The Veterinary Journal 185 (2010) 265–271

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The Veterinary Journal

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Review

Infertility and candidate gene markers for fertility in stallions: A review

Katrin Giesecke a, Harald Sieme b, Ottmar Distl a,*

a Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Bünteweg 17p, 30559 Hannover, Germanyb Clinic for Horses, Department of Reproductive Medicine, University of Veterinary Medicine, Bünteweg 15, 30559 Hannover, Germany

a r t i c l e i n f o

Article history:Accepted 27 July 2009

Keywords:StallionFertilityCandidate genesHorse genome assemblySingle nucleotide polymorphismsMicrosatellites

1090-0233/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tvjl.2009.07.024

* Corresponding author. Tel.: +49 511 9538875; faxE-mail address: ottmar.distl@tiho-hannover.de (O.

a b s t r a c t

Stallion fertility is of high economic importance for the horse industry. The discovery of molecular mech-anisms affecting fertility will be facilitated by the horse genome assembly and the development of noveltools for analysing complex genetic traits. Genetic markers in candidate genes, such as CRISP3, SPATA1and INHBA, in breeding stallions have been associated with pregnancy rate per oestrus in mares. Thispaper reviews candidate autosomal, X and Y genes for stallion fertility, including genes encoding hor-mones and their receptors of the hypothalamic-pituitary axis, proteins of the seminal plasma, proteinsinvolved in spermatozoa–ovum binding and genes influencing sexual development, as well as Y-specificgenes. Their chromosomal location and gene structure are described, based on the horse genome assem-bly EquCab2.0 and a resource for markers located within or in close vicinity to the candidate genes(including pre-designed primer sequences). The application of genetic markers in improving stallion fer-tility for breeding and management is discussed.

� 2009 Elsevier Ltd. All rights reserved.

Introduction

Stallion fertility is an economically important trait with a com-plex environmental and genetic background. Heritability estimatesfor stallion fertility vary from 0.03–0.15 for foaling rate per breed-ing season (Dohms, 2002; Hamann et al., 2005a). Pregnancy rateper oestrus (PRO) in mares is associated with breeding year andseason, breeding centre, age of mares, breeding history of mares,type of covering (natural or artificial insemination), breeding man-agement (number of coverings and time intervals between them),and type of semen (fresh within 24 h, fresh and shipped within48 h or frozen/thawed) (Hamann et al., 2005b).

Genetic markers may be useful in selection of breeding stal-lions. Studies in humans and mice have revealed a large numberof proteins involved in the mechanisms of male reproduction andthe cascade of fertilisation, but there are few reports of proteinswith an influence on fertility in stallions. Hamann et al. (2007) re-ported a significant association between a CRISP3-associated singlenucleotide polymorphism (SNP) in stallions and PRO in coveredmares. Significant associations of single markers and haplotypeswith least square means (LSM)-PRO and the embryonic and pater-nal component of breeding values (BVs) support a role for INHBAmutations in fertility of stallions (Giesecke et al., 2009a). We havealso found significant associations between fertility and a SPATA1-associated SNP (Giesecke et al., 2009b).

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: +49 511 9538582.Distl).

In this paper, we provide an overview of 37 candidate genesthat are potentially important in stallion fertility and discuss thedevelopment of genetic markers for stallion fertility based on thehorse genome assembly EquCab2.0.1 As many genes are known thatinfluence mammalian fertility in other species, especially humansand mice, further candidate genes in horses are likely to becomeknown in the future. The development of dense marker sets, includ-ing microsatellites or SNPs such as those provided on the EquineSNP50 Genotyping Beadchip (Illumina), may allow the roles ofrecognised candidate genes to be determined and may facilitateidentification of new genes associated with horse fertility.

Chromosomal anomalies and inborn defects

Intersexuality

In the presence of a Y chromosome in the zygote, the undiffer-entiated gonads develop into testes. The key factor in initiatingmale sexual development is testis determining factor encoded bythe SRY-gene (sex determining region of the Y chromosome) onthe Y chromosome. Abnormal development of the testes may re-sult in intersexuality (Kuiper and Distl, 2007). Few candidate genesfor intersexuality have been identified and tested in horses to date.Collecting a larger number of cases and matching controls will al-low the equine genome to be interrogated for linked and associ-ated genes involved in these sexual anomalies.

1 http://www.broad.mit.edu/ftp/pub/assemblies/mammals/horse/Equus2/.

266 K. Giesecke et al. / The Veterinary Journal 185 (2010) 265–271

The most frequent chromosomal abnormalities in stallions aresex chromosome mosaicism and sex-reversal syndromes. TheXXY-syndrome (Klinefelter’s syndrome), the unbalanced 65,XXYkaryotype and Y chromosome disomy are rare. Sex-reversal syn-drome results from a failure of differentiation of the gonads duringembryogenesis, so that chromosomal sex does not conform to go-nadal sex. Horses with the XY-sex-reversal mare syndrome showundifferentiated gonads or partial ovarian tissue and, in mostcases, have a deletion of the SRY-gene (Bugno et al., 2003).

The 64,XX sex-reversal condition is inherited as an autosomalrecessive trait and, in humans, is caused by defects in cortisol bio-synthesis. All 64,XX sex reversals are SRY-negative. In the absenceof the SRY-gene, SOX9 and FOXL2 may play a role in testiculardevelopment. XX horses exhibit stallion-like behaviour, testeswithout spermatogenesis, an enlarged clitoris or small penis andsome have ovarian tissue and other female organs (Constantet al., 1994; Milliken et al., 1995; Bannasch et al., 2007).

Mutations in the androgen receptor gene (AR) in humans lead totesticular feminisation (Krausz and Giachini, 2007), but there areno reports of mutations in the AR gene in the horse. Horses affectedby an AR-like syndrome have female external genitalia, high plas-ma testosterone concentrations and exhibit stallion-like behaviourand infertility. They have a 64,XY karyotype and SRY and ZFY genesare present (Howden, 2004; Switonski et al., 2005).

Autosomal defects

Only a few cases of autosomal deletions, autosomal transloca-tions and autosomal trisomy have been reported in the horse.Large chromosomal deletions or trisomies involving large chromo-somes are assumed to cause early embryonic losses (Lear et al.,2008). A stallion with a 64,XY,del(13)(qter) karyotype exhibitedabnormal spermatozoa with poor motility (Halnan et al., 1982).

Equine trisomy has been found for horse chromosomes 23, 26,27, 28, 30 and 31. These cases had multiple developmental defectsand some of them were infertile (Lear and Bailey, 2008). Only onecase of an autosomal chromosome translocation has been re-ported; this was a Thoroughbred stallion that had a 64,XY,t(1;30)karyotype, with a tandem fusion of chromosome 30 with ECA1pter(Long, 1996). A high incidence of repeated early embryonic losses(REEL) was observed in mares mated to this stallion.

Translocations in five mares included karyotypes of64,XX,t(1;21), 64,XX,t(16;22), 64,XX,t(4;13), 64,XX,t(1q;3q) and64,XX,t(1;16). Lear and Layton (2002) and Lear et al. (2008) foundREEL in all mares with autosomal chromosome translocations.

Cryptorchidism and monorchism

Cryptorchidism is classified according to the location of the af-fected testes into inguinal, incomplete abdominal and completeabdominal cryptorchidism. Most cases of cryptorchidism are uni-lateral (Aurich, 2005). Genetic and non-genetic factors may influ-ence testicular descent. Insulin-like factor 3 (encoded by INSL3)and its receptor leucine-rich repeat-containing G protein-coupledreceptor 8 (encoded by LGR8) are thought to be important signal-ling molecules in the control of testicular descent in humans andmice (Leeb et al., 2005).

During embryonic development, Leydig (interstitial) cells of thefetal testis produce insulin-like factor 3 (INSL3), while its receptor,leucine-rich repeat-containing G protein-coupled receptor 8(LGR8), is expressed by the gubernaculum. INSL3 is responsiblefor the process of abdominal testicular descent in humans (Agoul-nik, 2007). Mutations in INSL3 and LGR8 leading to cryptorchidismhave been identified in humans (Gorlov et al., 2002; Canto et al.,2003). Klonisch et al. (2003) demonstrated that INSL3 expressionwas up-regulated and LGR8 expression was down-regulated in

the cryptorchid testis of unilateral cryptorchid stallions. However,mutations affecting INSL3 or LGR8 genes have not been detected incryptorchid stallions. Since cryptorchidism is a common anomalyin stallions, whole genome linkage and association studies maybe useful in identification of genomic regions containing new can-didate genes.

Monorchism, where one testis is lacking, is rare in horses(Searle et al., 1999; Petrizzi et al., 2004). Another rare developmen-tal defect is hypospadia, where the urethra opens below the tip ofthe glans penis. Bleul et al. (2007) found hypospadia in a Friesianstallion with a ventrocaudal deviation of the shaft of the penisand an incomplete glans penis. A rare case of aplasia of the ductusdeferens was described by Estrada et al. (2003) in a 3 year oldQuarter stallion.

Development of the male reproductive tract

Androgen receptor

Androgen receptor (AR) is a ligand-dependent transcription fac-tor that belongs to the steroidal receptor class 1 family of nuclearreceptors (Callewaert et al., 2003). In stallions, AR is localised in epi-didymal and prostate cells, which are directly regulated by andro-gens (Hejmej et al., 2006). Parlevliet et al. (2006) detected AR inthe principal cells of the caput, corpus and cauda epididymidis ofstallions of different ages and concluded that AR and the mainandrogen testosterone are required for epididymal developmentand function. Hejmej et al. (2006) reported stronger immunostain-ing for AR in a cryptorchid horse compared to normal stallions.

Mutations in AR in humans lead to testicular feminisation, suchas androgen insensitivity syndrome (AIS) (Krausz and Giachini,2007). The length of a CAG repeat in exon 1 of the AR gene is asso-ciated with infertility in men (Shah et al., 2003). In horses, somecases of AIS are thought to be associated with mutations in AR(Crabbe et al., 1992; Pailhoux et al., 1995; Howden, 2004; Switon-ski et al., 2005).

Oestrogen

Oestradiol plays a key role in the maturation of the stallion epi-didymis during the pubertal transition. Parlevliet et al. (2006) de-tected an age-related increase in oestradiol-17b in theepididymis. The oestrogen receptor ESR2 is localised in the princi-pal cells of the caput, corpus and cauda epididymis in stallionsindependent of age, whereas ESR1 localisation is regional and agedependent (Parlevliet et al., 2006). Association studies in humanshave demonstrated several polymorphic sites in the two genesencoding ESR1 and ESR2, which have an influence on spermatozoalcount and cryptorchidism (Krausz and Giachini, 2007).

Testicular oestrogens, mainly oestradiol-17b, modulate releaseof luteinising hormone (LH) from the pituitary gland (Roser,1997). Stewart and Roser (1998) identified an increase in plasmaand testicular concentrations of oestradiol and oestrogen conju-gates in stallions with increasing age. Infertile stallions had signif-icantly lower plasma concentrations of oestradiol and oestrogenconjugates than fertile stallions.

Relaxin

The gene encoding relaxin (RLN) and three non-allelic relaxingenes (RLN1, RLN2 and RLN3) belong to the insulin gene superfam-ily. Relaxin plays key roles in the development of the male repro-ductive tract, the growth of the prostate gland and spermatozoalmotility (Samuel et al., 2003). The prostate gland is the mainsource of relaxin in the seminal plasma.

K. Giesecke et al. / The Veterinary Journal 185 (2010) 265–271 267

Hormonal influences on male reproduction

Genes encoding hormones involved in regulation of testiculardevelopment, sexual maturity and sexual activity are candidatesfor stallion fertility. Testicular function is dependent upon a func-tional hypothalamic-pituitary–testicular axis, which involvesgonadotrophin releasing hormone (GNRH), LH, follicle stimulatinghormone (FSH), testosterone, oestrogens and inhibins. Their local-isation on the horse genome is shown in Table 1 and similaritieswith orthologous genes in humans and mice are shown in Supple-mentary Table 1. Studies of candidate genes may be facilitatedusing the markers presented in Supplementary Tables 2 and 3.

Gonadotrophin releasing hormone and gonadotropins

GNRH is released by the hypothalamus in a pulsatile fashion,activating a G-protein-coupled receptor (GNRHR), which inducesrelease of LH (Leydig cell stimulating hormone) and FSH (Sertolicell stimulating hormone) from the anterior pituitary. FSH is essen-tial for spermatogenesis during puberty, whereas spermatogenesisin adults is promoted mainly by testosterone. Release of testoster-one and oestrogens from the Leydig cells of the testis is stimulatedby LH. FSH binds mostly to Sertoli cells and causes the release ofinhibin, activin and androgen binding protein. These testicular pro-teins and steroid hormones influence the release of GNRH, FSH andLH from the anterior pituitary gland via a negative feedback loop.

Inhibin, activin and follistatin

Inhibin, activin and follistatin (FST) belong to the transforminggrowth factor (TGF) b superfamily (Welt et al., 2002). In the stal-lion, FSH secretion is controlled by inhibin, in conjunction withoestrogen and testosterone (Roser, 1997). FST is produced in thetestis and inhibits the secretion of FSH by binding to activin. Plas-ma and intratesticular concentrations of inhibin in stallions areassociated with testicular maturation and fertility (Stewart andRoser, 1998). The intratesticular concentration of inhibin is a

Table 1Hormones of the hypothalamic-pituitary testicular axis as candidates for stallions’ fertility(ECA) and the size of the horse genomic sequence using EquCab2.0.

Candidate gene Gene identification (LOC) ECA Po

Sta

Beta actin (ACTB) ENSECAG00000015935 13 2,4Gamma actin (ACTG) ENSECAG00000018600 11 1,6

Alpha actinin 1 (ACTN1) ENSECAG00000019476 24 14

Alpha actinin 4 (ACTN4) ENSECAG00000021777 10 9,6Oestrogen receptor 1 (ESR1) ENSECAG00000022441 31 15

Oestrogen receptor 2 (ESR2) ENSECAG00000024947(fragment)

24 11

Follicle-stimulating hormone (FSHB) ENSECAG00000019116 7 96Follistatin (FST) ENSECAG00000017783 21 18Gonadotropin releasing hormone (GNRH) ENSECAG00000010664 2 54Gonadotropin releasing hormone receptor

(GNRHR)LOC100033874 3 67

Alpha inhibin (INHA) ENSECAG00000015864 6 9,0Beta inhibin A (INHBA) ENSECAG00000022448 4 12Beta inhibin B (INHBB) ENSECAG00000014285 18 10Insulin-like 3 (INSL3) ENSECAG00000016450

(fragment)21 2,6

Luteinising hormone (LHB) ENSECAG00000007548 10 18Leucine-rich repeat-containing G protein-

coupled receptor 8 (LGR8)ENSECAG00000014103 17 11

Prolactin (PRL) ENSECAG00000000114 20 20Prolactin receptor (PRLR) ENSECAG00000009483 21 29Prorelaxin (RLN1) ENSECAG00000013020 23 26

potential marker for early detection of fertility problems in youngstallions (Roser, 2008).

Genes encoding a and b inhibins (INHA, INHBA, INHBB), as wellas a, b and c actins (ACTN, ACTB, ACTG), are potential candidatesfor association analysis of fertility. In Hanoverian stallions, signif-icant associations of SNPs and their haplotypes within the inhibinbeta A (INHBA) gene have been demonstrated for LSM-PRO andthe embryonic and paternal component of BVs for PRO. The muta-tions identified in this gene result in altered transcription factorbinding sites and these mutations may regulate expression ofINHBA (Giesecke et al., 2009a).

Prolactin

Prolactin, produced by the PRL gene, is secreted by the pars dis-talis of the adenohypophysis in stallions in response to sexualstimulation (Thompson et al., 1996). Plasma concentrations of pro-lactin are positively correlated with day length and are highest installions during the breeding season (Gerlach and Aurich, 2000;Roser, 2008). Aurich et al. (2002) detected significant prolactin re-lease in stallions during the non-breeding season in response totreatment with the dopamine antagonist sulpiride, but no changein serum prolactin concentration after treatment with the opioidantagonist naloxone, suggesting that prolactin secretion is con-trolled by dopaminergic pathways. The interaction between pro-lactin, gonadotrophins and GNRH is modulated by photoperiodand melatonin, whereas prolactin release is not mediated bygonadotrophins (Henderson et al., 2008). Prolactin receptor (PRLR)is expressed by gonadotroph cells, which are embedded within lac-totroph clusters in the pars distalis of the adenohypophysis inhorses (Tortonese et al., 2001).

Spermatogenesis

Spermatogenesis is the development of spermatids in the testisfrom spermatogonia, which are derived from primordial germ cells(Johnson et al., 1997). Spermiogenesis is the differentiation of

, with their gene identification, localisation and position on the equine chromosome

sition (bp) Size (bp) Number of exons

rt End DNA RNA

63,585 2,465,463 1879 1098 628,565 1,630,637 2073 1604 (isoform 1)

654 (isoform 2)4 (isoform 1)4 (isoform 2)

,991,841 15,037,255 45,414 2819 (isoform 1)2753 (isoform 2)

22 (isoform 1)21 (isoform 2)

92,742 9,718,476 25,735 2241 24,081,987 15,329,542 247,556 3717 (isoform 1)

3508 (isoform 2)8 12

,029,301 11,082,098 52,798 1744 8

,163,676 96,165,734 2059 502 2,584,478 18,590,636 6159 2119 6,131,556 54,134,868 3313 308 3,130,453 67,151,298 20,888 1114 4

65,378 9,068,177 2800 1286 2,878,829 12,889,612 10,784 1445 2,834,802 10,838,204 3403 958 446,466 2,647,154 689 300 2

,963,376 18,964,111 736 510 2,005,845 11,078,391 72,547 2396 18

,481,551 20,491,085 9535 853 5,918,374 30,066,399 148,026 2200 10,699,707 26,704,126 4420 801 2

268 K. Giesecke et al. / The Veterinary Journal 185 (2010) 265–271

spermatids into spermatozoa. After spermatogenesis and spermio-genesis, spermatozoa are transported to the epididymis for matu-ration. A survey of these candidate genes is shown in Table 2 andSupplementary information for comparison with human andmouse genes, as well as markers for these genes, is shown inSupplementary Tables 1–3.

Spermatogenesis associated protein 1 (SPATA1) is thought to beinvolved in spermatogenesis, but its detailed function is still un-known. Strong linkage disequilibrium has been demonstrated foran intragenic SPATA1 SNP in Hanoverian stallions for the embry-onic component of BVs (Giesecke et al., 2009b). This marker chan-ged an SP1 binding site, but did not change the coding sequence orthe splice sites. Therefore, this intronic SPATA1 mutation is thoughtto confer improved fertility in stallions via regulation of geneexpression (Giesecke et al., 2009b).

Maturation of spermatozoa

Seminal plasma (SP) proteins derived from the epididymis andaccessory sex glands participate in post-testicular spermatozoalmaturation, where the spermatozoa acquire the ability for fertilisa-tion (capacitation) (Sostaric et al., 2008). During capacitation, sig-nal transducing pathways that initiate the acrosome reaction areactivated (Gadella et al., 2001; Neild et al., 2005). SP proteins con-sist of major and minor components in three main protein classes:fibronectin type II proteins, cysteine-rich secretory proteins(CRISPs) and spermadhesins (Töpfer-Petersen et al., 2005). Themost abundant proteins in the equine SP are the major fibronectintype II proteins SP-1 and SP-2 (Ekhlasi-Hundrieser et al., 2005,2007). The epididymal spermatozoa binding protein 1 (ELSPBP1)is a minor fibronectin type II protein involved in spermatozoalmaturation and capacitation (Ekhlasi-Hundrieser et al., 2007).

Brandon et al. (1999) found a significant correlation betweenthe concentrations of four seminal plasma proteins and fertilityin the stallion. Fertility was estimated as an individual breeding

Table 2Candidate genes involved in the development of genitals, sperm maturation and diverse stposition on the equine chromosome (ECA), as well as the size of the horse genomic seque

Candidate gene Gene identification ECA P

S

Development of genitalsAndrogen receptor (AR) ENSECAG00000010160 X 4

SpermatogenesisAurora kinase C (AURKC) ENSECAG00000021736 10 2Spermatogenesis associated 1 (SPATA1) ENSECAG00000016351 5 7

Sperm maturationAngiotensin-converting enzyme (ACE) ENSECAG00000012910 11 1Cysteine-rich secretory protein 1 (CRISP1) ENSECAG00000011049 20 4Cysteine-rich secretory protein 2 (CRISP2) ENSECAG00000019933 20 4Cysteine-rich secretory protein 3 (CRISP3) ENSECAG00000006482 20 4Epididymal sperm binding protein 1 (ELSPBP1) ENSECAG00000011903 10 1Seminal plasma protein 1 (SP1) ENSECAG00000023349 10 1Seminal plasma protein 2

(SPNEU/ SP2)ENSECAG00000024141 10 1

Spermatozoa-ovum-bindingAcrosin (ACR) ENSECAG00000013888 28 4Fertilin beta (ADAM2) ENSECAG00000000073 27 6

Calmegin (CLGN) ENSECAG00000017686 2 9Lactadherin (MFGE8) ENSECAG00000018875 1 9Phospholipase C f(zeta) (PLCz) ENSECAG00000011373 6 4

Sperm autoantigenic protein 17 (SP17) ENSECAG00000020689 7 3Zona pellucida protein (SP38) ENSECAG00000016511 4 1Zonadhesin (ZAN) ENSECAG00000016429 13 8

score for stallions by dividing the number of conceptions per cycleby the number of breedings for each stallion for four successivebreeding seasons. SP-1 was positively correlated with the breedingscore, whereas SP-2, SP-3 and SP-4 were negatively correlated.

A further family of SP proteins contains the equine cysteine-richsecretory proteins CRISP1, CRISP2 and CRISP3 (Töpfer-Petersenet al., 2005). CRISP1, expressed in the epididymis, binds to thespermatozoal surface, influencing spermatozoa–oocyte fusion.CRISP2 is mainly expressed in the testis, with lower expressionin the epididymis and seminal vesicles, and is thought to mediateinteractions between Sertoli cells and spermatocytes. CRISP3 is oneof the major seminal plasma proteins and is mainly expressed inthe ampulla of the vas deferens (Leeb et al., 2005).

The CRISP3 SNP AJ459965:c.+622 G > A (leading to the aminoacid substitution E208 K) has been associated with stallion fertil-ity (Hamann et al., 2007). The mean PRO in mares covered was onaverage 7% lower in CRISP3-heterozygous stallions than in homo-zygous stallions, although one stallion with a very low mean PROmainly contributed to this significant association. A haplotype-trait association could be found in the same Hanoverian stallionsfor three polymorphisms in CRISP3, suggesting that further muta-tions in CRISP3 may contribute to this association (Hamann et al.,2007).

The angiotensin-converting enzyme gene (ACE) encodes a so-matic isozyme found in blood and several tissues, including theepididymis, and a testis-specific isozyme localised in developingspermatids and mature spermatozoa (Hagaman et al., 1998). ACEcatalyses the conversion of angiotensin I into the active peptideangiotensin II, which acts as vasopressor and stimulates aldoste-rone, playing a role in the renin-angiotensin-system (Kondohet al., 2005). The activity of ACE in the spermatozoal plasma mem-brane of stallions was significantly higher than in the seminal plas-ma and there was also a significantly increased activity in thepostpubertal equine testis compared to the prepubertal testis (Ballet al., 2003).

eps of spermatozoa-ovum binding, with their gene identification (ID), localisation andnce, using EquCab2.0.

osition (bp) Size (bp) Number of exons

tart End DNA RNA

9,660,105 49,791,031 130,927 1668 8

6,107,597 26,110,989 3393 1041 89,186,712 79,222,754 36,043 1458 13

5,829,612 15,849,932 20,321 4002 297,841,108 47,871,684 30,577 1475 87,693,002 47,710,192 17,191 1384 107,721,076 47,745,394 24,319 1287 88,131,502 18,146,238 14,737 630 54,324,494 14,328,531 4038 639 64,346,137 14,349,564 3428 586 5

6,152,318 46,158,404 6087 1220 5,406,164 6,538,612 132,449 2226 (isoform 1)

2202 (isoform 2)21 (isoform 1)20 (isoform 2)

1,057,498 91,084,539 27,042 1904 144,313,226 94,323,091 9866 1433 85,571,949 45,612,094 40,146 2088 (isoform 1)

2001 (isoform 2)13 (isoform 1)15 (isoform 2)

3,256,379 33,267,160 10,782 567 59,849,416 19,977,483 128,068 977 7,732,910 8,760,977 28,068 6720 (isoform 1)

5502 (isoform 2)48 (isoform 1)33 (isoform 2)

K. Giesecke et al. / The Veterinary Journal 185 (2010) 265–271 269

Fertilisation

Zona pellucida–spermatozoa interaction

The zona pellucida protein 3 (ZP3), localised on the zona pel-lucida of the oocyte, mediates spermatozoa–zona pellucida adhe-sion. In mice, the ZP3 receptor is SP56 (Cohen and Wassarman,2001). In other mammals, the receptor for ZP3 is still unknown,but a possible candidate is b1,4-galactosyltransferase (Milleret al., 2002).

The spermatozoal membrane protein zonadhesin (ZAN) bindsto the zona pellucida of the ovum in several mammalian species(Gasper and Swanson, 2006). In stallions, ZAN is localised in thearea of the future acrosomal content of round spermatids and inthe luminal space of elongating spermatids and spermatozoa. Dif-ferences in the zonadhesin polypeptide between fertile and subfer-tile stallions have been identified (Bailey et al., 2006).

Lactadherin, also called sperm membrane-associated proteinP47 or milk fat globule-epidermal growth factor/factor 8 (MFG-E8), binds selectively to the zona pellucida of unfertilised oocytesand is necessary for spermatozoa–oocyte adhesion (Ensslin andShur, 2003).

Acrosin (ACR) is an intra-acrosomal protein involved in second-ary binding between the spermatozoa and zona pellucida-bindingproteins (Howes and Jones, 2002). It plays a crucial role inspermatozoa–oocyte binding, retaining the acrosome-reactedspermatozoa on the surface of the zona pellucida. The zona pellu-cida-binding protein SP38 is also thought to participate in second-ary binding between the acrosome-reacted spermatozoa and thezona pellucida (McLeskey et al., 1998). Sperm autoantigenicprotein (SP17) may also influence zona pellucida–spermatozoabinding (Richardson et al., 1994).

Spermatozoa–oocyte interaction

The spermatozoal surface protein fertilin b (ADAM2), a memberof the ADAM gene family, mediates the adhesion of the spermato-zoa to the oocyte membrane (Primakoff and Myles, 2000). A sper-matozoa-specific phospholipase C f (zeta) (PLCz) is responsible foroocyte development in several mammals (Swann et al., 2006). PLCzis thought to initiate the increase of inositol triphosphate produc-tion in mammalian eggs, causing intracellular Ca2+ oscillations. Theconcentration of PLCz was reduced in infertile stallions, suggestingthat expression of PLCz could be used as indicator for infertility(Gradil et al., 2006).

2 http://blast.ncbi.nlm.nih.gov/Blast.cgi.3 http://genome.ucsc.edu/cgi-bin/hgBlat.4 http://www.ensembl.org/index.html.5 http://www.ebi.ac.uk/tools/clustalw2/.6 http://www.broad.mit.edu/mammals/horse/snp/.

Equine Y chromosome

Based on studies in humans and mice, most genes involved insex determination, testicular development, spermatogenesis andother reproductive processes in stallions are thought to be locatedon the equine Y chromosome (ECAY) (Cederroth et al., 2007; Wil-helm et al., 2007). A detailed physical map of ECAY is availableand represents the most informative Y chromosome map amongmammalian species after the human and murine maps (Raudseppet al., 2004, 2007). The current ECAY map spans �10 Mb of theeuchromatic region, which includes the pseudoautosomal region(PAR) and the male specific region on the Y chromosome (MSY)(Chowdhary and Raudsepp, 2008). Thirty ECAY-associated geneshave been isolated by cDNA analysis using testis mRNA andmapped on the contig, with the aim to create a physical and func-tional map of ECAY (Raudsepp et al., 2007).

In a study of gene expression in the testes of three prepubertalcolts, the genes encoding dysferlin (DYS) on ECA15, down-regu-lated in ovarian cancer 1 (DOC1) on ECA8 and Golgi apparatus pro-

tein 1 (GLG1) on ECA3 were preferentially expressed inspermatogenesis inactive areas (Ing et al., 2004). In contrast, thegenes encoding outer dense fibre of sperm tails (ODF2) on ECA25and phosphodiesterase 3B (PDE3B) on ECA7 were highly expressedin spermatogenesis active areas.

Candidate gene approach

Candidate genes can be identified via their involvement in keyprocesses of reproduction in horses and other species. In the pres-ent study, we focussed on candidate genes which have been shownto play a role in stallion reproduction and for which a relevant rolein male reproduction in humans or mice has been demonstrated(Tables 1 and 2). Candidate genes can be analysed by employinglinkage and association analyses for gene-associated markers. SNPsand microsatellites are both suitable markers for candidate geneapproaches. It is an important precondition to compare human,murine and equine genome sequences using BLAST,2 BLAT3 or theEnsembl Genome Browser4 to identify sequences and localisationsof candidate genes.

We compared the similarity of published mRNAs of 37 candi-date genes among the horse (EquCab2.0), human (Human Gen-ome Build 36.3) and mouse (Mouse Genome Build 37.1)genome sequences using ClustalW25 (Supplementary Table 1).The identity scores were on average 78.7% between human andhorse mRNA and 71.6% between horse and mouse mRNA. Sinceidentity scores show high homology for most of the chosen candi-date genes, results from studies in humans and mice can be usedas a reference for equine gene analyses, including potential candi-date genes for equine fertility. In contrast, the mRNAs of the genesencoding the SP proteins SP1 and SP2 were compared with Bostaurus 4.0 and Sus scrofa genomes, since this protein family couldnot be identified other than in ungulates.

The SNP tables of the Broad Institute6 are useful for identifica-tion of intragenic SNP markers for fertility-related genes. We identi-fied intragenic SNP markers, as well as flanking and intragenic SNPmarkers, contained in the Equine SNP50 Genotyping Beadchip (Illu-mina) for these 37 selected candidate genes (Supplementary Table2). There were on average 9.36 intragenic SNPs per candidate gene,with a range from 0–46. Seven of the candidate genes featured nointragenic SNPs. Eleven genes had intragenic Equine SNP50 Genotyp-ing Beadchip-associated SNPs, with a range from 1–11 SNPs pergene. The average distance of the candidate genes flanking SNPson the Equine SNP50 Genotyping Beadchip to the candidate geneswas 51,749 bp, with a range from 267–210,755 bp. For ACR, no flank-ing SNP was available on the Equine SNP50 Genotyping Beadchipdownstream of the genomic sequence.

New microsatellites can be developed in the genomic regionsof candidate genes and the surrounding regions (SupplementaryTable 3). We generated permutation sequences with all varia-tions of di-, tri- and tetra-repeat motifs with a minimum of 15repeats and a maximum of 30 repeats. Then, we aligned thesesequences with the horse genome assembly (UCSC Genome Bio-informatics, version EquCab2.0, 2007) using the SSAHA2 package(Sequence Search and Alignment by Hashing Algorithm com-bined with the Cross-Match Sequence Alignment Program devel-oped by Phil Green at the University of Washington, version

270 K. Giesecke et al. / The Veterinary Journal 185 (2010) 265–271

1.0.1, The Wellcome Trust Sanger Institute, UK, 2007) anddeveloped candidate gene-associated microsatellites within adistance of one Mb.

All these newly developed microsatellites (ABGe171–ABGe341)have been deposited in GenBank and are accessible through NCBI7

or Ensembl.8 Equine PCR primers were designed using the Primer3programme9 after masking repetitive elements with RepeatMas-ker.10 Microsatellites are more robust for linkage and associationanalyses, since they display multiple alleles, whereas there is consid-erable variation in the degree to which biallelic SNPs are informativeamong populations and breeds. Microsatellites are preferable toSNPs for family-based linkage studies.

The pre-designed marker set for candidate genes of stallion fer-tility may be an effective starting point for further studies to testfor the possible influence of these candidates. However, the useof the marker set presented does not obviate the need for redesign-ing some PCR primers and further detailed interrogation of thehorse genome sequence.

A first step in deciphering a complex trait, such as stallion fer-tility, may be based on microsatellites and SNPs located withincandidate genes. Genotyping performed with the Illumina 50 KBeadchip may be complemented with highly informative microsat-ellites for specific candidate regions where SNPs are less informa-tive in the study population. In addition, after a genome-wideassociation analysis using the equine Illumina 50 K Beadchip, de-tailed analysis of positional candidate genes may be facilitatedthrough the pre-designed marker set.

Conclusions

Recent developments in equine genomics now provide novelpossibilities and tools for mapping fertility traits in the horseand for unravelling the causes of intersexuality and reproductiveinborn defects. The Equine SNP50 Genotyping Beadchip andexpression microarrays will greatly enhance studies aimed atunderstanding horse reproductive physiology and pathology.The large number of genome-wide equidistantly distributedmarkers offers the possibility for interrogation of the wholeequine genome for associations and linkage at high resolutionand this should greatly facilitate identification of causal genesfor stallion fertility and other complex traits, even those withlow heritability.

In this study, we have presented a large number of candidategene-associated SNPs and microsatellites. These markers may bea starting point for studying the genetic contribution of these can-didate genes in linkage and association analyses. Furthermore, weanalysed the number and distribution of SNPs available on theEquine SNP50 Genotyping Beadchip to give an idea of the powerof this novel tool for analysing fertility traits. At present, there havebeen only a few genetic studies on stallion fertility. Molecular ge-netic studies of candidate genes, such as CRISP3, SPATA1 and INHBA,are encouraging and, with the novel tools available for genome-wide association studies, our knowledge on genetic factors relatedto stallion fertility problems will be advanced.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

7 http://www.ncbi.nlm.nih.gov/nuccore/.8 http://www.ensembl.org/Equus_caballus/.9 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.

10 http://www.repeatmasker.org/.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.tvjl.2009.07.024.

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