The Veterinary Journal 185 (2010) 265271

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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
b

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

a r t i c l e

i n f o

Article history:
Accepted 27 July 2009

Keywords:
Stallion
Fertility
Candidate genes
Horse genome assembly
Single nucleotide polymorphisms
Microsatellites

a b s t r a c t
Stallion fertility is of high economic importance for the horse industry. The discovery of molecular mechanisms affecting fertility will be facilitated by the horse genome assembly and the development of novel
tools for analysing complex genetic traits. Genetic markers in candidate genes, such as CRISP3, SPATA1
and INHBA, in breeding stallions have been associated with pregnancy rate per oestrus in mares. This
paper reviews candidate autosomal, X and Y genes for stallion fertility, including genes encoding hormones and their receptors of the hypothalamic-pituitary axis, proteins of the seminal plasma, proteins
involved in spermatozoaovum binding and genes inuencing sexual development, as well as Y-specic
genes. Their chromosomal location and gene structure are described, based on the horse genome assembly 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 fertility for breeding and management is discussed.
2009 Elsevier Ltd. All rights reserved.

Introduction
Stallion fertility is an economically important trait with a complex environmental and genetic background. Heritability estimates
for stallion fertility vary from 0.030.15 for foaling rate per breeding season (Dohms, 2002; Hamann et al., 2005a). Pregnancy rate
per oestrus (PRO) in mares is associated with breeding year and
season, breeding centre, age of mares, breeding history of mares,
type of covering (natural or articial insemination), breeding management (number of coverings and time intervals between them),
and type of semen (fresh within 24 h, fresh and shipped within
48 h or frozen/thawed) (Hamann et al., 2005b).
Genetic markers may be useful in selection of breeding stallions. Studies in humans and mice have revealed a large number
of proteins involved in the mechanisms of male reproduction and
the cascade of fertilisation, but there are few reports of proteins
with an inuence on fertility in stallions. Hamann et al. (2007) reported a signicant association between a CRISP3-associated single
nucleotide polymorphism (SNP) in stallions and PRO in covered
mares. Signicant associations of single markers and haplotypes
with least square means (LSM)-PRO and the embryonic and paternal component of breeding values (BVs) support a role for INHBA
mutations in fertility of stallions (Giesecke et al., 2009a). We have
also found signicant associations between fertility and a SPATA1associated SNP (Giesecke et al., 2009b).

* Corresponding author. Tel.: +49 511 9538875; fax: +49 511 9538582.
E-mail address: ottmar.distl@tiho-hannover.de (O. Distl).
1090-0233/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2009.07.024

In this paper, we provide an overview of 37 candidate genes

that are potentially important in stallion fertility and discuss the
development of genetic markers for stallion fertility based on the
horse genome assembly EquCab2.0.1 As many genes are known that
inuence mammalian fertility in other species, especially humans
and mice, further candidate genes in horses are likely to become
known in the future. The development of dense marker sets, including microsatellites or SNPs such as those provided on the Equine
SNP50 Genotyping Beadchip (Illumina), may allow the roles of
recognised candidate genes to be determined and may facilitate
identication of new genes associated with horse fertility.

Chromosomal anomalies and inborn defects

Intersexuality
In the presence of a Y chromosome in the zygote, the undifferentiated gonads develop into testes. The key factor in initiating
male sexual development is testis determining factor encoded by
the SRY-gene (sex determining region of the Y chromosome) on
the Y chromosome. Abnormal development of the testes may result in intersexuality (Kuiper and Distl, 2007). Few candidate genes
for intersexuality have been identied and tested in horses to date.
Collecting a larger number of cases and matching controls will allow the equine genome to be interrogated for linked and associated genes involved in these sexual anomalies.
1

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

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K. Giesecke et al. / The Veterinary Journal 185 (2010) 265271

The most frequent chromosomal abnormalities in stallions are

sex chromosome mosaicism and sex-reversal syndromes. The
XXY-syndrome (Klinefelters syndrome), the unbalanced 65,XXY
karyotype and Y chromosome disomy are rare. Sex-reversal syndrome results from a failure of differentiation of the gonads during
embryogenesis, so that chromosomal sex does not conform to gonadal sex. Horses with the XY-sex-reversal mare syndrome show
undifferentiated gonads or partial ovarian tissue and, in most
cases, have a deletion of the SRY-gene (Bugno et al., 2003).
The 64,XX sex-reversal condition is inherited as an autosomal
recessive trait and, in humans, is caused by defects in cortisol biosynthesis. All 64,XX sex reversals are SRY-negative. In the absence
of the SRY-gene, SOX9 and FOXL2 may play a role in testicular
development. XX horses exhibit stallion-like behaviour, testes
without spermatogenesis, an enlarged clitoris or small penis and
some have ovarian tissue and other female organs (Constant
et al., 1994; Milliken et al., 1995; Bannasch et al., 2007).
Mutations in the androgen receptor gene (AR) in humans lead to
testicular feminisation (Krausz and Giachini, 2007), but there are
no reports of mutations in the AR gene in the horse. Horses affected
by an AR-like syndrome have female external genitalia, high plasma testosterone concentrations and exhibit stallion-like behaviour
and infertility. They have a 64,XY karyotype and SRY and ZFY genes
are present (Howden, 2004; Switonski et al., 2005).
Autosomal defects
Only a few cases of autosomal deletions, autosomal translocations and autosomal trisomy have been reported in the horse.
Large chromosomal deletions or trisomies involving large chromosomes are assumed to cause early embryonic losses (Lear et al.,
2008). A stallion with a 64,XY,del(13)(qter) karyotype exhibited
abnormal 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 defects
and some of them were infertile (Lear and Bailey, 2008). Only one
case of an autosomal chromosome translocation has been reported; 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 ve mares included karyotypes of
64,XX,t(1;21), 64,XX,t(16;22), 64,XX,t(4;13), 64,XX,t(1q;3q) and
64,XX,t(1;16). Lear and Layton (2002) and Lear et al. (2008) found
REEL in all mares with autosomal chromosome translocations.
Cryptorchidism and monorchism
Cryptorchidism is classied according to the location of the affected testes into inguinal, incomplete abdominal and complete
abdominal cryptorchidism. Most cases of cryptorchidism are unilateral (Aurich, 2005). Genetic and non-genetic factors may inuence testicular descent. Insulin-like factor 3 (encoded by INSL3)
and its receptor leucine-rich repeat-containing G protein-coupled
receptor 8 (encoded by LGR8) are thought to be important signalling molecules in the control of testicular descent in humans and
mice (Leeb et al., 2005).
During embryonic development, Leydig (interstitial) cells of the
fetal 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 responsible
for the process of abdominal testicular descent in humans (Agoulnik, 2007). Mutations in INSL3 and LGR8 leading to cryptorchidism
have been identied in humans (Gorlov et al., 2002; Canto et al.,
2003). Klonisch et al. (2003) demonstrated that INSL3 expression
was 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 in
cryptorchid stallions. Since cryptorchidism is a common anomaly
in stallions, whole genome linkage and association studies may
be useful in identication of genomic regions containing new candidate genes.
Monorchism, where one testis is lacking, is rare in horses
(Searle et al., 1999; Petrizzi et al., 2004). Another rare developmental defect is hypospadia, where the urethra opens below the tip of
the glans penis. Bleul et al. (2007) found hypospadia in a Friesian
stallion with a ventrocaudal deviation of the shaft of the penis
and an incomplete glans penis. A rare case of aplasia of the ductus
deferens was described by Estrada et al. (2003) in a 3 year old
Quarter stallion.
Development of the male reproductive tract
Androgen receptor
Androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the steroidal receptor class 1 family of nuclear
receptors (Callewaert et al., 2003). In stallions, AR is localised in epididymal and prostate cells, which are directly regulated by androgens (Hejmej et al., 2006). Parlevliet et al. (2006) detected AR in
the principal cells of the caput, corpus and cauda epididymidis of
stallions of different ages and concluded that AR and the main
androgen testosterone are required for epididymal development
and function. Hejmej et al. (2006) reported stronger immunostaining for AR in a cryptorchid horse compared to normal stallions.
Mutations in AR in humans lead to testicular feminisation, such
as androgen insensitivity syndrome (AIS) (Krausz and Giachini,
2007). The length of a CAG repeat in exon 1 of the AR gene is associated with infertility in men (Shah et al., 2003). In horses, some
cases of AIS are thought to be associated with mutations in AR
(Crabbe et al., 1992; Pailhoux et al., 1995; Howden, 2004; Switonski et al., 2005).
Oestrogen
Oestradiol plays a key role in the maturation of the stallion epididymis during the pubertal transition. Parlevliet et al. (2006) detected an age-related increase in oestradiol-17b in the
epididymis. The oestrogen receptor ESR2 is localised in the principal cells of the caput, corpus and cauda epididymis in stallions
independent of age, whereas ESR1 localisation is regional and age
dependent (Parlevliet et al., 2006). Association studies in humans
have demonstrated several polymorphic sites in the two genes
encoding ESR1 and ESR2, which have an inuence on spermatozoal
count and cryptorchidism (Krausz and Giachini, 2007).
Testicular oestrogens, mainly oestradiol-17b, modulate release
of luteinising hormone (LH) from the pituitary gland (Roser,
1997). Stewart and Roser (1998) identied an increase in plasma
and testicular concentrations of oestradiol and oestrogen conjugates in stallions with increasing age. Infertile stallions had significantly lower plasma concentrations of oestradiol and oestrogen
conjugates than fertile stallions.
Relaxin
The gene encoding relaxin (RLN) and three non-allelic relaxin
genes (RLN1, RLN2 and RLN3) belong to the insulin gene superfamily. Relaxin plays key roles in the development of the male reproductive tract, the growth of the prostate gland and spermatozoal
motility (Samuel et al., 2003). The prostate gland is the main
source of relaxin in the seminal plasma.

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Hormonal inuences on male reproduction

Genes encoding hormones involved in regulation of testicular
development, sexual maturity and sexual activity are candidates
for stallion fertility. Testicular function is dependent upon a functional hypothalamic-pituitarytesticular axis, which involves
gonadotrophin releasing hormone (GNRH), LH, follicle stimulating
hormone (FSH), testosterone, oestrogens and inhibins. Their localisation on the horse genome is shown in Table 1 and similarities
with orthologous genes in humans and mice are shown in Supplementary Table 1. Studies of candidate genes may be facilitated
using the markers presented in Supplementary Tables 2 and 3.

potential marker for early detection of fertility problems in young

stallions (Roser, 2008).
Genes encoding a and b inhibins (INHA, INHBA, INHBB), as well
as a, b and c actins (ACTN, ACTB, ACTG), are potential candidates
for association analysis of fertility. In Hanoverian stallions, significant associations of SNPs and their haplotypes within the inhibin
beta A (INHBA) gene have been demonstrated for LSM-PRO and
the embryonic and paternal component of BVs for PRO. The mutations identied in this gene result in altered transcription factor
binding sites and these mutations may regulate expression of
INHBA (Giesecke et al., 2009a).
Prolactin

Gonadotrophin releasing hormone and gonadotropins

GNRH is released by the hypothalamus in a pulsatile fashion,
activating a G-protein-coupled receptor (GNRHR), which induces
release of LH (Leydig cell stimulating hormone) and FSH (Sertoli
cell stimulating hormone) from the anterior pituitary. FSH is essential for spermatogenesis during puberty, whereas spermatogenesis
in adults is promoted mainly by testosterone. Release of testosterone and oestrogens from the Leydig cells of the testis is stimulated
by LH. FSH binds mostly to Sertoli cells and causes the release of
inhibin, activin and androgen binding protein. These testicular proteins and steroid hormones inuence the release of GNRH, FSH and
LH from the anterior pituitary gland via a negative feedback loop.
Inhibin, activin and follistatin
Inhibin, activin and follistatin (FST) belong to the transforming
growth factor (TGF) b superfamily (Welt et al., 2002). In the stallion, FSH secretion is controlled by inhibin, in conjunction with
oestrogen and testosterone (Roser, 1997). FST is produced in the
testis and inhibits the secretion of FSH by binding to activin. Plasma and intratesticular concentrations of inhibin in stallions are
associated with testicular maturation and fertility (Stewart and
Roser, 1998). The intratesticular concentration of inhibin is a

Prolactin, produced by the PRL gene, is secreted by the pars distalis of the adenohypophysis in stallions in response to sexual
stimulation (Thompson et al., 1996). Plasma concentrations of prolactin are positively correlated with day length and are highest in
stallions during the breeding season (Gerlach and Aurich, 2000;
Roser, 2008). Aurich et al. (2002) detected signicant prolactin release in stallions during the non-breeding season in response to
treatment with the dopamine antagonist sulpiride, but no change
in serum prolactin concentration after treatment with the opioid
antagonist naloxone, suggesting that prolactin secretion is controlled by dopaminergic pathways. The interaction between prolactin, gonadotrophins and GNRH is modulated by photoperiod
and melatonin, whereas prolactin release is not mediated by
gonadotrophins (Henderson et al., 2008). Prolactin receptor (PRLR)
is expressed by gonadotroph cells, which are embedded within lactotroph clusters in the pars distalis of the adenohypophysis in
horses (Tortonese et al., 2001).
Spermatogenesis
Spermatogenesis is the development of spermatids in the testis
from spermatogonia, which are derived from primordial germ cells
(Johnson et al., 1997). Spermiogenesis is the differentiation of

Table 1
Hormones of the hypothalamic-pituitary testicular axis as candidates for stallions fertility, with their gene identication, localisation and position on the equine chromosome
(ECA) and the size of the horse genomic sequence using EquCab2.0.
Candidate gene

Gene identication (LOC)

ECA

Position (bp)

Size (bp)

Number of exons

Start

End

DNA

RNA
6
4 (isoform 1)
4 (isoform 2)
22 (isoform 1)
21 (isoform 2)
24
8 12

Beta actin (ACTB)

Gamma actin (ACTG)

ENSECAG00000015935
ENSECAG00000018600

13
11

2,463,585
1,628,565

2,465,463
1,630,637

1879
2073

Alpha actinin 1 (ACTN1)

ENSECAG00000019476

24

14,991,841

15,037,255

45,414

Alpha actinin 4 (ACTN4)

Oestrogen receptor 1 (ESR1)

ENSECAG00000021777
ENSECAG00000022441

10
31

9,692,742
15,081,987

9,718,476
15,329,542

25,735
247,556

Oestrogen receptor 2 (ESR2)

ENSECAG00000024947
(fragment)
ENSECAG00000019116
ENSECAG00000017783
ENSECAG00000010664
LOC100033874

24

11,029,301

11,082,098

52,798

1098
1604 (isoform 1)
654 (isoform 2)
2819 (isoform 1)
2753 (isoform 2)
2241
3717 (isoform 1)
3508 (isoform 2)
1744

7
21
2
3

96,163,676
18,584,478
54,131,556
67,130,453

96,165,734
18,590,636
54,134,868
67,151,298

2059
6159
3313
20,888

502
2119
308
1114

2
6
3
4

ENSECAG00000015864
ENSECAG00000022448
ENSECAG00000014285
ENSECAG00000016450
(fragment)
ENSECAG00000007548
ENSECAG00000014103

6
4
18
21

9,065,378
12,878,829
10,834,802
2,646,466

9,068,177
12,889,612
10,838,204
2,647,154

2800
10,784
3403
689

1286
1445
958
300

2
2
4
2

10
17

18,963,376
11,005,845

18,964,111
11,078,391

736
72,547

510
2396

2
18

ENSECAG00000000114
ENSECAG00000009483
ENSECAG00000013020

20
21
23

20,481,551
29,918,374
26,699,707

20,491,085
30,066,399
26,704,126

9535
148,026
4420

853
2200
801

5
10
2

Follicle-stimulating hormone (FSHB)

Follistatin (FST)
Gonadotropin releasing hormone (GNRH)
Gonadotropin releasing hormone receptor
(GNRHR)
Alpha inhibin (INHA)
Beta inhibin A (INHBA)
Beta inhibin B (INHBB)
Insulin-like 3 (INSL3)
Luteinising hormone (LHB)
Leucine-rich repeat-containing G proteincoupled receptor 8 (LGR8)
Prolactin (PRL)
Prolactin receptor (PRLR)
Prorelaxin (RLN1)

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K. Giesecke et al. / The Veterinary Journal 185 (2010) 265271

spermatids into spermatozoa. After spermatogenesis and spermiogenesis, spermatozoa are transported to the epididymis for maturation. A survey of these candidate genes is shown in Table 2 and
Supplementary information for comparison with human and
mouse genes, as well as markers for these genes, is shown in
Supplementary Tables 13.
Spermatogenesis associated protein 1 (SPATA1) is thought to be
involved in spermatogenesis, but its detailed function is still unknown. Strong linkage disequilibrium has been demonstrated for
an intragenic SPATA1 SNP in Hanoverian stallions for the embryonic component of BVs (Giesecke et al., 2009b). This marker changed an SP1 binding site, but did not change the coding sequence or
the splice sites. Therefore, this intronic SPATA1 mutation is thought
to confer improved fertility in stallions via regulation of gene
expression (Giesecke et al., 2009b).
Maturation of spermatozoa
Seminal plasma (SP) proteins derived from the epididymis and
accessory sex glands participate in post-testicular spermatozoal
maturation, where the spermatozoa acquire the ability for fertilisation (capacitation) (Sostaric et al., 2008). During capacitation, signal transducing pathways that initiate the acrosome reaction are
activated (Gadella et al., 2001; Neild et al., 2005). SP proteins consist of major and minor components in three main protein classes:
bronectin type II proteins, cysteine-rich secretory proteins
(CRISPs) and spermadhesins (Tpfer-Petersen et al., 2005). The
most abundant proteins in the equine SP are the major bronectin
type II proteins SP-1 and SP-2 (Ekhlasi-Hundrieser et al., 2005,
2007). The epididymal spermatozoa binding protein 1 (ELSPBP1)
is a minor bronectin type II protein involved in spermatozoal
maturation and capacitation (Ekhlasi-Hundrieser et al., 2007).
Brandon et al. (1999) found a signicant correlation between
the concentrations of four seminal plasma proteins and fertility
in the stallion. Fertility was estimated as an individual breeding

score for stallions by dividing the number of conceptions per cycle

by the number of breedings for each stallion for four successive
breeding seasons. SP-1 was positively correlated with the breeding
score, whereas SP-2, SP-3 and SP-4 were negatively correlated.
A further family of SP proteins contains the equine cysteine-rich
secretory proteins CRISP1, CRISP2 and CRISP3 (Tpfer-Petersen
et al., 2005). CRISP1, expressed in the epididymis, binds to the
spermatozoal surface, inuencing spermatozoaoocyte fusion.
CRISP2 is mainly expressed in the testis, with lower expression
in the epididymis and seminal vesicles, and is thought to mediate
interactions between Sertoli cells and spermatocytes. CRISP3 is one
of the major seminal plasma proteins and is mainly expressed in
the ampulla of the vas deferens (Leeb et al., 2005).
The CRISP3 SNP AJ459965:c.+622 G > A (leading to the amino
acid substitution E208 K) has been associated with stallion fertility (Hamann et al., 2007). The mean PRO in mares covered was on
average 7% lower in CRISP3-heterozygous stallions than in homozygous stallions, although one stallion with a very low mean PRO
mainly contributed to this signicant association. A haplotypetrait association could be found in the same Hanoverian stallions
for three polymorphisms in CRISP3, suggesting that further mutations in CRISP3 may contribute to this association (Hamann et al.,
2007).
The angiotensin-converting enzyme gene (ACE) encodes a somatic isozyme found in blood and several tissues, including the
epididymis, and a testis-specic isozyme localised in developing
spermatids and mature spermatozoa (Hagaman et al., 1998). ACE
catalyses the conversion of angiotensin I into the active peptide
angiotensin II, which acts as vasopressor and stimulates aldosterone, playing a role in the renin-angiotensin-system (Kondoh
et al., 2005). The activity of ACE in the spermatozoal plasma membrane of stallions was signicantly higher than in the seminal plasma and there was also a signicantly increased activity in the
postpubertal equine testis compared to the prepubertal testis (Ball
et al., 2003).

Table 2
Candidate genes involved in the development of genitals, sperm maturation and diverse steps of spermatozoa-ovum binding, with their gene identication (ID), localisation and
position on the equine chromosome (ECA), as well as the size of the horse genomic sequence, using EquCab2.0.
Candidate gene

Gene identication

ECA

Position (bp)

Size (bp)

Number of exons

Start

End

DNA

RNA

Development of genitals
Androgen receptor (AR)

ENSECAG00000010160

49,660,105

49,791,031

130,927

1668

Spermatogenesis
Aurora kinase C (AURKC)
Spermatogenesis associated 1 (SPATA1)

ENSECAG00000021736
ENSECAG00000016351

10
5

26,107,597
79,186,712

26,110,989
79,222,754

3393
36,043

1041
1458

8
13

ENSECAG00000012910
ENSECAG00000011049
ENSECAG00000019933
ENSECAG00000006482
ENSECAG00000011903
ENSECAG00000023349
ENSECAG00000024141

11
20
20
20
10
10
10

15,829,612
47,841,108
47,693,002
47,721,076
18,131,502
14,324,494
14,346,137

15,849,932
47,871,684
47,710,192
47,745,394
18,146,238
14,328,531
14,349,564

20,321
30,577
17,191
24,319
14,737
4038
3428

4002
1475
1384
1287
630
639
586

29
8
10
8
5
6
5

Spermatozoa-ovum-binding
Acrosin (ACR)
Fertilin beta (ADAM2)

ENSECAG00000013888
ENSECAG00000000073

28
27

46,152,318
6,406,164

46,158,404
6,538,612

6087
132,449

Calmegin (CLGN)
Lactadherin (MFGE8)
Phospholipase C f(zeta) (PLCz)

ENSECAG00000017686
ENSECAG00000018875
ENSECAG00000011373

2
1
6

91,057,498
94,313,226
45,571,949

91,084,539
94,323,091
45,612,094

27,042
9866
40,146

Sperm autoantigenic protein 17 (SP17)

Zona pellucida protein (SP38)
Zonadhesin (ZAN)

ENSECAG00000020689
ENSECAG00000016511
ENSECAG00000016429

7
4
13

33,256,379
19,849,416
8,732,910

33,267,160
19,977,483
8,760,977

10,782
128,068
28,068

1220
2226
2202
1904
1433
2088
2001
567
977
6720
5502

Sperm maturation
Angiotensin-converting enzyme (ACE)
Cysteine-rich secretory protein 1 (CRISP1)
Cysteine-rich secretory protein 2 (CRISP2)
Cysteine-rich secretory protein 3 (CRISP3)
Epididymal sperm binding protein 1 (ELSPBP1)
Seminal plasma protein 1 (SP1)
Seminal plasma protein 2
(SPNEU/ SP2)

(isoform 1)
(isoform 2)

(isoform 1)
(isoform 2)

(isoform 1)
(isoform 2)

5
21
20
14
8
13
15
5
7
48
33

(isoform 1)
(isoform 2)

(isoform 1)
(isoform 2)

(isoform 1)
(isoform 2)

K. Giesecke et al. / The Veterinary Journal 185 (2010) 265271

Fertilisation
Zona pellucidaspermatozoa interaction
The zona pellucida protein 3 (ZP3), localised on the zona pellucida of the oocyte, mediates spermatozoazona pellucida adhesion. 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 (Miller
et al., 2002).
The spermatozoal membrane protein zonadhesin (ZAN) binds
to the zona pellucida of the ovum in several mammalian species
(Gasper and Swanson, 2006). In stallions, ZAN is localised in the
area of the future acrosomal content of round spermatids and in
the luminal space of elongating spermatids and spermatozoa. Differences in the zonadhesin polypeptide between fertile and subfertile stallions have been identied (Bailey et al., 2006).
Lactadherin, also called sperm membrane-associated protein
P47 or milk fat globule-epidermal growth factor/factor 8 (MFGE8), binds selectively to the zona pellucida of unfertilised oocytes
and is necessary for spermatozoaoocyte adhesion (Ensslin and
Shur, 2003).
Acrosin (ACR) is an intra-acrosomal protein involved in secondary binding between the spermatozoa and zona pellucida-binding
proteins (Howes and Jones, 2002). It plays a crucial role in
spermatozoaoocyte binding, retaining the acrosome-reacted
spermatozoa on the surface of the zona pellucida. The zona pellucida-binding protein SP38 is also thought to participate in secondary binding between the acrosome-reacted spermatozoa and the
zona pellucida (McLeskey et al., 1998). Sperm autoantigenic
protein (SP17) may also inuence zona pellucidaspermatozoa
binding (Richardson et al., 1994).
Spermatozoaoocyte interaction
The spermatozoal surface protein fertilin b (ADAM2), a member
of the ADAM gene family, mediates the adhesion of the spermatozoa to the oocyte membrane (Primakoff and Myles, 2000). A spermatozoa-specic phospholipase C f (zeta) (PLCz) is responsible for
oocyte development in several mammals (Swann et al., 2006). PLCz
is thought to initiate the increase of inositol triphosphate production in mammalian eggs, causing intracellular Ca2+ oscillations. The
concentration of PLCz was reduced in infertile stallions, suggesting
that expression of PLCz could be used as indicator for infertility
(Gradil et al., 2006).
Equine Y chromosome
Based on studies in humans and mice, most genes involved in
sex determination, testicular development, spermatogenesis and
other reproductive processes in stallions are thought to be located
on the equine Y chromosome (ECAY) (Cederroth et al., 2007; Wilhelm et al., 2007). A detailed physical map of ECAY is available
and represents the most informative Y chromosome map among
mammalian species after the human and murine maps (Raudsepp
et al., 2004, 2007). The current ECAY map spans 10 Mb of the
euchromatic region, which includes the pseudoautosomal region
(PAR) and the male specic region on the Y chromosome (MSY)
(Chowdhary and Raudsepp, 2008). Thirty ECAY-associated genes
have been isolated by cDNA analysis using testis mRNA and
mapped on the contig, with the aim to create a physical and functional map of ECAY (Raudsepp et al., 2007).
In a study of gene expression in the testes of three prepubertal
colts, the genes encoding dysferlin (DYS) on ECA15, down-regulated in ovarian cancer 1 (DOC1) on ECA8 and Golgi apparatus pro-

269

tein 1 (GLG1) on ECA3 were preferentially expressed in

spermatogenesis inactive areas (Ing et al., 2004). In contrast, the
genes encoding outer dense bre of sperm tails (ODF2) on ECA25
and phosphodiesterase 3B (PDE3B) on ECA7 were highly expressed
in spermatogenesis active areas.

Candidate gene approach

Candidate genes can be identied via their involvement in key
processes of reproduction in horses and other species. In the present study, we focussed on candidate genes which have been shown
to play a role in stallion reproduction and for which a relevant role
in male reproduction in humans or mice has been demonstrated
(Tables 1 and 2). Candidate genes can be analysed by employing
linkage and association analyses for gene-associated markers. SNPs
and microsatellites are both suitable markers for candidate gene
approaches. It is an important precondition to compare human,
murine and equine genome sequences using BLAST,2 BLAT3 or the
Ensembl Genome Browser4 to identify sequences and localisations
of candidate genes.
We compared the similarity of published mRNAs of 37 candidate genes among the horse (EquCab2.0), human (Human Genome 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 and
horse mRNA and 71.6% between horse and mouse mRNA. Since
identity scores show high homology for most of the chosen candidate genes, results from studies in humans and mice can be used
as a reference for equine gene analyses, including potential candidate genes for equine fertility. In contrast, the mRNAs of the genes
encoding the SP proteins SP1 and SP2 were compared with Bos
taurus 4.0 and Sus scrofa genomes, since this protein family could
not be identied other than in ungulates.
The SNP tables of the Broad Institute6 are useful for identication of intragenic SNP markers for fertility-related genes. We identied intragenic SNP markers, as well as anking and intragenic SNP
markers, contained in the Equine SNP50 Genotyping Beadchip (Illumina) for these 37 selected candidate genes (Supplementary Table
2). There were on average 9.36 intragenic SNPs per candidate gene,
with a range from 046. Seven of the candidate genes featured no
intragenic SNPs. Eleven genes had intragenic Equine SNP50 Genotyping Beadchip-associated SNPs, with a range from 111 SNPs per
gene. The average distance of the candidate genes anking SNPs
on the Equine SNP50 Genotyping Beadchip to the candidate genes
was 51,749 bp, with a range from 267210,755 bp. For ACR, no anking SNP was available on the Equine SNP50 Genotyping Beadchip
downstream of the genomic sequence.
New microsatellites can be developed in the genomic regions
of candidate genes and the surrounding regions (Supplementary
Table 3). We generated permutation sequences with all variations of di-, tri- and tetra-repeat motifs with a minimum of 15
repeats and a maximum of 30 repeats. Then, we aligned these
sequences with the horse genome assembly (UCSC Genome Bioinformatics, version EquCab2.0, 2007) using the SSAHA2 package
(Sequence Search and Alignment by Hashing Algorithm combined with the Cross-Match Sequence Alignment Program developed by Phil Green at the University of Washington, version

2
3
4
5
6

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

270

K. Giesecke et al. / The Veterinary Journal 185 (2010) 265271

1.0.1, The Wellcome Trust Sanger Institute, UK, 2007) and

developed candidate gene-associated microsatellites within a
distance of one Mb.
All these newly developed microsatellites (ABGe171ABGe341)
have been deposited in GenBank and are accessible through NCBI7
or Ensembl.8 Equine PCR primers were designed using the Primer3
programme9 after masking repetitive elements with RepeatMasker.10 Microsatellites are more robust for linkage and association
analyses, since they display multiple alleles, whereas there is considerable variation in the degree to which biallelic SNPs are informative
among populations and breeds. Microsatellites are preferable to
SNPs for family-based linkage studies.
The pre-designed marker set for candidate genes of stallion fertility may be an effective starting point for further studies to test
for the possible inuence of these candidates. However, the use
of the marker set presented does not obviate the need for redesigning some PCR primers and further detailed interrogation of the
horse genome sequence.
A rst step in deciphering a complex trait, such as stallion fertility, may be based on microsatellites and SNPs located within
candidate genes. Genotyping performed with the Illumina 50 K
Beadchip may be complemented with highly informative microsatellites for specic candidate regions where SNPs are less informative in the study population. In addition, after a genome-wide
association analysis using the equine Illumina 50 K Beadchip, detailed analysis of positional candidate genes may be facilitated
through the pre-designed marker set.
Conclusions
Recent developments in equine genomics now provide novel
possibilities and tools for mapping fertility traits in the horse
and for unravelling the causes of intersexuality and reproductive
inborn defects. The Equine SNP50 Genotyping Beadchip and
expression microarrays will greatly enhance studies aimed at
understanding horse reproductive physiology and pathology.
The large number of genome-wide equidistantly distributed
markers offers the possibility for interrogation of the whole
equine genome for associations and linkage at high resolution
and this should greatly facilitate identication of causal genes
for stallion fertility and other complex traits, even those with
low heritability.
In this study, we have presented a large number of candidate
gene-associated SNPs and microsatellites. These markers may be
a starting point for studying the genetic contribution of these candidate genes in linkage and association analyses. Furthermore, we
analysed the number and distribution of SNPs available on the
Equine SNP50 Genotyping Beadchip to give an idea of the power
of this novel tool for analysing fertility traits. At present, there have
been only a few genetic studies on stallion fertility. Molecular genetic studies of candidate genes, such as CRISP3, SPATA1 and INHBA,
are encouraging and, with the novel tools available for genomewide association studies, our knowledge on genetic factors related
to stallion fertility problems will be advanced.
Conict of interest statement
None of the authors of this paper has a nancial or personal
relationship with other people or organisations that could inappropriately inuence or bias the content of the paper.
7
8
9
10

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

Appendix A. Supplementary data

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

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