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04_[Zhihua_Jiang,_Troy_L._Ott]_Reproductive_Genomics_479_2010

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04_[Zhihua_Jiang,_Troy_L._Ott]_Reproductive_Genomics_479_2010

04_[Zhihua_Jiang,_Troy_L._Ott]_Reproductive_Genomics_479_2010

332 Genomics and Reproductive Biotechnology

immunized animals that respond effectively no severe inflammatory reactions were
to the vaccine for the desired period of time), observed at the injection site following
predictability of chemical structure, and immunization. An ongoing study is evaluat-
cost per dose. Many experimental LHRH ing the immune and biological responses to
antigens have successfully resulted in immu- this vaccine preparation in domestic cats,
nosterilization but the costs and time including fertility trials. Results from the
required for large scale production would preliminary study suggest that this vaccine
render the vaccine unmarketable. As previ- might be a promising tool for feral cat popu-
ously discussed, protein purification would lation control. Moreover, if approved for
be one of the major obstacles to large scale commercialization, the vaccine containing
production of many vaccine preparations. the non-purified recombinant protein would
However, protein purification might be be able to be marketed at a more reasonable
eliminated without diminishing the efficacy price compared with the purified protein.
of LHRH antigens.
14.7 Future research directions
In a recent preliminary study, the non-
purified form of the recombinant antigen In conclusion, the field of immunocontra-
ova-LHRH was tested in female domestic ception/sterilization offers vast opportuni-
cats. This recombinant fusion protein is ties for research and applications in human
found in inclusion bodies in the transformed and animal sciences. Currently, few veteri-
E. coli cells. In previous studies, ova-LHRH nary AF vaccines are commercially available
vaccines had the antigen purified by nickel (Table 14.1). One of the major obstacles to
chelation chromatography. However, gel approving novel AF vaccines for marketing
analyses indicated that this recombinant is low efficacy. Due to individual variations
fusion protein had basically the same molec- in immune response to AF vaccines, the per-
ular weight before and after purification, centage of treated animals that have satis-
suggesting that ova-LHRH is the predomi- factory biological response to immunization
nant protein in inclusion bodies of the trans- is often below the minimum required by
formed bacteria. Sera from cats vaccinated regulatory agencies. Improving immunoge-
with non-purified ova-LHRH had an increas- nicity without increasing toxicity still is one
ing anti-LHRH antibody activity following a of the biggest challenges in vaccine develop-
single immunization. The vaccine prepara- ment for fertility control. Future directions
tion contained a combination of encapsu- for AF vaccines will likely rely on genetic
lated ova-LHRH and CpG ODN for slow engineering to maximize immunogenicity
release plus the same antigen and adjuvant of antigens while eliminating the need for
in a water-in-oil emulsion, the idea being strong, potentially toxic adjuvants.
that the nonencapsulated antigen/adjuvant
mix would work as a primary injection, Acknowledgments
while the encapsulated counterpart would
mimic booster injections. The immune The author is grateful to Russell Dudley and
response observed approximately 8 months Dr. Jerry Reeves and Dr. Monica Stoops for
after a single injection of non-purified the valuable comments on the manuscript.
ova-LHRH is comparable with the results
obtained in other species after multiple
injections of purified ova-LHRH. Moreover,

Biotechnology and Fertility Regulation 333

Table 14.1 Past and present market-driven veterinary vaccines against reproductive antigens (adapted
from Meeusen et al. 2007).

Brand name (target Intended use Vaccine preparation Protocol Market2 Current
antigen) status

Antigen Adjuvant

SpayVac® (ZP) Ǩ deer, population porcine ZP Adjuvac™ single U.S. wildlife To be
control dose agencies approved
by the FDA
Vaxstrate™ (LHRH) Ǩ cattle, LHRH-OVA oil-based two Australia No longer
Improvac™ (LHRH) immuno-sterilization. LHRH1 water-soluble doses available
ǩ pigs, boar taint two Oceania, Latin Available
control doses America,3
S. Africa, Available
Equity™ (LHRH) Ǩ horses, estrous LHRH1 Quil-A-based two S. Korea
behavior control AdjuVac™ doses Australia, To be
GonaCon™; deer, population LHRH-KLH; single New Zealand approved
GonaCon-B™ control LHRH-blue Quil-A-based dose United States by the FDA
(LHRH) protein Available
Canine Gonadotropin ǩ dogs, treatment of LHRH-DT variable United States
Releasing Factor benign prostatic
immunother. (LHRH) hyperplasia

1No information provided by manufacturer on the LHRH molecule or carrier protein.
2Distributors, comments, and references:
SpayVac®: nonprofitable organization SpayVac( for Wildlife Inc., U.S.A. This vaccine was shown to reduce fertility in white-tailed deer
(Locke et al. 2007).
Vaxstrate: Websters Animal Health, Australia. This was the first commercially available LHRH vaccine. It was released in the Australian
market in the 1980s but was discontinued in 1996 due to low efficacy, high incidence of abscesses, and the need of two immunizations,
which was impractical for local husbandry practices. Scientific literature on this vaccine is scarce (Hoskinson et al. 1990).
Improvac™: Pfizer Animal Health, 3Brazil, Costa Rica, Guatemala, Mexico. Improvac™ efficiently eliminated boar taint when given at 8
and 4 weeks before slaughter (23–26 weeks of age). Improvac-immunized boars grew faster than intact boars during the 4-week period
following booster immunization, possibly due to reduced sexual and aggressive behaviors. Compared with barrows, immunized boars had
superior feed conversion and leaner carcasses (Dunshea et al. 2001).
Equity™: Pfizer Animal Health, Australia. The antigen is a synthetic LHRH molecule conjugated to a carrier protein. Estrous behavior was
suppressed for at least 3 months in mares receiving two immunizations with Equity™ (Elhay et al. 2007).
GonaCon™, GonaCon-B™: National Wildlife Research Center, U.S.A. GonaCon™ contains LHRH conjugated to KLH, while GonaCon-B™
contains LHRH-blue protein. A study compared the two formulations in female white-tailed deer; both reduced fertility but GonaCon-B™
had longer lasting contraceptive effects (Miller et al. 2008).
Canine Gonadotropin Releasing Factor immunotherapeutic: Pfizer Animal Health, U.S.A. This vaccine is used to suppress androgen
production as part of a treatment of intact male dogs diagnosed with benign prostatic hyperplasia (USDA).
AdjuVac™: adjuvant developed by the National Wildlife Research Center, U.S.A. Contains killed Mycobacterium avium (Miller et al.
2008).

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15

Proteomics of Male Seminal Plasma

Vera Jonakova, Jiri Jonak, and Marie Ticha

15.1 Introduction Jonakova and Ticha 2004; Calvete and Sanz
2007; Jonakova et al. 2007; Manjunath et al.
Mammalian fertilization is a unique event 2007; Tanphaichitr et al. 2007; Vadnais et al.
in which morphologically disparate gametes 2007). Participation of the seminal plasma
recognize each other, bind and fuse. This proteins of domestic animals in the indi-
event includes highly regulated biochemical vidual steps of the reproduction process is
interactions between molecules located on reviewed in this chapter.
the surface of both gametes as well as sub-
stances present in the natural environment Mammalian seminal plasma is a complex
of gametes both in the male and the female mixture of secretions mainly produced
reproductive organs. The following phases by the testis, the epididymis, and the male
of the reproduction process can be distin- accessory sex glands (seminal vesicles,
guished: binding of seminal plasma proteins ampulla, prostate, bulbourethral glands) and
to the sperm surface during ejaculation, contains a variety of different substances
interaction of sperm surface proteins with (amino acid, lipids, fatty acids, saccharides,
oviductal epithelial cells, sperm capacita- ions, peptides, and proteins). The complex
tion, gamete recognition, primary and sec- content of the seminal plasma is designed to
ondary binding of the sperm to the zona assure the successful fertilization of the
pellucida (ZP), acrosome reaction of sperm, oocyte by one of the spermatozoa present in
penetration of the sperm through the ZP of the ejaculum. Proteins represent an impor-
the ovum, and the fusion of the sperm and tant part of the high-molecular-weight sub-
the egg (reviewed in Evans and Kopf 1998; stances in the seminal plasma (Yanagimachi
Töpfer-Petersen et al. 1998, 2000, 2005, 1994; Henault and Killian 1996).
2008; Visconti et al. 1998; Töpfer-Petersen
1999; Wassarman 1999; Jansen et al. 2001; As far as the seminal plasma of domestic
Suarez 2001; Wassarman et al. 2001, 2005; animals is concerned, bull and boar pro-
teins belong to the most studied ones. A
number of papers concerning studies on the

339

340 Genomics and Reproductive Biotechnology

characterization of these protein molecules, plasma (BSP), the BSP proteins containing
as well as their properties and function, Fn-2 domains comprise about 65% of the
decrease in the following order: bovine total protein; homologous proteins in
(bull) >> porcine (boar) >> equine (stallion) > stallion and boar seminal plasma repre-
goat (buck) > ovine (ram) > poultry. sent only 20% and 1.1% of the total
protein, respectively (Manjunath et al.
15.2 Proteins of seminal plasma 2007). Proteins of CUB and Fn-2 struc-
tural groups are present in seminal plasma
15.2.1 Structure and properties both as non-modified polypeptide chains
and as differently glycosylated isoforms
The seminal plasma proteins of domestic as described for boar spermadhesins by
animals discussed in this review can be Calvete et al. (1993) and Solis et al. (1997)
approximately separated into three groups and for BSP proteins from bull seminal
according to their structural characteristics: plasma by (Manjunath and Sairam 1987).

1. Spermadhesins. They belong to a novel The third group of proteins detected
group of animal lectins. They form a sub- in seminal plasma need not be directly
group of a superfamily of proteins with a
single CUB domain (named after the pro- Table 15.1 Proteins of seminal plasma of various
teins in which it was first identified: C,
complement subcomponents C1r/C1s; species containing fibronectin type II domains and
U—uegf, urchin epidermal growth factor; homologous to boar spermadhesins.1
B—Bmp1, bone morphogenetic protein)
that has been found in a variety of devel- Species Fibronectin type II Spermadhesin
opmentally regulated processes (Bork and domain proteins proteins
Beckmann 1993).
Boar DQH (pB1) AWN family
2. Proteins containing Fibronectin type II Bull AQN family
(Fn-2) domains. They belong to a large Stallion BSP-A1/A2 (PDC-109) PSP-I/II
family of cell and matrix adhesion pro- Ram BSP-A3 aSFP
teins, which include seminal plasma pro- BSP-30kDa Z13
teins, fibronectins, and large cell surface Buck
receptors (Potts and Campbell 1994). A HSP1 HSP-7
list of well-characterized proteins from HSP2
various species of domestic animals HSP-12 15 kDa protein
(spermadhesins and Fn-2) is given in
Table 15.1. RSP-15 kDa BSFP
RSP-16 kDa
3. Different proteins exhibiting enzymatic, RSP-22 kDa
inhibitory, and other activities. Proteins RSP-24 kDa
belonging to the first two groups repre-
sent the major protein constituents of the GSP-15 kDa
mammalian seminal fluid. However, the GSP-20 kDa
relative abundance of these proteins GSP-22 kDa
varies in different species: in bull seminal
1 Data from the tables in Manjunath et al. (2007); references to
individual proteins are given in Chapter 5 and Tables 15.2–15.7.
DQH, AQN, AWN—boar seminal plasma proteins (designations
are in accordance with their N-terminal amino acid sequence).
PSP, porcine seminal plasma; BSP, bovine (bull) seminal plasma,
BSP-A1/A2 equals PDC-109; HSP, horse seminal plasma; RSP,
ram seminal plasma; GSP, goat seminal plasma; BSFP, buck
seminal fluid protein.

Proteomics of Male Seminal Plasma 341

involved in the reproduction process. They surface (Jonakova et al. 2000; Manaskova
may have a protective role (as, e.g., antioxi- et al. 2002, 2003). The protein coating layers
dant enzymes [Marti et al. 2007; Jelezarsky of sperm that are formed during ejaculation
et al. 2008]), participate in a modulation of are subject to remodeling in the female
activity of seminal plasma proteins in male reproductive tract.
and female reproductive tract (Meyer et al.
1997; Soubeyrand et al. 1997; Cibulkova et The aggregation state of the seminal
al. 2007), or participate in the inhibition of plasma proteins could be modulated by
enzymes affecting sperm function (Jonakova solute components, phosphorylcholine or
et al. 1992; Soubeyrand and Manjunath heparin, or by substances of the native envi-
1997; Jelinkova et al. 2003). The role of ronment of gametes as demonstrated for
several other proteins present in seminal PDC-109, the major protein of bull seminal
plasma in vivo, for example, lactoferrin, β- plasma (Gasset et al. 1997; Liberda et al.
microseminoprotein, RNAase dimer, is still 2001, 2002a; Talevi and Gualtieri 2001;
not clear. Jelinkova et al. 2004a).

The binding properties of homologous On the other hand, it was reported that the
proteins in different species do not always rates of heparin or phosphorylcholine binding
need to be similar; acidic Seminal Fluid to bull major seminal plasma proteins
Protein (aSFP ) from bull seminal plasma (Jelinkova et al. 2004a) and heparin binding
could serve as an example. This protein dis- to seminal plasma proteins of stallion (HSP—
plays about 50% amino acid sequence iden- horse seminal plasma) proteins (Calvete
tity with boar spermadhesins. Nevertheless, et al. 1995a) are significantly affected by the
it possesses neither carbohydrate nor ZP- extent and character of the seminal plasma
binding activity (Calvete and Sanz 2007). protein oligomerization. These association/
The representation of spermadhesins and dissociation processes thus result in changes
Fn-2 proteins in seminal plasma of various in their interaction properties as described,
investigated animals appears to significantly for example, in the case of PSP dimer from
differ. Human seminal plasma was found to boar seminal plasma (Calvete et al. 1995d;
be very low in spermadhesin-like proteins Campanero-Rhodes et al. 2006), or BSP pro-
(Kraus et al. 2001, 2005). teins (Jelinkova et al. 2004a).

It was shown that under physiological Proteins in a polydisperse form were
conditions the seminal plasma proteins of described to be present in bull seminal
boar, bull, and stallion form variable aggre- plasma (Manjunath and Sairam 1987;
gates (homo- and hetero-oligomers) differing Calvete et al. 1999). Changes in the poly-
in relative molecular mass, number of indi- dispersity of bull seminal proteins in the
vidual spermadhesins and fibronectin type II fertilization and the mechanism of sperm
domain proteins, and in interaction proper- capacitation by PDC-109 has been proposed
ties (Calvete et al. 1995a, 1997; Gasset et al. by Calvete and Sanz (2007) based on detailed
1997; Solís et al. 1998; Jonakova et al. 2000; structural studies of PDC-109 complexes
Manaskova et al. 2000, 2003; Jelinkova et al. with phosphorylcholine (Wah et al. 2002).
2004a).
Out of all domestic animals, a detailed
The aggregated forms of boar seminal proteomic analysis of only bull seminal
plasma proteins AQN, AWN, PSP, and DQH plasma has been published. The proteomic
make the protein coverage of the sperm approach involving 2-D and 1-D electro-
phoretic separation and mass spectroscopy

342 Genomics and Reproductive Biotechnology

analysis revealed the presence of about 250 were also found in seminal vesicles, pros-
protein spots, out of which 99 were identi- tate, and cauda epididymis.
fied (Kelly et al. 2006). A similar approach
was used to compare the protein composi- The mRNA transcripts of the DQH gene
tion of the accessory gland fluid from indi- were possible to detect and clone from boar
vidual Holstein bulls (Moura et al. 2006a,b). seminal vesicles (Plucienniczak et al. 1999),
but not from other reproductive organs,
15.2.2 Localization and expression such as testis, epididymis, or prostate
(Manaskova et al. 2007). The DQH cDNA
Spermadhesins AWN, AQN1, AQN3, PSP-I, derived amino acid sequence showed com-
and PSP-II cDNAs were amplified from total plete identity with the covalent structure of
RNA of porcine seminal vesicles (Ekhlasi- the boar DQH sperm surface protein, which
Hundrieser et al. 2002). This revealed, in the was determined by Edman degradation,
case of AWN, a 459 nucleotide open reading MALDI-MS, and post-source decay (PSD;
frame, comprising a signal peptide (amino Bezouska et al. 1999). The DQH protein
acids 1–20) and 133 amino acid residues consists of the N-terminal O-glycosylated
polypeptide, followed by 232 nucleotides of peptide followed by two fibronectin type II
the 3′-untranslated region. AWN cDNA repeats. This approach also allowed detec-
derived amino acid sequence differed in two tion of O-glycosidically linked carbohy-
positions, Tyr92 and Glu/Gln98, from that drates attached to Thr 10 of the isolated
obtained by direct sequencing of AWN1 N-terminal glycopeptide.
protein by Sanz et al. (1992b) who reported
Arg and His respectively, at these positions. The cDNA sequences of spermadhesins
The amino acid sequence deduced from the PSP-I, PSP-II obtained from the total RNA of
cDNA fragment encoding AQN1 protein porcine seminal vesicles were originally
showed the complete amino acid sequence described by Kwok et al. (1993). Amplified
identity to that determined from the mature PSP-I and PSP-II cDNA products could be
protein (Sanz et al. 1992a), whereas the also generated from total RNA of rete testis,
AQN3 cDNA deduced amino acid sequence caudal epididymis, seminal vesicles, and
differed in position Thr78 and Glu95 from prostate. A low expression of PSP-I mRNA
that obtained by protein sequencing (Gly78 was further detected in the testis, corpus,
and Asp95). The unidentified residue at and caput epididymis.
position 85 was shown to be a serine
residue. The presence of the 11-mer peptide, The porcine spermadhesin genes were
LNLXCGKEYV/LE, found in all mature located on pig chromosome 14q28–q29. The
porcine spermadhesins at positions 49–59, pig contains five closely linked spermadhe-
was also confirmed by cDNA sequencing. sin genes, whereas only two spermadhesin
Besides seminal vesicles, AWN transcripts genes are present in the cattle genome (Haase
were also detected in extracts from prostate et al. 2005).
and caudal epididymis. No PCR products
could be generated from RNA extracts of Using a monoclonal avian antibody
testis, rete testis, caput epididymis, corpus directed against purified porcine AWN and
epididymis, and bulbourethral glands (or a rabbit polyclonal antibody generated
liver). AQN1 and AQN3 specific transcripts against porcine AQN1, homologs of both
spermadhesins were detected in extracts of
seminal vesicles and prostate of the boar by
Western blot analysis (Jonakova et al. 1998;
Ekhlasi-Hundrieser et al. 2002). In stallion,

Proteomics of Male Seminal Plasma 343

in contrast, the seminal plasma protein plasma and immunodetected in the prostate
HSP-7, a homolog of the boar AWN sper- extract (Jeng et al. 2001; Manaskova et al.
madhesin, was found to be secreted in the 2002). Similarly, in humans, the homolo-
cauda epididymis and its localization on the gous protein (PSP94) was described as a pros-
ejaculated spermatozoa was shown to be on tate secretory protein (Ohkubo et al. 1995).
their equatorial segment (Reinert et al.
1997). Further studies revealed expression of In bull, expression products of the PDC-109
both PSP proteins in boar testis, caput, and gene, both the PDC-109 mRNA and the PDC-
corpus epididymis, and in bulbourethral 109 protein, the major seminal vesicle secre-
glands (García et al. 2008). tory protein, were detected in and isolated
from extracts of seminal vesicles (Scheit
Indirect immunofluorescence on tissue 1990). This finding confirmed and extended
sections from boar proved the presence of previous results showing immunoreactivity
AQN and AWN spermadhesins in the lumen of the seminal vesicle epithelium and of the
of epididymis, seminal vesicles, and prostate neck region and mid-piece of testicular and
(Veselsky et al. 1992, 1999), whereas signals ejaculated spermatozoa with antiserum
from PSP-I and PSP-II spermadhesins were against PDC-109 (Aumüller et al. 1988; Scheit
detected in the secretory tissues of corpus et al. 1988). Interestingly, neither the epididy-
epididymis, seminal vesicles, prostate, and mal epithelium nor the seminal vesicle tissue
Cowper’s glands but not in testes (Manaskova of the calf gave any reaction to the PDC-109
and Jonakova 2008). Both PSP proteins were antibody (Scheit et al. 1988). Finally, Wempe
also detected in extracts from boar epididy- et al. (1992) reported preparation of the aSFP
mis, seminal vesicles, prostate, and Cowper’s cDNA from extracts of bull seminal vesicle
glands (Ekhlasi-Hundrieser et al. 2002; tissue. Expression and localization of acrosin
Manaskova et al. 2002; García et al. 2008; inhibitor in boar reproductive tract is
Manaskova and Jonakova 2008). The AQN described in Davidova et al. (2009).
and AWN antibodies interacted with the
acrosomal region of both epididymal and 15.3 Function of seminal
ejaculated boar spermatozoa (Veselsky et al. plasma proteins
1999). An interaction of PSP-I and PSP-II
antibodies was observed with the acrosomal Proteins of seminal plasma participate in
head region and the mid-piece of the epididy- almost all phases of the reproduction process;
mal spermatozoa but only with the acroso- not only do they affect the properties and
mal head region of the ejaculated sperm thus the behavior of sperm in both the male
(Manaskova and Jonakova 2008). Besides, and the female reproductive tracts, but they
the PSP-II antibody stained the principal also modulate a natural environment in
piece of the flagellum of the ejaculated sper- which individual steps of the complex
matozoa (Manaskova and Jonakova 2008). process proceed. Major seminal plasma pro-
teins are mostly multifunctional substances;
In boar, the AQN, AWN, and PSP sper- their function is not limited to one phase of
madhesins and the DQH sperm surface the reproduction process only; it is frequently
protein were also detected on the surface of more complex and not yet fully understood.
epididymal spermatozoa (Jonakova et al.
1998; Manaskova et al. 2007; Manaskova Detailed studies mostly performed with
and Jonakova 2008), ß-microseminoprotein bull and boar seminal plasma proteins
(ß-MSP) was isolated from the seminal

344 Genomics and Reproductive Biotechnology

showed that these proteins are involved in the membrane destabilizing change that
or at least might participate in the following encompasses the first stages of capacitation.
steps of the reproduction process: remodeling Sperm activation can be delayed or even
of sperm surface, establishment of the ovi- reversed by co-incubation with membrane
ductal reservoir, modulation of capacitation, proteins of the tubal lining, isthmic fluid,
gamete interaction, sperm membrane protec- or specific tubal glycosaminoglycans, such
tion, sperm destruction, and modulation of as hyaluronan (Rodriguez-Martinez 2007).
sperm motility. They can also influence the Hyaluronan, on the other hand, increased
antimicrobial activity of the seminal fluid capacitation in the post-ovulation period
and function as enzyme inhibitors (reviewed (Tienthai et al. 2004).
in, e.g., Töpfer-Petersen et al. 1998; Jansen
et al. 2001; Jonakova and Ticha 2004; Calvete Formation of the sperm oviductal reser-
and Sanz 2007; Manjunath et al. 2007). voir belongs to one of the saccharide-medi-
ated events of the fertilization process and
A correlation of structure and function of is probably species-specific. The following
ungulate seminal plasma proteins was molecules are involved in the attachment of
recently discussed by Calvete and Sanz. They sperm to the oviduct and in the sperm
showed that the sperm membrane remodel- release to meet an oocyte: (1) glycosylated
ing events occurring in the female genital components of the oviductal epithelium, (2)
tract are essentially conserved although dif- constituents of oviductal fluid, and (3) pro-
ferent seminal plasma proteins participate in teins localized on the sperm surface.
these steps in different species (Calvete and
Sanz 2007). In cow, the oviductal reservoir is formed
by the binding of sperm to L-fucose-
15.3.1 Establishment of the containing glycoconjugates on the surface of
oviductal reservoir oviductal epithelium cells (Lefebvre et al.
1995, 1997; Revah et al. 2000; Suarez
In many mammals when sperm cells reach 2001; Suarez and Ignotz 2001). The L-fucose-
the uterotubal junction–isthmus of the ovi- binding molecule that promotes bull sperm
ductal tract, its epithelium cells trap the attachment to the oviductal epithelium was
spermatozoa to form a sperm reservoir. The identified as PDC-109 (BSP-A1/A2), a protein
main function of the reservoir is to maintain secreted by seminal vesicles and associated
a given population of spermatozoa viable for with the sperm plasma membrane upon
an extensive period of time, until ovulation, ejaculation (Gwathmey et al. 2001, 2003).
and to prevent polyspermy (Suarez 1998, Two other proteins of bovine seminal plasma
2001, 2002, 2007, 2008). (BSP-30-kDa and BSP-A3) enhance the sperm
binding to oviductal cells (Gwathmey et al.
The functional sperm reservoir ensures 2006). Binding of epididymal bull sperm to
that suitable numbers of viable and poten- the epithelium is low (Gwathmey et al.
tially fertile spermatozoa are available for 2003). L-Fucose-binding molecules are lost
fertilization at the ampullary isthmic junc- during capacitation and, at the same time,
tion. In vivo, the most viable spermatozoa D-mannose-binding sites are uncovered for
in the preovulatory sperm reservoir are the interaction with the ovum (Revah et al.
uncapacitated. Capacitation rates signifi- 2000; Ignotz et al. 2001).
cantly increase after ovulation. Bicarbonate
appears to be a common primary effector of Annexins isolated from the apical plasma
membrane of bovine oviductal epithelium

Proteomics of Male Seminal Plasma 345

were suggested to be candidates for bull The loss of binding affinity could be
sperm receptors in the sperm oviductal res- explained by a release of sperm coating pro-
ervoir formation (Ignotz et al. 2007). teins during heparin-induced capacitation
(Gwathmey et al. 2003). The role of the con-
Contrary to the bovine model, the forma- stituents of oviductal fluid both of the
tion of the porcine oviductal sperm reservoir protein and the glycosaminoglycan (e.g.,
was shown to comprise a participation of hyaluronic acid) nature on these processes
D-mannosyl residues (Green et al. 2001); a cannot be neglected (Liberda et al. 2006;
detailed study showed a high affinity of Rodriquez-Martinez 2007).
sperm to oligomannosyl residues (Wagner
et al. 2002). As epididymal spermatozoa 15.3.2 Modulation of capacitation
showed significantly lower capability to
bind to oviductal epithelium than ejaculated Sperm capacitation is a gradual multistep
sperm, participation of the components of event of the reproduction process occurring
seminal plasma especially of sperm coating in the female reproductive tract (Yanagimachi
proteins in this binding process was sug- 1994; Rodríguez-Martínez et al. 2005). It
gested (Petrunkina et al. 2001). The presence involves a release of sperm from the sperm
of highly mannosylated structures on reservoir (Fazeli et al. 1999), removal of deca-
porcine oviductal epithelium that could be pacitation substances, mainly of adsorbed
recognized by boar AQN1 spermadhesin epidydimal and seminal plasma proteins,
has been demonstrated (Ekhlasi-Hundrieser from the sperm surface, reorganization of
et al. 2005a, 2008) as well as the affinity sperm membrane as a result of the promotion
of boar spermadhesins to yeast mannan of membrane lipid disorder with consequent
(Jelinkova et al. 2004b). Heparin-binding protein relocation, and so on (Yanagimachi
proteins of boar seminal plasma and espe- 1994; Tienthai et al. 2004).
cially AQN1 protein displayed the strongest
interaction with the oviductal epithelium Participation of seminal plasma proteins
that was inhibited by yeast mannan (Liberda in sperm capacitation has been studied in
et al. 2006). On the other hand a glycopro- detail only with major seminal proteins and
tein (SPG) isolated from porcine oviductal in bull. BSP proteins (BSP-A1/A2 [PDC-109],
cells containing Gal-ß1-3-GalNAc disaccha- BSP-A3, and BSP-30K), which are secreted by
ride chain was also described to bind to boar seminal vesicles, are adsorbed to the sperm
sperm (Marini and Cabada 2003; Teijeiro surface upon ejaculation (Manjunath et al.
et al. 2007). 1994a). These proteins are specifically bound
to phosphorylcholine containing phospho-
Contrary to the data concerning the par- lipids present in the sperm membrane
ticipation of seminal plasma proteins in the (Desnoyers and Manjunath 1992). In addi-
process of sperm reservoir formation, much tion to the phosphorylcholine-binding activ-
less information is available about their ity, BSP proteins interact with high-density
fate in the course of sperm release from lipoprotein (HDL; Manjunath et al. 1989;
the oviductal epithelium and capacitation Thérien et al. 1997, 2001) and glycosamino-
(Rodríguez-Martínez et al. 2005; Suarez glycans (GAGs, e.g., heparin; Thérien et al.
2007). Capacitated bull sperm showed a 2005). Both types of these substances
reduced binding of sperm to the oviductal (HDL and GAGs) are physiological inducers
epithelium, as well as to the saccharide of sperm capacitation and are present in
ligands (Revah et al. 2000; Ignotz et al. 2001).

346 Genomics and Reproductive Biotechnology

oviductal and follicular fluids. BSP proteins 1990; Yanagimachi 1994; Wassarman et al.
potentiate sperm capacitation induced by 2005). These restrictions are attributed to
either HDL or heparin (Thérien et al. 1997, the presence of receptors for spermatozoa on
2001). In addition, BSP binding to sperm the ovum surface. The ability of sperm to
induces cholesterol and choline phospho- interact with the egg surface can be detected
lipid efflux from sperm (Thérien et al. 1998, by using solubilized ZP(Gwatkin 1977). The
1999) and thus modulates the capacitation majority of experimental data using solubi-
of bull sperm cells (Manjunath and Therien lized ZP or its components implicate their
et al. 2002; Tannert et al. 2007a,b). O-linked and N-linked oligosaccharide
chains in the primary sperm binding. It has
Based on the results of these studies, two been estimated that about 75–80% of sperm–
types of mechanism of the participation of ZP binding is of the lectin-like nature, while
BSP proteins in sperm capacitation mediated the remaining ones are based on the protein-
either by HDL or GAGs were proposed protein interactions (Litscher et al. 1995;
(Manjunath et al. 2007). Clark and Dell 2006).

Only a limited amount of information is The mammalian glycoprotein egg enve-
available on the role of seminal plasma pro- lope has been the most extensively studied
teins from other species. Boar seminal in the case of mouse (Wassarman et al. 2005)
plasma contains low amounts of a homolo- and much less attention has been paid to
gous protein of the BSP family, named pB1 other species. The murine ZP3 glycoprotein
(DQH). This protein, as well as BSP-A1/A2 is supposed to be the primary sperm receptor
proteins from bull seminal plasma, potenti- (Wassarman 1999; Tanphaichitr et al. 2007);
ated boar epididymal sperm capacitation in the pig: a heterodimer complex of ZPB
(Lusignan et al. 2007). and ZPC glycoprotein is suggested to partici-
pate in the primary binding (Yurewicz et al.
The heterodimer of boar spermadhesins 1998).
PSP-I/PSP-II (present in post-sperm-rich
fraction of boar ejaculate) acts as leukocyte While there are only a few glycoprotein
chemoattractant both in vitro and in vivo, components of ZP that were shown to be
contributing to the phagocytosis of those involved in the primary gamete interaction,
spermatozoa not reaching the sperm reser- a large number of protein molecules were
voir (Rodríguez-Martínez et al. 2005). described to possess the ZP-binding ability
(Tanphaichitr et al. 2007). This group of sub-
15.3.3 Gamete interaction stances involves mostly sperm membrane-
bound proteins, and in the case of the porcine
Sperm-ovum interaction occurs in two model, proteins from seminal plasma. The
sequential steps. It starts with the primary boar spermadhesins were shown to be tightly
binding of acrosome intact sperm to the ZP, bound to the sperm membrane of in vitro
which is followed by the secondary binding capacitated spermatozoa, and not removed
of acrosome-reacted sperm to the ZP (Bleil by the capacitation process (Sanz et al. 1993;
et al. 1988). Primary mammalian sperm Dostalova et al. 1994; Calvete et al. 1995b).
binding to the ovulated egg is not strictly Sperm-egg binding test and other experi-
species-specific, but the glycoprotein enve- mental data demonstrated that intact pro-
lope of the ovum is supposed to represent a teins on the sperm surface (e.g., AQN1,
significant barrier to many, if not most, het- AWN1, DQH) are required for the primary
erospecific interactions in vitro (Wassarman

Proteomics of Male Seminal Plasma 347

binding of the sperm with the ZP of the the sequencing of its cDNA (Kwok et al.
ovum (Veselsky et al. 1992, 1999; Dostalova 1994). Proteinase inhibitors may also protect
et al. 1995; Ensslin et al. 1995; Calvete et al. spermatozoa from a proteolytic damage.
1996a; Rodríguez-Martinez et al. 1998; Spermadhesins (AQN, AWN) attached to
Manaskova et al. 2000, 2007; Caballero et al. the sperm head at ejaculation are acceptor
2005). molecules for SAAI. The attachment of the
inhibitor to surface molecules of the sperm
There exists a list of many other candi- can stabilize its binding site for the ZP of
dates of sperm components involved in the the oocyte (Sanz et al. 1992c). Formation
gamete primary binding and this step of the of a complex between SAAI and AQN1
fertilization process remains unresolved. spermadhesin was proven by gel chromatog-
raphy (Jelinkova et al. 2003), by Western
15.3.4 Seminal plasma proteins as blotting (in this case also with AWN sper-
enzyme inhibitors madhesin), and by the detection of SAAI-
AQN1 complex on the surface of boar
Seminal plasma also contains proteins that capacitated spermatozoa (Sanz et al. 1992c).
regulate the activity of enzymes occurring It is thought that SAAI protects the ZP
in the ejaculate. Proteinase inhibitors are binding sites of spermadhesins on the sperm
present in all tissues and body fluids. They surface against proteolytic degradation from
interfere with the activity of the proteinases the moment of ejaculation until the sperm-
and thus maintain the homeostasis. In the egg encounter (Sanz et al. 1992c; Jonakova
male reproductive tract, proteolytic enzymes et al. 1995).
occur in the sperm acrosome, in the epididy-
mal fluid, and in the seminal plasma. The Besides proteinase inhibitors, the presence
main role of proteinase inhibitors is the of an inhibitor of another enzyme was
inactivation of prematurely released acrosin shown. Major BSP proteins from bovine
from occasionally damaged spermatozoa, seminal plasma inhibited the activity of
and thus protecting the male and the female bovine seminal phospholipase A2 (PLA2) that
genital tract against proteolytic degradation. has been shown to be a platelet-activating
The presence of several proteinase inhibitors factor acetylhydrolase (PAF-AH). BSP pro-
in the seminal plasma of different species teins modulate PLA2 activity and therefore,
has been described. Serine proteinase inhibi- phospholipid metabolism. They may act as
tors, of Kazal type, belong to the most spermatozoa-stabilizing agents by prevent-
studied inhibitors from the seminal plasma ing premature lipolysis of the sperm surface
of domestic animals, such as boar (Fritz (Manjunath et al. 1994b; Soubeyrand et al.
et al. 1976; Jonakova and Cechova 1985; 1997; Soubeyrand and Manjunath 1997;
Jonakova et al. 1991b, 1992; Jelinkova et al. Soubeyrand et al. 1998).
2003) or bull (Cechova and Jonakova 1981;
Meloun et al. 1983, 1985). The acrosin inhib- 15.4 In vitro effects of seminal
itor isolated from boar seminal plasma plasma proteins
(SPAI) (Fritz et al. 1976) is structuraly related
to the sperm-associated acrosin inhibitor Seminal plasma contains various compo-
(SAAI) isolated from boar spermatozoa and nents, including those of a protein nature,
sequenced (Jonakova et al. 1991b, 1992). The that are beneficial and/or detrimental not
protein sequence of SAAI was confirmed by

348 Genomics and Reproductive Biotechnology

only to the sperm function but also to sperm found to be deleterious when the frozen-
storage in vitro. thawed spermatozoa activity to penetrate
oocytes was checked (Caballero et al. 2004,
In vitro handling of spermatozoa in pre- 2008).
paration for artificial insemination, involv-
ing processes such as dilution, cooling, 15.5 Properties of major
freezing, re-warming and sperm sexing by proteins of seminal plasma
flow cytometric sorting, may modify the of domestic animals
proteins bound to the sperm surface, and
thus the sperm membranes may be destabi- Proteins of seminal plasma isolated from
lized (Maxwell et al. 2007; de Graaf et al. bull, boar, stallion, ram, buck, and poultry
2008). are listed according to their origin in Tables
15.2–15.7. The Tables also summarize their
Mammalian sperm preservation in extend- relative molecular mass and basic character-
ers containing egg yolk and milk has been istics. As previously mentioned, literature
used for a long time. In the case of bull data on boar, bull, and stallion seminal
sperm, the mechanism of the protective plasma proteins predominate. The seminal
action of these substances was investigated. plasma protein primary structures were
Upon binding to sperm surface the phospho- determined for BSP proteins and spermad-
lipid-binding proteins present in bull seminal hesins from bull, spermadhesins and DQH
plasma (BSP proteins) induce cholesterol and protein from boar, and HSP proteins and
phospholipid removal from sperm mem- spermadhesin from stallion (Tables 15.2–
brane and its destabilization in the course of 15.4); in some cases the structure of saccha-
storage. Sequestration of BSP proteins by ride chains was also determined (e.g.,
their interaction with low-density lipopro- O-linked saccharide chains of PDC-109 from
teins (LDL) of egg yolk was suggested as bull [Calvete et al. 1994c; Gerwig et al.
a major mechanism of sperm protection 1996], N-linked oligosaccharides of PSP-I/
by LDL (Bergeron and Manjunath 2006; PSP-II spermadhesins from boar seminal
Manjunath et al. 2007). The mechanism of plasma [Nimtz et al. 1999]).
sperm protection by skimmed milk was sug-
gested to involve BSP protein-casein micelle Investigation of the crystal structure of
interactions (Bergeron et al. 2006, 2007). heterodimer PSP-I/PSP-II from boar seminal
plasma (Romero et al. 1997; Varela et al.
In the case of boar seminal plasma pro- 1997) and PDC-109 (Wah et al. 2002), aSFP
teins, the heparin-binding proteins (HBP) (Romão et al. 1997; Romero et al. 1997),
exert opposite effects on viability, motility, and ribonuclease (Mazzarella et al. 1993;
and mitochondrial activity of highly diluted Vitagliano et al. 1998) from bull seminal
spermatozoa compared with PSP-I/PSP-II plasma was a subject of other studies.
spermadhesins. The addition of the HBP
had a detrimental effect on these parame- Binding properties of individual proteins
ters, whereas PSP-I/PSP-II heterodimer con- are summarized in Tables 15.2–15.3. The
tributed to maintaining the functionality following interactions of seminal proteins
of the highly diluted boar spermatozoa that participate in the formation of sperm
(Centurion et al. 2003) and improved the in coating layers belong to the most studied
vivo fertilizing ability of sex-sorted boar ones:
spermatozoa (García et al. 2006, 2007). On
the other hand, the PSP-I/PSP-II effect was

Table 15.2 Major proteins isolated from boar seminal plasma.

Protein type Protein Relative Binding properties, References

molecular mass inhibition

Spermadhesin PSP-I, 14,000 Hep− Parry et al. 1992; Rutherfurd et al. 1992;
PSP-II 16,000 Calvete et al. 1993, 1995d; Solis et al.
1997, 1998
AQN1 13,000 Hep+; saccharide-, Jonakova et al. 1991a, 1998; Sanz et al.
AQN3 12,000 ZP-binding 1991,1992a; Calvete et al. 1993,1996a;
Jelinkova et al. 2004b
AWN 14,000–16,000 Hep+; saccharide-, Sanz et al. 1992b; Calvete et al. 1994a;
family ZP-binding Jonakova et al. 1998; Jelinkova et al.
2004b
Fibronectin type II DQH 13,000 Hep+; P-choline-,
domains (Fn-2 (pB1) mannan-, Sanz et al. 1993; Calvete et al. 1997;
type protein) (pAIF) ZP-binding Jonakova et al. 1998; Bezouska et al.
1999; Liberda et al. 2002a; Jelinkova
et al. 2004b; Manaskova et al. 2007

Proteinase SPAI 12,000 Acrosin Fritz et al. 1976; Jonakova et al. 1991b,
inhibitors SAAI 8,000 Acrosin, trypsin 1992; Jelinkova et al. 2003; Davidova
et al. 2009

Others ß-MSP 10,000 Fernlund et al. 1994; Manaskova et al.
2002; Wang et al. 2005
Lactoferrin 70,000 Roberts and Boursnell 1975

ZP, zona pellucida; P-choline, phosphorylcholine; Hep+, heparin-binding protein; Hep−, non-heparin-binding protein; ß-MSP, ß-
microseminoprotein; SAAI, sperm-associated acrosin inhibitor; SPAI, seminal plasma acrosin inhibitor.

Table 15.3 Major proteins isolated from bull seminal plasma.

Protein type Protein Relative Binding properties, References

molecular mass inhibition

Fibronectin type II BSP-A1/A2 13,000 Hep+; gelatin-, Esch et al. 1983; Manjunath and
domains (Fn-2 PDC-109 26,000 P-choline, Sairam 1987; Manjunath and
type protein) mannan-binding Thérien 2002; Liberda et al. 2002b
BSP-A3 Hep+; gelatin-, Manjunath and Sairam 1987;
P-choline-, Manjunath and Thérien 2002;
BSP-30 mannan-binding Seidah et al. 1987
Hep+; gelatin-, Calvete et al. 1996b,c; Liberda et al.
P-choline-, 2002b
mannan-binding

Spermadhesin aSFP 14,000 Einspanier et al. 1991, 1994
Z13 Tedeschi et al. 2000
Hep−

Proteinase BUSI I 9,000 Acrosin, trypsin, Cechova and Jonakova 1981;
inhibitors BUSI II 6,000
elastase,cathepsin G, Meloun et al. 1983, 1985

Acrosin, trypsin

Others PAF-AH 60,000 Soubeyrand et al. 1997;
RNAase dimer 29,000 Soubeyrand and Manjunath 1997;
Mannan-binding Soubeyrand et al. 1998
Di Donato and D’Alessio 1981;
D’Alessio et al. 1991; Calvete
et al. 1996b; Liberda et al. 2002b

Hep+, heparin-binding protein; Hep−, non-heparin-binding protein; PAF-AH, platelet-activating factor acetylhydrolase with phospholipase
A2 (PLA2) activity (Soubeyrand et al. 1998); ZP, zona pellucida; P-choline, phosphorylcholine; BUSI I, bull seminal plasma inhibitor I; BUSI
II, bull seminal plasma inhibitor II; BSP, bovine seminal plasma proteins; aSFP, acidic seminal fluid protein.

349

Table 15.4 Major proteins isolated from stallion seminal plasma.

Protein type Protein Relative Properties References
molecular mass

Fibronectin type II HSP-1 14,000 Hep+; gelatin-, Calvete et al. 1994b, 1995a,c;
15,000 P-choline-binding Ekhlasi-Hundrieser et al. 2005b
domains (Fn-2 Hep+; gelatin-, Calvete et al. 1994b;
P-choline-binding Ekhlasi-Hundrieser et al. 2005b
type protein) HSP-2 Calvete et al. 1994b; Greube
P-choline-binding et al. 2004
HSP-12
(EQ-12)

Spermadhesin HSP-7 15,000 Saccharide-, ZP-binding Reinert et al. 1996

Others CRISP proteins Magdaleno et al. 1997;
(HSP-3) Schambony et al. 1998
HPK 29,000 D-galactose-, Carvalho et al. 2002
Lactoferrin 25,000 sperm-binding Inagaki et al. 2002
D-gal-binding Sabeur and Ball 2007
protein

Hep+, heparin-binding protein; Hep−, non-heparin-binding protein; ZP, glycoproteins of zona pellucida; P-choline, phosphorylcholine; HPK,
horse prostate kallikrein; HSP, horse seminal plasma protein.

Table 15.5 Characterized proteins from ram seminal plasma.

Protein type Protein Properties References
Bergeron et al. 2005
Fibronectin type II RSP-15 kDa Gelatin-binding
domains (Fn-2 RSP-16 kDa Barrios et al. 2005;
type protein) RSP-22 kDa Gelatin-binding Cardozo et al. 2008
Gelatin-binding, Hep+ Bergeron et al. 2005
RSP-24 kDa Gelatin-binding, Hep+ Upreti et al. 1999
P14 Barrios et al. 2005;
Cardozo et al. 2008
Spermadhesin 15.5 kDa protein1 Hep+
Others Phospholipase A2 (PLA2)
P20

1 Major ram seminal plasma protein.
Hep+, heparin-binding protein; RSP, ram seminal plasma protein.

Table 15.6 Characterized proteins from buck seminal plasma.

Protein type Protein Relative Binding properties References
molecular mass

Fibronectin type II GSP-14 14,000 Gelatin-binding, Hep− Villemure et al. 2003
domains (Fn-2 type GSP-15 15,000 Gelatin-binding, Hep− Teixeira et al. 2002; 2006
protein) GSP-20 20,000 Gelatin-binding, Hep+
GSP-22 22,000 Gelatin-binding, Hep+
Spermadhesin
BSFP 12,500 Hep−

Others Phospholipase A Sias et al. 2005

BSFP, buck seminal fluid protein; Hep+, heparin-binding protein; Hep−, non-heparin-binding protein; GSP-14, GSP-15, GSP-20, GSP-22,
goat seminal plasma proteins.

350

Proteomics of Male Seminal Plasma 351

Table 15.7 Characterized proteins from seminal plasma of poultry.

Protein Relative Properties References
molecular mass

Chicken, roaster Proteinase inhibitor 6,000 Acrosin inhibitor Lessley and Brown 1978
(Gallus Acid phosphatase Crystallization Dumitru and Dinischiotu 1994
domesticus) SMIF 78,000 Antibacterial activity Mohan et al. 1995
UPSEBP Fragment of prosaposin Hammerstedt et al. 2001
28,000
Turkey TSPE –30,000+ Not identical with acrosin Thurston et al. 1993
(Meleagris
gallopavo) 38,000
–44,000

SMIF, Universal primary sperm-egg binding protein; UPSEBP, Sperm motility inhibiting factor; TSPE, Turkey seminal plasma protease.

• interaction with different types of detected, out of these 3-5 interacted with
glycoconjugates, antibodies against BSP-A1/A2 proteins (from
bull seminal plasma; Jobim 2005). The same
• interaction with membrane phospholipids, electrophoretic method was used to assess
• interactions between proteins. monthly variations in ram seminal plasma
proteins (Cardozo et al. 2006).
Saccharide-based interactions of seminal
plasma proteins play an important role in Properties of the buck seminal plasma
the interaction of sperm with glycoconju- proteins are summarized in Table 15.6. The
gates present in the female reproductive protein composition of the buck seminal
tract; binding sperm to the oviductal epithe- plasma seems to be similar to that of bull
lium or primary sperm binding to ZP belong and stallion. It contains proteins with Fn-2
to the most investigated ones. The phosphor- domain (GSP; Villemure et al. 2003) that
ylcholine-binding activity of seminal plasma are characterized by their gelatin-binding
proteins is responsible for their adsorption ability, and it also contains a protein of sper-
to the sperm membrane and participation madhesin family (BSFP;Teixeira et al. 2002,
in sperm membrane modulation during 2006). Buck gene encoding BSFP protein was
capacitation. Interactions between proteins characterized and its expression along the
participate in the arrangement and remodel- genital tract was investigated (Melo et al.
ing of sperm-coating layers and modulate 2008, 2009).
binding properties or other activities of
protein monomer forms (compared above). Only a limited amount of information in
the literature is available on the character-
Properties of the proteins isolated from ization of proteins obtained from poultry
ram seminal plasma are summarized in Table seminal plasma. These studies mostly
15.5. Similarly as in the case of buck seminal concern chicken and turkey seminal plasma
plasma (Table 15.6), their differential binding proteins. A list of proteins isolated from
affinities to heparin and gelatin were used for those sources and at least partially charac-
their separation (Bergeron et al. 2005). The terized is presented in Table 15.7. Similarly
ram proteins belong to the Fn-2 type and to as in the case of mammalian proteins, the
the spermadhesin family. The protein profile removal of surface-associated proteins from
of ram seminal plasma was investigated chicken sperm affected the sperm function
using 2D PAGE. More than 20 spots were in vivo, especially migration in the female

352 Genomics and Reproductive Biotechnology

reproductive tract (Thurston et al. 1993; sophisticated support in our attempts to
Steele et al. 1996). reduce infertility and improve fertility in
breeding populations of agriculturally impor-
15.6 Future research directions tant animals, as well as in human popula-
tion. Proteomic studies on seminal plasma
Mammalian seminal plasma is a very proteins can thus also contribute to an
complex fluid containing both the low- assessment of animal and human fertility
molecular and the high-molecular compo- by monitoring changes in their reproductive
nents, and in this chapter, attention was tracts and to the improvement of the condi-
paid to the protein constituents. The physi- tions of mammalian sperm preservation.
ological functions of a large number of
seminal plasma proteins have not yet been References
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well characterized. Future research in this M., and Scheit, K.H. 1988. Binding of a
field will probably be directed to detailed major secretory protein from bull seminal
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This approach can contribute to better Immunocytochemical localization and
knowledge of changes in protein structure biochemical characterization of two
and protein modifications, which may alter seminal plasma proteins that protect ram
their properties and help explain some steps spermatozoa against cold shock. Journal
in fertilization physiology and pathology. of Andrology 26(4): 539–549.

Proteomics also provides a tool for under- Bergeron, A., Villemure, M., Lazure, C.,
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seminal plasma proteins will provide a prevent lipid loss from the sperm mem-
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Proteomics of Male Seminal Plasma 353

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16

Evolutionary Genomics of Sex Determination in
Domestic Animals

Eric Pailhoux and Corinne Cotinot

16.1 Introduction for Temperature-dependent Sex Determina-
tion, has been intensely investigated and
In vertebrates, sex is set up at fertilization described in different reptiles (reviewed in
and depends on the sex chromosome received Pieau and Dorizzi 2004). In these TSD
from the heterogametic parent (XY/XX species, the egg incubation temperature
system when male is heterogametic; ZZ/ seems directly linked to steroid hormone
ZW when female is heterogametic). Even if production and more precisely to the enzyme
zygotes are genetically different, no sex dif- P450 aromatase (CYP19 gene). This gene is
ference has been clearly evidenced before directly responsible for male to female ste-
gonad differentiation occurs. Thus, individ- roidogenesis reversal by converting andro-
uals of both sexes seem morphologically gens into estrogens (reviewed in Conley and
identical during early development. The Hinshelwood 2001).
first sign of sexual dimorphism appears
when the undifferentiated gonad engages its Steroid hormone treatments have been
differentiation onto a testis or an ovary, fol- shown to be critical in sex differentiation of
lowing a sex-determining signal. In hetero- numerous vertebrates. With the exception of
gametic vertebrates, this genetic signal is placental mammals, an estrogen or an anti-
located on sex chromosomes. Although aromatase treatment could reverse the
dependent on the considered species, this genetically or temperature-dependent prede-
signal could be more or less influenced by termined sex of the gonad (Scheib 1983;
environmental factors such as temperature, Elbrecht and Smith 1992; Guiguen et al.
steroid hormones, or population constitu- 1999; Krisfalusi and Cloud 1999; Pieau et al.
tion (Figure 16.1). 1999; Coveney et al. 2001).

Gonad differentiation depending on the Population constitution and/or social
temperature of egg incubation, called TSD factors influencing sex determination have
been described in some fish hermaphroditic

367

368 Genomics and Reproductive Biotechnology

Figure 16.1 Phylogeny displaying the different sex chromosome systems of major vertebrate groups (TSD,
Temperature Sex-Dependent). Divergence times are derived from Veyrunes et al. (2008).

species (Fishelson 1970). As a general feature, Gorski 1984; Gorski 1984; McEwen 1992);
a perturbation of social interactions in a sex-specific liver metabolisms (Gustafsson
given subpopulation results in a complete et al. 1983; Roy and Chatterjee 1983; Robins
sex reversal of one or several individuals 2005); or secondary sexual characteristics
(reviewed in Baroiller et al. 1999). such as horn development in different species
(Toledano-Díaz et al. 2007) or feathers in
Following the determining switch, early some birds (Wilson et al. 1987). Sex-specific
differentiating gonads will secrete sexual features closely or distantly linked to the
hormones controlling the development of initial sex-determining signal are numerous
different sexual features of the species. This and diverse, even in the way human males
hormonal control has been clearly demon- and females chew gum (Gerstner and
strated for some mammalian traits such as Parekh 1997). Intrigued or not by the abun-
the genital tract development (Jost 1947); dant diversity of sexual dimorphisms,
sex-specific brain features (Arnold and

Sex Determination in Domestic Animals 369

humans have developed many ways of under- expressed for a few hours in Sertoli cell pre-
standing sex differences that originate from cursors in the XY gonads between embry-
sex determination. onic days 10.5 and 12.5 (Koopman et al.
1990; Hacker et al. 1995; Albrecht and Eicher
Most data on sex determination and 2001). Both the spatial and temporal regula-
gonad differentiation have been obtained on tions of Sry levels are critical to correctly
humans and mice. In this review, we will direct the testis differentiation (Salas-Cortes
present the main commonalities in mam- et al. 1999; Bullejos and Koopman 2001;
malian sex differentiation, and then species- Sekido et al. 2004). Low or delayed expres-
specific features will be discussed for sions lead to the development of an ovotes-
domestic mammals (mainly pig, sheep, and tis, a gonad containing a mixture of male
goat) and briefly for nonmammal vertebrates and female tissues (Bullejos and Koopman
(mainly chicken). 2005; Taketo et al. 2005).

16.2 State of knowledge of SRY contains a high-mobility group
sex differentiation (HMG)-box DNA-binding domain character-
istic of the SOX gene family of transcription
16.2.1 Mammalian sex determination: factors. Consistent with the importance of
The key role of SRY the DNA-binding function of SRY, most
sex-reversed mutations occur within the
Our present knowledge of mammalian HMG domain (Harley et al. 1992; Mitchell
sex determination is based on studies per- and Harley 2002). Furthermore, this domain
formed over 50 years ago, on Klinefelter and is the only conserved feature across mam-
Turner syndromes in humans and mice that malian SRY. Regions outside this domain
revealed the dominant Y chromosome factor have evolved rapidly and present a large
in male differentiation. Individuals with variability across species (Whitfield et al.
Turner’s syndrome are XO and are pheno- 1993).
typically females, whereas individuals with
Klinefelter’s syndrome are XXY and pheno- Since its discovery, a variety of mecha-
typically males (Ford et al. 1959; Jacobs and nisms has been proposed by which SRY
Strong 1959). This identified the Y chromo- might initiate the testis differentiation from
some as the factor that engenders maleness early bipotential gonads: (1) as a repressor of
and generated a long quest for identifying a repressor of male development (McElreavey
the testis-determining factor (TDF). In 1990, et al. 1993), (2) through effects on local chro-
studies in human XX male patients led to matin structure (Pontiggia et al. 1994), (3)
the discovery of SRY (sex-determining region through a role in mRNA splicing (Ohe et al.
of the Y; Sry in mice) as the primary testis- 2002), and (4) as a transcriptional activator
determining factor (Sinclair et al. 1990). of one or more critical male-specific targets,
through partner proteins (Dubin and Ostrer
Primary sex determination in mammals 1994; Poulat et al. 1997; Thevenet et al.
appears to be focused on the cell-fate deci- 2005). Recent works have shown that Sry
sion that occurs in the supporting cell binds to multiple Sox9 elements located
lineage precursor when the cells chose to within a gonad-specific enhancer in mice,
differentiate into Sertoli or follicular (granu- supporting a model for the positive regula-
losa) cells. In mice, Sry is transiently tion of Sox9 expression in the male mouse
gonad (Sekido and Lovell-Badge 2008).

370 Genomics and Reproductive Biotechnology

16.2.2 The cascade after the switch induces nuclear localization of Fgfr2 in
Sertoli cell precursors. Moreover, it has been
The first gene known to be expressed down- shown that Sox9 is required for Fgfr2 nuclear
stream of SRY is SOX9, a closely related localization and, conversely, that Fgfr2 is
family member. Sox9 is expressed in various important for the maintenance of Sox9
tissues during embryogenesis (Ng et al. expression. These results suggest that Fgfr2
1997) and in Sertoli cells of male gonads and Sox9 regulate each other through a
(Morais da Silva et al. 1996). In contrast to Sox9–Fgf9–Fgfr2 positive signaling loop
Sry, Sox9 is well conserved among mammals (Kim et al. 2007; Bagheri-Fam et al. 2008).
and also in vertebrates that have another sex
chromosome system such as birds, reptiles, The ability of extracellular signals to
and fishes. recruit cells to the testis developmental
pathway has previously been hypothesized
Mutations or deletions of the Sox9 gene by XX-XY chimera experiments. These
lead to male-to-female sex reversal (Wagner studies have shown that in XX↔XY chime-
et al. 1994; Foster 1996; Chaboissier et al. rical mouse embryos, the ratio of XX to XY
2004; Smyk et al. 2007) whereas duplication cells is ∼50:50 in all tissues, the only excep-
or overexpression of Sox9 is responsible for tion being Sertoli cells. These cells were
female-to-male sex reversal (Huang et al. found to be more than 90% XY, indicating
1999; Bishop et al. 2000; Vidal et al. 2001). that the differentiation of Sertoli cells needs
These studies indicate that Sox9 can replace the presence of the Y chromosome (Palmer
Sry, even leading to fertile males when and Burgoyne 1991). However, these experi-
expressed at sufficient levels in XY Sry null ments also imply that Sry is not essential for
embryos (Qin and Bishop 2005). In mice, the the differentiation of all Sertoli cells. XX
expression of Sry is turned off just after that cells were recruited to differentiate into
Sox9 reached a critical threshold. Sertoli cells contributing almost one-tenth
of the total number of Sertoli cells. In addi-
Several extracellular signaling pathways tion to the cell-autonomous mechanism, at
have been involved in recruiting the cells of least one noncell-autonomous mechanism
the gonad to the testis pathway; among exists to ensure the differentiation of a suf-
these are prostaglandin D2(PGD2) and ficient number of Sertoli cells, above the
Fibroblast growth factor 9 (FGF9). Both estimated threshold of 20% to guarantee
induce Sox9 expression in XX cells in vitro testis differentiation (Burgoyne et al. 1988;
and promote Sertoli cell differentiation Patek et al. 1991).
(Malki et al. 2005, 2007; Wilhelm et al. 2005,
2007). In the absence of Fgf9 in KO mice, Sry Proliferation is also a critical event for
is expressed normally and Sox9 expression testis development. The use of proliferation
is initiated but rapidly silenced. Subsequently inhibitors or the disruption of Fgf9 pathway
the cells of the XY Fgf9−/− gonads express leads to male-to-female sex reversal (Schmahl
genes characteristic of the female pathway et al. 2000, 2004; Kim et al. 2006). It has also
(Colvin et al. 2001). These data indicate that been shown that the insulin receptor tyro-
Fgf9 is necessary for the maintenance of sine kinase family, comprising Ir, Igf1r, and
Sox9 expression and promotes testis differ- Irr, is required for the appearance of male
entiation in vivo (Schmahl et al. 2004; Kim gonads and thus for male sexual differentia-
et al. 2006). Sox9 initiates Fgf9 transcription, tion. XY mice that are mutant for all three
and Fgf9 maintains Sox9 expression and receptors develop ovaries and show a female

Sex Determination in Domestic Animals 371

phenotype. Reduced expression of both Sry cells induce migration of cells from the
and the early testis-specific marker Sox9 mesonephros into the gonad. The migrating
indicated that the insulin signaling pathway cells contribute to precursors of the peritu-
is required for male sex determination (Nef bular myoid and vascular cell lineages
et al. 2003). (Martineau et al. 1997; Capel et al. 1999;
Tilmann and Capel 1999). Differentiation of
Based on the established role of the peritubular myoid cells and the consequent
platelet-derived growth factor (PDGF) family formation of testis cords are regulated by
of ligands and receptors in cell migration, Desert hedgehog (Dhh), a signaling protein
proliferation, and differentiation in various produced by Sertoli cells (Clark et al. 2000;
organ systems, the role of PDGF in testis Pierucci-Alves et al. 2001). It has been shown
organogenesis has been investigated. Pdgfr- that Leydig cells, the male steroidogenic
α−/− XY gonads displayed disruptions in cells, differentiate under the action of
the organization of the vasculature and in Dhh and platelet-derived growth factor
the partitioning of interstitial and testis A (PdgfA), two factors produced by Sertoli
cord compartments. Closer examination cells (Yao et al. 2002; Brennan et al. 2003).
revealed severe reductions in characteristic Consequently, Sertoli cells appear as the
XY proliferation, mesonephric cell migra- conductor of testis differentiation.
tion, and fetal Leydig cell differentiation.
This work identified PDGF signaling through All vertebrate males have testes that are
the alpha receptor as an important event similar in anatomy. Despite a variety of sex
downstream of Sry in testis organogenesis chromosome systems, a large number of
and Leydig cell differentiation (Brennan genes acting in the differentiation of testes
et al. 2003). and male genitalia are conserved in verte-
brates. The primary switch controlling sex
Once the fate of supporting cell precursor determination is highly divergent across
is determined by Sry, feedback loops rein- species, but it seems that the pathways
forcing the male pathway are initiated. Sox9 downstream of the switch call on the same
and Fgf9 upregulate each other and generate factors. Nevertheless the combination of
the first cellular events toward Sertoli cell these factors or their spatiotemporal expres-
fate. In addition, extracellular signals work sion can vary between species.
to recruit other cells in the male pathway.
Defects in these signaling loops could 16.2.3 The ovarian pathway
explain disorders in sexual development
such as ovotestis formation and ambiguous In contrast to the male, the molecular bases
genitalia. of mammalian female sex determination are
poorly understood. Indeed female sexual
The critical event in testis organogenesis development was considered for a long time
is the specification of somatic cell lineages as a passive process due to the fact that
including Sertoli cells, peritubular myoid female external genitalia can be established
cells, and Leydig cells. Specification of these in the absence of a gonad whereas two active
lineages is crucial for the establishment of factors (testosterone and AMH) are needed
testis morphology and the production of hor- to promote male sexual development (Jost
mones. Autonomous expression of Sry in 1947; Josso et al. 1993). In the last decade,
somatic cells and production of extracellular three factors have been isolated having
factors in the XY gonad lead to differentia-
tion of Sertoli cells. Differentiating gonadal

372 Genomics and Reproductive Biotechnology

essential roles in ovary determination: premature ovarian failure with only partial
WNT4, FOXL2, and RSPO1. secondary sex reversal (Schmidt et al. 2004;
Uda et al. 2004; Ottolenghi et al. 2005). In
Wnt4 is expressed in the bi-potential addition, forced expression of Foxl2 impairs
gonad of both sexes and is then downregu- testis tubule differentiation in XY trans-
lated in the testis and upregulated in the genic mice (Ottolenghi et al. 2007). This
ovary at 11.5 dpc (Vainio et al. 1999; Yao result is consistent with an anti-testis role
et al. 2004b). Several Wnt receptors are of Foxl2.
expressed in somatic cells of the gonads
such as Fzd6. They might mediate auto- Although Wnt4 and Foxl2 are indepen-
crine/paracrine signaling for Wnt4 in cells dently expressed, they show complementary
that are engaged in sex determination. The phenotypes in ovary morphogenesis, with
inactivation of Wnt4 leads to incomplete sex Wnt4 being required in early stroma differ-
reversal with early production of testoster- entiation and oocyte survival and FoxL2
one and male internal genitalia in XX mice being involved in supporting cell lineage
(Vainio et al. 1999; Chassot et al. 2008). differentiation. The Wnt4−/−/Foxl2−/− double
Germ cells started oogenesis before degener- knockout ovaries produce testis-like tubules
ating, and somatic supporting cells acquired and spermatogonia (Ottolenghi et al.
partial testis-like features lately (Vainio 2007). This demonstrates that female sex-
et al. 1999; Yao et al. 2004b)s. In the absence determining genes are required to suppress
of Wnt4, Fgf9 and Sox9 expression are tran- an alternative male fate in the ovary.
siently upregulated in the XX gonad. This
suggests that Wnt4 normally represses the Recently, mutations in the R-Spondin 1
male pathway in female gonad and that addi- (RSPO1) gene have been identified in human
tional factors are needed to reinforce the XX patients with testis development (Parma
female pathway. et al. 2006). This is the first human muta-
tion that results in complete female-to-male
Based on genetic analysis of natural muta- sex reversal. RSPO1 has also been shown to
tions in goats (detailed below) or XX male activate the canonical β-catenin signaling
human patients, two other genes have been pathway, which raises the possibility that
proposed as candidate female sex-determin- Wnt4 and Rspo1 act cooperatively to block
ing genes: FoxL2 and RSPO1 (Pailhoux et al. the male pathway in XX gonads (Kim et al.
2001a; Parma et al. 2006). FoxL2 expression 2006, 2008; Chassot et al. 2008). Rspo1
is uniquely female, undetectable in XY knockout mice show masculinized gonads.
gonads of all tested species (Cocquet et al. Molecular analyses demonstrate an absence
2002; Pisarska et al. 2004; Uda et al. 2004). of female-specific activation of Wnt4 and as
In mice, it is activated at 12.5 dpc in the fetal a consequence XY-like vascularization and
ovary and increases in level steadily until steroidogenesis. Moreover, germ cells of XX
early postnatal life, with a maximum of Rspo1−/− knockout embryos show changes in
expression in supporting cells of primordial cellular adhesions and a failure to enter XX
follicles. FOXL2 has also been identified as specific meiosis (Chassot et al. 2008). Sex
the gene mutated in human patients with cords develop around birth, when Sox9
a syndromic form of premature ovarian becomes strongly activated. These experi-
failure called BPES (Crisponi et al. 2001). ments demonstrate a balance between Sox9
Experimental ablation of Foxl2 in mice gives and β-catenin activation to determine the

Sex Determination in Domestic Animals 373

fate of the gonad, with Rspo1 acting as a birth (McLaren 1988). The timing of germ
crucial regulator of the canonical β-catenin cell entry into meiosis appears to be based
signaling required for female development. on an intrinsic clock. By generating XX/XY
recombinant aggregates in culture, it has
Parallel to gene inactivation, multiple been shown that the physical presence of
types of differential transcriptome analyses germ cells inhibits initiation of the testis
have been used to identify genes involved in pathway. The developmental stage when
testicular and ovarian differentiation, includ- germ cells from XX gonads inhibit the male
ing cDNA microarray (Grimmond et al. 2000; pathway is temporally correlated with the
Boyer et al. 2004; Nef et al. 2005; Small et al. time needed for germ cells to spontaneously
2005; Olesen et al. 2007), differential display enter meiosis (Yao et al. 2003). Thus, it has
(Nordqvist 1995; Nordqvist and Töhönen been proposed that once germ cells commit
1997; Töhönen et al. 1998), and representa- to meiosis, they reinforce ovarian fate by
tional difference analysis (Perera et al. 2001; antagonizing the testis pathway.
Adams and McLaren 2002). A large amount
of expressional data has been obtained; The development of germ cells in concor-
however, assigning roles for genes in particu- dance with sexual fate of the somatic cells
lar morphological pathways has been a rate- in the gonad seems important not only for
limiting difficulty. The quantity of the data fertility but also for contributing to the fate
obtained likely reflects the complexity of the of the gonad and for participating in the
rapid and overlapping changes that occur maintenance of a testis or an ovary.
during testis determination and differentia-
tion. It also shows clearly that initiation and 16.2.5 The critical balance
maintenance of ovarian pathway involves
the active regulation of many genes and is not A model (Figure 16.2) has been proposed to
a passive/default developmental process. explain the SRY-negative XX female-to-male
sex reversal existence in which SRY could
16.2.4 Sexual dimorphism of germ cells repress a repressor of male development,
called “Z.” Based on this model, mutations
The developmental fate of primordial germ in “Z” could lead to derepression of the male
cells in the mammalian gonad depends on pathway in XX gonads (McElreavey et al.
their environment. In the XY gonad, Sry 1993). The sum of the current studies sug-
induces a cascade of molecular and cellular gests that multiple redundant anti-testis
events leading to the organization of testis activities (“Z factors”) are deployed in fetal
cords. Germ cells are sequestered inside ovaries. It seems likely that activation of the
testis cords by 12.5 dpc where they arrest in Wnt4/Rspo1 and FoxL2 pathways antago-
mitosis. In contrast to male gonad, germ nizes the establishment of Sox9 in support-
cells are crucial for the formation and main- ing cell precursors. This finding leads to a
tenance of ovarian structures. In the absence new model of sex determination in which
of germ cells, ovarian follicles do not assem- the fate of somatic cells in the gonad depends
ble, and when germ cells are lost, ovarian on the predominance of Sox9 versus Wnt4/
follicles rapidly degenerate (McLaren 1988). Rspo1 and FoxL2 downstream signals (Kim
By 13.5 dpc, germ cells in the XX gonad enter et al. 2006). In mammals, SRY normally acts
meiosis and they arrest in prophase I by as the testis determinant by promoting

374 Genomics and Reproductive Biotechnology

Figure 16.2 Schematic representation of genes involved in gonad differentiation in mammals. The upper
numbers indicate the developmental stages in goat (bold) and mouse (italic). Sry and Foxl2 in lower cases
present when these genes are expressed in mouse, time points that seem to be delayed compared with
the goat ones.

SOX9 expression. However, this outcome 16.3 Sex differentiation in
can also be promoted by loss of RSPO1 or domestic mammals
FOXL2. Within the bi-potential gonad, the
somatic cells seem to be highly plastic and Since the discovery of SRY in 1990, much
can differentiate as cells of the ovary or cells progress has been made in the understanding
of the testis during the window of fetal sex of sex differentiation in mammals, espe-
determination. There is also evidence that cially by studying numerous naturally
XX cells can trans-differentiate to male cell occurring sex-reversed mutants (mainly in
fate under certain conditions in adult life. human) or produced by specific gene target-
Perhaps the critical balance between these ing (in mouse). According to the current
signaling pathways helps to explain the knowledge on sex differentiation in different
underlying “bi-potential” property of the vertebrate species, some mechanisms appear
gonadal cells and suggests a molecular to be well conserved and some others more
mechanism by which this balance is tipped variable between species. In this section we
in one or another direction to regulate the will highlight, using the goat as a model,
fate of gonadal cells (DiNapoli and Capel two features appearing variable between
2008). mouse and other mammals: (1) the SRY gene

Sex Determination in Domestic Animals 375

and (2) the ovarian differentiating pathway. other than testis determination in non-
Two additional examples will also be pre- rodent species. To partially answer this, we
sented in pig and sheep in order to illustrate showed that the goat SRY gene is able to
differences in sexual differentiation between induce testis differentiation in XX mouse
mammals. despite the poor conservation of SRY
between both species and despite the goat-
16.3.1 SRY conservation across species type expression profile of the transgene
(Pannetier et al. 2006a).
The SRY gene has been equated as the testis-
determinant mainly in human and mouse by Thus, SRY must be expressed at the begin-
mutation analysis and mouse transgenesis ning of XY gonad differentiation in order to
(reviewed in Polanco and Koopman 2007). determine the testis fate of the gonad.
Furthermore, SRY orthologs, located on the However, as this gene is present only in XY
Y chromosome, have been identified for individuals, one can imagine that its strict
numerous mammalian species except in spatiotemporal regulation would not be
monotremes (Wallis et al. 2007) and in some absolutely required, should its “mis-expres-
rare cases of rodents such as voles (Just sion” (non-gonadic or gonadic after sex dif-
et al. 1995) and Japanese rats (Soullier et al. ferentiation) have no detrimental effects.
1998; Sutou et al. 2001). SRY studies in dif- Accordingly, goat SRY was found expressed
ferent species have pointed out an unex- in non-gonadic tissues and in gonads of all
pected feature of poor conservation of this stages in transgenic XX and XY mice and no
master gene at the structural (Tucker and additional phenotype was observed aside
Lundrigan 1993; Whitfield et al. 1993; Payen from the XX sex reversal one (Pannetier
and Cotinot 1994) and expressional levels. et al. 2006a).
In contrast to mice, SRY gonadal expression
persists many days after Sertoli cells dif- 16.3.2 The goat as model for early
ferentiation in pig (Daneau et al. 1996; ovarian differentiation
Parma et al. 1999), sheep (Payen et al. 1996),
dog (Meyers-Wallen 2003), human (Salas- Based on previous isolation of key factors for
Cortés et al. 1999; Hanley et al. 2000), goat testis differentiation by linkage analyses and
(Pannetier et al. 2006a), and tamar (Harry et positional cloning (Gessler et al. 1990;
al. 1995). In tamar, SRY expression was Sinclair et al. 1990; Pelletier et al. 1991;
detected in different non-gonadic tissues Foster et al. 1994; Wagner et al. 1994) and
(Harry et al. 1995) and in goat, SRY was according to the Z hypothesis (McElreavey
found expressed from as early as the first et al. 1993), we have attempted to isolate
sign of genital ridges formation to adulthood key ovarian differentiating factors by study-
(Pannetier et al. 2006a). According to these ing XX sex-reversal pathologies. Apart from
results it appears that depending on the con- human, such pathologies have been described
sidered species, SRY expression is more or in at least four mammalian domestic species:
less focused on the crucial period preceding dog, horse, pig, and goat (Pailhoux et al.
Sertoli cell differentiation with a strict regu- 1994; Meyers-Wallen et al. 1999; Buoen
lation in mouse compared with other species. et al. 2000; Pailhoux et al. 2005). In goat, as
It could directly reflect species-specific SRY the polled trait was shown to be associated
regulation or SRY implication in processes with XX sex reversal (Asdell 1944), we used

376 Genomics and Reproductive Biotechnology

families based on heterozygous polled males second point on long-range regulation will
in order to localize and clone the Polled not be treated here.
Intersex Syndrome (PIS) mutation (Vaiman
et al. 1996; Schibler et al. 2000; Pailhoux 16.3.3 FOXL2 seems to be the major
et al. 2001a). This PIS mutation, responsible PIS-regulated gene
for both traits, polled (dominant) and XX sex
reversal (recessive), was shown to be an 11.7- Following the discovery of FOXL2 as the
kb deletion located on goat chromosome 1 gene responsible for Blepharophimosis Ptosis
(Pailhoux et al. 2001a). This 11.7-kb DNA Epicanthus inversus Syndrome (BPES, MIM
fragment encompassed no gene or part of #110100; Crisponi et al. 2001) and its poten-
gene but exerted transcriptional regulatory tial involvement in XX sex reversal in PIS−/−
effects on at least three genes located in the goats, Foxl2 invalidation has been performed
vicinity, PIS regulated transcript number 1 in mouse (Schmidt et al. 2004; Uda et al.
(PISRT1), Promoter FOXL2 inverse comple- 2004). In contrast to goat, XX Foxl2−/− mice
mentary (PFOXic), and Forkhead box L2 developed premature ovarian failure (POF)
(FOXL2). Among these genes, only FOXL2 with a blockage in the first steps of follicle
appears to be a classical one encoding a tran- formation, as observed in XX BPES type 1
scription factor. Indeed, PISRT1 encodes a patients heterozygous for FOXL2 mutations
poly-adenylated mono-exonic 1.5-kb tran- (De Baere et al. 2003). In these XX Foxl2−/−
script devoid of open-reading frame and mice, the sole sign of XX sex reversal appears
PFOXic, a putative but nonconserved only 2 days after birth and consists of an
protein. All these three genes are transcrip- overexpression of Sox9 in the somatic granu-
tionally controlled by the PIS region. Their losa cells (Ottolenghi et al. 2005). One of our
expression depends on the PIS genotype and major goals has thus been to understand the
on the considered tissue. In the female origin of the phenotype discrepancy between
gonads, the three genes are expressed from XX Foxl2−/− mice with POF and XX PIS−/−
the beginning of ovarian formation (34 days goats with sex reversal. A first hypothesis
post-coïtum [dpc] in goat) until adulthood in could be that other PIS-regulated genes could
normal PIS+/+ and PIS+/− animals, but their sustain the sex-reversal phenotype besides
expression is loss in PIS−/− XX gonads. In the FOXL2. Alternatively, the phenotype dis-
horn buds of both sexes, these three genes crepancy could result from species-specific
are not expressed under a PIS+/+ wild-type differences in the role of FOXL2. Following
genotype, but their expression is turn on in the first hypothesis and based on the spatio-
PIS+/− and PIS−/− horn buds. temporal expression profile of PISRT1 that
was shown to be decoupled from that of
Since the characterization of the mutation FOXL2 on the earliest stages of ovarian
in 2001, studies have been developed in development and after birth (Pailhoux et al.
order (1) to understand the role of each PIS- 2001a), PISRT1 was first considered as a
regulated genes and (2) to decipher the potential anti-testis Z gene. However,
molecular mechanism involved in the long- PISRT1 expression ectopically restored in
range regulation of these genes by the 11.7- XX PIS−/− goat gonads had no effect on the
kb PIS region (FOXL2 and PFOXic lie at sex-reversal phenotype (Boulanger et al.
more than 300 kb apart from the PIS region). 2008). According to this result and to the fact
As results have mainly been accumulated on that PFOXic was shown to be involved in
the role of each PIS-regulated gene, the

Sex Determination in Domestic Animals 377

FOXL2 local regulation via a bidirectional goat there is a period of around 20 days
promoter (Pannetier et al. 2005), FOXL2 before germ cell meiosis during which ovar-
remains the sole “acting” gene of the PIS ian-specific genes such as FOXL2 and CYP19
locus and might be responsible for both PIS- are turned on and consequently estrogen
associated phenotypes. FOXL2 invalidation production begins (Mauléon et al. 1977;
in goat is currently in progress in order to Pannetier et al. 2006b). This period seems to
highlight its species-specific function. have no equivalent in mouse ovarian devel-
opment and this difference could account for
16.3.4 FOXL2 as a female the phenotype discrepancy observed.
steroidogenic factor
16.3.5 Early ovarian organization
One important feature of FOXL2 is its ability in goat
to increase CYP19 gene expression at the
transcriptional level (Pannetier et al. 2006b). Following the recent discovery of a new gene
It was demonstrated by our team in goat RSPO1 involved in human XX sex reversal
following the observation that CYP19 gene associated with palmoplantar hyperkera-
expression was drastically decreased in early tosis (PPK; Parma et al. 2006), the four
developing XX PIS−/− gonads, as a conse- R-spondin genes have been studied in goat
quence of the PIS-regulated genes extinction (Kocer et al. 2008). From this study it appears
(Pailhoux et al. 2002). Thereafter, an impres- that FOXL2 and RSPO1 are expressed by
sive work demonstrated the crucial role of two different somatic cell types in early
FOXL2 in the control of female steroidogen- developing ovaries and that FOXL2 extinc-
esis orientation in the nonmammalian fish tion in XX PIS−/− gonads does not primarily
species tilapia (Wang et al. 2007). In this affect RSPO1 expression (Kocer et al. 2008).
study, FOXL2 was shown to act with Ad4BP/ Indeed, at 40 dpc, 5 days after FOXL2 extinc-
SF-1 on different promoters of key steroido- tion, RSPO1 remains expressed in XX PIS−/−
genic gene (including CYP19) in order to gonads even if these gonads begin to express
increase estrogen production. This close rela- SOX9 and show clear histological signs of
tion of FOXL2 with estrogen production masculinization (Pailhoux et al. 2002; Kocer
was also evidenced in other nonmammalian et al. 2008). Conclusively, the two unique
species such as birds, reptiles, and other genes, RSPO1 and FOXL2, characterized
fishes, suggesting an ancient and conserved today for their involvement in mammalian
mechanism (Baron et al. 2004; Govoroun et al. XX sex reversal, act on two different ovarian
2004; Hudson et al. 2005; Liu et al. 2007b; pathways. Efforts must now be developed in
Nakamoto et al. 2007; Rhen et al. 2007). order to decipher putative cross talking
between these two pathways.
The following observations on the pheno-
type discrepancy observed between Foxl2−/− According to immuno-histological studies,
mice and PIS−/− goat may help in its it seems clear now that ovarian differentia-
understanding: (1) In contrast to goat, mouse tion in goat begins at the same time as testis
fetal ovaries are not steroidogenically active differentiation (34–36 dpc). The first sign of
before meiosis; (2) Foxl2/FOXL2 expression ovarian differentiation consists of germ cell
starts around 12.5 dpc (1 day before germ cell location in the cortical area just under the
meiosis) in mouse and at 34 dpc (∼20 days coelomic epithelium. By contrast, germ cells
before meiosis) in goat. Consequently, in occupy all the medullar region of the XY

378 Genomics and Reproductive Biotechnology

Figure 16.3 Schematic representation of a goat Figure 16.4 Schematic representation of a goat
testis at developmental stages 40–45 dpc. Endothelial ovary at developmental stages 40–45 dpc. Endo-
and fibroblastic cells are not represented. thelial and fibroblastic cells are not represented.

testes (Figure 16.3). Moreover, at these early gonadal regionalization—following their
stages of ovarian development, two somatic migration in the female gonads, the germ
cell types exist (Figure 16.4). One type cells localize and stay under the coelomic
expresses RSPO1 and WNT4 and is mainly epithelium—and (2) early steroidogenesis—
located in the cortical area of the gonad in estrogens are produced by the medulla part
close relation with the germ cells. The other of the female gonads before meiosis. Inter-
one expresses FOXL2, produces estrogens, estingly, both features have been described
and is mainly localized in the medulla part of in different nonmammalian vertebrates and
the ovary (Pannetier et al. 2006b; Kocer et al. seem to be part of conserved mechanisms of
2008). Under this scheme it is interesting to gonad development. Under this scheme, the
notice that in XX PIS mutant animals the mouse species might have delayed (regional-
affected somatic cells are those expressing ization) or switched off (early estrogen pro-
FOXL2, consequently those producing ste- duction) these features.
roids. In XX PIS−/− gonads, these steroidogeni-
cally active cells will trans-differentiate into Early ovarian regionalization in cortex
Sertoli-like cells that express SOX9 and AMH containing germ cells and medulla has been
but are devoided of steroidogenic activity. described in reptiles (Pieau et al. 1999, and
references therein) and chicken (Smith and
16.3.6 Conservation of goat ovarian Sinclair 2004, and references therein). In the
differentiation features red-eared slider turtle Trachemys scripta,
before gonadal differentiation, the undiffer-
By contrast to mouse, early developing goat entiated gonads of both sexes contain primi-
ovaries show two main features: (1) early tive sex cords in their medulla part and the
germ cells are located outside these cords

Sex Determination in Domestic Animals 379

just under the coelomic epithelium. At later developmental stages at which the germ
stages, following gonadal differentiation, cells show a differential localization between
germ cells enter the sex cords in the medulla sexes (cortical in female, medullar in male).
of male gonads and stay outside the sex cords If this event occurs as expected during
in the cortex of female gonads. Concomitantly, the undifferentiated period, it will be of
sex cords increase in size and pursue their great interest to decipher the cellular and
development in testes but regress in ovaries molecular mechanisms sustaining this sex-
(Pieau et al. 1999; Yao et al. 2004a). dimorphic feature. Moreover, the role of
estrogens produced before germ cell meiosis
In chicken, early regionalization has been should also be investigated.
described since the undifferentiated stages
(Stahl and Carlon 1973). Then, gonad differen- More generally, different pieces of evi-
tiation depends upon which component, the dence seem to indicate that the major differ-
cortex or the medulla, develops and maintains ences between domestic mammals and the
the germ cells (Smith and Sinclair 2004). mouse model lie on the germinal lineage
Chicken ovary development seems to be development, especially before meiosis.
closely similar to that of goat ovaries. In Future studies will be developed in order
chicken the medulla part of the left gonad to determine the role of germ cells in
expresses FOXL2, CYP19 and produces estro- sex differentiation of domestic mammals,
gens; the cortical area contains the germ cells especially small ruminants such as sheep
that are in close relation with somatic cells and goat.
expressing RSPO1 and WNT4 (Nakabayashi
et al. 1998; Smith et al. 2008). Also interesting 16.3.8 The pig species as
in this species is the asymmetric ovarian a counterexample
development: the left ovary develops but the
right regresses following an absence of cortical Early developing ovaries of pigs also show
development. Importantly, it has been shown compartmentalization with a cortical area
that even if estrogens are produced by both left containing germ cells and a medulla part
and right ovaries, the estrogen receptor is (Pelliniemi 1975, 1985). The difference in
expressed unilaterally in the cortical area this species is the fact that the medulla part
of the left gonad (Nakabayashi et al. 1998). does not express CYP19 and consequently
According to this observation, the role of cannot produce estrogens (Parma et al. 1999;
estrogens in cortical cell proliferation, includ- Pailhoux et al. 2001b). Furthermore, in XY
ing the germinal lineage, appears likely. This pigs, testes expressed CYP19 since the begin-
early ovarian estrogen production might rep- ning of Sertoli cell differentiation and trace
resent the endocrine link between both ovarian amounts of estrone has been detected as
somatic cell types, those expressing FOXL2/ early as when testosterone secretion starts
CYP19 in the medulla and those expressing (Raeside et al. 1993; Parma et al. 1999).
RSPO1/WNT4 in the cortex. According to the results in pig and mouse,
it seems clear that some eutherian mammals
16.3.7 Perspectives on sex could develop ovaries without estrogen
differentiation in goat production before germ cell meiosis.
Consequently for sex differentiation mecha-
One of our future prospects on the goat nisms, some species-specific differences
species will be to precisely determine the exist and mechanisms appearing highly

380 Genomics and Reproductive Biotechnology

conserved should be reconsidered in a present at the primary follicle stage for fol-
given species of interest in order to avoid liculogenesis to proceed normally because
misunderstanding. mutations in the BMP-15 gene in ewes and
women cause the arrest of primary follicle
16.3.9 Mono-ovulatory/poly-ovulatory growth (Galloway et al. 2000; Otsuka et al.
folliculogenesis 2000; McNatty et al. 2003; Hanrahan et al.
2004). Whereas ewes homozygous for BMP-
Folliculogenesis is the development of the 15 mutations are infertile, heterozygous
follicle from the primordial stage through a ewes exhibit an increased ovulation quota
series of morphologically defined stages: (Galloway et al. 2000; McNatty et al. 2003;
primary, secondary, antral, and subsequently Hanrahan et al. 2004). It has been hypothe-
culminating in the Graafian or preovulatory sized that the poly-ovulatory nature of mice
mature follicle. The development of ovarian might be associated with the lack (or very
follicle has been differentiated as a two- low level) of functional Bmp-15 mature
phase process: the initial recruitment of the protein during folliculogenesis (Moore et al.
follicle from the primordial pool to pre-antral 2004). However, mice overexpressing Bmp-
follicles and the cyclic recruitment of the 15 suffer from an early onset of acyclicity.
growing follicles, involving gonadotropin- This indicates that the lack of Bmp-15 in
dependent stages of rapid growth from pre- wild type mice during early folliculogenesis
antral to mature Graafian follicles. is important in restraining follicle develop-
ment to prevent a premature decline in the
On the basis of gene-targeting studies, ovarian follicle pool (McMahon et al. 2008).
several factors have been shown to play an
important role in the transition from resting This example of phenotype difference in
primordial follicles to the growing phase mice and ewes lacking BMP-15 illustrates
(Kuroda et al. 1988; Huang et al. 1993; multiple differences existing in gonadal
Carabatsos et al. 1998; Elvin et al. 1999). development within mammals. It thus rein-
Among those are GDF9 and BMP-15, two forces the idea that knockout studies in
oocyte-secreted factors. Both BMP-15 and mice could not reflect all human mutation
GDF9 are known to be important determi- phenotypes and that domestic animals
nants of ovulation quota and litter size, might represent alternative pertinent models
whereas homozygous mutations lead to for understanding ovarian function.
infertility with an arrest at the primary stage
of folliculogenesis (Galloway et al. 2000; 16.4 Sex determination in
McNatty et al. 2003; Hanrahan et al. 2004). nonmammal domestic species

The first indication that BMP-15 may In nonmammal vertebrate and in mono-
have distinct functions in mono-ovulatory tremes, SRY orthologs have not been
versus poly-ovulatory species came with the detected and sex-determining signals seem
development of the Bmp-15 null mouse. to be different from that of therian mammals
Unlike the Gdf9 null mice, mice lacking and seem to be variable between species.
Bmp-15 exhibit normal folliculogenesis but By contrast, sex-differentiating key genes
are sub-fertile due to defects in ovulation such as SOX9 for the male pathway and
and early embryonic development (Yan et al. FOXL2 for the female one were found highly
2001). In mono-ovulatory sheep and human
species, bioactive mature BMP-15 must be

Sex Determination in Domestic Animals 381

conserved in all studied vertebrates, from homology with the ancestral therian X chro-
fishes, batrachians, reptiles, and birds to mosome but display strong homology with
mammals (Kent et al. 1996; Morais da Silva the bird ZZ/ZW system (Veyrunes et al.
et al. 1996; Western et al. 1999; Choudhary 2008).
et al. 2000; Takase et al. 2000; Vaillant et al.
2001; Valleley et al. 2001; Pask et al. 2002; In chicken, there is presently no clear evi-
Loffler et al. 2003; Zhou et al. 2003; Baron dence in favor of a male Z dosage effect or a
et al. 2004; Govoroun et al. 2004; Baron female W dominant effect as a primary sex-
et al. 2005; Rodríguez-Marí et al. 2005; determining signal. Studies of ZZW triploid
Nakamoto et al. 2006; Takada et al. 2006; animals suggest that the W chromosome
Liu et al. 2007a; Rhen et al. 2007; Wotton carries a female determinant that can be
et al. 2007; Alam et al. 2008; Ijiri et al. 2008). antagonized by the dosage of a Z-linked
Moreover, for SOX9, a Drosophila equiva- male-determinant (Smith and Sinclair 2004,
lent (Sox100B) has been shown to be involved and references therein). Importantly, the
in the determination of the male-specific Z-linked gene DMRT1 supports the Z-dosage
somatic gonadal precursors, supporting a model carrying a testis-determinant gene.
throughout conservation of this gene for Indeed, orthologs of DMRT1 have been
testis differentiation (DeFalco et al. 2003). shown to be involved in different aspects of
sexual differentiation, from invertebrates
Conclusively, upregulation of SOX9 and (Drosophila and Caenorhabditis) to human
downregulation of female genes such as (Raymond et al. 1998). In fishes, reptiles,
FOXL2 and RSPO1 are well-conserved pre- birds, and mammals, DMRT1 appears to be
requisites for testis differentiation. By con- involved in testes development, at different
trast, the ways by which each species levels depending on the considered species
controls these genes seem more species- (reviewed in Ferguson-Smith 2007). In the
specific (Wilkins 1995). medaka fish Oryzias latipes, a DMRT1
ortholog called DMRT1bY/DMY has been
In chicken, sex is genetically determined described on the male Y chromosome and is
but, on the opposite, with therian mammals, considered as the testis-determining gene in
female is the heterogametic sex (ZW) while this species (Matsuda et al. 2002; Nanda et
the male is homogametic (ZZ). The avian al. 2002). Indeed DMRT1bY is expressed
sex chromosome (ZW) has been shown to be exclusively in testis and mutations of this
completely different from the mammalian gene cause sex reversal (Kondo et al. 2006).
XY system. Indeed, both systems evolve
from different ancestral autosomes and do In addition to the Z-dosage hypothesis
not have any gene in common (Graves and with DMRT1 as a strong putative male-
Shetty 2001). Interestingly is the situation determining gene in chicken, some W
in monotremes with the platypus Ornitho- female-specific genes strengthen the female
rynchus anatinus as example. It possesses a W-dominant model. The small W chromo-
complex male heterogametic system with some (like the Y chromosome in mammals)
five male-specific Y chromosomes and five has degenerated during evolution, suggest-
X chromosomes; the female harbors two ing that it carried a female sex-determining
copies of the five X (Rens et al. 2004; gene (reviewed in Stiglec et al. 2007). The
McMillan et al. 2007). It has recently been chicken W chromosome encompasses few
shown that in contrast with some previous genes (∼30–40) and many of them have
reports, platypus sex chromosomes share no homologs on the Z chromosome (reviewed


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