487
Journal of Cell Science 102, 487-494 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
Expression and function of ras proto-oncogene proteins in human sperm
cells
RAJESH K. NAZ*, KHALIQ AHMAD
Department of Obstetrics and Gynecology, Reproductive Immunology and Molecular Biology Laboratories, Albert Einstein College of
Medicine, Bronx, NY 10461, USA
and PAUL KAPLAN
Department of Obstetrics and Gynecology, Perinatal and Fertility Laboratory, Mount Sinai School of Medicine, New York, NY 10029, USA
*Author for correspondence at: Ullmann Research Building, Room no. 123, Department of Obstetrics and Gynecology, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Summary
The presence and role of c-ras proteins were investigated
in mature human sperm cells. The \-H-ras monoclonal
antibody (mAb) against the c-ras protein, p21, reacted
specifically with the acrosomal region of methanol-fixed
as well as unfixed-live capacitated and non-capacitated
human sperm cell in the indirect immunofluorescence
technique. The v-H-ras mAb predominantly recognized
c-ras protein of 21 kDa on the Western blot of lithium
diiodosalicylate (LlS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb
affected the sperm cell function in the human sperm
penetration assay. The antibody significantly reduced
the acrosome reaction and release of acrosin activity
from the sperm cells. There was no effect of the mAb on
percentage motility, although the mAb significantly
affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and
beat frequency, the motility parameters involved in the
hyperactivation phenomenon of sperm cells leading to
capacitation and acrosome reaction. These results
suggest that the c-ras or c-ras-like proteins are present in
mature sperm cell and may have a role in capacitation
and/or acrosome reaction of human sperm cell.
Introduction
cellular homologues constitute a family of three human
genes, namely c-H-ras, c-K-ras and c-N-ras, which
encode a remarkably well-conserved protein of 21 kDa,
designated as p21 (Shimizu et al., 1983). The p21 has
been shown to bind guanine nucleotides, has GTPase
activity in vitro, and is associated with the plasma
membrane of various eukaryotic cells (Barbacid, 1987;
Cantley et al., 1991). Interestingly, ras proteins are
structurally and functionally analogous to the G
protein, suggesting that ras proteins may participate in
the transduction of signals across the cellular membrane
(Gilman, 1987; Hall, 1990).
The mechanisms involved in the signal transduction
pathways leading to capacitation and acrosome reaction
in spermatozoa are not yet clearly understood. A
guanine nucleotide binding regulatory protein (Go) has
been identified in sea urchin spermatozoa with a
possible role in receptor-effector coupling (Kopf, 1989).
Among the mammalian species, Gi-like proteins (similar to Go proteins of sea urchin sperm) have been
identified in the mouse sperm (the only mammalian
Proto-oncogenes are a group of normal genes that play
various important roles in regulation of cell proliferation and function (Cantley et al., 1991). A number of
these proto-oncogenes especially c-abl, pim-l, c-mos,
int-1, c-raf, c-ras, c-fos, c-jun and c-myc are expressed
in a stage-specific pattern during spermatogenesis in the
testes (Ponzetto and Wolgemuth, 1985; Propst et al.,
1987; Wolfes et al., 1989), suggesting that they may
have an important function in germ-cell development.
The expression product of one of these proto-oncogenes, the c-myc protein, is present on the mature
mammalian sperm with a possible role in sperm cell
function (Naz et al., 1991a). Our laboratory is investigating the presence of expression products of other
proto-oncogenes in the mammalian sperm cell and their
possible role in sperm cell function.
The ras genes were first identified as the viral
oncogenes of the Harvey (v-H-ras) and Kirsten (v-Kras) rat sarcoma viruses (Shih et al., 1980). Human
Key words: sperm, c-ras proto-oncogene, Western blot,
capacitation, fertilization.
488
R. K. Naz and others
species studied to date) and have been implicated in the
physiological cascade leading to the acrosome reaction
(Endo et al., 1987). In lieu of these findings, and
because of the significant structural and functional
homology between p21 and G proteins, the present
study was conducted to investigate the presence and
possible role of c-ras proteins in human sperm cell
function, especially during the development of its
fertilizing capacity.
Materials and methods
Spermatozoa collection
Human spermatozoa were collected from healthy fertile
donors. Semen was liquefied and analyzed for volume, sperm
concentration, percentage and progressive motility, and the
presence of anti-sperm antibodies using the immuno-bead test
(IBT) (Naz, 1987; Naz et al., 1990; Naz et al., 1991a). Only
those semen samples that had sperm concentration of >50 x
106 sperm/ml, percentage motility of >60%, progressive
motility of >+3 (on a scale of 0 to +5), contamination of
immature germ cells and immune cells of < 1 % , and were
negative in IBT, were used to collect a pure swim-up sperm
population. Sperm were washed (twice) by centrifugation
(500 g, 10 min) with Ham's F-10 medium (pH 7.4, osmolarity
280 mosmol/kg) supplemented with 5% BSA (Ham's/BSA
medium). Motile sperm were collected by the swim-up
procedure (Naz, 1987) and the sperm in the swim-up were
analyzed for sperm concentration, motility, and any contamination. The pure population of motile sperm in the overlay
was washed twice with PBS (pH 7.4, osmolarity 280
mosmol/kg) and pooled from different swim-up samples; the
pellet was stored at —70°C for protein extraction.
The human testes were collected, with permission, as
autopsy tissue from a 35-year-old white male without any
terminal disease, who died accidently, and the testes material
was stored at — 70°C until use. Seminal plasmas were collected
from fertile men (n=3) who demonstrated normal semen
analysis, as described above. Vasectomized seminal plasmas
were collected from vasectomized men (n=2) who had
undergone vasectomy between 1979 and 1981. The time
interval from vasectomy and collection of seminal plasma
varied from 48 to 52 months. Both were determined to be
azoospermic by means of semen analysis.
Indirect immunofluorescence technique (IFT)
IFT was performed on methanol-fixed and unfixed viable noncapacitated as well as capacitated sperm cells by the
procedure described elsewhere (Naz et al., 1984a,b, 1991a,b).
Motile sperm cells collected by the swim-up procedure were
washed 3 times with PBS by centrifugation (500 g, 10 min) and
used for the IFT procedure. For IFT on methanol-fixed cells,
the sperm were air-dried (2 x 104 sperm/well) on 10-well
slides (Roboz Surgical Instrument Co., Inc., Washington,
DC) at room temperature, fixed in methanol for 30 min, and
air-dried again. The slides were then rinsed in PBS, blocked
with PBS containing 5% BSA for 45 min, and then the 10 fi\ of
monoclonal antibody in PBS (10 ^jg/100 ,al) was added to the
wells and incubated for 1.5 h at room temperature in a moist
chamber. After thorough washing in PBS, the FITC-labeled
goat anti-IgG (1:40 dilution, Cappel Labs., Malvern, PA) was
added and incubated as above for 1.5 h. After the slides were
washed in PBS, they were mounted in PBS containing 80%
glycerol. Samples were maintained at 4CC until examined.
The capacitation was induced by incubating (8 h, 37°C in 5%
CO2 and 95% air mixture) the swim-up sperm (10 x lO6
sperm/ml) in Ham's F-medium containing 5% BSA. The
capacitated sperm were washed twice with PBS, methanolfixed and studied for antibody reactivity as described above.
For IFT on unfixed viable cells, the swim-up sperm were
washed twice with PBS and incubated (37°C for 2 h) with
monoclonal antibody (4 x 106 sperm/ml, 6 ,ug/ml of the
antibody), washed twice in PBS, and incubated (37°C for 1 h
30 min) with FITC-labeled goat anti-rat immunoglobulin
(1:40 dilution containing 0.02% sodium azide to avoid
capping). The cells were washed, mounted and examined as
described above. The sperm cells incubated with control rat
myeloma ascites fluid and c-ras monoclonal antibody immunoabsorbed with c-ras peptide served as control.
For immunoabsorption experiments, the \-H-ras monoclonal antibody (described below) was reacted with 5-, or 10fold (by weight) excess of peptide in PBS overnight at 4°C; the
reaction mixture was centrifuged and the supernatants were
tested as described above.
The v-H-ras (Ab-1) monoclonal antibody used in the
present study was derived by fusion of rat spleen cells with Y3
Ag 1.2.3 rat myeloma cells (Clone Y13-259, Oncogene
Science, Inc., Manhasset, NY; azide-free, Cat. no. OPO1L).
v-H-ras rat IgGi monoclonal antibody reacts with phosphorylated and non-phosphorylated forms of v-H-ras and v-K-ras
p21s, and also reacts with p21 translational products of H-, Kand N-ras human oncogenes (Furth et al., 1987). This
antibody neutralizes the biological and biochemical activities
of H, K and N-ras p21 by binding to residues Glu63, Ser65,
Ala66, Met67, Glu70 and Arg73. For immunoneutralization
experiments, the ras 15 amino acid peptide (peptide 1)
including amino acids 62 through 7 6 ( E E Y S A M R D Q Y M
R T G E) of the ras protein was obtained from Oncogene
Science, Inc. (Cat. no. PP08). The rat IgGi purified from rat
ascitesfluidwas used as antibody control in these experiments
(Binding Site, Inc., San Diego, CA, Cat. no. ME273).
Western blot procedure
The antigenic specificity of the c-ras monoclonal antibody was
evaluated by the Western blot procedure (Naz et al., 1984a,b,
1986). Briefly, lithium diiodosalicylate (LlS)-solubilized preparations of human, sperm and testis, seminal plasma from
fertile men and seminal plasma from vasectomized men were
run (30-50 jug protein per lane) in the slab SDS-PAGE (10%
gel), transferred to nitrocellulose paper, and reacted with the
antibody (20 |Ug/l0 ml of the incubation buffer). The reacted
antigens were localized by first reacting the antibody-reacted
strips with alkaline-phosphatase-conjugated rabbit anti-rat
antibodies (heavy- and light-chain-specific; Cooper Biomedical Inc., Malvern, PA) and then with nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate as a substrate as
described elsewhere (Naz et al., 1991a). Sperm extracts were
prepared by dissolving washed sperm cell membrane proteins
in lithium diiodosalicylate (0.3 M LIS, 0.05 M Tris-HCI, pH
8.0, containing 1 mM phenylmethylsulfonyl fluoride (PMSF)
and 5 mM soybean trypsin inhibitor) at room temperature for
30 min and at 4°C for 2 h. The control rat myeloma ascites
igG! served as control for the antibody.
Sperm penetration assay
The zona-free hamster ova/human sperm penetration assay
was performed by the method of Yanagimachi et al. (1976) as
described elsewhere (Naz et al., 1991b). Superovulation was
induced in adult female golden hamsters by i.p. injection of 30
i.u. eCG (Sigma Chemical Co., St. Louis, MO) on Day 1 of
the cycle. After 55-72 h, 25 i.u. hCG (Sigma Chemical Co.)
was administered i.p. The animals were killed 15-17 h after
c-ras proto-oncogene and sperm cell function
hCG injection and mature unfertilized ova were collected
from the oviducts. The ova were separated from the
surrounding cumulus cells by incubation with 0.2% hyaluronidase in Biggers, Whitten and Whittingham medium (BWW)
and from the zonae pellucidae by treatment with 0.1%
pancreatic trypsin in BWW. The zona-free ova were washed
twice in BWW and placed in the center of tissue culture
dishes.
Semen from fertile men was allowed to liquefy for 15-30
min at 37°C and the swim-up sperm population was collected
as described above. The sperm cells in the swim-up were
washed with BWW supplemented with 1% BSA (fraction V,
no. A-7906, Sigma Chemical Co.), adjusted to 5 x 106 to 10 x
106 motile sperm/ml and then allowed to incubate for 5-6 h at
37°C (in 5% CO2 and 95% air mixture) with the c-ras antibody
(10 /xg/100 ,ul of the sperm suspension) or the control rat
antibody or the equivalent volume of the PBS. After
incubation, the sperm were washed to remove the unreacted
antibody and co-incubated with zona-denuded hamster
oocytes (30-40 egg/treatment in each assay) for 3-4 h. The
oocytes were removed, washed thoroughly, fixed with 3%
glutaraldehyde and stained with acetocarmine solution.
Penetration was determined by the presence of a swollen
sperm head with discernible tail in the cytoplasm of the ovum.
Motility on sperm before and after incubation with ova was
recorded. The assays were repeated at least 2-8 times using 5
different fertile donors and each sample was tested with at
least 68-305 oocytes.
Assessment of acrosomal status
The motile sperm collected in BWW medium by the swim-up
procedure (5xlO6 to lOxlO6 motile sperm/ml, >75% motility, +3 to +4 forward progression) were incubated with vH-ras monoclonal antibody (final protein concentration, 10
iUg/100 /A) at 37°C for 5 h. The controls were treated with PBS
containing the same concentration of rat control ascites IgGi
antibody or BSA. After incubation, the spermatozoa were
centrifuged, washed with PBS, and treated with calcium
ionophore A23187 (Sigma) for 1 h to induce the acrosome
reaction (10 ^M final ionophore concentration) (Byrd et al.,
1989). The acrosomal status was assessed using the triple-stain
procedure described by Jager et al. (1984). Briefly, the
ionophore-treated sperm were washed twice in PBS and then
incubated for 37°C for 15 min with 2% Trypan blue (Sigma)
(1:1, v/v) in PBS. The sperm were washed twice, fixed in 3%
glutaraldehyde in 0.1 M cacodylate buffer for 60 min and then
washed twice again in PBS. A drop of suspension was placed
on a glass slide and allowed to air-dry overnight. The slides
were stained in 0.8% Bismarck Brown (Sigma) in deionized
water (pH 1.8 with 2 M HO) rinsed in deionized water, and
stained for 45 min (at room temperature) in 0.8% Rose
Bengal (Sigma) in 0.1 M cacodylate buffer (pH 6.0). The
slides were then washed in deionized water, dehydrated in an
ethanol series, cleared in xylene and mounted with Permount
and a coverslip. A total of 200 sperm per slide were evaluated
and recorded as either alive or dead and the alive sperm were
evaluated as acrosome-intact or acrosome-not intact (reacted)
sperm.
Assessment of acrosin activity
After incubation with the antibody for 5 h and then treating
with the calcium ionophore A23187 for 1 h to induce the
acrosome reaction as described above, the sperm cells were
centrifuged and the acrosin activity was determined in the
supernatant and cells by the method described by Kennedy et
al. (1989). Briefly, 90 [A of the supernatant or the pelleted
sperm cells suspended in 90 /il of PBS, were mixed with 1 ml
489
of the reaction mixture (1 mg/ml of N-benzoyl-dl-arginine-pnitraniline (BAPNA) hydrochloride in 0.055 M HEPES
buffer, 0.055 M NaCl, 10% (v/v) dimethyl sulfoxide, 0.1%
(v/v) Triton X-100) and incubated for 3 h at room temperature. The reaction was stopped by the addition of 100 (A of 0.5
M benzamidine and the absorbance was measured at 405 nm.
Acrosin activity was expressed as ^i.u./l06 spermatozoa. The
daily variability of the assay was normalized using a
cryopreserved partially purified human acrosin extract prepared as described elsewhere (Howe et al., 1991).
Sperm motion analysis
After incubation with the antibody for 5 h as described above,
a sample (7 (A) of the sperm suspension was placed into a
Makler chamber (Sefi-Medical Instruments, Israel) and
motion characteristics were determined using a computerized
semen analyzer (Cell soft Cyro Resources, NY) as described
elsewhere (Naz et al., 1991a). The following parameter
settings were used throughout the study: 30 Hz, 3 frames
minimum sampling for motility, 15 frames minimum sampling
for both velocity and amplitude of the lateral head movement
(ALH) measurements, 10 (im/s threshold velocity, minimum
linearity of 2.5 for ALH measurement, and cell size range of 4
to 40 pixels with a magnification calibration of 0.688 ^m/pixel.
Statistical analysis
The significance of differences between treated and untreated
controls was based on the unpaired Student's /-test.
Results
Detection of c-ras proteins in sperm cells
Analysis by indirect IFT
The ras protein was expressed in human sperm cells.
The c-ras monoclonal antibody demonstrated binding,
predominantly with acrosomal regions of methanolfixed (Fig. la) as well as unfixed-viable (Fig. lc) human
non-capacitated sperm cells in IFT. The antibody
reacted in the similar acrosomal regions of the methanol-fixed human sperm cells that had been capacitated
for 8 h (Fig. lb). The absorption (antibody.antigen
ratio, 1:10) of the c-ras monoclonal antibody with the
reactive peptide completely absorbed out the immunoactivity of the antibody with the sperm cells (Fig. le).
The control rat ascites fluid IgG as well as the peptideabsorbed antibody did not react with unfixed-viable and
methanol-fixed, capacitated or non-capacitated human
sperm cells.
Analysis by Western blot procedure
The c-ras monoclonal antibody reacted with a protein
band of 21 kDa corresponding to p21 of c-ras protein on
a Western blot of LIS-solubilized human sperm cells
(Fig. 2, lane a). There was also another band of 18 ± 2
kDa, below the 21 kDa band, recognized by the c-ras
antibody on the blots of sperm cells (Fig. 2, lane a). The
antibody absorbed with the peptide or the control rat
ascites fluid-IgG (of the same concentration and the
same isotype specificity) did not react with either of
these bands on the blots of human sperm cells (Fig. 2,
lane a'). On the Western blot of LIS-solubilized human
testes, the c-ras monoclonal antibody recognized three
specific bands, a band of 21 kDa (corresponding to p21)
490
R. K. Naz and others
Fig. 1. Epifluorescent
photomicrographs
demonstrating the
immunofluorescent reaction
pattern of the c-ras
monoclonal antibody. The
antibody predominantly
recognized the acrosomal
regions of non-capacitated,
unfixed-live (c) as well as
methanol-fixed (a) human
spermatozoa. The same
binding pattern was observed
on methanol-fixed or unfixedlive capacitated human sperm
cells (b). The c-ras monoclonal
antibody absorbed with the
corresponding peptide, or the
control rat ascites fluid IgG,
did not react with the unfixedlive or methanol-fixed
capacitated or non-capacitated
human sperm cell (e). The
phase-contrast picture in e has
been included for comparison
(d). a-e, X36O.
and two other bands of 18 ± 2 kDa and 16 ± 2 kDa,
respectively (Fig. 2, lane b). There was another nonspecific band visible in the region of 65 kDa (Fig. 2, lane
b), which was also recognized by the control rat ascites
fluid-IgG (Fig. 2, lane b'). The antibody absorbed with
the peptide or the control rat ascites fluid IgG did not
react with either of these bands on the blots of human
testes (Fig. 2, lane b'). On the Western blot of seminal
plasma of fertile men, the c-ras monoclonal antibody
recognized a major band of 21 kDa (corresponding to
p21), besides a few minor bands of various molecular
identities (Fig. 2, lane c). The 21 kDa band was not
recognized by the antibody absorbed with the peptide
or the control rat ascites fluid IgG on the blot of seminal
plasma (Fig. 2, lane c'). On the Western blots of
seminal plasma from vasectomized men (devoid of
antisperm antibodies), the c-ras monoclonal antibody
did not recognize the specific band of 21 kDa,
corresponding to p21 (Fig. 2, lane d).
Effects of c-ras antibody on human sperm cell
function
Effects on human sperm penetration assay (SPA)
To investigate the role of c-ras proteins in human sperm
cell function, we studied the effects of c-ras monoclonal
antibody on human sperm penetration of zona-free
hamster oocytes (SPA). The c-ras antibody significantly
(P<0.001) decreased the percentage of ova penetrated
compared to untreated control (Table 1). Immunoabsorption of the antibody with corresponding peptide
caused a dose-dependent reduction in the fertilizationinhibitory activity of the antibody; absorption with the
peptide (antibody:antigen ratio of 1:10 (w/w)), completely neutralized the antibody activity. The rat ascites
fluid immunoglobulins of the same isotype specificity
(IgGx) as the c-ras monoclonal antibody did not affect
penetration rates (Table 1). There was no visible affect
of the c-ras monoclonal antibody on the sperm motility
(percentage and progressive).
Effects on acrosomal status of sperm
The v-H-ras monoclonal antibody significantly inhibited
capacitation and the acrosome reaction of the spermatozoa. On incubation with the antibody and subsequent
treatment with the ionophore, the sample demonstrated a significantly (P=0.005) higher percentage of
acrosome-intact and a lower percentage of acrosome-
c-ras proto-oncogene and sperm cell function
491
on the percentage of motile sperm in the sample, upto 5
h of the observation period (Table 3). Similarly, there
was no significant effect of the antibody on velocity of
the sperm cells. However, the v-H-ras antibody significantly (P=0.004 to 0.005) increased the linearity, and
decreased the ALH and beat frequency of the sperm
cells, compared to the sperm treated with rat ascites
IgGj or PBS-BSA controls.
Discussion
a
a'
Fig. 2. Reaction of the c-ras monoclonal antibody with
spermatozoal, testicular and seminal plasma proteins on
Western blots. The c-ras monoclonal antibody reacted
specifically with a band of 21 kDa (arrow) and a minor
band of 18 ± 2 kDa on the blots of LIS-solubilized human
sperm cells (lane a). The antibody recognized a specific
band of 21 kDa and two minor bands of 18 ± 2 kDa and
16 ± 2 kDa, respectively, on blots of LIS-solubilized
human testes (lane b). On the blots of seminal plasma
from fertile men (lane c), but not on the blots of seminal
plasma from vasectomized men (lane d), the c-ras
monoclonal antibody recognized a specific band of 21 kDa,
besides several minor bands of various molecular identities.
The c-ras antibody absorbed with the peptide, and the
control rat ascites fluid IgG did not react with the specific
band of 21 kDa and other minor specific bands on the
blots of LIS-solubilized human sperm (lane a'), LISsolubilized human testes (lane b') and seminal plasma from
fertile men (lane c').
reacted sperm, compared to the samples treated with
rat-ascites IgGj or PBS-BSA (Table 2).
Effects on acrosin concentration
Again, on incubation with the v-H-ras antibody and
subsequent treatment with the ionophore the sperm
sample demonstrated a significantly (/>=0.005 to 0.007)
lower quantity of acrosin concentration released in the
supernatant and a higher quantity of acrosin concentration in the sperm cells, compared to the samples
treated with the rat-ascites I g d or PBS-BSA (Table 2).
Effects on sperm motility characteristics
The v-H-ras antibody demonstrated no significant effect
Our results demonstrate that the human sperm cell has
c-ras proteins recognized by the v-H-ras monoclonal
antibody. The c-ras proteins were predominantly
observed in the acrosomal region of the sperm cell. The
c-ras proteins have been localized in the inner side of
the plasma membrane of various mammalian cells
(Barbacid, 1987). The primary translational product of
ros oncogenes is synthesized in the cytosol and
attachment to the plasma membrane requires a posttranslational modification that involves the acylation of
Cysl86 by palmitic acid (Barbacid, 1987; Fujiyama and
Tamanoi, 1986). In our study, we found that the
antibody binds to both methanol-fixed as well as
unfixed live sperm, indicating the surface localization of
the c-ros proteins. The binding pattern did not change
after capacitation of the sperm cells. Besides various
other physiological changes, capacitation involves shedding of the seminal plasma coating proteins from the
sperm surface (Yanagimachi, 1988). Though it is
difficult to rule out the presence of residual seminal
plasma proteins after capacitation, our immunofluorescence may indicate that the ras proteins are intrinsic
membrane proteins present on the sperm surface rather
than absorbed from the seminal plasma. The normal
seminal plasma (free of sperm) from fertile men showed
the presence of p21 protein, corresponding to c-ras
proteins in the Western blot procedure. However, the
seminal plasma collected from vasectomized men (not
exposed to sperm cells) did not reveal the presence of a
p21 protein band, suggesting that the source of the p21
protein band in the normal seminal plasma may be
the sperm cells that are digested/degraded/acrosomereacted during their transit through the male genital
tract. This was further confirmed by the presence of cras proteins in human testes blot, thus indicating that
the c-ras proteins present on the sperm cell are
synthesized during spermatogenesis in the testes.
v-H-ras monoclonal antibody recognizes the translational product of all three ros human oncogenes,
namely the H-, K- and N-ras proto-oncogenes, each of
which encode a protein of 21 kDa with slight differences
in sequence (Barbacid, 1987; Shimizu et al., 1983). At
this time, we do not know if the sperm cell has
expression products of one or all three (H-, K- and N-)
ras proto-oncogenes. In a preliminary study, we found
that the monoclonal antibody specifically directed
against the c-N-ras protein does not bind to human
sperm cells and nor does it inhibit human sperm
penetration of zona-free hamster oocytes. These results
492
R. K. Naz and others
Table 1. Effects of v-H-ras antibody on human sperm penetration of zona-free hamster oocytes
Antibody absorbed"
(antibody:antigen ratio)
Ova tested
(no.)
Ova penetrated (%)
(mean ± s.d.)
1. \-H-ras monoclonal antibody
10
10
10
None
1:5
1:10
284
119
68
31.6 ± 17.9b
45.6 ± 16.1"
91.2 ± 5.3e
2. Rat ascites IgGi
10
None
86
86.5 ± 3.4f
3. PBS, control
None
305
95.5 ± 7.2C
Treatment (,ug/lOO fi\)
a
For neutralization experiments, the antibody was reacted with 5- or 10-fold (by weight) excess of peptide in PBS (0.025 M sodium
phosphate buffer, pH 7.4, containing 0.15 M sodium chloride) overnight at 4°C; the reaction mixture was centrifugcd and the supernatant
tested as described in Materials and methods.
b
vs. c , P<0.001; d vs. b, P=0.03; e vs. b, P<0.001; f vs. c , insignificant.
Table 2. Effects of v-H-ras antibody on the acrosome reaction of human sperm
Acrosin activity"
Acrosome status of sperm (%)
Treatment (10 /ig/100 (A)
1. v-H-ras monoclonal antibody
2. Rat ascites IgGi
3. Control
Acrosome-intact
(mean ± s.d.)
Acrosome-reacted
(mean ± s.d.)
Supernatant
(mean ± s.d.)
Cells
(mean ± s.d.)
43.50 ± 2.38b
33.20 ± 4.21°
28.20 ± 6.57d
39.25 ± 2.87b
53.20 ± 2.77°
55.80 ± 4.82d
31.60 ± 8.90b
48.60 ± 10.26°
50.00 ± 6.00d
43.00 ± 9.75b
19.00 ± 4.00°
20.80 ± 9.04d
Assays («=5) were performed on various days using sperm collected from four different fertile men.
Acrosin activity was expressed as jui.u. of acrosin/106 sperm cells.
vs. c or d , ^=0.005 to 0.007; c vs. d , insignificant.
a
b
Table 3. Effects of v-H-ras antibody on human sperm motility parameters
Motility characteristics (mean ± s.d.)
Treatment (10 //g/100 /d)
1. v-H-ras monoclonal antibody
2. Rat ascites igG!
3. Control
% Motility
(mean ± s.d.)
78.40 ± 7.21
79.82 ± 5.48
82.30 ± 6.32
Velocity
55.84 ± 4.50
56.28 ± 4.52
57.35 ± 5.72
ALHa
Linearity
5.01 ± 0.41
4.12 ± 0.17c
3.98 ± 0.28d
b
Beat frequency
b
2.98 ± 0.48
3.51 ± 0.33c
3.62 ± 0.48d
11.28 ± 1.69"
12.98 ± 1.02c
12.86 ± 0.62d
Assays («=9) were performed on various days using sperm collected from eight different fertile men.
a
ALH, amplitude of lateral head displacement.
b
vs. c or d , P=0.004 to 0.005; c vs. d , insignificant.
suggest that probably the H- and K-ras proteins and not
the c-N-ras protein, may be the predominant molecules
expressed on the human sperm cell. The \-H-ras
monoclonal antibody specifically recognized an additional band of 18 ± 2 kDa (besides the 21 kDa ras
protein) on the Western blot of sperm, and the antibody
specifically recognized two bands of 18 ± 2 kDa and 16
± 2 kDa, respectively (besides recognizing the 21 kDa
ras protein), on the Western blot of testes. Emerging
evidence suggests that ras genes are members of a
supergene family and the genes, e.g. rho gene,
exhibiting sequence homology to the ras gene family
have been identified in a variety of eukaryotic cells. The
16 ± 2 and 18 ± 2 kDa proteins may be the expression
products of ras-related genes. These proteins seem to
have sequence homology with the p21 ras protein and
are specifically recognized by the v-H-ras monoclonal
antibody.
The biological function of ras genes in mammalian
cells is poorly understood. The available data indicate
that the ras genes may play a dual role in proliferation
and in the cellular differentiation process (Barbacid,
1987). The structural and functional similarity with G
proteins has raised the possibility that ras proteins may
also participate in the transduction of mitogenic signals
(Barbacid, 1987; Cantley et al., 1991; Hall, 1990).
Recent studies have suggested that proteins of the ras
family may also play a general role in controlling
movement of proteins within the cell (Chardin et al.,
1989; Paterson et al., 1990). The mechanism by which
this control occurs is unknown, but it would probably
involve actin filament rearrangements.
Mammalian fertilization is a complex process requiring the spermatozoon to undergo a cascade of
events before it can fuse with the egg plasma membrane. These chains of events include capacitation,
acrosome reaction, and binding and penetration
through the zona pellucida of the oocyte, which
c-ras proto-oncogene and sperm cell function
subsequently cleaves, develops and implants. Capacitation, leading to the acrosome reaction, is a physiological change necessary for mammalian fertilization
(Yanagimachi, 1988). The mechanisms involved in
sperm capacitation and the acrosome reaction are not
clearly understood at the molecular level. However, the
sperm cell has to be capacitated before it can fertilize
the oocyte. In our study, the v-H-ras monoclonal
antibody reduced human sperm penetration of zonafree hamster eggs (SPA), indicating that the c-ras
proteins may have a role in sperm cell function. These
effects of the antibody suggest that c-ras proteins may
be involved in capacitation and/or the acrosome
reaction. Indeed, the sperm sample treated with the
antibody demonstrated a decrease in the percentage of
acrosome-reacted sperm and an increase in the percentage of acrosome-intact sperm, when induced to acrosome react in the presence of calcium ionophore. These
results were further confirmed by a decrease in the
acrosin activity in the supernatant, and an increase in
the acrosin activity in the sperm cells, further indicating
that the antibody is inhibiting the acrosome reaction.
Since capacitation is a prerequisite for the sperm cell to
undergo the acrosome reaction, the ionophore-induced
reduction in the acrosome reaction in the antibodytreated sperm cells suggest a blockage in the capacitation process. The antibody did not affect the
percentage motility although it did affect the various
motility characteristics of human sperm cells, namely
the linearity, ALH and beat frequency. The linearity,
ALH and beat frequency have been reported as
important contributors towards the estimation of
hyperactivation of sperm cells (Mortimer et al., 1984;
Robertson et al., 1988). Hyperactivation is considered
an integral part of capacitation, preceding the acrosome
reaction and sperm binding to zona pellucida (Mortimer et al., 1984; Robertson et al., 1988).
The exact mechanism of inhibition of capacitation by
the antibody needs further investigation. The ras
proteins have significant sequence homology to the
guanine nucleotide-binding inhibitory proteins, which
have been shown to have a role in the acrosome
reaction of the sperm cell in the mouse (Endo et al.,
1987; Kopf, 1989). For several receptors, including
those coupled to G proteins, phosphorylation by
endogenous protein kinases represents an important
mode of regulation. We have shown that tyrosine
phosphorylation plays an important role in the development of the fertilizing capacity of human sperm cells
(Naz et al., 1991b). Our laboratory recently demonstrated that the c-myc protein (Naz et al., 1991a), but
not the expression products of c-erfe-B-1 and c-erb-B2/HER 2 proto-oncogenes (Naz and Ahmad, 1992) may
be involved in the signal transduction pathway leading
to capacitation and/or the acrosome reaction. The exact
mode of interaction between c-ras proteins, c-myc
protein and various other growth factors and their
receptors, and their participation in a physiological
cascade leading to capacitation and the acrosome
reaction, needs further study.
In conclusion, our data suggest that the human sperm
493
cell has c-ras- or c-ras-like proteins that are recognized
by the v-H-ras monoclonal antibody, and these proteins
may be directly or indirectly involved in some step that
is vital for capacitation and/or the acrosome reaction.
This investigation is the first to report the presence of
expression products of c-ras proto-oncogene on sperm
cells and their possible role in human sperm cell
function. Further studies are needed to investigate the
exact signal transduction pathway by which c-ras
proteins are involved in the capacitation and/or acrosome reaction of the sperm cell. Besides enabling us to
understand the molecules and physiological mechanisms involved in sperm differentiation (capacitation and
acrosome reaction), these studies may have a clinical
application in male-factor-mediated human infertility.
At present, we are investigating the expression of c-ras
proteins in sperm cells from infertile men who have
defective sperm motility and/or are incapable of
fertilizing the oocytes in the human in vitro fertilization/embryo transfer (IVF-ET) program, and studying
the modulation of sperm motility and function by
various guanosine nucleotides.
We sincerely thank Valerie Neumaier and Laura Keller for
superb technical help, and Daniela Pulitano for excellent
typing assistance. This work was supported by NIH grant
HD24425 to R.K.N.
References
Barbacid, M. (1987). ras genes. Annu. Rev. Biochem. 56, 779-827.
Byrd, W., Tsu, J. and Wolf, D. (1989). Kinetics of spontaneous and
induced acrosomal loss in human sperm incubated under
capacitating and non-capacitating conditions. Gamete Res. 22, 109122.
Cantley, L. C , Auger, K. R., Carpenter, C , Duckworth, B.,
Graziani, A., Kapeller, R. and Soltoff, S. (1991). Oncogenes and
signal transduction. Cell 64, 281-302.
Chardin, P., Boquet, P., Madaule, P., Popoff, M. R., Rubin, E. J. and
Gill, D. M. (1989). The mammalian G protein rho C is ADPribosylated by Clostridium botulinum exoenzyme C3 and affects
actin microfilaments in Vero cells. EMBO J. 8, 1087-1092.
Endo, Y., Lee, M. A. and Kopf, G. S. (1987). Evidence for a role of a
guanine nucleotide-binding regulatory protein in the zona
pellucida-induced mouse sperm acrosome reaction. Develop. Biol.
119, 210-216.
Fujiyama, A. and Tamanoi, F. (1986). Processing and fatty acid
acylation of RAS1 and RAS2 proteins in Saccharomyces
ccrevisiae. Proc. Nat. Acad. Sci. U.S.A. 83, 1266-1270.
Furth, M. E., Davis, L. J., Fleurdelys, B. and Scolnick, E. M. (1987).
Monoclonal antibodies to the p21 products of the transforming
gene of Harvey murine sarcoma virus and of the cellular ras gene
family. J. Virol. 43, 294-304.
Gilman, A. G. (1987). G proteins: transducers of receptor-generated
signals. Annu. Rev. Biochem. 56, 615-649.
Hall, A. (1990). The cellular functions of small GTP-binding proteins.
Science 249, 635-640.
Howe, S., Grider, S., Lynch, D. and Fink, L. (1991). Antisperm
antibody binding to human acrosin: a study of patients with
unexplained infertility. Fertil. Steril. 55, 1176-1182.
Jager, S., Kuiken, J. and Kremer, J. (1984). Triple staining of human
sperm: technical aspects. Arch. Androl. 12, 53-58.
Kennedy, W. P., Kaminiski, J. M., Vauder Ven, H. H., Jeyendran, R.
S., Reid, D. S., Blackwell, J., Bielfled, P. and Zaneveld, L. J. D.
(1989). A simple clinical assay to evaluate the acrosin activity of
human spermatozoa. J. Androl. 10, 21-27.
Kopf, G. S. (1989). Mechanisms of signal transduction in mouse
spermatozoa. Ann. N.Y. Acad. Sci. 564, 289-302.
494
R. K. Naz and others
Mortimer, D., Courtot, A. M., Giovangrandi, Y., Jevlin, C. and
David, G. (1984). Human sperm motility after migration into and
incubation in synthetic media. Gamete Res. 9, 131-144.
Naz, R. K. (1987). Involvement of the fertilization antigen (FA-1) in
involuntary immunoinfertility in humans. J. Clin. Invest. 80, 13751383.
Naz, R. K. and Ahmad, K. (1992). Presence of expression products of
c-erbB-1 and c-erbB-2/HER 2 genes on mammalian sperm cell and
effects of their regulation on fertilization. /. Reprod. Immunol. 21,
223-239.
Naz, R. K., Ahmad, K. and Kumar, G. (1991a). Presence and role of
c-myc proto-oncogene product in mammalian sperm cell function.
Biol. Reprod. 44, 842-850.
Naz, R. K., Ahmad, K. and Kumar, R. (1991b). Role of membrane
phosphotyrosine proteins in human spermatozoal function. J. Cell
Sci. 99, 157-165.
Naz, R. K., Alexander, N. J., Isahakia, M. and Hamilton, H. S.
(1984a). Monoclonal antibody against human germ cell
glycoprotein that inhibits fertilization. Science 225, 342-344.
Naz, R. K., Ellaurie, M., Phillips, T. M. and Hall, J. (1990).
Antisperm antibodies in human immunodeficiency virus infection:
effects on fertilization and embryonic development. Biol. Reprod.
42, 859-868.
Naz, R. K., Phillips, T. M., Rosenblum, B. B. and Menge, A. C.
(1986). Characterization of fertilization antigen-1 for the
development of a contraceptive vaccine. Proc. Nat. Acad. Sci. USA
83, 5713-5717.
Naz, R. K., Rosenblum, B. B. and Menge, A. C. (1984b).
Characterization of a membrane antigen from rabbit testis and
sperm isolated by using monoclonal antibodies and effect of its
antiserum of fertility. Proc. Nat. Acad. Sci. USA 81, 857-861.
Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K. and
Hall, A. (1990). Microinjection of recombinant p21 r h o induces rapid
changes in cell morphology. J. Cell Biol. I l l , 1001-1007.
Ponzetto, C. and Wolgemuth, D. J. (1985). Haploid expression of a
unique c-abl transcript in the mouse sperm germ line. Mol. Cell.
Biol. 5, 1791-1794.
Propst, F., Rosenberg, M. P., Iyer, A., Kaul, K. and Vande Woude,
G. F. (1987). c-mos proto-oncogene RNA transcripts in mouse
tissues: structural features, development regulation, and
localization in specific cell types. Mol. Cell. Biol. 7, 1629-1637.
Robertson, L., Wolf, D. P. and Tash, J. S. (1988). Temporal changes
in motility parameters related to acrosomal status: identification
and characterization of populations of hyperactivated human
sperm. Biol. Reprod. 39, 797-805.
Shih, T. Y., Papageorge, A. G., Stokes, P. E., Weeks, M. O. and
Scolnick, E. M. (1980). Guanine nucleotide-binding and
autophosphorylating activities associated with the p21 lrc protein of
Harvey murine sarcoma virus. Nature 287, 686-691.
Shimizu, K., Goldfarb, M., Suard, Y., Perucho, M., Li, Y., Kamata,
T., Feramisco, J., Stavnezer, E., Fogh, J. and Wigler, M. H.
(1983). Three human transforming genes are related to the viral ras
oncogenes. Proc. Nat. Acad. Sci. USA 80, 2112-2116.
Signal, I. S., Gibbs, J. B., D'Alonzo, J. S. and Scolnick, E. J. (1986).
Identification effector residues and neutralizing epitope of Ha-rasencoded p21. Proc. Nat. Acad. Sci. USA 83, 4725-4729.
Wolfes, H., Kogawa, K., Millette, C. F. and Cooper, G. M. (1989).
Specific expression of nuclear proto-oncogenes before entry into
meiotic prophase of spermatogenesis. Science 245, 740-743.
Yanagimachi, R. (1988). Mammalian fertilization. In The Physiology
of Reproduction (ed. Dnobil, E. and Neill, J.D.), pp. 135-186.
Raven Press, New York.
Yanagimachi, R., Yanagimachi, H. and Rogers, B. J. (1976). The use
of zona-free animal ova as a test-system for the assessment of the
fertilizing capacity of human spermatozoa. Biol. Reprod. 45, 471476.
(Received 28 January 1992 - Accepted 18 March 1992)