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Seminal Plasma Proteins: Functions and Interaction With Protective Agents During Semen Preservation
Seminal Plasma Proteins: Functions and Interaction With Protective Agents During Semen Preservation
Seminal Plasma Proteins: Functions and Interaction With Protective Agents During Semen Preservation
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Introduction
Seminal plasma is a complex mixture of secretions from the testes, epididymides and accessory
sex glands (seminal vesicles, ampulla, prostate, bulbourethral glands). Despite its physiologi-
cal significance as the carrier of spermatozoa to the female reproductive tract, the biochemical
characteristics and physiological roles of the various seminal plasma proteins are poorly under-
stood. While some seminal plasma proteins appear to be beneficial to sperm functions such as
motility, capacitation, acrosome reaction, viability and/or sperm-egg interaction, others seem
to be detrimental, particularly to sperm preservation including cryopreservation (for a review
see Manjunath and Therien, 2002; Bergeron and Manjunath, 2006). In this review, we describe
BSP proteins
In bovine, the major protein fraction of seminal plasma (30-50 mg/ml) constitutes a family of
closely related proteins named BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (BSP proteins)
(Manjunath, 1984; Manjunath and Sairam, 1987. These proteins are the secretory products of
the seminal vesicles. The biochemical properties and structure of these proteins have been
studied in detail (Manjunath, 1984; Manjunath and Sairam, 1987; Manjunath et al., 1987;
Seidah, Manjunath, Rochemont, Sairam and Chretien, 1987; Manjunath, Baillargeon, Marcel,
Seidah, Chretien and Chapdelaine, 1988; Leblond, Desnoyers and Manjunath, 1993; Desnoyers,
Therien and Manjunath, 1994). They are small acidic proteins with apparent molecular weights
of 15-16 kDa (BSP-A1, -A2 and -A3) and 28-30 kDa (BSP-30-kDa), which are all glycoproteins
with the exception of BSP-A3. BSP-A1 and BSP-A2 (also called PDC-109) originate from the
same gene but differ in the degree of glycosylation (glycoforms). All of these proteins contain
two homologous type II domains similar to those found in the gelatin-binding domains of
fibronectin (called Fn2 domains) and an amino-terminal extension that is variable between
each protein (Fig. 1) (reviewed in Manjunath and Therien, 2002; Fan, Lefebvre and Manjunath,
2006). The crystal structure of BSP-A1/-A2 (PDC-109) has been determined (Wah, Fernandez-
Tornero, Sanz, Romero and Calvete, 2002), allowing us to create homology models of BSP-A3
and BSP-30kDa (Fig. 2). Homologues of BSP proteins have been isolated and characterised in
seminal plasma or seminal vesicle secretions from numerous other mammals, namely boar and
stallion (Menard, Nauc, Lazure, Vaillancourt and Manjunath, 2003; Calvete, Mann, Schafer,
Sanz, Reinert, Nessau, Raida and Topfer-Petersen, 1995; Calvete, Raida, Gentzel, Urbanke,
Sanz and Topfer-Petersen, 1997; Sanz, Calvete, Mann, Gabius and Topfer-Petersen, 1993),
goat (Villemure, Lazure and Manjunath, 2003), bison (Boisvert, Bergeron, Lazure and Manjunath,
2004), ram (Bergeron, Villemure, Lazure and Manjunath, 2005) and water buffalo (unpub-
lished). All of these species contain 3-4 molecular forms, except boar, which contains one form
(Table 1). BSP-like antigens are also present in rat, mouse and hamster seminal vesicle fluid as
well as in human seminal plasma (Leblond et al., 1993). The concentration of BSP homologues
in seminal plasma varies from species to species (Table 2). At the genetic level, the cloning
and sequencing of the cDNA corresponding to BSP proteins has been reported (Salois, Menard,
Paquette and Manjunath, 1999; Kemme and Scheit, 1988). In a recent study, three new BSP
related genes (BSPH4, BSPH5 and BSPH6) were discovered in bovine (Fan et al., 2006). In
addition, an exhaustive genomic search indicated the presence of BSP-related DNA sequences
in human, rat, mouse, chimpanzee, dog, horse and rabbit (Fan et al., 2006). Molecular evolu-
tionary analysis revealed that all BSP-related sequences could be grouped into three sub-fami-
lies: BSPH4, which includes BSP-A1/-A2, BSP-A3 and BSP-30 kDa and is expressed in seminal
vesicles, BSPH5 and BSPH6, which are expressed in the epididymis and testis (Fig. 3). It is
becoming increasingly evident that BSP proteins and their homologous proteins represent a
new emerging superfamily in mammals.
BSP-A1/-A2 proteins
Q DE
D G
1 V R N R K H
S V Y
F F I Y G G K
P P F
T F K
D F Y 80
E V F L 40 T C C V
C P S M W M E
P W S G H V K W
E C A A C S T 109
T E C 60 Y Q Y G C C
Q R D S
D S K L I K T Y
D E L W K
G 20 D R S
P A D Y V G P N A W
P Y D K D R
A EL 100
BSP-A3 protein
E D
S N
L
Q V
I
Q L G N K K
D 1 P F I Y Y 40 80
P F P F I Y E G K
K F F S
V D V Y
E F L S C C N M F
K C L T W Y D
K W G H L K T T
K C C T A C
N S Y K N S 100 S I C C 115
D D Y
D K L G I K Y
P L W K
A K D R S S W
A D Y T G
T N Y D E D G V
20 S
G A E 60
BSP-30-kDa protein
80
G K K
Y K
F T Y 120
Y I Y R K K S
F
P
M F P F
A V W F Y
L L V S C C R
C T
W R 140 F E
A W N K R E
S
C C T P C S
P F E E S H C C 158
S K R D V R T Y
G L
E L W K
D N T W
F T E Q G S A
100 Y N Y D R D K
V
A
N 60 40
L D
N A K S L L S P F P K D D Y P M E E Q P Y I T R
T P P G M A D P
K P E L P T E T Y D L P P E I Y T T T F L
S 20
G
P
D P I D G
1
Figure 1. Primary structure of BSP proteins. The carbohydrate moieties are indicated by
the symbol (•). Modified from Manjunath and Therien (2002).
Figure 2. Ribbon representation of homology models of BSP proteins. The potential ligand,
phosphorylcholine, is represented in ball and stick structure. The figure was prepared with
Swiss-PbdViewer 3.7b2 (Guex and Peitsch, 1997) and MOLMOL 2K.2 (Koradi, Billeter
and Wuthrich, 1996). The BSP-A1/-A2 (PDC-109) template is from the Research
Collaboratory for Structural Bioinformatics Protein Data Bank (1H8P), in which chains A
and B correspond to 2 monomers, because this protein forms dimers when associated
with phosphorylcholine.
Table 1. BSP protein homologues isolated from seminal plasma of various species.
100/100/99 pBSPH1
hBSPH1
99/84/89
81/ 73/ 100 mBSPH1 BSPH5
rBSPH1
64/ 53/-
BSPH5
BSPH6
0.2
Figure 3. Neighbour-joining trees of two Fn2 domains of BSP-related proteins. BSP pro-
teins and their relatives are separated into three subfamilies: BSPH4, BSPH5 and BSPH6.
Bootstrap values from 1000 replicates for neighbour-joining (1st number), 1000 replicates
for maximum parsimony (2nd number) and 100 replicates for maximum likelihood (3rd
number) are indicated at the nodes and were used to assess the robustness of the trees.
Only bootstrap values larger than 50% are shown and the bold numbers indicate the three
main branches. Genetic distance is indicated as the number of substitutions per amino
acid site. BSP protein, bovine seminal plasma protein; BSPH, BSP protein homologue;
SP1/2, equine seminal plasma protein 1/2; pB1, boar seminal plasma protein pB1; CDK105,
canine gene; EP52c1, epididymal protein 52 from rabbit. The small letter preceding each
abbreviation represents the name of the species (h, Homo sapiens; p, Pan troglodytes; m,
Mus musculus; r, Rattus norvegicus) from which the orthologous protein originates. Modi-
fied from Fan et al. (2006).
the total phospholipids of the sperm membrane (Parks, Arion and Foote, 1987). Further studies
indicated that BSP proteins bind to high-density lipoproteins (HDL) (Manjunath et al., 1988;
Manjunath, Marcel, Uma, Seidah, Chretien and Chapdelaine, 1989; Therien, Soubeyrand and
Manjunath, 1997; Therien, Bousquet and Manjunath, 2001), and glycosaminoglycans (GAGs)
such as heparin (Chandonnet, Roberts, Chapdelaine and Manjunath, 1990), heparan sulfate and
chondroitin sulfate (Therien, Bergeron, Bousquet and Manjunath, 2005). HDL and GAGs are
components of follicular and oviductal fluids and are physiological inducers of sperm capacita-
tion. Furthermore, BSP proteins potentiate capacitation induced by follicular fluid-HDL and -
GAGs (Therien et al., 1997, Therien et al., 2001). It was shown that epididymal sperm exposed
to BSP proteins followed by incubation with GAGs or HDL exhibit a higher incidence of
capacitation compared to GAGs or HDL alone. In addition, BSP proteins mediate cholesterol
efflux from sperm, which is also accompanied by choline phospholipid efflux (Therien, Moreau
and Manjunath, 1998; Therien, Moreau and Manjunath, 1999). The removal of cholesterol from
the sperm membrane is deemed to be essential during capacitation.
The studies described above led us to propose that BSP proteins participate in the sperm
membrane lipid modification events that occur during capacitation in the female reproductive
tract (Manjunath and Therien, 2002; Therien et al., 2001, Therien et al., 2005). Two distinct
mechanisms, both mediated by BSP proteins but one induced by HDL and the other by GAGs,
were deduced (Fig. 4). In brief, at ejaculation, sperm are mixed with accessory gland secretions
containing BSP proteins, contributed by the seminal vesicles. During this brief exposure, BSP
proteins remove some cholesterol (first cholesterol efflux) accompanied by the release of some
phospholipids. This lipid efflux may slightly destabilise the sperm membrane (priming). At the
same time, BSP proteins coat the sperm surface via their interaction with the membrane cho-
line phospholipids. Within the next 10-20 min, sperm travel through the cervical mucus into
the uterus, leaving behind most of the seminal plasma. During this time, bound BSP proteins
may prevent the free movement of phospholipids, thereby stabilising the sperm membrane
(arrested state). BSP protein-coated sperm then travel through the female genital tract and reach
the oviduct, the site of fertilisation, where they encounter HDL and/or GAGs. With HDL-
induced capacitation, oviductal- or follicular fluid-HDL remove BSP proteins from the sperm
membrane. As a result, sperm membrane lipids are free to move. In addition, HDL may induce
a second efflux of cholesterol. Since cholesterol is recognized to have a stabilising effect on
membranes (Yeagle, 1985), its efflux would be expected to provoke further reorganization or
destabilization of the membrane and trigger some unknown signal transduction pathways. Such
events could regulate the surface expression of sperm zona pellucida receptors, and adhesion
to the zona pellucida could trigger the acrosome reaction. With GAG-induced capacitation,
sperm-bound BSP proteins may play a role, via their interaction with GAGs, in the uptake of
Ca2+, intracellular alkalinization and protein tyrosine phosphorylation (Therien et al., 2001;
Therien et al., 2005). The role of tyrosine phosphorylation in capacitation in various species
(human, bovine, murine, porcine) has been investigated in detail (Galantino-Homer, Visconti
and Kopf, 1997; Leclerc, de Lamirande and Gagnon, 1996; Aitken, Harkiss, Knox, Paterson and
Irvine, 1998; Tardif, Dube, Chevalier and Bailey, 2001; Osheroff, Visconti, Valenzuela, Travis,
Alvarez and Kopf, 1999; de Lamirande, Leclerc and Gagnon, 1997; Visconti, Westbrook,
Chertihin, Demarco, Sleight and Diekman, 2002; Naz and Rajesh, 2004; Baldi, Luconi,
Bonaccorsi, Muratori and Forti, 2000). HDL-induced capacitation, in contrast to GAG-induced
capacitation, is not associated with an increase in tyrosine phosphorylation (Lane, Therien,
Moreau and Manjunath, 1999).
The first functional role identified for BSP proteins is that they promote the capacitation of
bull sperm, which involves a complex series of events and is schematised in Figure 4 (Therien,
Bleau and Manjunath, 1995; Therien et al., 1997, 1998a, 2005; Manjunath and Therien, 2002).
Recently, two additional functions were proposed for BSP proteins. The group of Yu et al. (Yu,
Zhao, Zhao, Chen, Liu, Zhang, Fu, Zong, Yu and Guan, 2003) demonstrated that, in vitro, the
activity of protein kinase C (PKC) and of tyrosine protein kinase (TPK) are inhibited by PDC-
109 (BSP-A1/-A2) and proposed that the inhibition of PKC may serve to prevent the premature
acrosome reaction of sperm in the female reproductive tract. The Suarez group demonstrated
that BSP proteins mediate the binding of sperm to the oviductal epithelium and proposed that
they are involved in prolonging sperm survival during storage and in the maintaining sperm
motility in the oviduct (Ignotz, Lo, Perez, Gwathmey and Suarez, 2001; Gwathmey, Ignotz
and Suarez, 2003; Gwathmey, Ignotz, Mueller, Manjunath and Suarez, 2006). Therefore, BSP
proteins play multiple roles in sperm functions and have a beneficial effect on sperm fertility.
Ejaculation
First cholesterol efflux
BSP proteins
Heparin-binding protein
Sperm membrane
cholesterol
Ejaculated sperma tozoa phospholipid
AC
A
PK-A
CAPACITATION
ram also bind to LDL (Menard et al., 2003; Villemure et al., 2003; Boisvert et al., 2004;
Bergeron et al., 2005), the mechanism of sperm protection by egg yolk may be the same for all
mammals.
Conclusions
Elucidating the mechanisms occurring during fertilisation is fundamental to resolve causes of
infertility, develop contraceptives and sperm storage media, and optimise in vitro fertilisation.
One of the key events regulating fertility is sperm capacitation, which is a complex and multi-
step phenomenon. It occurs in the female reproductive tract and requires factors present in the
oviduct. Despite more than 50 years of research and efforts by hundreds of scientists, the
molecular events involved in sperm capacitation are still being investigated. Our laboratory
has identified a family of lipid-binding proteins of seminal plasma (BSP) proteins that play a
key role in this complex process. Based on our studies aimed at investigating the effects of
purified BSP proteins, isolated follicular fluid-HDL and -GAGs on epididymal sperm, we have
proposed the sequence of events shown in Figure 4. This investigation also unravelled another
important mechanism that is sperm protection by egg yolk during storage (Fig. 5). While a brief
exposure of sperm to BSP proteins (as in natural mating) is beneficial to sperm (promotes
capacitation), a continuous exposure to BSP proteins (as in the case of semen collected for
artificial insemination) is detrimental. However, the detrimental effect of seminal plasma BSP
proteins is abolished or minimised when they associate with LDL, the protective agent present
in egg yolk extender. This discovery may lead to improvements in the existing protocols for
semen preservation and in developing novel protocols for the preservation of semen from
domestic, farm, zoo and endangered animals as well as from human. The existence of benefi-
cial and detrimental factors for sperm in seminal plasma has been known for many years.
However, the fact that the same family of proteins acts like a double edged-sword that is both
beneficial and detrimental to sperm depending on the context (capacitation or preservation) is
surprising. We hope that this review will create renewed interest in the significance of seminal
plasma proteins as well as their effect on sperm storage.
Acknowledgements
This review, which is based on the plenary talk by PM to the 10th International Symposium on
Spermatology, was possible due to a “IHDCYH Lectureship in Child and Reproductive Health”
awarded by the Institute of Human Development, Child and Youth Health of the Canadian
Institutes of Health Research. This work was supported by grants from the Canadian Institutes of
Health Research, Natural Sciences and Engineering Research Council of Canada, Lalor Founda-
tion, World Health Organization, Semex Alliance, Cattle Breeding Research Council and
L’Alliance Boviteq (Canada). We appreciate the collaboration of Mr Yves Brindle, Centre
d’Insémination Artificielle du Québec. We thank all the graduate students, postdoctoral fel-
lows and research assistants who contributed to the progress of this research since the project
was initiated in 1984.
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