BIOLOGY OF REPRODUCTION 49, 1202-1207 (1993)
Fertility-Associated Proteins in Holstein Bull Seminal Plasma'
GARYJ. KILLIAN, 2 DAVID A. CHAPMAN, and LEE A. ROGOWSKI
Dairy Breeding Research Center, Department of Dairy and Animal Science, Pennsylvania State University
University Park, Pennsylvania 16802
ABSTRACT
This study was undertaken to determine whether bovine seminal plasma contained protein markers associated with bull
fertility, and whether these markers were of value in predicting bull fertility. Seminal plasma was obtained from 35 Holstein bulls
of known fertility. Two-dimensional PAGE of seminal plasma samples indicated that two proteins (26 kDa, pi 6.2; 55 kDa, pI 4.5)
predominated in higher-fertility bulls, and two proteins (16 kDa, pi 4.1; 16 kDa, pi 6.7) predominated in lower-fertility bulls.
Densitometry data for these proteins in individual samples were combined for bulls grouped by fertility level. Average density
of the 26-kDa protein was significantly greater n seminal plasma of high-fertility bulls, and high-fertility seminal plasma also
contained more of the 55-kDa protein than that of average- and below average-fertility bulls. Below average- and low-fertility
bull seminal plasma had significantly more of both 16-kDa proteins than that of average- and high-fertility bulls. A regression
model was developed to predict bull fertility using the four fertility-associated protein densities. A plot of actual bull fertility
versus that calculated by this model was linear and positively correlated (r = 0.89). These findings indicate that bull seminal
plasma contains fertility-associated proteins that are predictive of bull fertility.
INTRODUCTION
Reports for several species suggest that seminal plasma
contains factors that may influence male fertility. These
studies are. generally based on comparisons of seminal
plasma composition between males of differing fertility [16] or the isolation of factors from seminal plasma that facilitate or inhibit sperm capacitation, fertilization, or related
events [7-14].
We developed an interest in this topic after surveying
the fertility of reproductively normal dairy bulls primarily
housed at artificial breeding cooperatives in Pennsylvania
and New York. In vivo fertility data summarized for 131
bulls with normal semen parameters, as assessed by laboratory evaluation, indicated that the fertility of the bull population followed a distribution ranging from 8.5 percentage
points below to 5.4 points above average fertility, designated as zero (Fig. 1). This relatively small range of fertility
differences among bulls in the normal population was distinguishable because fertility data were available for individual bulls based on more than 1000 breedings by artificial insemination. We are not aware of any other species
for which such data are available on individual sires. These
circumstances provide a unique animal model for the study
of factors contributing to the relative fertility of the normal
male.
The objective of the present study was to determine
whether seminal plasma contained protein markers associated with relative fertility in a population of reproductively normal bulls, and whether these markers were of value
Accepted August 6, 1993.
Received June 15, 1993.
'Partial support provided by the Commonwealth of Pennsylvania's Ben Franklin
Partnership Program, Atlantic Breeders Cooperative, Sire Power, Inc., Eastern Artificial Insemination Cooperative and USDA Grant 90-37240-5512.
2
Correspondence. FAX: (814) 863-0833.
in predicting bull fertility. In addition, we compared the
molecular masses and isoelectric focusing points for the
fertility-associated proteins we detected to bovine seminal
plasma proteins characterized by other investigators.
MATERIALS AND METHODS
Samples of seminal plasma were obtained for protein
analyses from 35 mature Holstein bulls of known reproductive history. These bulls were a subset of those represented in Figure 1 and were maintained at Atlantic Breeders Cooperative (14 bulls), Eastern Artificial Insemination
Cooperative (16 bulls), and Sire Power Inc. (5 bulls).
Fertility data on each bull were provided by the respective artificial insemination (Al) centers that housed the bulls
and were based on breeding records for more than 1000
inseminations using frozen semen. Because the method of
preparing fertility data varied slightly among the AI centers,
data for each bull were expressed as a percentage point
deviation from the average fertility for all production bulls
at that AI center sampled during the same period. To maximize the possibility of detecting differences among bulls
associated with fertility, the bulls on which seminal plasma
analyses were conducted were selected to include adequate numbers of bulls in each fertility level separated by
vertical lines in Figure 1. This selection process also was
influenced by the availability of samples from the cooperating AI centers. For the 35 bulls supplying seminal plasma
for analysis, the percentage point deviations ranged from
6.6 to +4.5, with average fertility designated as 0.0.
Bulls providing seminal plasma were subjected to a regular semen collection schedule, dictated by the AI center,
which typically amounted to one to three ejaculates per week.
A single ejaculate was analyzed from each bull without regard to month, season, or order in a sequence of ejaculates.
1202
1203
FERTILITY PROTEINS IN SEMINAL PLASMA
30
V)
-J
-J
ZD
I
I
25
_
20
_
I
66% (i836)
J:
_
m
U0
15
bi
z
10
_
!
*
5% (
·- :i
_
I21% (28)
5
8% (10)
n
A)
0
__
-10
7
I
i
-8
-6
-4
iI:
-2
0
2
__
I - _
mrrYlnrr\mllnr\l
11.IkalduJctduldllUl,
6
8
PERCENTAGE POINT DEVIATION FROM AVERAGE ()
FIG. 1. Plot of number of bulls versus fertility
production dairy bulls, housed at Al centers in the Northeast.
Bull fertilities
are expressed as a percentage point deviation frorn the average fertility of
the bulls at the Al center where the bulls were hou sed. Average fertility for
purposes of this study was designated as equal to
The percentage and
number (in parentheses) of bulls grouped by fertililty0.0.level
are separated by
a vertical dotted line on the plot.
The date a seminal plasma sample was t;aken fell within the
same time interval used to calculate the fertility of the bull.
Within 15 min of ejaculate collectionn, semen was centrifuged (1000 x g, 15 min), and the siupernatant seminal
plasma was transferred to cryovials (#7 2,694,007 Sardstedt,
Princeton, NJ) for storage in liquid nitro gen. Samples were
shipped in liquid nitrogen from the Al center to our laboratory, where they remained in liquid nitrogen storage until
analyzed. After being thawed at ambient temperature, seminal plasma samples were recentrifuged (10 000 x g, 60
min at 5C), assayed for protein concentration [15], divided
into aliquots, and refrozen in liquid nitrogen. Immediately
before electrophoresis was performed, samples were thawed
at ambient temperature.
One-dimensional PAGE of seminal plasma was performed under denaturing conditions by methods previously described [16-18]. Seminal plasma samples were
subjected to two-dimensional PAGE according to the method
of O'Farrell [19] as modified for use in our laboratory [20].
The isoelectric focusing tube gels contained a mixture of
ampholytes consisting of 0.4 ml of pH 3-7 and 0.1 ml of
pH 3-10 (Serva, Heidelberg, Germany) to establish a pH
gradient. Tube gels were prefocused at a constant voltage
of 200 V for 15 min, 300 V for 30 min, and 400 V for 30
min. Samples of seminal plasma consisting of 400-500 Rig
protein in less than 50 pl were solubilized in urea and beta-
. .
rr·ll·trr
IVI,
I
n r·r\nl I rrr\l,·m
-
r\r
~_
II
d IIIIU VOIUIIIt; VI IU
I
t
-
L
-A
U.
IUaCU
onto the gel. Tube gels then were electrofocused for 20 h
at 375 V, followed by 1 h at 800 V. After electrofocused gels
were extruded from the tubes, they were placed on 4%
stacking gels covering the top of 10-17.5% acrylamide gradient slab gels, with 1% molten agarose used to seal them
in position. Molecular mass standards (Sigma Chemical Co.,
St. Louis, MO) were electrophoresed in the second dimension along with the proteins originating from the isoelectric
focusing gel. The standards were myosin (205 kDa), 3-galactosidase (116 kDa), phosphorylase B (97 kDa), BSA (66
kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).
Gels were stained overnight in 0.125% Coomassie Brilliant Blue R-250 dye (Sigma), 50% methanol, and 10% acetic
acid in distilled water. Gels were destained in 25% methanol and 10% acetic acid, and photographed with LPD-4
positive film (Kodak, Rochester, NY). One positive film of
each gel for each bull was scanned with a Bio-Rad model
""
"~""'^"^'"'
'/.'.,U-lli.JtIiIlUttl
4
I
"'^"""
IIkIaU,
i
1
"^~'""""
lIkUtlV
1
III
""""
LI,
"'"
IN
I),
'"'"'
1IIT L;I-
faced with a Dell system 310 computer. These data were
used to prepare a map of the proteins present in each gel
and to objectively estimate the amount of each protein.
The fertility-associated protein densities were adjusted
by subtracting the average background for each gel image
from the protein densities of interest. Densities for each
fertility-associated protein were then compiled, and average ( SE) protein densities were calculated for bulls grouped
by fertility level.
Densities of the four fertility-associated proteins of each
bull also were used as a database to develop a model to
predict bull fertility. Several regression models were evaluated to determine which provided the best empirical prediction of fertility. Evaluations were made on log and square
root transformations of protein densities. The criteria for
comparing models were the calculated R-squared, adjusted
R-squared, C-P, and multicollinearity values [21].
RESULTS
Preliminary studies with two different ejaculates from each
of three bulls indicated similar protein profiles on one- and
two-dimensional electrophoresis gels between ejaculates
from the same bull and virtually identical profiles between
replicate gels of the same ejaculate. Therefore, only one
ejaculate was evaluated from each bull in subsequent studies. Densitometry data of one-dimensional SDS-PAGE of
seminal plasma from bulls of differing fertility did not provide consistent evidence for differences in protein banding
patterns associated with fertility. Although visual inspection
of two-dimensional gels of seminal plasma from the 35 bulls
indicated some variation in protein maps among individuals, four different polypeptides appeared to occur in association with bull fertility level. Two proteins (26 kDa, pI
6.2; 55 kDa, p 4.5) occurred with greater frequency and
density in bulls of higher fertility (Fig. 2, top), and two pro-
1204
KILLIAN ET AL.
FIG. 2. a and b) Representative two-dimensional polyacrylamide gels of seminal plasma proteins from bulls of
higher (top) and lower (bottom) fertility. Locations of fertility-associated proteins are indicated with arrows. (a) 55 kDa,
4.1 pi; (b) 26 kDa, 6.2 pi; (c) 16 kDa, 6.7 pi and (d) 16 kDa, 4.1 pl.
teins (16 kDa, p 4.1; 16 kDa, p 6.7) were more prominent
in bulls of lower fertility (Fig. 2, bottom). On the basis of
these preliminary observations, we subjected all two-dimensional gels to analysis by video densitometry to obtain
quantitative data on the polypeptides of interest. The density and area of the individual polypeptides on a gel were
determined for each bull sample, and these quantitative data
were summarized for bulls combined by fertility level into
1205
FERTILITY PROTEINS IN SEMINAL PLASMA
45
6
40
35
5
>-
z
30
I-
4
z
w
w
z
z
3
7j
E-
20
0
0
a_
25
CL
W
2
15
10
l
0
5
I
/1
LF
BA
k
AF
K\\1
HF
I
VA
IIII
II
LF
BA
AF
n
-\\
LF
HF
FIG. 3. Average densities ( SE) of the high-fertility proteins determined in two-dimensional gels of seminal plasma for bulls grouped by fertility. LF, -5.5, n = 6; BA, -2.8, n = 7; AF, +0.5, n = 11; HF, +2.7, n =
11.
four groups. Seminal plasma from bulls in the high-fertility
group (HF), ranging from 2.0-4.5 percentage points above
average fertility, had significantly more (p < 0.05) of the
26-kDa protein than did seminal plasma from bulls in the
average-fertility (AF; +1.4 to -0.2 percentage points), below-average-fertility (BA; -2.3 to -3.8), and low-fertility (LF;
-4.3 to -6.6) groups (Fig. 3). HF bull seminal plasma also
had more of the 55-kDa protein than seminal plasma of AF
and BA bulls.
Plasma from BA and LF bulls had significantly more (p
< 0.05) of the 16-kDa, pI 4.1 protein than did AF and HF
bull seminal plasma (Fig. 4); HF seminal plasma had significantly more (p < 0.05) of the protein than did AF seminal plasma. LF bull seminal plasma had significantly more
of the 16-kDa, pI 6.7 protein than did AF, BA, and HF plasma,
and BA bull seminal plasma had significantly more (p <
0.05) of the 16-kDa, pI 6.7 protein than did plasma of AF
and HF bulls.
The regression model that provided the best empirical
prediction of fertility based on the four fertility-associated
proteins in seminal plasma was:
Fertility = -2.12 + 3.58- - 0.90\V/ + 1.61-D +
V
0.35VS - 1.69Vi
where fertility represents the predicted percentage point
deviation from the average fertility of bulls at an Al center,
and D, through D4 represent the densities of the fertilityassociated proteins: 26 kDa, pI 6.2; 16 kDa, pI 4.1; 55 kDa,
pI 4.5; 16 kDa, pI 6.7, respectively. The model used square
root transformations of the protein densities. Although the
BA
AF
HF
LF
BA
AF
HF
FIG. 4. Average densities ( SE) of the low-fertility proteins determined
in two-dimensional gels of seminal plasma for bulls grouped by fertility as
described in Figure 3 legend.
main effect of the square root of D4 was not significant, it
was retained in the model because of a significant interaction between the square roots of D, and D4.
Estimated fertility values for each bull were generated
by comparison to a data set that included seminal plasma
6 -
N =
5
0
R = 0.89
z
0
0
4- I
2- I
a
0
0- I
m
E-
0
0
-2 -4
-
U
-6 00
Q
-OI
f[
-8
I
-6
-4
I
I
-2
0
2
4
6
ACTUAL BULL DEVIATION
FIG. 5. Plot of the actual bull fertility (percentage point deviation from
the mean) versus the calculated fertility values for the 35 bulls undergoing
analysis of seminal plasma by two-dimensional PAGE.
1206
KILLIAN ET AL.
fertility protein data from the 34 other bulls in the study.
A plot of actual in vivo fertility versus calculated fertility
(Fig. 5) was linear, and there was a positive correlation (r
= 0.89) indicating that the predictive model was valid over
the range of bull fertilities studied.
Calculated and actual fertility values differed by an average of 1.19
0.15 percentage points over all bulls. Average percentage point differences (mean SE) between the
calculated and actual fertility values among fertility groups
were minimal (HF, 1.30 + 0.34; AF, 1.07
0.29; BA, 0.83
+ 0.18; LF, 1.59 + 0.28).
DISCUSSION
The present study clearly suggests the existence of four
fertility-associated proteins in bovine seminal plasma that
appear to be of value in predicting small differences in relative fertility among bulls. The bulls studied represented a
population of reproductively normal males with normal semen parameters. Because similar numbers of sperm from
each bull were used to inseminate the females, the effect
of sperm numbers on fertility was similar among bulls. Although studies with other species have reported the presence of factors in seminal plasma related to in vivo fertility
[1, 3-5], the males being compared were typically categorized as fertile or infertile, with the infertile males often
oligo- or azoospermic. Because the absence of or reduction
in sperm numbers in the infertile male could contribute to
infertility, the biological significance of differences in seminal plasma composition associated with fertility was unclear.
Antifertility factors from seminal plasma have been described for several species and include decapacitation factors purified to various degrees [22], human antifertility factor 1 (AF1) [10, 13, 23], rabbit acrosome stabilizing factor
(ASF) [9], and bull seminal plasmin (SPLN) [12]. Generally,
these factors are believed to inhibit sperm capacitation, the
acrosome reaction, or acrosomal enzymes and ultimately
to interfere with fertilization.
Recent studies [14, 24] have suggested that heparin-binding proteins in bull seminal plasma are taken up by cauda
epididymidal sperm membranes. Because other studies have
indicated that the ability of sperm to bind heparin and other
glycosaminoglycans is correlated with semen quality and
fertility [6, 25-27], heparin-binding proteins in seminal plasma
may positively influence fertility.
Although the results of the present study demonstrate a
high correlation between the amount of certain seminal
plasma proteins and bull fertility, we do not know whether
this relationship is causal or coincidental. Furthermore, it
is important to note that the differences in amount of fertility-associated proteins we observed in a gel based on
staining intensity may reflect post-translational modifications of proteins rather than more or less of a protein, or
different proteins, per se.
The fertility-associated proteins we detected in bull seminal plasma have biochemical similarities to bull seminal
plasma proteins characterized by other investigators. Miller
et al. [14] described a group of 15-17-kDa seminal plasma
heparin-binding proteins that had pis of 4.1-6.0 and were
associated with the sperm membrane. These proteins are
similar to the two 16-kDa proteins we observed with pis of
4.1 and 6.7. Likewise, the 26-kDa, 6.2 pI fertility-associated
protein we observed in seminal plasma is within the range
of basic heparin-binding proteins Miller et al. [14] described for seminal plasma at 24 kDa.
The biochemical characteristics of a group of acidic bovine seminal plasma proteins at 16 kDa have been studied
in detail [28-31]. Esch et al. [28] named three 16-kDa seminal plasma proteins BSPI, BSPII and BSPIII. These proteins
have amino acid sequences [32] identical to those of seminal plasma proteins BSPA3, BSPA2, and BSPA, respectively,
studied by Manjunath et al. [31]. Furthermore, BSPII and
BSPIII, collectively referred to as PDC-109 [28], have considerable amino acid sequence homology [32] with the major protein (MP) synthesized by the seminal vesicle [29].
Interestingly, BSPA, BSPA2, and BSPA3 apparently bind heparin [33], and PDC-109 (BSPA,/A 2) is exclusively bound by
the sperm midpiece [34], where it inhibits calcium uptake
by bovine sperm [35]. The fact that the 16-kDa acidic BSP
proteins bind heparin and influence calcium uptake by sperm
raises the possibility that they are the 16-kDa heparin-binding proteins described by Miller et al. [14, 36] and associated with sperm fertility.
In addition to the proteins already mentioned, numerous enzymes and protease inhibitors also have been detected in bovine seminal plasma [37]. However, with the
exception of phospholipase A2, these do not have molecular masses and isoelectric points consistent with the fertility-associated proteins we detected in seminal plasma. Purified phospholipase A2 from bovine seminal plasma and
seminal vesicle secretions exists in two forms [38]. One form
has 15- and 16-kDa subunits with a p of 4.7. The other
form has subunits of 16 kDa and 60 kDa with multiple pI
values from 3.75-5.0. The molecular masses and pls of the
subunits of the purified phospholipase [38] and the 16-kDa,
pi 4.1 and 55-kDa, p 4.5 fertility-associated proteins we
identified in seminal plasma are sufficiently similar to suggest that the fertility-associated proteins may be related to
phospholipase A2. Because phospholipase has been shown
immunohistochemically to be present on the surface of
ejaculated bovine sperm acrosome, post-acrosome, and
middle piece, but not on epididymal sperm [39], it is likely
that some phospholipase is adsorbed from seminal plasma
to the sperm surface.
Given the role that has been proposed for phospholipase in sperm capacitation and the acrosome reaction [40],
it is possible that the phospholipase in seminal plasma that
FERTILITY PROTEINS IN SEMINAL PLASMA
is adsorbed by sperm could account for the relatively small
differences in bull fertility we observed in conjunction with
the fertility-associated proteins.
It was beyond the scope of the present study to biochemically and functionally characterize the fertility-associated proteins we detected by electrophoresis in two-dimensional polyacrylamide gels of bovine seminal plasma.
Nevertheless, the similarities between the fertility-associated proteins we observed and those biochemically characterized by other investigators provide impetus for further
studies to link biochemistry with the biological function of
proteins in seminal plasma.
ACKNOWLEDGMENTS
The assistance of the staff at the Dairy Breeding Research Center is greatly appreciated. We also thank Atlantic Breeders Cooperative, Lancaster, PA; Sire Power,
Inc., Tunkhannock, PA; and Eastern Artificial Insemination Cooperative, Ithaca, NY,
for providing samples of seminal plasma and fertility data.
REFERENCES
1. Constentino MJ, Emilson LBV, Cockett ATK. Prostaglandins in semen and their
relationship to male fertility: a study of 145 men. Fertil Steril 1984; 41:88-94.
2. Sandowski T, Rogers BJ. Two-dimensional electrophoretic patterns of seminal
plasma proteins from fertile and infertile men. Biol Reprod 1985; 32(suppl 1):102.
3. Jeyendran RS, Van der vern HH, Rosecrans R, Perez-Pelaez M, Al-Hasani S, Zaneveld LJD. Chemical constituents of human seminal plasma: relationship to fertility. Andrologia 1989; 21:423-428.
4. Panidis D, Rousso D, Pappas C, Kalogeropoulos A. Seminal plasma transferrin:
does it help in the diagnosis of fertility? J Obstet Gynecol 1991; 11:211-214.
5. Autiero M, Sansone G, Abreccia P. Relative ratios of lactoferrin, albumin, and acid
phosphatase seminal levels as sperm quality markers in fertile and infertile men.
J Androl 1991; 12:191-200.
6. Kandell RL, Bellin ME, Hajokins HE, Ax RL. Bull fertility was related to distribution of heparin binding proteins in sperm membrane and seminal plasma. J
Androl 1992; 13(suppl 1):30.
7. Dukelow WR, Cheinoff HN, Williams WL. Properties of decapacitation factor and
presence on various species. J Reprod Fertil 1967; 14:393-399.
8. Hunter AG, Nornes HO. Characterization and isolation of a sperm coating antigen from rabbit seminal plasma with capacity to block fertilization. J Reprod
Fertil 1969; 20:419-427.
9. Eng LE, Oliphant G. Rabbit sperm reversible decapacitation by membrane stabilization with a highly purified glycoprotein from seminal plasma. Biol Reprod
1978; 19:1083-1094.
10. Reddy JM, Stark RA, Zaneveld LJD. A high molecular weight antifertility factor
from human seminal plasma. J Reprod Fertil 1979; 57:437-446.
11. Gaur RD, Talwar GP. Further studies on the fertility promoting factor from human seminal plasma. IntJ Fertil 1975; 20:133-136.
12. Shivaji S, Bhargava PM. Antifertility factors of mammalian seminal fluid. BioAssays
1987; 7:13-17.
13. Audhya T, Reddy J, Zaneveld LJD. Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma. Biol
Reprod 1987; 36:511-521.
14. Miller DJ, Winer MA, Ax RL. Heparin-binding proteins from seminal plasma bind
to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990;
42:899-915.
15. Lowry OH, Rosebrough WJ, Farr AL, Randall RJ. Protein measurement with Folin
phenol reagent. J Biol Chem 1951; 193:265-275.
16. Laemmli UK. Cleavage of structural proteins during assembly of the head of the
bacteriophage T. Nature (Lond) 1970; 277:680-685.
1207
17. Hames BD. An introduction to polyacrylamide gel electrophoresis. In: Hames B,
Rickwood D (eds.), Gel Electrophoresis, A Practical Approach. Washington, DC:
IRL Press; 1981: 1-86.
18. McNutt T, Rogowski L,Vasilatos-Younken R, Killian G. Uptake of oviductal fluid
proteins by the bovine sperm membrane during in vitro capacitation. Mol Reprod Dev 1992; 33:313-323.
19. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol
Chem 1975; 250:4007-4021.
20. Shabanowitz RB, Killian GJ. Two-dimensional electrophoresis of proteins in principal cells, spermatozoa and fluid associated with the rat epididymis. Biol Reprod
1987; 36:753-768.
21. NeterJ, Wasserman W, Kutner MH. Applied Linear Statistical Models. Homewood,
IL: RD Irwin Inc.; 1985.
22. Shivaji S, Scheit KH, Bhargava PJ. Proteins of Seminal Plasma. New York: John
Wiley and Sons, Inc.; 1990: 332-333.
23. Reddy J, Audhya T, Goodpasture JC, Zaneveld LJD. Properties of a highly purified
antifertility factor from human seminal plasma. Biol Reprod 1982; 27:1076-1083.
24. Nass SJ, Miller DJ, Winer MA, Ax RL. Male accessory sex glands produce heparinbinding proteins which bind to cauda epididymal spermatozoa and are testosterone-dependent. Mol Reprod Dev 1990; 25:237-246.
25. Ax RL, Lenz RW. Glycosaminoglycans as probes to monitor differences in fertility
of bulls. J Dairy Sci 1987; 70:1477-1486.
26. Vasquez JM, Winer MA, Ax RL, Boone WR. Correlation of human spermatozoa
heparin binding with the zona-free hamster egg in vitro penetration assay. Am
J Obstet Gynecol 1989; 160:20-26.
27. Blottner S, Nehring H, Torner H. Individual differences in capacitation of bull
spermatozoa by heparin in vitro. Relationship to fertility. Theriogenology 1990;
34:619-628.
28. Esch FS, Ling NC, Bohlen P, Ying SY, Guillemin R. Primary structure of PDC-109,
a major protein constituent of bovine seminal plasma. Biochem Biophys Res
Commun 1983; 113:861-867.
29. Kemme M, Madiraju MVVS, Kroules E, Zimmer M, Scheit KH. The major protein
of bull seminal plasma is a secretory product of seminal vesicles. Biochim Biophys Acta 1986; 884:282-290.
30. Manjunath P, Sairam MR Purification and biochemical characterization of three
major acidic proteins (BSP-AI, BSP-A2 and BSP-A3) from bovine seminal plasma.
Biochem J 1987; 241:685-692.
31. Manjunath P, Baillargeon L, Marcel YL, Seidh NG, Chretien M, Chapdelaine A
Diversity of novel proteins in gonadal fluids. In: Mckems KW, Chretien M (eds.),
Molecular Biology of Brain and Endocrine Peptidergic Systems. New York: Plenum
Press; 1988: 259-273.
32. Shivaji S, Scheit KH, Bhargava PJ. Proteins of Seminal Plasma. New York: John
Wiley and Sons, Inc.; 1990: 393-409.
33. Chandonnet L, Roberts KD, Chapdelain A, Manjunath P. Identification of heparinbinding proteins in bovine seminal plasma. Mol Reprod Dev 1990; 26:313-318.
34. Aumuller G, Vesper M, SeitzJ, Kemme M, Scheit KH. Binding of a major secretory
protein from bull seminal vesicles to bovine spermatozoa. Cell Tissue Res 1988;
252:377-384.
35. Vijayaraghavan S, Manjunath P, Hoskins DP. The seminal vesicle protein BSP Al/
A2 binds to bovine epididymal sperm and inhibits calcium uptake. Bio Reprod
1989; 40(suppl 1):142 (abstract 283).
36. Miller DJ, First NL, Ax RL. Isolation and characterization of seminal fluid proteins
that bind heparin. In: Mahesh VB, Dhindsa DS, Anderson E, Kalra SP (eds.), Regulation of Ovarian and Testicular Function. Adv Exp Med Biol 1987: 219:597601.
37. Shivaji S, Scheit KH, Bhargava PJ. Proteins of Seminal Plasma. New York: John
Wiley and Sons, Inc.; 1990: 196-303.
38. Ronkko S, Risto L, Vanha-Pertula T. Phospholipase A2 in the reproductive system
of the bull. Int J Biochem 1991; 23:595-603.
39. Ronkko S. Immunohistochemical localization of phospholipase A2 in the bovine
seminal vesicle and on the surface of the ejaculated spermatozoa. Int J Biochem
1992; 24:869-876.
40. Langlais L, Roberts KD. A molecular membrane model of sperm capacitation and
the acrosome reaction of mammalian spermatozoa. Gamete Res 1985; 12:183224.