Fluorescence in situ hybridisation of human X and Y centromeric sequences

Nhi Hin

ABSTRACT – Centromeres are regions on chromosomes that play an essential role during the segregegation

of chromosomes during meiosis. Different chromosomes have different centromeric sequences. In the present study,

fluorescence in situ hybridisation is used to visualise X and Y chromosomes in human metaphase cells through the

use of fluorescent probes that bind specific X chromosome and Y chromosome centromeric sequences. Findings

are consistent with current knowledge; fluorescent probes were used to successfully identify X and Y chromosomes,

allowing female and male cells to be distinguished. Additionally, evidence for X-inactivation in female somatic

cells was found through the presence of Barr bodies, while the ‘X-shaped’ appearance of metaphase

chromosomes is consistent with the process of sister chromatid cohesion.

I N T R O D U C T I O N

In eukaryotes, DNA is packaged into chromosomes which are

replicated and segregated into daughter cells during cell division.

The centromeres are regions on the chromosomes which are essential

in ensuring that this process occurs accurately. Centromeres are

epigenetically defined in most eukaryotes, meaning that specific

DNA sequences are not required. Instead, chromatin organisation,

centromere-associated proteins and histone variants characterise

centromeres. For example, a common feature of most eukaryotic

centromeres is the presence of nucleosomes containing the histone

H3 variant centromere protein A (CENP-A) (McKinley & Cheeseman

2016; Yoda et al. 2000) (see Figure 1).

The underlying DNA sequences of centromeres are not well

conserved across different chromosomes and species (Lichter et al.

1988). In the present study, a technique known as fluorescent in situ

hybridisation (FISH) is used to identify and visualise the unique

centromeric sequences on the human X and Y chromosomes. In FISH,

a probe with a fluorophore attached is made to have a DNA

sequence which is complementary to the specific target DNA

sequence. The probe is introduced into target cells and allowed to

anneal with the target DNA. Although high-homology probe-target

DNA hybrids are formed, the probe may also bind weakly to other

DNA sequences, resulting in low and medium homology DNA

hybrids. Subsequent high and low stringency washes are important

in removing these hybrids, leaving only the high-homology probe-

target DNA hybrids. When the cell spreads are visualised under UV, areas of fluorescence indicate locations

where the probe is bound, with higher intensity corresponding to regions of high-homology DNA hybrids. A

summary of the process of FISH in regards to the present study is shown in Figure 2.

Figure 1. Epifluorescent micrograph

of human chromosome using

fluorescence in situ hybridisation

(FISH) probes specific to

nucleosomes containing major form

of histones (green) and the histone

H3 variant CENP-A (red). CENP-A is

an epigenetic modification localised

at the centromeres. (Image Credit:

Yoda et al., 2008)

E X P E R I M E N T A L

As stated in the University of Adelaide Genetics III Practical Manual (Grutzner 2016) without modification.

Figure 2. Usage of fluorescent in situ hybridisation to visualise human X and Y centromeric

sequences. Adapted from the University of Adelaide Genetics II I Practical Manual (2016)

and O’Connor (2008).

R E S U L T S & D I S C U S S I O N

DAPI staining allows visualisation of the chromosomes

in Figure 3 through preferential binding to AT-rich

regions of DNA (heterochromatin) (Heng & Tsui 1993).

More intense blue fluorescence suggests a high

concentration of heterochromatin relative to

euchromatin, which is less dense and has lower AT-

content. Figures 1A and 1B show that all 46

chromosomes are accounted for in each human

metaphase cell. Furthermore, Fluorescence in situ

Hybridisation (FISH) allows for visualisation of specific

DNA sequences through hybridisation of a probe with

a fluorophore attached to specific target DNA in cells.

In this study, two different probes are used that

recognise and bind specifically to the X chromosome

(green fluorescence) and Y chromosome (red

fluorescence) centromeric sequences.

Each human female somatic cell has two X-

chromosomes.

There are three cells visible in the female cell spread

in Figure 3A. In each cell, two green signals indicate

Probe has DNA sequence complementary to centromeric sequence of the X (or Y) chromosome in human cells.

Fluorophore is attached to probe DNA.

Probe and target DNA are denatured.

Allow probe to hybridise to target DNA, and washes to remove low/medium homology hybrids.

Visualise X (or Y) centromeric sequences under UV light.

1. Obtain short term lymphocyte cells from human blood samples. 2. Culture lymphocytes in presence phytohaemagglutinin to stimulate cell proliferation. 3. Use colchicine to arrest cell division. 4. Treat with hypotonic KCl to facilitate ‘spreading out’ of metaphase chromosomes.

5. Fixate cells onto glass slides with 3:1 methanol: acetic acid.

the presence of two X-chromosomes, one maternally-

inherited and one paternally-inherited. These green

signals are due to high homology binding of probe

DNA to complementary target DNA on the

centromeric sequence of X chromosomes. Each

chromosome has a unique centromeric sequence

(Schueler et al., 2001), resulting in only medium to

low-homology binding between the probe and

centromeric sequences of these other chromosomes.

Consequently, subsequent high and low stringency

washes after hybridisation are sufficient to remove

these lower-homology hybrids and result in only two

green signals per cell.

Figure 3. Epifluorescent micrograph showing Fluorescence in situ hybridisation (FISH) of human X

and Y centromeric sequences on metaphase chromosomes in human short -term lymphocytes.

Centromeric probes are specif ic to chromosome X (green) and chromosome Y (red), and DAPI (blue) is

used to visualise karyotypes. (A) Female metaphase chromosome spread. White arrows indicate Barr

bodies. (B) Male metaphase chromosome spread.

Each human male somatic cell has one X-chromosome

and one Y-chromosome.

In Figure 3B, two male cells are visible, each with an intense

fluorescent green signal signifying a maternally-inherited

X chromosome and a fluorescent red signal indicating one

paternally-inherited Y chromosome. Several other

chromosomes show weak green signals at their centromeres,

although no other chromosomes appear to show red

fluorescence. This suggests that the stringency of the washes

was sufficient in removing the majority of low and medium

homology hybrids between probe and target DNA, and

the low salt concentration and high hybridisation

temperature created a high stringency environment.

However, centromeric sequences consist of repetitive DNA

sequences (Grady et al. 1992), and it is possible that the

centromeric sequences of some other chromosomes may be

similar enough to the X-chromosome centromeric sequence

such that the probe can still bind there with medium

homology and not be readily washed off. This leads to

weak green signals on the other chromosomes, although

none of these signals are as intense as the signals indicating

high homology X-chromosome and probe hybrids.

Each human female somatic cell has a Barr body,

suggesting X-inactivation has occurred.

In the cells which do not yet show condensed chromosomes,

one of the two green signals is clearly located at the

nuclear periphery (Figure 3A). The intensity of this signal

A B

suggests highly dense DNA, indicative of a heterochromatic

Barr body. X-inactivation occurs in all female somatic cells

to ensure correct expression levels of X-chromosome genes

(“dosage compensation”). One of the two X-chromosomes

is randomly and independently ‘chosen’ to be inactivated

so that only one X-chromosome is functional per adult

female diploid cell (Lyon, 2003). It is well-established that

X-chromosome inactivation is mediated by a key regulator

called the X-chromosome-inactivation centre (Xic), which is

responsible for initiating choice, counting and cis-

inactivation in each female somatic cell (Brockdorrf, 2011).

The Xic region encodes a long non-coding RNA transcript

(Xist), which is expressed exclusively from the inactive X-

chromosome. Avner and Heard (2001) used FISH to

visualise Xist RNA expression in human female embryonic

stem cells undergoing differentiation (Figure 4), where it

can be seen that Xist RNA acts in cis to coat the X-

chromosome to be inactivated, leading to formation of the

Barr body at the nuclear periphery, while the Xist gene on

the active X is silenced. This means that only one X-

chromosome will be expressed per cell, and these findings

are consistent with the present study.

Figure 4. RNA fluorescence patterns of Xist RNA expression in female embryonic stem cells

undergoing differentiation, visualised using RNA Fluorescence in s itu hybridisation.

Undifferentiated female embryonic stem cells show low expression of Xist (indicated by two distinct

Xist RNA signals; left). Upon differentiation, Xist RNA from one of the two alleles coats the X-

chromosome to be inactivated (centre). In fully differentiated cells (right), Xist RNA coats the inactive

X chromosome and the Xist gene on the active X is si lenced (Avner & Heard, 2001).

The X-chromosome is larger than the Y-chromosome.

Each of the chromosomes has a distinct size and sequence,

meaning that techniques such as chromosome banding

would be useful in visualising their distinct euchromatin

and heterochromatin patterns and hence identify them.

However, it is difficult to distinguish most of the

chromosomes in Figure 3 due to the mostly similar shape

and size of the chromosomes, aside from the X and Y

chromosomes. The Y-chromosome is noticeably smaller

than the X-chromosome, and this is consistent with its

evolution from an autosome, which is thought to involve

large deletions (Bachtrog, 2013). Consequently, while the

human X chromosome contains 150 Mb of euchromatin

and around 800 protein-coding genes, the Y chromosome

contains approximately 23 Mb of euchromatin and 78

protein-coding genes (Bachtrog, 2013). Despite this,

many genome sequencing and transcriptome analysis

studies have confirmed that many Y-chromosome genes

have male-specific function and are necessary for sex-

determination in mammals (Carvalho et al., 2000; Gatti

& Pimpinelli, 1983).

Metaphase chromosomes have an ‘X-shaped’

appearance, consistent with sister chromatid cohesion.

In both cell spreads in Figure 3, the metaphase

chromosomes are condensed and have an ‘X-shaped’

appearance consistent with sister chromatid cohesion.

According to Klein et al. (1999), cohesins play a central

role in sister chromatid cohesion, formation of axial

elements, and recombindation. In summary, after DNA

replication in S-phase, the Cohesin protein complex

associates with replicated DNA. Polo and Aurora B

kinases soon phosphorylate the Cohesin protein Scc3,

which inactivates and displaces Cohesin along the

chromatids. However, cohesin remains bound at the

centromeres since the Shugoshin protein associates with

and protects centromeric cohesin (Watanabe & Kitajima,

2005). This results in th characteristic ‘X’ shape of the

condensed metaphase chromosomes.

Improvements

Chromosome banding techniques (e.g. G banding, R

banding) could be employed to help identify individual

chromosomes, which each have unique heterochromatin

and euchromatin patterning. Creating a higher stringency

environment than the one used in the male cell spread to

reduce non-specific binding of the probe DNA could be

accomplished by possibly leaving the high stringency

wash for a longer time, reducing the salt concentration or

increasing the temperature, although this would have to

be done carefully to avoid washing off high-homology

hybrids as well. To ensure correct identification of Barr

bodies, a probe specific to Xist and with a different

fluorescence wavelength could be used to visualise the

Barr body.

C O N C L U S I O N

Fluorescence in situ hybridisation was used to visualise X

and Y chromosomes in human metaphase cells, with results

being consistent with expected results and reported

literature. Each human somatic cell undergoing mitosis

had 46 chromosomes. Fluorescent probes were used to

successfully identify X and Y chromosomes, allowing

female and male cells to be distinguished. Additionally,

evidence for X-inactivation in female somatic cells was

found through the presence of one Barr body per cell,

while the ‘X-shaped’ appearance of metaphase

chromosomes is consistent with the process of sister

chromatid cohesion. Improvements could be implemented

to further explore these concepts.

R E F E R E N C E S

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counting, choice and initiation’, Nature Reviews Genetics, vol. 2,

no. 1, pp.59-67.

Bachtrog, D 2013, ‘Y-chromosome evolution: emerging insights

into processes of Y-chromosome degeneration’, Nature

Reviews Genetics, vol. 14, no. 2, pp.113-124.

Brockdorff, N 2011, ‘Chromosome silencing mechanisms in X-

chromosome inactivation: unknown

unknowns’, Development, vol. 138, no. 23, pp.5057-5065.

Carvalho, AB, Lazzaro, BP & Clark, AG 2000, ‘Y

chromosomal fertility factors kl-2 and kl-3 of Drosophila

melanogaster encode dynein heavy chain

polypeptides’, Proceedings of the National Academy of

Sciences, vol. 97, no. 24, pp.13239-13244.

Gatti, M & Pimpinelli, S 1983, ‘Cytological and genetic

analysis of the Y chromosome of Drosophila

melanogaster’, Chromosoma, vol. 88, no. 5, pp.349-373.

Grady, DL, Ratliff, RL, Robinson, DL, McCanlies, EC, Meyne, J

& Moyzis, RK 1992, ‘Highly conserved repetitive DNA

sequences are present at human centromeres’, Proceedings of

the National Academy of Sciences, vol. 89, no. 5, pp.1695-

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Human X and Y Centromere-Associated Sequences to

Metaphase Chromosomes’, practical notes for Genetics 3111,

University of Adelaide, viewed 20 April 2016,

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ac%20Handbook%202016.pdf>.

Heng, HH & Tsui LC 1993, ‘Modes of DAPI banding and

simultaneous in situ hybridisation’, Chromosoma, vol. 102, no.

5, pp. 325-332.

Klein, F, Mahr, P, Galova, M, Buonomo, SB, Michaelis, C,

Nairz, K & Nasmyth, K 1999, ‘A central role for cohesins in

sister chromatid cohesion, formation of axial elements, and

recombination during yeast meiosis’, Cell, vol. 98, no. 1,

pp.91-103.

Lichter, P, Cremer, T, Borden, J, Manuelidis, L & Ward, DC

1988, ‘Delineation of individual human chromosomes in

metaphase and interphase cells by in situ suppression

hybridization using recombinant DNA libraries’, Human

genetics, vol. 80, no. 3, pp.224-234.

Lyon, MF 2003, ‘The Lyon and the LINE hypothesis’, Seminars

in Cell & Developmental Biology, vol. 14, no. 6, pp. 313-318.

O’Connor, C 2008, ‘Fluorescence in situ hybridization (FISH)’,

Nature Education, vol. 1, no. 1, p. 171.

Schueler, MG, Higgins, AW, Rudd, MK, Gustashaw, K &

Willard, HF 2001, ‘Genomic and genetic definition of a

functional human centromere’, Science, vol. 294, no. 5540,

pp.109-115.

University of Adelaide 2016, ‘Genetics III Practical Manual’.

Watanabe, Y & Kitajima, TS 2005, ‘Shugoshin protects

cohesin complexes at centromeres’, Philosophical Transactions

of the Royal Society of London B: Biological Sciences, vol. 360,

no. 1455, pp.515-521.

Immunostaining of the Synaptonemal Complex in Male Mouse Meiotic Cells

Nhi Hin

A B S T R A C T – Synaptonemal complex protein SCP3 plays an important role in formation of the

synaptonemal complex in mammallian cells during prophase I. In the present study, immunostaining of SCP3 was

used to visualise the synaptonemal complex in male mouse meiotic cells and hence identify several important

features of these cells. SCP3 was found to localise between homologous chromosomes to facilitate crossing-over

and synapsis. SCP3 patterning was found to be dynamic and useful for identifying cells at particular stages of

prophase I such as the pachytene and diplotene stages. In addition, SCP3 staining revealed the failure of X

and Y chromosomes to pair up during prophase I, resulting in the formation of a sex body which is

heterochromatic and transcriptionally inactive.

I N T R O D U C T I O N

Meiosis is a specialised form of cell division that

occurs in sexually-reproducing organisms and

involves a single round of DNA replication followed

by two rounds of chromosome segregation to produce

haploid gametes. During meiotic prophase, a protein

structure called the synaptonemal complex (SC) forms

between homologous chromosomes to enable

synapsis, recombination and subsequent disjunction of

homologous chromosomes (Cohen et al. 2006). The SC

comprises two lateral elements which are formed

along the axis joining a pair of sister chromatids and

a central element that connects them. Assembly of the

SC is used to define stages of meiotic prophase

(Fig.1A-D). First, short filaments of axial elements like

synaptonemal complex protein 3 (SCP3) assemble

along the homologous chromosomes in leptotene

(Fig.1A). As axial elements lengthen during zygotene,

they begin to synapse through becoming connected

by the central element (Fig.1B). Synapsis unfurls along

the axis of all paired chromosomes with the exception

of X and Y sex chromosomes in male spermatocytes,

which only pair up at their pseudoautosomal regions,

located at the chromosome ends. Pachytene nuclei

display complete synapsis with the complete

formation of the SC (Fig.1C). Finally, recombination

with a non-sister chromatid from the homologous

chromosome generates chiasmata during late

pachytene, which physically link chromosomes

together after the SC disassembles during diplotene

(Fig.1D) (Henderson & Keeney, 2005).

In mammals, three protein components of the SC have

been identified: SCP1, SCP2 and SCP3, while other

proteins such as histones also play crucial roles (Fig.2).

SCP1 forms the transverse filaments of the central

element while SCP2 and SCP3 are components of the

lateral element (Baarends et al. 2003). According

Yuan et al. (2000), SCP3 is required for axial element

formation in spermatocytes as Scp3 null mutations in

male mice resulted in infertility. It was found that

apoptosis occurred at the zygotene stage, likely as

the unpaired chromosomal regions were detected by

a checkpoint mechanism. Overall, correct formation of

the lateral element and presence of SCP3 is required

for completion of synapsis, making it a suitable

marker for visualisation of the SC in the present study.

In this study, immunostaining is used to visualise the SC

by allowing a primary anti-SCP3 antibody to bind to

SCP3 in mouse testis cells undergoing meiosis. A

secondary antibody with fluorophore attached is then

allowed to bind to the primary antibody, allowing

visualisation of the presence and location of the SC

under UV light. A summary of the process and aims of

the experiment is shown in Figure 3.

Figure 1. Schematic overview of assembly of the synaptonemal complex (SC). During

leptotene, axial elements (blue and pink) lengthen (A) and become connected by the central

element (green bars) (B) . The axial/lateral (SCP2, SCP3) and central elements (SCP1) extend

bidirectionally until complete synapsis occurs along each pair of homologous chromosomes (C) .

The SC disassembles during late pachytene. Chiasmata are the sites of crossing -over and

keep chromosomes attached during diplotene (D). Image Credit: Morelli and Cohen (2005).

1. Isolate meiotic cells from male mice testis

2. Fix cells onto slides. Meiotic cells will be in variety of stages at this stage.

3. Stain slides with primary anti-SCP3 mouse antibody and secondary anti-mouse IgG antibody to visualise only

meiotic cells in prophase.

From SCP3 appearance, identify prophase substage.

4. Stain with DAPI to visualise regions of heterochromatin (sex bodies and centromeres) to aid in identification of

chromosomes.

Figure 2. Summary of the steps and aims of experiment .

E X P E R I M E N T A L

As stated in the University of Adelaide Genetics III Lab Manual without modifications (Grutzner 2016).

R E S U L T S & D I S C U S S I O N

Figure 3. Meiotic prophase cell spreads from 3-week old male mice stained with anti-SCP3

antibody (red signal) to visualise the synaptonemal complex protein SCP3 and DAPI (blue

signal) to visualise chromatin. Arrows indicate XY sex body (A) Pachytene stage . Image:

Arnold, Scott & Srimayee (2015). (B) Diplotene stage . Image: Qikun Wang (2016).

The Synaptonemal Complex is fully formed at the

pachytene stage of meiotic prophase I.

Prophase I is the first stage of meiosis and includes

five phases: leptotene, zygotene, pachytene,

diplotene and diakinesis. During Prophase I,

homologous chromosomes pair up, synapse and cross-

over. Synaptonemal complex protein 3 (SCP3) is a

major structural protein within the lateral element of

the synaptonemal complex and can be used to

visualise the presence of synaptonemal complex

structures between homologous chromosomes during

prophase I (Figure 3). A primary anti-mouse SCP3

antibody is used that recognises and binds SCP3,

while a fluorescent secondary antibody is then

introduced to bind the primary antibody. Figure 4A

shows the 21 chromosomes present in a single

spermatocyte nucleus during the pachytene stage of

meiosis I. The synaptonemal complex structures

between all chromosomes are continuous and uniform

lines, suggesting they are fully formed and able to

facilitate synapsis and crossing-over between the

aligned non-sister chromatids of the homologous

chromosomes.

It is expected that the XY bivalent (arrow) would

remain largely unpaired and unsynapsed due to low

homology between X and Y chromosomes in mammals.

This is as the Y chromosome is significantly smaller and

possesses Y-specific male-determining genes.

Consequently, pairing up and crossing over in the XY

bivalent only occurs at the pseudoautosomal regions

located at the chromosome ends (Baarends et al.

2003).

A B

Epigenetic silencing of unpaired chromosomes

during meiotic prophase.

A general mechanism in cells called the Meiotic

Silencing of Unsynapsed Chromosomes (MSUC) is

responsible for epigenetically silencing any

chromosomes that do not pair with their homologous

partners (Turner et al. 2005). This is particularly

important for ensuring correct segregation of

chromosomes into daughter cells and minimising the

risk of aneuploidy in future generations. Meiotic Sex

Chromosome Inactivation (MSCI) is a specific case of

MSUC that occurs during pachytene in male sperm

cells. It is responsible for silencing the XY bivalent by

forming a sex body, preventing the unpaired XY

regions from activating cell cycle checkpoints that

would halt meiosis (Turner, 2007).

MSCI involves various modifications associated with

the DNA double-strand-break repair response and

chromatin silencing mechanism. This involves the

translocation of ATR along DNA loops, where it

phosphorylates H2AX (γH2AX) (Fernandex-Capetillo

et al., 2003). γH2AX localises to DSBs in leptotene

nuclei, where it recruits other recombination proteins.

This leads to other histone modifications including

methylation of histone 3 at lysine 9 (H3K9me),

increased ubiquitination of H2A, and DNA-repair

proteins like ATR bound to unpaired parts of the XY

chromatin (Turner et al. 2005). These modifications

establish and maintain MSCI by allowing the XY

bivalent to take the form of a sex body composed of

dense heterochromatin.

Presence of sex body supports meiotic sex

chromosome silencing in pachytene and diplotene.

The results from the present study are consistent with

the importance of the inactivation of X and Y

chromosomes during male meiotic prophase

described in the literature. Figure 3A shows that the

sex body is sequestered towards the nuclear

periphery of the pachytene nucleus. Regions of

intense DAPI staining indicate dense regions of DNA

often associated with AT-rich heterochromatin. In

Figure 4A, there is strong DAPI staining surrounding

the XY sex body, which is consistent with its

heterochromatic state. The strong DAPI staining at

other locations may indicate the centromeres of the

other chromosomes.

This DAPI staining pattern can also be used to identify

the X and Y chromosomes in the diplotene stage

(Figure 4B). The chromosomes indicated by the arrow

are surrounded by an intense region of DAPI staining,

suggesting the DNA is in a heterochromatic state that

is consistent with the sex body. The intensity of the

stain suggests the DNA is highly condensed to

minimise access by transcription proteins like DNA

polymerases. This ensures that the sex body is

transcriptionally silent at this stage.

According to Turner et al. (2007), the X and Y

chromosomes are still transcriptionally active during

leptotene and zygotene. Although no clear images of

cells at these stages were obtained in the present

study, it would be expected that the sex body would

not be visible in these cells.

The synaptonemal complex starts to degrade

during the diplotene stage of meiotic prophase I.

In diplotene, the appearance of the synaptonemal

complex is less uniform compared to pachytene, as

the paired homologous chromosomes begin to

separate to allow some transcription of DNA while

remaining attached at the regions where synapsis

and crossing-over have occurred (Baarends et al.

2003). These thicker regions on Figure 3B represent

the chiasmata where crossing over has occurred.

Figure 5. Pachytene cell spread from 30-

day-old male mice immmunostained with anti-

SCP3 antibody and anti-uH2A antibody

(Baarends et al., 2003).

Comparison of X-inactivation and Meiotic Sex

Chromosome Inactivation

Both X-inactivation and MSCI occur are epigenetic

modifications that implicate the sex chromosomes and

produce heterochromatic sex bodies. However, their

mechanisms differ. X-inactivation occurs in female

cells during early development, permanently

inactivating one X-chromosome per female somatic

cell. This modification occurs independently in each

cell and is inherited by all daughter cells (Penny et al.

1996). In contrast, MSCI occurs to silence the X and Y

chromosomes temporarily throughout

spermatogenesis in male meiotic cells. While X-

inactivation occurs to ensure correct gene dosage,

MSCI occurs to prevent the unpaired X and Y

chromosomes from triggering cell cycle termination at

checkpoints.

X-inactivation is initiated by an X-encoded RNA

called Xist which acts in cis to coat the X-chromosome

to be inactivated, leading to histone modifications

such as methylation, ubiquitylation and deacetylation

that contribute to formation of heterochromatin.

However, Penny et al. (1996) showed that although

Xist is essential for X-inactivation, it is not required for

MSCI. Instead, MSCI heavily implicates the histone

variant H2AX. According to Turner et al. (2007),

H2AX is rapidly phosphorylated at the zygotene-

pachytene transition on the X and Y chromosomes to

form γH2AX, which then recruits DNA repair proteins

and histone modifiers associated with

heterochromatin formation. A study by Mahadvaiah

et al. (2001) supports this, as H2AX-null mice meiotic

cells underwent complete arrest.

Experimental Errors

There was difficulty in obtaining images from the cell

spreads prepared by our group, resulting in the need

to use other groups’ images. Our group’s images did

not show any DAPI or antibody staining, suggesting

errors in the meiotic cell preparation. It is suspected

that there may be an issue with the

paraformaldehyde fixative as the slides were not dry

even after 60 minutes. Because of this, the meiotic

cells may have been removed by the subsequent rinse,

leaving no material for staining. Some others who

used the same paraformaldehyde solution also

reported similar issues.

Improvements

The experiment should be repeated using fresh

solutions of paraformaldehyde to determine if this

was the cause of error. Additionally, to ensure correct

identification of the X and Y chromosomes, future

experiments may use another antibody with a

different fluorescence frequency to visualise

ubiquitinated H2A (uH2A), which would be enriched

at the XY sex body. In a similar experiment by

Baarends et al. (2003), this technique was used to

visualise the XY sex body (Figure 5). It would be

expected that uH2A would not be present at

leptotene and zygotene while it would become more

intense during pachytene and diplotene.

C O N C L U S I O N

Synaptonemal complex protein SCP3 plays an

important role in formation of the synaptonemal

complex in mammallian cells during prophase I. In the

present study, immunostaining of SCP3 was used to

visualise the synaptonemal complex in male mouse

meiotic cells. SCP3 was found to localise between

homologous chromosomes to facilitate crossing-over

and synapsis. SCP3 patterning was found to be

dynamic and useful for identifying cells at particular

stages of prophase I such as the pachytene and

diplotene stages. In addition, SCP3 staining revealed

the failure of X and Y chromosomes to pair up during

prophase I, resulting in the formation of a sex body

which is heterochromatic and transcriptionally inactive.

R E F E R E N C E S

Baarends, WM & Grootegoed, JA 2003, ‘Chromatin

dynamics in the male meiotic prophase’, Cytogenetic and

genome research, vol. 103, no. 3, pp.225-234.

Baarends, WM, Wassenaar, E, Hoogerbrugge, JW, van

Cappellen, G, Roest, HP, Vreeburg, J, Ooms, M,

Hoeijmakers, JH & Grootegoed, JA (2003), ‘Loss of HR6B

ubiquitin-conjugating activity results in damaged

synaptonemal complex structure and increased crossing-

over frequency during the male meiotic prophase’,

Molecular and Cellular Biology, vol. 23, no. 4, pp.1151-

1162.

Cohen, PE, Pollack, SE & Pollard, JW 2006, ‘Genetic

analysis of chromosome pairing, recombination, and cell

cycle control during first meiotic prophase in

mammals’, Endocrine Reviews, vol. 27, no. 4, pp.398-426.

Fernandez-Capetillo, O, Mahadevaiah, SK, Celeste, A,

Romanienko, PJ, Camerini-Otero, RD, Bonner, WM,

Manova, K, Burgoyne, P & Nussenzweig, A 2003, ‘H2AX

is required for chromatin remodeling and inactivation of

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