the MDC1-independent recruitment of DDR factors tounsynapsed axes of sex chromosomes. The other is the MDC1-dependentchromosome-wide spreading of DDR proteins to the entire chromatin.

Recently, a new outlook on sex chromosome inactivation was proposedby Page et al. (2012). Based on the spatiotemporal dynamics of transcrip-tional activation/inactivation and MSCI marks, the authors suggest thatMSCI functions in maintenance or reinforcement of a transcriptionallyinactive state globally persisting from leptonema to mid-pachynema. Inother words, transcriptional silencing of sex-linked genes is a result ofspecific lack of reactivation of transcriptionally silent chromatin of X andY chromosomes and not silencing of previously active chromosomes.

4.9. Checkpoint control

Meiosis involves coordination of many cellular processes in order to achievecorrect chromosome number in gametes. Failure to do so can have disas-trous consequences for the developing embryo. Aneuploidies are generally


lethal in utero or cause genetic diseases in surviving embryos. As in themitotic cycle, there are specialized meiotic checkpoints, preventing gener-ation of abnormal gametes. Cells undergoing meiotic division have tocoordinate two major processes: DSB repair and chromosome synapsis.Errors in either can activate meiotic checkpoints that either halt the cellcycle to allow more time for repair or activate apoptosis to remove defectivecells. In mammals, the meiotic checkpoint is often called the pachytenecheckpoint because it prevents cells from exiting pachynema before recom-bination and synapsis are completed. However, it can be also separated intorecombination (DNA damage) and synapsis checkpoints, as seen in Trip13and Spo11 mutants, respectively (Baudat et al., 2000; Li and Schimenti,2007; Romanienko and Camerini-Otero, 2000). In males, the recombina-tion and synapsis checkpoints have the same outcome—elimination ofdefective cells in pachynema (epithelial stage IV) (Hamer et al., 2008a). Infemales, both are less stringent and cells often complete meiosis I, althoughwith frequent aneuploidies (Hunt and Hassold, 2002). The obvious differ-ence between the two sexes is the presence of the heterologous pair ofchromosomes, X and Y. As described in the previous section, pairing andsequestration of the X and Y to the silencing environment of the sex bodyare essential for meiosis. Failure to silence sex-linked genes also results inpachytene stage IV arrest (Heard and Turner, 2011; Royo et al., 2010).Interestingly, recombination and synapsis defects often go together withMSCI failure; therefore, it has been proposed that it is the MSCI defect thatcauses the meiotic arrest at stage IV (pachynema). An exception comes fromthe Trip13mutant (Li and Schimenti, 2007), which displays normal synapsisand sex body formation but has persistent DNA breaks and male meiosisarrests at epithelial stage IV. Spo11 mutants arrest at the same stage, butbeing deficient in DSB formation, the arrest is attributed to asynapsis and/orMSCI failure.

The mechanism behind the synapsis checkpoint (whether or not it isdirectly attributed to failed MSCI) is unclear. Chromosome cores that failedto synapse are coated with BRCA1. This recruits the ATR kinase, which inturn phosphorylates histone H2AX, leading to meiotic silencing of unsy-napsed chromatin (MSUC) (Burgoyne et al., 2009; Turner et al., 2005),affecting autosomes in a manner analogous to MSCI. Thus, one idea is thattranscriptional silencing of unsynapsed chromosomes may affect meioticprogression if the silenced chromosome segment contains genes required formeiotic processes. How asynapsis affects meiotic progression has been asubject of many studies (Burgoyne et al., 2009; Manterola et al., 2009;Turner et al., 2005). It is clear now that extensive asynapsis leads to MSCIfailure, but how much asynapsis would be detrimental to the meiotic cell?A recent study addressed this question by increasing the size of autosomalasynapsis and analyzing MSUC response and its consequence on meiosis(Homolka et al., 2012). Using mice bearing an autosomal translocation


T(16;17)43H (Forejt et al., 1980) and the t12 haplotype, a naturally occurringvariant of chromosome 17 carrying four tandem inversions (Schimenti, 2000),authors were able to manipulate the size of asynapsed chromosomes. Theyshowed that the strength of MSUC response and the severity of spermatogenicdefect are proportional to the extent of asynapsis. They were also able to showthat the MSUC response precedes MSCI and therefore could interfere withMSCI. However, the authors conclude that because MSUC is initiated in latezygonema/early pachynema and epithelial stage IV arrest happens at mid/latepachytene, it is unlikely that MSUC/asynapsis is the cause of arrest; rather it isthe interference with MSCI. However, it is still not clear if there is a singularcheckpoint response to meiotic defects or the pachytene arrest is the sum ofMSCI, recombination, and synapsis checkpoints.

It is now widely accepted that in mammals, female meiotic checkpoints areless stringent and thus responsible for chromosome aneuploidies. However, itis not understood why. Work in C. elegans might suggest an explanation. Infemale and male worms, the same checkpoint is activated in response tomeiotic errors; however, the downstream outcomes differ. Females activateapoptotic responses to eliminate damaged oocytes, while males prefer toactivate repair response ( Jaramillo-Lambert et al., 2010). If a similar mecha-nism exists in mammals, however in reverse, it could explain sexually dimor-phic phenotypes of meiotic mutants. Meiotic defects in spermatocytes activatea meiotic checkpoint that results in apoptosis. In females, the same defectswould also activate a meiotic checkpoint; however, the first response would beto salvage the oocyte and repair the defects. The choice of downstreamresponses makes sense if we take into account that all four meiotic productsin males become sperm and only one becomes an oocyte in females. Addi-tionally, waves of spermatogenesis produce millions of sperm throughout thereproductive life of the male, whereas female meiosis takes place only once andfemale is born with a set number of oocytes. Therefore, investing in repairinstead of elimination may be beneficial for reproductive success.

Despite the tremendous progress in the understanding of the meioticcheckpoint in yeast and worms (MacQueen and Hochwagen, 2011), theactual mechanisms of mammalian checkpoint control are not known.Orthologs of yeast meiotic checkpoint genes often do not exhibit check-point activity in mouse (Atm/Tel1 and Trip13/Pch2; Li and Schimenti,2007; Roig et al., 2010; Xu et al., 1996) or play essential roles in somaticcells, thus confounding the analysis. It is possible that the proper genes haveyet to be tested.

4.10. Small RNAs

Since their discovery, small noncoding RNAs have been intensively studiedin various organisms and cellular processes (Li and Liu, 2011). Small RNAshave been also identified in the mouse germ line, the most predominant of


which are piRNAs (PIWI-interacting small RNAs) in male germ cells(Castaneda et al., 2011). However, miRNA, piRNAs, and siRNAs werealso identified in mouse oocytes (Aravin et al., 2008; Kuramochi-Miyagawaet al., 2001; Murchison et al., 2007; Suh et al., 2010; Tang et al., 2007;Watanabe et al., 2008). The importance of piRNA for meiotic progressionhas been revealed by mouse mutants defective in their biogenesis;spermatocytes lacking piRNAs arrest at pachynema (Frost et al., 2010;Kuramochi-Miyagawa et al., 2004; Soper et al., 2008; Zheng et al., 2010).piRNAs allow recognition and silencing of TEs by DNA methylation(Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008). piRNAs are25–32 nt in length and are defined by their association with PIWI proteins.The exact mechanism of piRNA biogenesis is not yet known, but it hasbeen proposed that they are produced in a “ping-pong” amplification cycle(Aravin et al., 2007; Brennecke et al., 2007; Gunawardane et al., 2007). Inmouse, piRNAs are generated from long, single-stranded RNA precursors,often encoded by clusters of repetitive intergenic sequences, that are pro-cessed to functional size small RNAs. There seem to be two independentcellular compartments in mouse germ cells where piRNAs are generated.The piRNA-body or pi-body is where presumably the primary piRNAs aresynthesized, and the piRNA-processing body or piP-body is where thesecondary piRNAs are generated (van der Heijden et al., 2010). Immuno-localization studies of piRNA pathway proteins showed MOV10L1, MILI,GASZ, and TDRD1 in pi-bodies, whereas MAEL, MIWI2, and TDRD9were found in piP-bodies.

Analyses of mouse mutants deficient for different PIWI and PIWI-associated proteins have revealed some aspects of piRNA biogenesis inmice. MILI is essential for most piRNA synthesis; it binds primary piRNAsand is required for piP-body formation and secondary piRNA amplification(Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008). Absence ofMOV10L1 completely abrogated piRNA synthesis, indicating its functionin generating primary piRNA or their loading to MILI (Frost et al., 2010;Zheng et al., 2010). GASZ (Germ cell protein with Ankyrin repeats, Sterilealpha motif, and leucine Zipper) deficiency results in downregulation andmislocalization of pi-body components MILI and TDRD1 and reduction ofprimary and secondary piRNAs (Ma et al., 2009). MAEL is required forformation of piP-body and timely production of primary piRNAs (Soperet al., 2008). TDRD9 and MIWI2 interact in the piP-body and are requiredfor secondary piRNA biogenesis (Aravin et al., 2008; Kuramochi-Miyagawaet al., 2008; Shoji et al., 2009). Recently, a mitochondrial proteinMITOPLDhas been shown to be important for generation of primary piRNA andcorrect localization of p-body and piP-body components (Watanabe et al.,2011). piRNA biogenesis components are usually found in regions close tomitochondria and used to be called intermitochondrial cement. The Mitopldmutant reveals that contact with mitochondria is critical for piRNA


biogenesis. A lack of aforementioned proteins leads to loss or reduction ofpiRNAs and failure to silence TEs, resulting in synapsis defects in spermato-cytes. Although piRNAs were also found in oocytes, female meiosis isunaffected by PIWI protein/piRNAs deficiency, possibly reflecting differenttimings of meiosis with respect to DNA remethylation. There is only limitedinformation about other small RNA functions in female germ cells. Loss ofDicer and Ago2 (involved in miRNA and siRNA biogenesis) shows post-prophase I phenotypes; mutant oocytes display severe spindle and chromo-some segregation defects (Kaneda et al., 2009; Murchison et al., 2007; Suhet al., 2010; Tam et al., 2008).

4.11. Cell cycle regulation and exit from prophase I

Cell cycle regulation is probably one of the least understood aspects ofmeiosis. After DNA replication, meiocytes enter prolonged G2 phase, duringwhich meiosis-specific processes, synapsis and recombination, take placeleading to a formation of linkages between homologous chromosomes.This physical connection between homologs (chiasmata) ensures propercoalignment of chromosomes at the meiosis I spindle (Bascom-Slack et al.,1997). To form these linkages, meiotic cells endure hundreds of DNA DSBsthat need to be repaired in the process of meiotic recombination, in order toachieve at least one chiasma per chromosome. The aforementioned meioticcheckpoints ensure that cells do not exit meiotic G2 (prophase I) until allbreaks are repaired. However, regulation of prophase I exit is sexuallydimorphic. In males, spermatocytes progress to metaphase I (MI) immedi-ately following completion of CO recombination and then undergo thesecond meiotic division. In females, oocytes arrest at G2 (diplotene/diakine-sis) around birth and progress to MI after ovulation. The second meioticdivision takes place after fertilization.

The signature events of G2/MI transition are disassembly of the SC,phosphorylation of histone H3 on Ser10, and chromosome condensation.Desynapsis requires activity of the chaperone heat-shock protein HSPA2that localizes to the SC. In its absence, SYCP1 fails to be removed from theCE (Dix et al., 1997; Sun et al., 2010). Other factors involved in this earlystage of G2/MI transition remain unknown. Phosphorylation of histoneH3Ser10 seems to be regulated by AURKs kinases. MPF (metaphase-promoting factor) and cyclin-dependent kinases (CDKs) regulate later stagesof the G2/MI transition (post desynapsis)(Sun and Handel, 2008).

The key factors in cell cycle regulation—cyclins and CDKs—have beenextensively studied for their role in meiosis (Wolgemuth, 2008, 2011;Wolgemuth and Roberts, 2010). CDK1 is the master regulator of themammalian cell cycle and is indispensable for embryo development. Defi-ciency for cyclins A2 and B1 also causes embryonic lethality, confoundingstudies of their potential roles in meiosis. Of many cyclins expressed in


meiotic cells, only cyclin A1 (Ccna1) has been shown to be essential for meioticdivision, while cyclin E2 (Ccne2) deficiency causes reduced fertility, butwithout clear-cut arrest (Geng et al., 2003; Salazar et al., 2003, 2005). CyclinA1 is a catalytic partner of CDK2, and mutants of both proteins arrest beforemeiosis I. However, Cdk2-deficient mice display a more severe phenotype.Cdk2-null spermatocytes arrest in pachynema with abnormal synapsis andrecombination (Viera et al., 2009), while Ccna1 mutant spermatocytes prog-ress to diplonema (Nickerson et al., 2007). Therefore, CDK2, possibly withits other partners, plays additional roles during prophase I.

Interestingly, a microtubule-associated protein MTAP2 has been recentlyreported essential for exit from prophase I.Mtap mutant spermatocytes failedto undergo G2/MI transition in vivo and in vitro (following okadaic acidtreatment) despite acquiring all marks of the transition competence (Sunand Handel, 2011). Thus, this is the first genetic evidence linking micro-tubules and microtubule-associated proteins in meiotic cell cycle regulation.

4.12. Protein modification during prophase I:Phosphorylation, SUMOylation, ubiquitination,methylation, acetylation, etc.

Covalent modifications such as phosphorylation, SUMOylation, methyla-tion, and acetylation play an important role in regulating protein activity,conformation, or cellular localization. Meiotic DNA damage responseinvolves cascade of phosphorylation events mediated by ATM/ATRkinases and their downstream effector kinases. Probably, the best knownphosphorylation target is the histone H2AX phosphorylated by ATM inresponse to DSBs. Histone H2AX is also phosphorylated by ATR in theprocess of MSCI and MSUC. Many components of the SC are alsophosphorylated during meiosis. SYCP2/3, HORMAD1/2, SMC3/1b,STAG3, and REC8 show spatiotemporal phosphorylation patterns andhave been proposed to label chromosome axes according to their synapsisor repair status (Fukuda et al., 2012). The importance of protein phosphor-ylation during meiosis is best illustrated by regulation of IS:IH recombina-tion bias in yeast, which is crucial for formation of COs (see Section 4.6).Phosphorylations of Hop1 and Rad54 prevent recombination betweensister chromatids (Callender and Hollingsworth, 2010; Carballo et al.,2008; Wu et al., 2010). Unlike in other model organisms, it is difficult tostudy function of specific phosphorylation sites by mutagenesis in mammals;however, their roles can be inferred from immunolocalization studies usingvarious meiotic mutants (Fukuda et al., 2012).

SUMO (small ubiquitin-like modifier) has emerged as a potential regu-latory mechanism involved in meiotic processes. Conjugation of SUMOinvolves E1-activating enzyme (Sae1–Sae2), E2-conjugating enzyme(Ubc9), and multiple often tissue-specific E3 ligases (Wang and Dasso,


2009). SC assembly in budding yeast is orchestrated by SUMO E3 ligaseZip3. Further, AE component Red1 and CE component Zip1 containSUMO-interacting motifs (SIMs), and Red1 has been shown to beSUMOylated (Cheng et al., 2006; de Carvalho and Colaiacovo, 2006).Inmammals, the role of SUMO inmeiosis remains enigmatic. However, theonly SUMO E2-conjugating enzyme—UBC9 (UBE2I)—has been shownto localize to the SC and SUMO1/2/3 has been detected in spermatocytes atthe XY body and heterochromatic chromocenters (La Salle et al., 2008;Rogers et al., 2004), implicating SUMO in meiotic processes. Unfortu-nately, Ube2i-null embryos die early after postimplantation, confoundinganalysis of its role in meiosis (Nacerddine et al., 2005). On the other hand,Sumo1�/� mice are fertile, suggesting that either SUMO1 is dispensableduring meiosis or SUMO2/3 can compensate in its absence (Zhang et al.,2008). One study detected SUMO1 conjugates in the SC of human pachy-tene spermatocytes and showed SUMO1 modification of SYCP1 andSYCP2 but not of SYCP3 in human testicular extracts (Brown et al.,2008). In the mouse, SUMO conjugates are mainly detected at the XYbody suggesting its role in MSCI. Although SUMO-modified proteins areabundantly detected in spermatocytes, their identity and function are largelyunknown. The identity of SUMOylation targets is determined by E3 ligases.PIAS family proteins have been shown to possess E3 ligase activity, and theyare expressed in spermatocytes. However, there are no reports suggesting theirrole during meiosis. Recently, a novel putative meiosis-specific E3 ligase hasbeen identified in mouse (Strong and Schimenti, 2010). CCNB1IP1 containsRING domain typical of E3 ligases and interacts with SUMO2 and proteinswith consensus SUMOylation site. Ccnb1ip1mutant mice are infertile due toCO failure, suggesting a role for SUMOylated proteins in maturation ofrecombination intermediates. For recent review of SUMO function in malereproduction see Vigodner, 2011.

Ubiquitination has been studied in many aspects of cell biology: cellcycle control (Mocciaro and Rape, 2012), DNA replication and damagerepair (Ghosh and Saha, 2012), and gametogenesis (Baarends et al., 1999b,2000). Ubiquitin conjugates are mainly found at the XY body and unsy-napsed chromosomes in spermatocytes and can be detected using FK2antibodies (Fujimuro and Yokosawa, 2005). One of the best known targetsof ubiquitination, histone H2A, is also enriched in the sex body (Baarendset al., 1999a). Similar to the SUMO pathway, attachment of ubiquitinrequires a hierarchical multistep process involving E1, E2, and E3 enzymes.There are two E1 activation enzyme genes in the mouse, Ube1x and Ube1y,present on the X and Y chromosomes, respectively. Interestingly, the Ycopy was lost in the primate lineage. Their functions in meiosis remainunknown due to lack of mutant mouse models. However, if similar toSUMO pathway, E1 deficiency could cause broad developmental defectsleading to embryo lethality. One of the E2-conjugating enzymes HR6B is


required for spermatogenesis, while HR6A seems to be dispensable or com-pensated by HR6B (Baarends et al., 2003, 2007; Mulugeta Achame et al.,2010; Roest et al., 1996, 2004).Hr6b-null males are infertile due to abnormalpostmeiotic sperm differentiation. HR6B has been shown to control histonemodifications in the XY body (Baarends et al., 2007;Mulugeta Achame et al.,2010). Specificity of ubiquitination targets is determined by E3 ligases. TheE3 ligase UBR1 is dispensable for meiosis and Ubr1-null mice are fertile(Kwon et al., 2001), while Ubr2-deficient males are infertile and femalesare subfertile and partially lethal (An et al., 2010; Kwon et al., 2003). Malesterility has been shown to result from failure to silence sex-linked genes (Anet al., 2010) although synapsis defects were also observed in certain mousestrain backgrounds (Kwon et al., 2003). An ortholog of yeast E3 ligaseRAD18Sc can be detected in pachytene and diplotene spermatocytes at theXY body and colocalizes with HR6B (van der Laan et al., 2004). Rad18knockdown mutants obtained by expression of shRNA suggest a role forRAD18 together with HR6B in signaling of chromosome pairing defectsthat leads to transcriptional silencing and thus a role in MSCI/MSUC (vander Laan et al., 2004). Ubiquitinated histone H2A (ubiH2A) has been linkedto transcriptional silencing of X-linked genes (MSCI) (Baarends et al., 2005).Interestingly, mutant spermatocytes deficient for E3 ligase RNF8 shownormal MSCI and meiotic progression despite the absence of ubiH2A andubiquitin conjugates in the sex body (Lu et al., 2010; Ma et al., 2011).

Histone modifications show dynamic spatiotemporal patterns duringprophase I, suggesting an important role during meiosis. Histone H3Lysine4 trimethylation and dimethylation (H3K4me3 and H3K4me2) andLysine9 acetylation (H3K9ac) and histone H4 hyperacetylation are asso-ciated with recombination hot spots (see Section 4.5) (Buard et al., 2009).Various histone modifications mark pericentromeric chromatin H3K9me2,H3K9me3, H3K27me1, H4K20me3, H4K5ac, H4K16Ac (H3K27me1)(Khalil and Driscoll, 2010; Khalil et al., 2004; Namekawa et al., 2006).Certain histone marks are either specific to the sex body (H3K9me2) orexcluded from sex body (H4K16ac, H3K9ac, H3K27me1, H3K27me3)(Khalil et al., 2004; Namekawa et al., 2006). The dynamics of histonedeacetylation and methylation within XY body and pericentromericchromatin correlates with transcriptional inactivation.

4.13. Concluding remarks

In recent years, there has been tremendous progress in understandingprocesses governing meiosis and recombination. We have just started tounderstand the regulation of recombination partner choice that has a hugeimpact on CO and DSB repair. We obtained more insight into regulation ofDSB formation and the molecular signatures of recombination hot spots.New and exciting processes have also been uncovered including the


involvement of small RNAs and SUMO conjugates. However, our under-standing of mammalian meiosis still lags well behind that of simpler eukary-otic model organisms such as yeast and worms. Studying meiosis inmammals is complicated for many reasons. The existence of two sexes,with one being heterogametic, creates an additional obstacle in the form ofthe MSCI. MSCI in males is still poorly understood, and its disruption almostalways accompanies synapsis and recombination defects, thus confoundingmeiotic analysis. Female meiosis takes place during a narrow window of fetaldevelopment, necessitating (in mice) the sacrifice of pregnant females andtheir entire litter. Genetic analysis utilizing compound mutations is inefficientdue to infertility of single homozygous mutants, not mentioning the expensesrelated tomouse husbandry. Finally, the mammalian system lacks biochemicaland genetic assays commonly used in yeast or worms. Therefore, derivationof functional gametes in vitro from ES or iPS cells is highly anticipated.Providing that it fully replicates in vivo processes, it could serve as an easierand faster system to study mammalian meiosis. Despite all the drawbacks,mouse meiosis and recombination are being slowly elucidated. New meioticgenes have been identified using different approaches, and mutants have beenanalyzed shedding light on mammalian meiotic regulation. The KnockoutMouse Project is greatly facilitating this genetic avenue of research.


Appendix

Table 5.A1 Summary of phenotypes of mouse mutants exhibiting meiotic defects

Gene

symbol Male phenotype Female phenotype References

Atm Arrest between the

zygotene/

pachytene, sterile

Meiotic arrest, ovary

degeneration,

sterile

Xu et al. (1996),

Goetz et al. (1984)

Brca1 Normal synapsis,

persistent DSBs,

no crossover,

sterile

Fertile Hakem et al. (1997)

Brca2 Arrest prior to

pachytene,

persistent DSBs,

sterile

Reduced numbers of

oocytes

Sharan et al. (2004)

Ccnb1ip Normal synapsis and

pachytene

progression,

crossover failure,

sterile

Oocyte loss, sterile Ward et al. (2007),

Strong and

Schimenti (2010)

Dmc1 Arrest at a zygotene-

like stage, no

synapsis,

persistent DSBs,

sterile

Zygotene arrest,

ovaries devoid of

oocytes, sterile

Pittman et al. (1998),

Yoshida et al.

(1998)

Exo1 Arrest at metaphase

I, sterile

Nondisjunction at

first meiotic

division, sterile

Wei et al. (2003)

Eif4g3 Normal synapsis and

crossover, meiotic

prophase exit

defect, sterile

Fertile Sun et al. (2010)

Fkbp6 Pachytene arrest,

sterile

Fertile Crackower et al.

(2003)

H2afx Pachytene arrest of

spermatocytes,

sex body

formation failure,

no crossover,

sterile

Fertile Celeste et al. (2002),

Fernandez-

Capetillo et al.

(2003)


Table 5.A1 (Continued)

Gene


Hop1 Zygotene/

pachytene arrest,

defective synapsis

and

recombination,

sterile

Zygotene/pachytene

arrest, defective

synapsis and

recombination,

sterile

Petukhova et al.

(2003)

Hspa2 Pachytene arrest due

to desynapsis

defects, sterile

Fertile Dix et al. (1996)

Hormad1 Zygotene/

pachytene arrest,

synapsis and

recombination

failure, sterile

Zygotene/pachytene

arrest, synapsis and

recombination

failure, sterile

Daniel et al. (2011),

Shin et al. (2010)

Mei1 Leptotene/zygotene

arrest, defective

synapsis, lack of

DSBs, sterile

Oocyte loss, sterile Libby et al. (2002),

Libby et al. (2003),

Reinholdt and

Schimenti (2005)

Mei4 Leptotene/zygotene

arrest, DSB

formation failure,

sterile

Oocyte loss, sterile Kumar et al. (2010)

Mlh1 Normal synapsis,

crossover failure,

sterile

Normal synapsis,

crossover failure,

sterile

Edelmann et al.

(1996), Baker et al.

(1996)

Mlh3 Normal synapsis,

crossover failure,

sterile

Normal synapsis,

crossover failure,

sterile

Lipkin et al. (2002)

Mre11 Fertile Severely reduced

fertility

Theunissen et al.

(2003)

Msh4 Zygotene/

pachytene arrest,

synapsis and

recombination

failure, sterile

Zygotene/pachytene


recombination

failure, sterile

Kneitz et al. (2000)

Msh5 Zygotene/

pachytene arrest,

synapsis and

recombination

failure, sterile

Zygotene/pachytene


recombination

failure, sterile

de Vries et al. (1999)

(Continued)


Gene


Prdm9/

Meisetz

Pachytene arrest,

synapsis and

recombination

defects, sterile

Pachytene arrest,

synapsis and

recombination

defects, sterile

Hayashi et al. (2005)

Rad21l Zygotene arrest,

synapsis and

recombination

failure, sterile

Fertile, develop age-

dependent sterility

Herran et al. (2011),

Ishiguro et al.

(2011)

Rec8 Abnormal SC

formation

between sister

chromatids rather

than between

homologous

chromosomes,

sterile

Meiotic arrest and

oocyte depletion,

sterile

Bannister et al.

(2004), Xu et al.

(2005)

Smc1b Pachytene arrest,

short SCs,

abnormal sister

chromatid

cohesion, lack of

crossover, sterile

Similar defects to

those seen in

males, oocytes

progress past

pachytene but are

aneuploid, sterile

Revenkova et al.

(2004)

Spo11 Do not form DSBs,

mostly

nonhomologous

and partial

synapsis, no

recombination,

sterile

Do not form DSBs,

mostly

nonhomologous

and partial

synapsis, no

recombination,

some oocytes

present in the

ovary, infertile

Baudat et al. (2000),

Romanienko and

Camerini-Otero

(2000)

Sun1 Zygotene/

pachytene arrest,

synapsis and

recombination

failure, sterile

Zygotene/pachytene


recombination

failure, sterile

Ding et al. (2007)

Syce1 Pachytene-like

arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Pachytene-like

arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Bolcun-Filas et al.

(2009)




Gene



arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Pachytene-like

arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Bolcun-Filas et al.

(2007)


arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Pachytene-like

arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

Schramm et al.

(2011)

Sycp1 Pachytene-like

arrest, AE fully

formed, synapsis,

and

recombination

failure, sterile

Pachytene-like

arrest, AE fully

formed, synapsis

and

recombination

failure, sterile

de Vries et al. (2005)

Sycp2 Zygotene/

pachytene arrest,

abnormal AE,

synapsis and

recombination

failure, sterile

Subfertile, high

aneuploidy rates

Yang et al. (2006)

Sycp3 Zygotene/

pachytene arrest,

abnormal AE,

synapsis and

recombination

failure, sterile

Subfertile, high

aneuploidy rates

Yuan et al. (2000)

Tex11/

Zip4h

Relatively normal

synapsis,

decreased

numbers of

crossovers,

achiasmate

chromosome, cell

death at anaphase,

sterile

Reduced fertility due

to higher

aneuploidies

Adelman and Petrini

(2008), Yang et al.

(2008b)

(Continued)


Gene


Tex12 Pachytene-like

arrest, AE fully

formed,

synapsis and

recombination

failure, sterile

Pachytene-like

arrest, AE fully

formed,

synapsis and

recombination

failure, sterile

Hamer et al.

(2008a,b)

Tex15 Zygotene/

pachytene arrest,

DSB and synapsis

failure, sterile

Fertile Yang et al. (2008a)

Trip13 Normal synapsis and

crossover,

persistent DSB

(non-CO), sterile

Normal synapsis and

crossover,

persistent DSB

(non-CO), sterile

Li and Schimenti

(2007), Roig et al.

(2010)


REFERENCES

Adams, I.R., McLaren, A., 2002. Sexually dimorphic development of mouse primordialgerm cells: switching from oogenesis to spermatogenesis. Development 129, 1155–1164.

Adelfalk, C., Janschek, J., Revenkova, E., Blei, C., Liebe, B., Gob, E., et al., 2009. CohesinSMC1beta protects telomeres in meiocytes. J. Cell Biol. 187, 185–199.

Adelman, C.A., Petrini, J.H., 2008. ZIP4H (TEX11) deficiency in the mouse impairsmeiotic double strand break repair and the regulation of crossing over. PLoS Genet. 4,e1000042.

An, J.Y., Kim, E.A., Jiang, Y., Zakrzewska, A., Kim, D.E., Lee, M.J., et al., 2010. UBR2mediates transcriptional silencing during spermatogenesis via histone ubiquitination.Proc. Natl. Acad. Sci. U.S.A. 107, 1912–1917.

Andersen, S.L., Sekelsky, J., 2010. Meiotic versus mitotic recombination: two differentroutes for double-strand break repair: the different functions of meiotic versus mitoticDSB repair are reflected in different pathway usage and different outcomes. Bioessays 32,1058–1066.

Anderson, E.L., Baltus, A.E., Roepers-Gajadien, H.L., Hassold, T.J., de Rooij, D.G.,van Pelt, A.M., et al., 2008. Stra8 and its inducer, retinoic acid, regulate meiotic initiationin both spermatogenesis and oogenesis in mice. Proc. Natl. Acad. Sci. U.S.A. 105,14976–14980.

Aravin, A.A., Sachidanandam, R., Girard, A., Fejes-Toth, K., Hannon, G.J., 2007. Devel-opmentally regulated piRNA clusters implicate MILI in transposon control. Science 316,744–747.

Aravin, A.A., Sachidanandam, R., Bourc’his, D., Schaefer, C., Pezic, D., Toth, K.F., et al.,2008. A piRNA pathway primed by individual transposons is linked to de novo DNAmethylation in mice. Mol. Cell 31, 785–799.

Arnold, C.N., Xia, Y., Lin, P., Ross, C., Schwander, M., Smart, N.G., et al., 2011. Rapididentification of a disease allele in mouse through whole genome sequencing and bulksegregation analysis. Genetics 187, 633–641.


Baarends, W.M., Hoogerbrugge, J.W., Roest, H.P., Ooms, M., Vreeburg, J.,Hoeijmakers, J.H., et al., 1999a. Histone ubiquitination and chromatin remodeling inmouse spermatogenesis. Dev. Biol. 207, 322–333.

Baarends, W.M., Roest, H.P., Grootegoed, J.A., 1999b. The ubiquitin system in gameto-genesis. Mol. Cell. Endocrinol. 151, 5–16.

Baarends, W.M., van der Laan, R., Grootegoed, J.A., 2000. Specific aspects of the ubiquitinsystem in spermatogenesis. J. Endocrinol. Invest. 23, 597–604.

Baarends, W.M., Wassenaar, E., Hoogerbrugge, J.W., van Cappellen, G., Roest, H.P.,Vreeburg, J., et al., 2003. Loss of HR6B ubiquitin-conjugating activity results indamaged synaptonemal complex structure and increased crossing-over frequency duringthe male meiotic prophase. Mol. Cell. Biol. 23, 1151–1162.

Baarends, W.M., Wassenaar, E., van der Laan, R., Hoogerbrugge, J., Sleddens-Linkels, E.,Hoeijmakers, J.H., et al., 2005. Silencing of unpaired chromatin and histone H2Aubiquitination in mammalian meiosis. Mol. Cell. Biol. 25, 1041–1053.

Baarends, W.M., Wassenaar, E., Hoogerbrugge, J.W., Schoenmakers, S., Sun, Z.W.,Grootegoed, J.A., 2007. Increased phosphorylation and dimethylation of XY bodyhistones in the Hr6b-knockout mouse is associated with derepression of the X chromo-some. J. Cell Sci. 120, 1841–1851.

Baker, S.M., Plug, A.W., Prolla, T.A., Bronner, C.E., Harris, A.C., Yao, X., et al., 1996.Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat.Genet. 13, 336–342.

Baltus, A.E., Menke, D.B., Hu, Y.C., Goodheart, M.L., Carpenter, A.E., de Rooij, D.G.,et al., 2006. In germ cells of mouse embryonic ovaries, the decision to enter meiosisprecedes premeiotic DNA replication. Nat. Genet. 38, 1430–1434.

Bannister, L.A., Reinholdt, L.G., Munroe, R.J., Schimenti, J.C., 2004. Positional cloningand characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8.Genesis 2000 (40), 184–194.

Barchi, M., Roig, I., Di Giacomo, M., de Rooij, D.G., Keeney, S., Jasin, M., 2008. ATMpromotes the obligate XY crossover and both crossover control and chromosome axisintegrity on autosomes. PLoS Genet. 4, e1000076.

Bascom-Slack, C.A., Ross, L.O., Dawson, D.S., 1997. Chiasmata, crossovers, and meioticchromosome segregation. Adv. Genet. 35, 253–284.

Bass, H.W., Riera-Lizarazu, O., Ananiev, E.V., Bordoli, S.J., Rines, H.W., Phillips, R.L.,et al., 2000. Evidence for the coincident initiation of homolog pairing and synapsis duringthe telomere-clustering (bouquet) stage of meiotic prophase. J. Cell Sci. 113, 1033–1042.

Baudat, F., de Massy, B., 2007. Cis- and trans-acting elements regulate the mouse Psmb9meiotic recombination hotspot. PLoS Genet. 3, e100.

Baudat, F., Manova, K., Yuen, J.P., Jasin, M., Keeney, S., 2000. Chromosome synapsis defectsand sexually dimorphic meiotic progression in mice lacking Spo11. Mol. Cell 6, 989–998.

Baudat, F., Buard, J., Grey, C., Fledel-Alon, A., Ober, C., Przeworski, M., et al., 2010.PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice.Science 327, 836–840.

Bellani, M.A., Romanienko, P.J., Cairatti, D.A., Camerini-Otero, R.D., 2005. SPO11 isrequired for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest ofAtm-/- spermatocytes. J. Cell Sci. 118, 3233–3245.

Bellani, M.A., Boateng, K.A., McLeod, D., Camerini-Otero, R.D., 2010. The expressionprofile of the major mouse SPO11 isoforms indicates that SPO11beta introduces doublestrand breaks and suggests that SPO11alpha has an additional role in prophase in bothspermatocytes and oocytes. Mol. Cell. Biol. 30, 4391–4403.

Bellve, A.R., Cavicchia, J.C., Millette, C.F., O’Brien, D.A., Bhatnagar, Y.M., Dym, M.,1977. Spermatogenic cells of the prepuberal mouse. Isolation and morphological charac-terization. J. Cell Biol. 74, 68–85.


Biemont, C., Vieira, C., 2006. Genetics: junk DNA as an evolutionary force. Nature 443,521–524.

Bogdanov, Y.F., Grishaeva, T.M., Dadashev, S.Y., 2007. Similarity of the domain structureof proteins as a basis for the conservation of meiosis. Int. Rev. Cytol. 257, 83–142.

Bolcun-Filas, E., Costa, Y., Speed, R., Taggart, M., Benavente, R., De Rooij, D.G., et al.,2007. SYCE2 is required for synaptonemal complex assembly, double strand breakrepair, and homologous recombination. J. Cell Biol. 176, 741–747.

Bolcun-Filas, E., Hall, E., Speed, R., Taggart, M., Grey, C., de Massy, B., et al., 2009.Mutation of the mouse Syce1 gene disrupts synapsis and suggests a link between synapto-nemal complex structural components and DNA repair. PLoS Genet. 5, e1000393.

Bolcun-Filas, E., Bannister, L.A., Barash, A., Schimenti, K.J., Hartford, S.A., Eppig, J.J.,et al., 2011. A-MYB (MYBL1) transcription factor is a master regulator of male meiosis.Development 138, 3319–3330.

Bowles, J., Knight, D., Smith, C., Wilhelm, D., Richman, J., Mamiya, S., et al., 2006.Retinoid signaling determines germ cell fate in mice. Science 312, 596–600.

Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R., et al., 2007.Discrete small RNA-generating loci as master regulators of transposon activity in Dro-sophila. Cell 128, 1089–1103.

Brown, P.W., Hwang, K., Schlegel, P.N., Morris, P.L., 2008. Small ubiquitin-relatedmodifier (SUMO)-1, SUMO-2/3 and SUMOylation are involved with centromericheterochromatin of chromosomes 9 and 1 and proteins of the synaptonemal complexduring meiosis in men. Hum. Reprod. 23, 2850–2857.

Buard, J., Barthes, P., Grey, C., de Massy, B., 2009. Distinct histone modifications defineinitiation and repair of meiotic recombination in the mouse. EMBO J. 28, 2616–2624.

Burgoyne, P.S., Mahadevaiah, S.K., Turner, J.M., 2009. The consequences of asynapsis formammalian meiosis. Nat. Rev. Genet. 10, 207–216.

Callender, T.L., Hollingsworth, N.M., 2010. Mek1 suppression of meiotic double-strandbreak repair is specific to sister chromatids, chromosome autonomous and independent ofRec8 cohesin complexes. Genetics 185, 771–782.

Carballo, J.A., Johnson, A.L., Sedgwick, S.G., Cha, R.S., 2008. Phosphorylation of the axialelement protein Hop1 byMec1/Tel1 ensures meiotic interhomolog recombination. Cell132, 758–770.

Castaneda, J., Genzor, P., Bortvin, A., 2011. piRNAs, transposon silencing, and germlinegenome integrity. Mutat. Res. 714, 95–104.

Celeste, A., Petersen, S., Romanienko, P.J., Fernandez-Capetillo, O., Chen, H.T.,Sedelnikova, O.A., et al., 2002. Genomic instability in mice lacking histone H2AX.Science (New York, N.Y.) 296, 922–927.

Chalmel, F., Rolland, A.D., Niederhauser-Wiederkehr, C., Chung, S.S., Demougin, P.,Gattiker, A., et al., 2007. The conserved transcriptome in human and rodent malegametogenesis. Proc. Natl. Acad. Sci. U.S.A. 104, 8346–8351.

Chen, Y.T., Venditti, C.A., Theiler, G., Stevenson, B.J., Iseli, C., Gure, A.O., et al., 2005.Identification of CT46/HORMAD1, an immunogenic cancer/testis antigen encoding aputative meiosis-related protein. Cancer Immun. 5, 9.

Cheng, C.H., Lo, Y.H., Liang, S.S., Ti, S.C., Lin, F.M., Yeh, C.H., et al., 2006. SUMOmodifications control assembly of synaptonemal complex and polycomplex in meiosis ofSaccharomyces cerevisiae. Genes Dev. 20, 2067–2081.

Costa, Y., Speed, R., Ollinger, R., Alsheimer, M., Semple, C.A., Gautier, P., et al., 2005.Two novel proteins recruited by synaptonemal complex protein 1 (SYCP1) are at thecentre of meiosis. J. Cell Sci. 118, 2755–2762.

Crackower, M.A., Kolas, N.K., Noguchi, J., Sarao, R., Kikuchi, K., Kaneko, H., et al.,2003. Essential role of Fkbp6 in male fertility and homologous chromosome pairing inmeiosis. Science (New York, N.Y.) 300, 1291–1295.


Daniel, K., Lange, J., Hached, K., Fu, J., Anastassiadis, K., Roig, I., et al., 2011. Meiotichomologue alignment and its quality surveillance are controlled by mouse HORMAD1.Nat. Cell Biol. 13, 599–610.

de Carvalho, C.E., Colaiacovo, M.P., 2006. SUMO-mediated regulation of synaptonemalcomplex formation during meiosis. Genes Dev. 20, 1986–1992.

De La Fuente, R., Baumann, C., Fan, T., Schmidtmann, A., Dobrinski, I., Muegge, K.,2006. Lsh is required for meiotic chromosome synapsis and retrotransposon silencing infemale germ cells. Nat. Cell Biol. 8, 1448–1454.

De Muyt, A., Vezon, D., Gendrot, G., Gallois, J.L., Stevens, R., Grelon, M., 2007.AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana.EMBO J. 26, 4126–4137.

de Vries, S.S., Baart, E.B., Dekker, M., Siezen, A., de Rooij, D.G., de Boer, P., et al., 1999.Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male andfemale meiosis. Genes Dev. 13, 523–531.

de Vries, F.A., de Boer, E., van den Bosch, M., Baarends, W.M., Ooms, M., Yuan, L., et al.,2005. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombina-tion, and XY body formation. Genes Dev. 19, 1376–1389.

Ding, X., Xu, R., Yu, J., Xu, T., Zhuang, Y., Han, M., 2007. SUN1 is required for telomereattachment to nuclear envelope and gametogenesis in mice. Dev. Cell 12, 863–872.

Dix, D.J., Allen, J.W., Collins, B.W., Mori, C., Nakamura, N., Poorman-Allen, P., et al.,1996. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis,and male infertility. Proc. Natl. Acad. Sci. U.S.A. 93, 3264–3268.

Dix, D.J., Allen, J.W., Collins, B.W., Poorman-Allen, P., Mori, C., Blizard, D.R., et al.,1997. HSP70-2 is required for desynapsis of synaptonemal complexes during meioticprophase in juvenile and adult mouse spermatocytes. Development 124, 4595–4603.

Dobson, M.J., Pearlman, R.E., Karaiskakis, A., Spyropoulos, B., Moens, P.B., 1994.Synaptonemal complex proteins: occurrence, epitope mapping and chromosome dis-junction. J. Cell Sci. 107, 2749.

Dresser, M.E., Moses, M.J., 1979. Silver staining of synaptonemal complexes in surfacespreads for light and electron microscopy. Exp. Cell Res. 121, 416–419.

Edelmann, W., Cohen, P.E., Kane, M., Lau, K., Morrow, B., Bennett, S., et al., 1996.Meiotic pachytene arrest in MLH1-deficient mice. Cell 85, 1125–1134.

Elson, A., Wang, Y., Daugherty, C.J., Morton, C.C., Zhou, F., Campos-Torres, J., et al.,1996. Pleiotropic defects in ataxia-telangiectasia protein-deficient mice. Proc. Natl.Acad. Sci. U.S.A. 93, 13084–13089.

Fawcett, D.W., 1956. The fine structure of chromosomes in the meiotic prophase ofvertebrate spermatocytes. J. Biophys. Biochem. Cytol. 2, 403–406.

Fernandez-Capetillo, O., Mahadevaiah, S.K., Celeste, A., Romanienko, P.J., Camerini-Otero, R.D., Bonner, W.M., et al., 2003. H2AX is required for chromatin remodelingand inactivation of sex chromosomes in male mouse meiosis. Dev. Cell 4, 497–508.

Forejt, J., Capkova, J., Gregorova, S., 1980. T(16:17)43H translocation as a tool in analysis ofthe proximal part of chromosome 17 (including T-t gene complex) of the mouse. Genet.Res. 35, 165–177.

Fraune, J., Schramm, S., Alsheimer, M., Benavente, R., 2012. The mammalian synaptonemalcomplex: Protein components, assembly and role in meiotic recombination. Exp. Cell Res.15, 1340–1346

Frost, R.J., Hamra, F.K., Richardson, J.A., Qi, X., Bassel-Duby, R., Olson, E.N., 2010.MOV10L1 is necessary for protection of spermatocytes against retrotransposons byPiwi-interacting RNAs. Proc. Natl. Acad. Sci. U.S.A. 107, 11847–11852.

Fujimuro, M., Yokosawa, H., 2005. Production of antipolyubiquitin monoclonal antibodiesand their use for characterization and isolation of polyubiquitinated proteins. MethodsEnzymol. 399, 75–86.


Fukuda, T., Daniel, K., Wojtasz, L., Toth, A., Hoog, C., 2010. A novel mammalianHORMA domain-containing protein, HORMAD1, preferentially associates with unsy-napsed meiotic chromosomes. Exp. Cell Res. 316, 158–171.

Fukuda, T., Pratto, F., Schimenti, J.C., Turner, J.M., Camerini-Otero, R.D., Hoog, C.,2012. Phosphorylation of chromosome core components May serve as axis marks for thestatus of chromosomal events during mammalian meiosis. PLoS Genet. 8, e1002485.

Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J.E., Bhattacharya, S., et al., 2003.Cyclin E ablation in the mouse. Cell 114, 431–443.

Getun, I.V., Wu, Z.K., Khalil, A.M., Bois, P.R., 2010. Nucleosome occupancy landscapeand dynamics at mouse recombination hotspots. EMBO Rep. 11, 555–560.

Ghosh, S., Saha, T., 2012. Central role of ubiquitination in genome maintenance: DNAreplication and damage repair. ISRN Mol. Biol. 2012, doi:10.5402/2012/146748.

Gill, M.E., Hu, Y.C., Lin, Y., Page, D.C., 2011. Licensing of gametogenesis, dependent onRNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells.Proc. Natl. Acad. Sci. U.S.A. 108, 7443–7448.

Goetz, P., Chandley, A.C., Speed, R.M., 1984. Morphological and temporal sequence ofmeiotic prophase development at puberty in the male mouse. J. Cell Sci. 65, 249–263.

Goldberg, M., Stucki, M., Falck, J., D’Amours, D., Rahman, D., Pappin, D., et al., 2003.MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421,952–956.

Goodier, J.L., Kazazian, H.H., Jr., 2008. Retrotransposons revisited: the restraint andrehabilitation of parasites. Cell 135, 23–35.

Goodpasture, C., Bloom, S.E., 1975. Visualization of nucleolar organizer regions in mam-malian chromosomes using silver staining. Chromosoma 53, 37–50.

Grey, C., Baudat, F., de Massy, B., 2009. Genome-wide control of the distribution ofmeiotic recombination. PLoS Biol. 7, e35.

Guillon, H., Baudat, F., Grey, C., Liskay, R.M., de Massy, B., 2005. Crossover andnoncrossover pathways in mouse meiosis. Mol. Cell 20, 563–573.

Gunawardane, L.S., Saito, K., Nishida, K.M., Miyoshi, K., Kawamura, Y., Nagami, T.,et al., 2007. A slicer-mediated mechanism for repeat-associated siRNA 50 end formationin Drosophila. Science 315, 1587–1590.

Habu, T., Taki, T., West, A., Nishimune, Y., Morita, T., 1996. The mouse and humanhomologs of DMC1, the yeast meiosis-specific homologous recombination gene, have acommon unique form of exon-skipped transcript in meiosis. Nucleic Acids Res. 24,470–477.

Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., et al., 2002.Epigenetic reprogramming in mouse primordial germ cells. Mech. Dev. 117, 15–23.

Hakem, R., de la Pompa, J.L., Elia, A., Potter, J., Mak, T.W., 1997. Partial rescue ofBrca1 (5-6) early embryonic lethality by p53 or p21 null mutation. Nat. Genet. 16,298–302.

Hamer, G., Novak, I., Kouznetsova, A., Hoog, C., 2008a. Disruption of pairing and synapsisof chromosomes causes stage-specific apoptosis of male meiotic cells. Theriogenology 69,333–339.

Hamer, G., Wang, H., Bolcun-Filas, E., Cooke, H.J., Benavente, R., Hoog, C., 2008b.Progression of meiotic recombination requires structural maturation of the centralelement of the synaptonemal complex. J. Cell Sci. 121, 2445–2451.

Hayashi, K., Yoshida, K., Matsui, Y., 2005. A histone H3 methyltransferase controlsepigenetic events required for meiotic prophase. Nature 438, 374–378.

Heard, E., Turner, J., 2011. Function of the sex chromosomes in mammalian fertility. ColdSpring Harb. Perspect. Biol. 3, a002675.

Hernandez-Hernandez, A., Rincon-Arano, H., Recillas-Targa, F., Ortiz, R., Valdes-Quezada, C., Echeverria, O.M., et al., 2008. Differential distribution and association of


repeat DNA sequences in the lateral element of the synaptonemal complex in ratspermatocytes. Chromosoma 117, 77–87.

Hernandez-Hernandez, A., Vazquez-Nin, G.H., Echeverria, O.M., Recillas-Targa, F.,2009. Chromatin structure contribution to the synaptonemal complex formation. Cell.Mol. Life Sci. 66, 1198–1208.

Herran, Y., Gutierrez-Caballero, C., Sanchez-Martin, M., Hernandez, T., Viera, A.,Barbero, J.L., et al., 2011. The cohesin subunit RAD21L functions in meiotic synapsisand exhibits sexual dimorphism in fertility. EMBO J. 30, 3091–3105.

Heyting, C., Moens, P.B., van Raamsdonk, W., Dietrich, A.J., Vink, A.C., Redeker, E.J.,1987. Identification of two major components of the lateral elements of synaptonemalcomplexes of the rat. Eur. J. Cell Biol. 43, 148.

Heyting, C., Dietrich, A.J., Moens, P.B., Dettmers, R.J., Offenberg, H.H., 1989. Synapto-nemal complex proteins. Genome 31, 81.

Hiraoka, Y., Dernburg, A.F., 2009. The SUN rises on meiotic chromosome dynamics. Dev.Cell 17, 598–605.

Holloway, J.K., Booth, J., Edelmann, W., McGowan, C.H., Cohen, P.E., 2008. MUS81generates a subset of MLH1-MLH3-independent crossovers in mammalian meiosis.PLoS Genet. 4, e1000186.

Holloway, J.K., Morelli, M.A., Borst, P.L., Cohen, P.E., 2010. Mammalian BLM helicase iscritical for integrating multiple pathways of meiotic recombination. J. Cell Biol. 188,779–789.

Homolka, D., Jansa, P., Forejt, J., 2012. Genetically enhanced asynapsis of autosomal chroma-tin promotes transcriptional dysregulation and meiotic failure. Chromosoma 121, 91–104.

Hunt, P.A., Hassold, T.J., 2002. Sex matters in meiosis. Science 296, 2181–2183.Ichijima, Y., Ichijima, M., Lou, Z., Nussenzweig, A., Camerini-Otero, R.D., Chen, J.,

et al., 2011. MDC1 directs chromosome-wide silencing of the sex chromosomes in malegerm cells. Genes Dev. 25, 959–971.

Ishiguro, K., Kim, J., Fujiyama-Nakamura, S., Kato, S., Watanabe, Y., 2011. A newmeiosis-specific cohesin complex implicated in the cohesin code for homologous pairing.EMBO Rep. 12, 267–275.

Jaramillo-Lambert, A., Harigaya, Y., Vitt, J., Villeneuve, A., Engebrecht, J., 2010. Meioticerrors activate checkpoints that improve gamete quality without triggering apoptosis inmale germ cells. Curr. Biol. 20, 2078–2089.

Kaneda, M., Tang, F., O’Carroll, D., Lao, K., Surani, M.A., 2009. Essential role forArgonaute2 protein in mouse oogenesis. Epigenetics Chromatin 2, 9.

Kauppi, L., Barchi, M., Baudat, F., Romanienko, P.J., Keeney, S., Jasin, M., 2011. Distinctproperties of the XY pseudoautosomal region crucial for male meiosis. Science 331,916–920.

Keeney, S., 2008. Spo11 and the formation of DNA double-strand breaks in meiosis.Genome Dyn. Stab. 2, 81–123.

Keeney, S., Giroux, C.N., Kleckner, N., 1997. Meiosis-specific DNA double-strand breaksare catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375.

Kelly, W.G., Aramayo, R., 2007. Meiotic silencing and the epigenetics of sex. ChromosomeRes. 15, 633–651.

Khalil, A.M., Driscoll, D.J., 2010. Epigenetic regulation of pericentromeric heterochroma-tin during mammalian meiosis. Cytogenet. Genome Res. 129, 280–289.

Khalil, A.M., Boyar, F.Z., Driscoll, D.J., 2004. Dynamic histone modifications mark sexchromosome inactivation and reactivation during mammalian spermatogenesis. Proc.Natl. Acad. Sci. U.S.A. 101, 16583–16587.

Kim, K.P., Weiner, B.M., Zhang, L., Jordan, A., Dekker, J., Kleckner, N., 2010. Sistercohesion and structural axis components mediate homolog bias of meiotic recombina-tion. Cell 143, 924–937.


Kneitz, B., Cohen, P.E., Avdievich, E., Zhu, L., Kane, M.F., Hou, H., Jr., et al., 2000.MutS homolog 4 localization to meiotic chromosomes is required for chromosomepairing during meiosis in male and female mice. Genes Dev. 14, 1085–1097.

Kolas, N.K., Yuan, L., Hoog, C., Heng, H.H., Marcon, E., Moens, P.B., 2004. Male mousemeiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align byhomology but fail to synapse and have possible impaired specificity of chromatin loopattachment. Cytogenet. Genome Res. 105, 182–188.

Kouznetsova, A., Novak, I., Jessberger, R., Hoog, C., 2005. SYCP2 and SYCP3 arerequired for cohesin core integrity at diplotene but not for centromere cohesion at thefirst meiotic division. J. Cell Sci. 118, 2271–2278.

Kumar, R., Bourbon, H.M., de Massy, B., 2010. Functional conservation of Mei4 formeiotic DNA double-strand break formation from yeasts to mice. Genes Dev. 24,1266–1280.

Kumar, R., De Massy, B., 2011. Initiation of meiotic recombination in mammals. Genes 1,521–549.

Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y., Fujita, Y.,et al., 2001. Two mouse piwi-related genes: miwi and mili. Mech. Dev. 108, 121–133.

Kuramochi-Miyagawa, S., Kimura, T., Ijiri, T.W., Isobe, T., Asada, N., Fujita, Y., et al.,2004. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.Development 131, 839–849.

Kuramochi-Miyagawa, S., Watanabe, T., Gotoh, K., Totoki, Y., Toyoda, A., Ikawa, M.,et al., 2008. DNA methylation of retrotransposon genes is regulated by Piwi familymembers MILI and MIWI2 in murine fetal testes. Genes Dev. 22, 908–917.

Kwon, Y.T., Xia, Z., Davydov, I.V., Lecker, S.H., Varshavsky, A., 2001. Construction andanalysis of mouse strains lacking the ubiquitin ligase UBR1 (E3alpha) of the N-end rulepathway. Mol. Cell. Biol. 21, 8007–8021.

Kwon, Y.T., Xia, Z., An, J.Y., Tasaki, T., Davydov, I.V., Seo, J.W., et al., 2003. Femalelethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of theN-end rule pathway. Mol. Cell. Biol. 23, 8255–8271.

La Salle, S., Mertineit, C., Taketo, T., Moens, P.B., Bestor, T.H., Trasler, J.M., 2004.Windows for sex-specific methylation marked by DNA methyltransferase expressionprofiles in mouse germ cells. Dev. Biol. 268, 403–415.

La Salle, S., Sun, F., Zhang, X.D., Matunis, M.J., Handel, M.A., 2008. Developmentalcontrol of sumoylation pathway proteins in mouse male germ cells. Dev. Biol. 321,227–237.

La Salle, S., Palmer, K., O’Brien, M., Schimenti, J., Eppig, J., Handel, M.A., 2011. Spata22,a novel vertebrate-specific gene, is required for meiotic progress in mouse germ cells.Biol. Reprod. 86, doi: 10.1095/biolreprod.111.095752.

Lange, J., Pan, J., Cole, F., Thelen, M.P., Jasin, M., Keeney, S., 2011. ATM controls meioticdouble-strand-break formation. Nature 479, 237–240.

Lardenois, A., Gattiker, A., Collin, O., Chalmel, F., Primig, M., 2010. GermOnline 4.0 is agenomics gateway for germline development, meiosis and the mitotic cell cycle. Database2010, doi: 10.1093/database/baq030.

Lee, J., Iwai, T., Yokota, T., Yamashita, M., 2003. Temporally and spatially selective loss ofRec8 protein from meiotic chromosomes during mammalian meiosis. J. Cell Sci. 116,2781.

Lees-Murdock, D.J., De Felici, M., Walsh, C.P., 2003. Methylation dynamics of repetitiveDNA elements in the mouse germ cell lineage. Genomics 82, 230–237.

Li, L., Liu, Y., 2011. Diverse small non-coding RNAs in RNA interference pathways.Methods Mol. Biol. 764, 169–182.

Li, X.C., Schimenti, J.C., 2007. Mouse pachytene checkpoint 2 (trip13) is required forcompleting meiotic recombination but not synapsis. PLoS Genet. 3, e130.


Li, X.C., Bolcun-Filas, E., Schimenti, J.C., 2011. Genetic evidence that synaptonemalcomplex axial elements govern recombination pathway choice in mice. Genetics 189,71–82.

Libby, B.J., De La Fuente, R., O’Brien, M.J., Wigglesworth, K., Cobb, J., Inselman, A.,et al., 2002. The mouse meiotic mutation mei1 disrupts chromosome synapsis withsexually dimorphic consequences for meiotic progression. Dev. Biol. 242, 174–187.

Libby, B.J., Reinholdt, L.G., Schimenti, J.C., 2003. Positional cloning and characterizationof Mei1, a vertebrate-specific gene required for normal meiotic chromosome synapsis inmice. Proc. Natl. Acad. Sci. U.S.A. 100, 15706–15711.

Liebe, B., Alsheimer, M., Hoog, C., Benavente, R., Scherthan, H., 2004. Telomereattachment, meiotic chromosome condensation, pairing, and bouquet stage durationare modified in spermatocytes lacking axial elements. Mol. Biol. Cell 15, 827.

Liebe, B., Petukhova, G., Barchi, M., Bellani, M., Braselmann, H., Nakano, T., et al., 2006.Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptoteneand bouquet stages. Exp. Cell Res. 312, 3768–3781.

Lin, Y., Gill, M.E., Koubova, J., Page, D.C., 2008. Germ cell-intrinsic and -extrinsic factorsgovern meiotic initiation in mouse embryos. Science 322, 1685–1687.

Lipkin, S.M., Moens, P.B., Wang, V., Lenzi, M., Shanmugarajah, D., Gilgeous, A., et al.,2002. Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat. Genet. 31,385–390.

Liu, J.G., Yuan, L., Brundell, E., Bjorkroth, B., Daneholt, B., Hoog, C., 1996. Localizationof the N-terminus of SCP1 to the central element of the synaptonemal complex andevidence for direct interactions between the N-termini of SCP1 molecules organizedhead-to-head. Exp. Cell Res. 226, 11.

Lou, Z., Minter-Dykhouse, K., Wu, X., Chen, J., 2003. MDC1 is coupled to activatedCHK2 in mammalian DNA damage response pathways. Nature 421, 957–961.

Lu, L.Y., Wu, J., Ye, L., Gavrilina, G.B., Saunders, T.L., Yu, X., 2010. RNF8-dependenthistone modifications regulate nucleosome removal during spermatogenesis. Dev. Cell18, 371–384.

Ma, L., Buchold, G.M., Greenbaum, M.P., Roy, A., Burns, K.H., Zhu, H., et al., 2009.GASZ is essential for male meiosis and suppression of retrotransposon expression in themale germline. PLoS Genet. 5, e1000635.

Ma, T., Keller, J.A., Yu, X., 2011. RNF8-dependent histone ubiquitination during DNAdamage response and spermatogenesis. Acta Biochim. Biophys. Sin. 43, 339–345.

MacQueen, A.J., Hochwagen, A., 2011. Checkpoint mechanisms: the puppet masters ofmeiotic prophase. Trends Cell Biol. 21, 393–400.

Maleki, S., Neale, M.J., Arora, C., Henderson, K.A., Keeney, S., 2007. Interactionsbetween Mei4, Rec114, and other proteins required for meiotic DNA double-strandbreak formation in Saccharomyces cerevisiae. Chromosoma 116, 471–486.

Manterola, M., Page, J., Vasco, C., Berrios, S., Parra, M.T., Viera, A., et al., 2009. A highincidence of meiotic silencing of unsynapsed chromatin is not associated with substantialpachytene loss in heterozygous male mice carrying multiple simple Robertsonian trans-locations. PLoS Genet. 5, e1000625.

Maratou, K., Forster, T., Costa, Y., Taggart, M., Speed, R.M., Ireland, J., et al., 2004.Expression profiling of the developing testis in wild-type and Dazl knockout mice. Mol.Reprod. Dev. 67, 26–54.

Matsuda, Y., Habu, T., Hori, T., Morita, T., 1996. Chromosome mapping of the mousehomologue of DMC1, the yeast meiosis-specific homologous recombination gene.Chromosome Res. 4, 249–250.

Mays-Hoopes, L.L., Bolen, J., Riggs, A.D., Singer-Sam, J., 1995. Preparation ofspermatogonia, spermatocytes, and round spermatids for analysis of gene expressionusing fluorescence-activated cell sorting. Biol. Reprod. 53, 1003–1011.


Meistrich, M.L., Bruce, W.R., Clermont, Y., 1973. Cellular composition of fractions ofmouse testis cells following velocity sedimentation separation. Exp. Cell Res. 79, 213–227.

Metzler-Guillemain, C., de Massy, B., 2000. Identification and characterization of anSPO11 homolog in the mouse. Chromosoma 109, 133–138.

Meuwissen, R.L., Offenberg, H.H., Dietrich, A.J., Riesewijk, A., van Iersel, M., 1992.A coiled-coil related protein specific for synapsed regions of meiotic prophase chromo-somes. EMBO J. 11, 5091.

Mihola, O., Trachtulec, Z., Vlcek, C., Schimenti, J.C., Forejt, J., 2009. A mouse speciationgene encodes a meiotic histone H3 methyltransferase. Science 323, 373–375.

Mills, R.E., Bennett, E.A., Iskow, R.C., Devine, S.E., 2007. Which transposable elementsare active in the human genome? Trends Genet. 23, 183–191.

Mocciaro, A., Rape, M., 2012. Emerging regulatory mechanisms in ubiquitin-dependentcell cycle control. J. Cell Sci. 125, 255–263.

Molnar, M., Bahler, J., Sipiczki, M., Kohli, J., 1995. The rec8 gene of Schizosaccharomycespombe is involved in linear element formation, chromosome pairing and sister-chromatid cohesion during meiosis. Genetics 141, 61.

Morita, T., Yoshimura, Y., Yamamoto, A., Murata, K., Mori, M., Yamamoto, H., et al.,1993. A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiaeRAD51 genes. Proc. Natl. Acad. Sci. U.S.A. 90, 6577–6580.

Moses, M.J., 1956. Chromosomal structures in crayfish spermatocytes. J. Biophys. Biochem.Cytol. 2, 215–218.

Moses, M.J., 1977. Synaptonemal complex karyotyping in spermatocytes of the Chinesehamster (Cricetulus griseus). I. Morphology of the autosomal complement in spreadpreparations. Chromosoma 60, 99–125.

Mulugeta Achame, E., Wassenaar, E., Hoogerbrugge, J.W., Sleddens-Linkels, E.,Ooms, M., Sun, Z.W., et al., 2010. The ubiquitin-conjugating enzyme HR6B isrequired for maintenance of X chromosome silencing in mouse spermatocytes andspermatids. BMC Genomics 11, 367.

Murchison, E.P., Stein, P., Xuan, Z., Pan, H., Zhang, M.Q., Schultz, R.M., et al., 2007.Critical roles for Dicer in the female germline. Genes Dev. 21, 682–693.

Nacerddine, K., Lehembre, F., Bhaumik, M., Artus, J., Cohen-Tannoudji, M., Babinet, C.,et al., 2005. The SUMO pathway is essential for nuclear integrity and chromosomesegregation in mice. Dev. Cell 9, 769–779.

Namekawa, S.H., Park, P.J., Zhang, L.F., Shima, J.E., McCarrey, J.R., Griswold, M.D., et al.,2006. Postmeiotic sex chromatin in the male germline of mice. Curr. Biol. 16, 660–667.

Nasmyth, K., Haering, C.H., 2009. Cohesin: its roles and mechanisms. Annu. Rev. Genet.43, 525–558.

Nickerson, H.D., Joshi, A., Wolgemuth, D.J., 2007. Cyclin A1-deficient mice lack histoneH3 serine 10 phosphorylation and exhibit altered aurora B dynamics in late prophase ofmale meiosis. Dev. Biol. 306, 725–735.

Noguchi, J., Ozawa, M., Nakai, M., Somfai, T., Kikuchi, K., Kaneko, H., et al., 2008.Affected homologous chromosome pairing and phosphorylation of testis specific histone,H2AX, in male meiosis under FKBP6 deficiency. J. Reprod. Dev. 54, 203–207.

Novak, I., Wang, H., Revenkova, E., Jessberger, R., Scherthan, H., Hoog, C., 2008.Cohesin Smc1beta determines meiotic chromatin axis loop organization. J. Cell Biol.180, 83–90.

Offenberg, H.H., Schalk, J.A., Meuwissen, R.L., van Aalderen, M., Kester, H.A., 1998.SCP2: a major protein component of the axial elements of synaptonemal complexes ofthe rat. Nucleic Acids Res. 26, 2572.

Oh, S.D., Lao, J.P., Hwang, P.Y., Taylor, A.F., Smith, G.R., Hunter, N., 2007. BLMortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichro-matid joint molecules. Cell 130, 259–272.


Ollinger, R., Alsheimer, M., Benavente, R., 2005. Mammalian protein SCP1 forms synap-tonemal complex-like structures in the absence of meiotic chromosomes. Mol. Biol. Cell16, 212–217.

Ollinger, R., Childs, A.J., Burgess, H.M., Speed, R.M., Lundegaard, P.R., Reynolds, N.,et al., 2008. Deletion of the pluripotency-associated Tex19.1 gene causes activation ofendogenous retroviruses and defective spermatogenesis in mice. PLoS Genet. 4, e1000199.

Page, J., de la Fuente, R., Manterola, M., Parra, M.T., Viera, A., Berrios, S., et al., 2012.Inactivation or non-reactivation: what accounts better for the silence of sex chromo-somes during mammalian male meiosis? Chromosoma. 121, 307–326.

Pang, A.L., Taylor, H.C., Johnson, W., Alexander, S., Chen, Y., Su, Y.A., et al., 2003.Identification of differentially expressed genes in mouse spermatogenesis. J. Androl. 24,899–911.

Pang, A.L., Johnson, W., Ravindranath, N., Dym, M., Rennert, O.M., Chan, W.Y., 2006.Expression profiling of purified male germ cells: stage-specific expression patterns relatedto meiosis and postmeiotic development. Physiol. Genomics 24, 75–85.

Pangas, S.A., Yan, W., Matzuk, M.M., Rajkovic, A., 2004. Restricted germ cell expressionof a gene encoding a novel mammalian HORMA domain-containing protein. GeneExpr. Patterns 5, 257–263.

Panizza, S., Mendoza, M.A., Berlinger, M., Huang, L., Nicolas, A., Shirahige, K., et al.,2011. Spo11-accessory proteins link double-strand break sites to the chromosome axis inearly meiotic recombination. Cell 146, 372–383.

Parvanov, E.D., Ng, S.H., Petkov, P.M., Paigen, K., 2009. Trans-regulation of mousemeiotic recombination hotspots by Rcr1. PLoS Biol. 7, e36.

Parvanov, E.D., Petkov, P.M., Paigen, K., 2010. Prdm9 controls activation of mammalianrecombination hotspots. Science 327, 835.

Petukhova, G.V., Romanienko, P.J., Camerini-Otero, R.D., 2003. The Hop2 protein has adirect role in promoting interhomolog interactions during mouse meiosis. Dev. Cell 5, 927.

Pittman, D.L., Cobb, J., Schimenti, K.J., Wilson, L.A., Cooper, D.M., Brignull, E., et al.,1998. Meiotic prophase arrest with failure of chromosome synapsis in mice deficient forDmc1, a germline-specific RecA homolog. Mol. Cell 1, 697–705.

Plug, A.W., Peters, A.H., Keegan, K.S., Hoekstra, M.F., de Boer, P., Ashley, T., 1998.Changes in protein composition of meiotic nodules during mammalian meiosis. J. CellSci. 111, 413–423.

Prieto, I., Suja, J.A., Pezzi, N., Kremer, L., Martinez, A.C., 2001. Mammalian STAG3 is acohesin specific to sister chromatid arms in meiosis I. Nat. Cell Biol. 3, 761.

Reinholdt, L.G., Schimenti, J.C., 2005. Mei1 is epistatic to Dmc1 during mouse meiosis.Chromosoma 114, 127–134.

Revenkova, E., Eijpe, M., Heyting, C., Hodges, C.A., Hunt, P.A., Liebe, B., et al., 2004.Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatidcohesion and DNA recombination. Nat. Cell Biol. 6, 555–562.

Roest, H.P., van Klaveren, J., de Wit, J., van Gurp, C.G., Koken, M.H., Vermey, M., et al.,1996. Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in micecauses male sterility associated with chromatin modification. Cell 86, 799–810.

Roest, H.P., Baarends, W.M., de Wit, J., van Klaveren, J.W., Wassenaar, E.,Hoogerbrugge, J.W., et al., 2004. The ubiquitin-conjugating DNA repair enzymeHR6A is a maternal factor essential for early embryonic development in mice. Mol.Cell. Biol. 24, 5485–5495.

Rogers, R.S., Inselman, A., Handel, M.A., Matunis, M.J., 2004. SUMO modified proteinslocalize to the XY body of pachytene spermatocytes. Chromosoma 113, 233–243.

Roig, I., Dowdle, J.A., Toth, A., de Rooij, D.G., Jasin, M., Keeney, S., 2010. MouseTRIP13/PCH2 is required for recombination and normal higher-order chromosomestructure during meiosis. PLoS Genet. 6, e1001062.


Romanienko, P.J., Camerini-Otero, R.D., 1999. Cloning, characterization, and localiza-tion of mouse and human SPO11. Genomics 61, 156–169.

Romanienko, P.J., Camerini-Otero, R.D., 2000. The mouse Spo11 gene is required formeiotic chromosome synapsis. Mol. Cell 6, 975.

Rossi, P., Dolci, S., Sette, C., Capolunghi, F., Pellegrini, M., Loiarro, M., et al., 2004.Analysis of the gene expression profile of mouse male meiotic germ cells. Gene Expr.Patterns 4, 267–281.

Royo, H., Polikiewicz, G., Mahadevaiah, S.K., Prosser, H., Mitchell, M., Bradley, A., et al.,2010. Evidence that meiotic sex chromosome inactivation is essential for male fertility.Curr. Biol. 20, 2117–2123.

Salazar, G., Liu, D., Liao, C., Batkiewicz, L., Arbing, R., Chung, S.S., et al., 2003.Apoptosis in male germ cells in response to cyclin A1-deficiency and cell cycle arrest.Biochem. Pharmacol. 66, 1571–1579.

Salazar, G., Joshi, A., Liu, D., Wei, H., Persson, J.L., Wolgemuth, D.J., 2005. Induction ofapoptosis involving multiple pathways is a primary response to cyclin A1-deficiency inmale meiosis. Dev. Dyn. 234, 114–123.

Scherthan, H., 2003. Knockout mice provide novel insights into meiotic chromosome andtelomere dynamics. Cytogenet. Genome Res. 103, 235–244.

Scherthan, H., Eils, R., Trelles-Sticken, E., Dietzel, S., Cremer, T., Walt, H., et al., 1998.Aspects of three-dimensional chromosome reorganization during the onset of humanmale meiotic prophase. J. Cell Sci. 111, 2337–2351.

Scherthan, H., Sfeir, A., de Lange, T., 2011. Rap1-independent telomere attachment andbouquet formation in mammalian meiosis. Chromosoma 120, 151–157.

Schimenti, J., 2000. Segregation distortion of mouse t haplotypes the molecular basisemerges. Trends Genet. 16, 240–243.

Schramm, S., Fraune, J., Naumann, R., Hernandez-Hernandez, A., Hoog, C., Cooke, H.J.,et al., 2011. A novel mouse synaptonemal complex protein is essential for loading ofcentral element proteins, recombination, and fertility. PLoS Genet. 7, e1002088.

Schultz, N., Hamra, F.K., Garbers, D.L., 2003. A multitude of genes expressed solely inmeiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc.Natl. Acad. Sci. U.S.A. 100, 12201–12206.

Schwartz, E.K., Heyer, W.D., 2011. Processing of joint molecule intermediates bystructure-selective endonucleases during homologous recombination in eukaryotes.Chromosoma 120, 109–127.

Sha, J., Zhou, Z., Li, J., Yin, L., Yang, H., Hu, G., et al., 2002. Identification of testisdevelopment and spermatogenesis-related genes in human and mouse testes using cDNAarrays. Mol. Hum. Reprod. 8, 511–517.

Sharan, S.K., Pyle, A., Coppola, V., Babus, J., Swaminathan, S., Benedict, J., et al., 2004.BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development(Cambridge, England) 131, 131–142.

Shima, J.E., McLean, D.J., McCarrey, J.R., Griswold, M.D., 2004. The murine testiculartranscriptome: characterizing gene expression in the testis during the progression ofspermatogenesis. Biol. Reprod. 71, 319–330.

Shin, Y.H., Choi, Y., Erdin, S.U., Yatsenko, S.A., Kloc, M., Yang, F., et al., 2010.Hormad1 mutation disrupts synaptonemal complex formation, recombination, andchromosome segregation in mammalian meiosis. PLoS Genet. 6, e1001190.

Shinohara, A., Ogawa, H., Matsuda, Y., Ushio, N., Ikeo, K., Ogawa, T., 1993. Cloning ofhuman, mouse and fission yeast recombination genes homologous to RAD51 and recA.Nat. Genet. 4, 239–243.

Shoji, M., Tanaka, T., Hosokawa, M., Reuter, M., Stark, A., Kato, Y., et al., 2009. TheTDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing inthe mouse male germline. Dev. Cell 17, 775–787.


Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., et al., 2011.A conditional knockout resource for the genome-wide study of mouse gene function.Nature 474, 337–342.

Smagulova, F., Gregoretti, I.V., Brick, K., Khil, P., Camerini-Otero, R.D., Petukhova, G.V.,2011. Genome-wide analysis reveals novel molecular features of mouse recombinationhotspots. Nature 472, 375–378.

Smirnova, N.A., Romanienko, P.J., Khil, P.P., Camerini-Otero, R.D., 2006. Gene expres-sion profiles of Spo11-/- mouse testes with spermatocytes arrested in meiotic prophase I.Reproduction 132, 67–77.

Soper, S.F., van der Heijden, G.W., Hardiman, T.C., Goodheart, M., Martin, S.L.,de Boer, P., et al., 2008. Mouse maelstrom, a component of nuage, is essential forspermatogenesis and transposon repression in meiosis. Dev. Cell 15, 285–297.

Stewart, G.S., Wang, B., Bignell, C.R., Taylor, A.M., Elledge, S.J., 2003. MDC1 is amediator of the mammalian DNA damage checkpoint. Nature 421, 961–966.

Stracker, T.H., Theunissen, J.W., Morales, M., Petrini, J.H., 2004. TheMre11 complex andthe metabolism of chromosome breaks: the importance of communicating and holdingthings together. DNA Repair 3, 845–854.

Strong, E.R., Schimenti, J.C., 2010. Evidence implicating CCNB1IP1, a RING domain-containing protein required for meiotic crossing over in mice, as an E3 SUMO ligase.Genes 1, 440–451.

Su, Y.-Q., Sugiura, K., Sun, F., Pendola, J., Cox, G., Handel, M.A., et al., 2012. MARF1regulates essential oogenic processes in mice. Science 335, 1496–1499.

Suh, N., Baehner, L., Moltzahn, F., Melton, C., Shenoy, A., Chen, J., et al., 2010.MicroRNA function is globally suppressed in mouse oocytes and early embryos. Curr.Biol. 20, 271–277.

Sun, F., Handel, M.A., 2008. Regulation of the meiotic prophase I to metaphase I transitionin mouse spermatocytes. Chromosoma 117, 471–485.

Sun, F., Handel, M.A., 2011. A mutation in Mtap2 is associated with arrest of mammalianspermatocytes before the first meiotic division. Genes 2, 21–35.

Sun, F., Palmer, K., Handel, M.A., 2010. Mutation of Eif4g3, encoding a eukaryotictranslation initiation factor, causes male infertility and meiotic arrest of mouse sperma-tocytes. Development (Cambridge, England) 137, 1699–1707.

Suzuki, A., Saga, Y., 2008. Nanos2 suppresses meiosis and promotes male germ celldifferentiation. Genes Dev. 22, 430–435.

Tam, O.H., Aravin, A.A., Stein, P., Girard, A., Murchison, E.P., Cheloufi, S., et al., 2008.Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.Nature 453, 534–538.

Tang, F., Kaneda, M., O’Carroll, D., Hajkova, P., Barton, S.C., Sun, Y.A., et al., 2007.Maternal microRNAs are essential for mouse zygotic development. Genes Dev. 21,644–648.

Tarsounas, M., Pearlman, R.E., Gasser, P.J., Park, M.S., Moens, P.B., 1997. Protein-proteininteractions in the synaptonemal complex. Mol. Biol. Cell 8, 1405.

Terasawa, M., Ogawa, H., Tsukamoto, Y., Shinohara, M., Shirahige, K., Kleckner, N.,et al., 2007. Meiotic recombination-related DNA synthesis and its implications for cross-over and non-cross-over recombinant formation. Proc. Natl. Acad. Sci. U.S.A. 104,5965–5970.

Theunissen, J.W., Kaplan, M.I., Hunt, P.A., Williams, B.R., Ferguson, D.O., Alt, F.W.,et al., 2003. Checkpoint failure and chromosomal instability without lymphomagenesisin Mre11(ATLD1/ATLD1) mice. Mol. Cell 12, 1511–1523.

Tsubouchi, T., Zhao, H., Roeder, G.S., 2006. The meiosis-specific zip4 protein regulatescrossover distribution by promoting synaptonemal complex formation together withzip2. Dev. Cell 10, 809–819.


Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., et al., 1996.Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl.Acad. Sci. U.S.A. 93, 6236–6240.

Turner, J.M., 2007. Meiotic sex chromosome inactivation. Development 134, 1823–1831.Turner, J.M., Mahadevaiah, S.K., Fernandez-Capetillo, O., Nussenzweig, A., Xu, X.,

Deng, C.X., et al., 2005. Silencing of unsynapsed meiotic chromosomes in the mouse.Nat. Genet. 37, 41–47.

van der Heijden, G.W., Castaneda, J., Bortvin, A., 2010. Bodies of evidence—compart-mentalization of the piRNA pathway in mouse fetal prospermatogonia. Curr. Opin. CellBiol. 22, 752–757.

van der Laan, R., Uringa, E.J., Wassenaar, E., Hoogerbrugge, J.W., Sleddens, E., Odijk, H.,et al., 2004. Ubiquitin ligase Rad18Sc localizes to the XY body and to other chromo-somal regions that are unpaired and transcriptionally silenced during male meioticprophase. J. Cell Sci. 117, 5023–5033.

Viera, A., Rufas, J.S., Martinez, I., Barbero, J.L., Ortega, S., Suja, J.A., 2009. CDK2 isrequired for proper homologous pairing, recombination and sex-body formation duringmale mouse meiosis. J. Cell Sci. 122, 2149–2159.

Vigodner, M., 2011. Roles of small ubiquitin-related modifiers in male reproductivefunction. Int. Rev. Cell Mol. Biol. 288, 227–259.

Wang, Y., Dasso, M., 2009. SUMOylation and deSUMOylation at a glance. J. Cell Sci. 122,4249–4252.

Ward, J.O., Reinholdt, L.G., Motley, W.W., Niswander, L.M., Deacon, D.C., Griffin, L.-B., et al., 2007. Mutation in mouse hei10, an e3 ubiquitin ligase, disrupts meiotic crossingover. PLoS Genet. 3, e139.

Watanabe, Y., Nurse, P., 1999. Cohesin Rec8 is required for reductional chromosomesegregation at meiosis. Nature 400, 461.

Watanabe, T., Totoki, Y., Toyoda, A., Kaneda, M., Kuramochi-Miyagawa, S., Obata, Y.,et al., 2008. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts inmouse oocytes. Nature 453, 539–543.

Watanabe, T., Chuma, S., Yamamoto, Y., Kuramochi-Miyagawa, S., Totoki, Y.,Toyoda, A., et al., 2011. MITOPLD is a mitochondrial protein essential for nuageformation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375.

Wei, K., Clark, A.B., Wong, E., Kane, M.F., Mazur, D.J., Parris, T., et al., 2003. Inactiva-tion of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancersusceptibility, and male and female sterility. Genes Dev. 17, 603–614.

Wilkins, A.S., Holliday, R., 2009. The evolution of meiosis from mitosis. Genetics 181,3–12.

Wojtasz, L., Daniel, K., Roig, I., Bolcun-Filas, E., Xu, H., Boonsanay, V., et al., 2009.Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins,are depleted from synapsed chromosome axes with the help of TRIP13 AAA-ATPase.PLoS Genet. 5, e1000702.

Wolgemuth, D.J., 2008. Function of cyclins in regulating the mitotic and meiotic cell cyclesin male germ cells. Cell Cycle 7, 3509–3513.

Wolgemuth, D.J., 2011. Function of the A-type cyclins during gametogenesis and earlyembryogenesis. Results Probl. Cell Differ. 53, 391–413.

Wolgemuth, D.J., Roberts, S.S., 2010. Regulating mitosis and meiosis in the male germ line:critical functions for cyclins. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 1653–1662.

Wu, C.I., Xu, E.Y., 2003. Sexual antagonism and X inactivation—the SAXI hypothesis.Trends Genet. 19, 243–247.

Wu, S.M., Baxendale, V., Chen, Y., Pang, A.L., Stitely, T., Munson, P.J., et al., 2004.Analysis of mouse germ-cell transcriptome at different stages of spermatogenesis bySAGE: biological significance. Genomics 84, 971–981.


Wu, H.Y., Ho, H.C., Burgess, S.M., 2010. Mek1 kinase governs outcomes of meioticrecombination and the checkpoint response. Curr. Biol. 20, 1707–1716.

Xu, Y., Ashley, T., Brainerd, E.E., Bronson, R.T., Meyn, M.S., Baltimore, D., 1996.Targeted disruption of ATM leads to growth retardation, chromosomal fragmentationduring meiosis, immune defects, and thymic lymphoma. Genes Dev. 10, 2411–2422.

Xu, H., Beasley, M.D., Warren, W.D., van der Horst, G.T., McKay, M.J., 2005. Absence ofmouse REC8 cohesin promotes synapsis of sister chromatids in meiosis. Dev. Cell 8,949–961.

Yang, F., Wang, P.J., 2009. The Mammalian synaptonemal complex: a scaffold and beyond.Genome Dyn. 5, 69–80.

Yang, F., De La Fuente, R., Leu, N.A., Baumann, C., McLaughlin, K.J., Wang, P.J., 2006.Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synap-sis during male meiosis. J. Cell Biol. 173, 497–507.

Yang, F., Eckardt, S., Leu, N.A., McLaughlin, K.J., Wang, P.J., 2008a. Mouse TEX15 isessential for DNA double-strand break repair and chromosomal synapsis during malemeiosis. J. Cell Biol. 180, 673–679.

Yang, F., Gell, K., van der Heijden, G.W., Eckardt, S., Leu, N.A., Page, D.C., et al., 2008b.Meiotic failure in male mice lacking an X-linked factor. Genes Dev. 22, 682–691.

Yoshida, K., Kondoh, G., Matsuda, Y., Habu, T., Nishimune, Y., Morita, T., 1998. Themouse RecA-like gene Dmc1 is required for homologous chromosome synapsis duringmeiosis. Mol. Cell 1, 707–718.

Yuan, L., Liu, J.G., Zhao, J., Brundell, E., Daneholt, B., Hoog, C., 2000. The murine SCP3gene is required for synaptonemal complex assembly, chromosome synapsis, and malefertility. Mol. Cell 5, 73.

Zamudio, N., Bourc’his, D., 2010. Transposable elements in the mammalian germline: acomfortable niche or a deadly trap? Heredity 105, 92–104.

Zeng, W., Baumann, C., Schmidtmann, A., Honaramooz, A., Tang, L., Bondareva, A.,et al., 2011. Lymphoid-specific helicase (HELLS) is essential for meiotic progression inmouse spermatocytes. Biol. Reprod. 84, 1235–1241.

Zhang, F.P., Mikkonen, L., Toppari, J., Palvimo, J.J., Thesleff, I., Janne, O.A., 2008. Sumo-1 function is dispensable in normal mouse development. Mol. Cell. Biol. 28, 5381–5390.

Zheng, K., Xiol, J., Reuter, M., Eckardt, S., Leu, N.A., McLaughlin, K.J., et al., 2010.Mouse MOV10L1 associates with Piwi proteins and is an essential component of thePiwi-interacting RNA (piRNA) pathway. Proc. Natl. Acad. Sci. U.S.A. 107,11841–11846.

Zickler, D., Kleckner, N., 1998. The leptotene-zygotene transition of meiosis. Annu. Rev.Genet. 32, 619.

Zickler, D., Kleckner, N., 1999. Meiotic chromosomes: integrating structure and function.Annu. Rev. Genet. 33, 603.

[International Review of Cell and Molecular Biology] International Review of Cell and Molecular...

Documents

Transcript of [International Review of Cell and Molecular Biology] International Review of Cell and Molecular...