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Original Studies |
Dipartimento di Fisiopatologia Clinica, Unità di Andrologia, Università di Firenze, I-50139 Firenze, Italy
Address all correspondence and requests for reprints to: Elisabetta Baldi, Ph.D., Dipartimento di Fisiopatologia Clinica, Unità di Andrologia, Università di Firenze, Viale Pieraccini 6, I-50139 Florence, Italy. E-mail: e.baldi{at}mednuc2.dfc.unifi.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-glucuronide-[125I]iodotyramine as
tracer. Computer analysis of competition curves using different
steroids as competitors indicated the presence of two distinct binding
sites for P. The high affinity site (Kd in the nanomolar
range) appears to be specific for P, whereas the low affinity one
(Kd in the micromolar range) binds with equal affinity
11ß-hydroxyprogesterone (11ßOHP) and 17-hydroxyprogesterone
(17OHP). A significant correlation exists among affinity constants
(as determined by binding studies) and EC50 values for the
effects of P, 11ßOHP, and 17OHP on intracellular Ca2+
in fura-2-loaded spermatozoa, strongly indicating the involvement of
P-binding sites in the biological effect of the steroid. In particular,
dose-response curves for P were biphasic, with an EC50 in
the nanomolar range and another in the micromolar range. Conversely,
curves for 11ßOHP and 17OHP were monophasic, with an
EC50 just in the micromolar range. Ligand blot analysis of
sperm total lysates performed with peroxidase-conjugated P revealed the
presence of two binding proteins of 54 and 57 kDa that were specific
for P. Indeed, peroxidase-conjugated P binding was blocked by the
simultaneous presence of the unconjugated steroid. Using c262
antibody, which is directed against the P-binding domain of genomic
receptor, we detected two proteins of similar molecular mass (54 and 57
kDa), whereas using antibodies directed against the DNA-binding and
N-terminal domains of the genomic P receptors, the two proteins were
not detected. In addition, p54 and p57 appear to be mostly localized in
sperm membranes and virtually absent in the cytoplasm. The involvement
of these proteins in the biological effects of P is indicated by the
strong inhibitory effect of c262 on P-induced acrosome reaction of
capacitated human spermatozoa. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acidA
(GABAA) receptor/Cl- channel complex in the
effects of P has been suggested (for review, see Ref.8), leading to
the hypothesis of the existence of at least two different receptors for
P, one mediating the influx of calcium and the other mediating the
efflux of chloride (9). However, although data obtained in mice (10)
suggest that the GABAA receptor/Cl- channel
complex can account for both P-induced chloride and calcium fluxes,
results obtained in human spermatozoa seem to exclude the involvement
of GABAA receptor/Cl- channel complex
involvement in the increase in intracellular Ca2+
([Ca2+]i) caused by P (11, 12, 13), with the
exception of a recent report (14). Several findings suggest that the putative receptor(s) for P in spermatozoa is located on the membrane surface. BSA-conjugated P, which is unable to cross the plasma membrane, produces the same effects as P (15, 16). Moreover, immunohistochemical studies using fluorescein isothiocyanate (FITC)-labeled BSA-conjugated P (17, 18), peroxidase-conjugated P (13), and an antibody directed against the C-terminal domain of the genomic P receptor (19) localize P-binding sites on human sperm heads. Further, sperm P-binding sites are sensitive to treatment with trypsin and aggregate after binding with the agonist (20), suggesting similarities with other surface receptors. Despite all of these studies showing P binding to the surface of ejaculated spermatozoa, the molecular, pharmacological, and kinetic characteristics of this putative receptor(s) have not been clearly elucidated. Possible candidates as sperm P receptors include a 94- to 97-kDa protein that is tyrosine phosphorylated upon stimulation with the steroid (21, 22, 23, 24) and GABAA receptor-like/Cl channels with apparent masses of 50 and 75 kDa (reviewed in Ref.8). Using an antibody directed against the C-terminal binding domain of the P genomic receptor, Sauber et al. (19) recently identified a sperm protein with an apparent mass of 50–52 kDa as a possible nongenomic sperm P receptor.
In the present study, we used different approaches to identify and
characterize nongenomic P receptors on human sperm surface. By using
progesterone-11-glucuronide-[125I]iodotyramine as the
labeled ligand, we identified two distinct P-binding sites on human
sperm plasma membrane. By ligand and Western blot analysis we initiated
the molecular characterization of P-binding proteins. We identified two
proteins of 54 and 57 kDa molecular mass that specifically bind P. In
addition, we reexamined the possible involvement of GABA receptors in
the effect of P by evaluating the ability of this agonist to displace P
binding as well as its effects on sperm
[Ca2+]i.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Percoll was obtained from Pharmacia LKB (Uppsala, Sweden). Human
serum albumin-free human tubal fluid (HTF) was purchased from Irvine
(Santa Ana, CA).
Progesterone-11-glucuronide-[125I]iodotyramine (2666
Ci/mmol) and enhanced chemiluminescence Western blotting detection
reagent (ECL) were obtained from Amersham (Aylesbury, UK). Mifepristone
(RU486) was provided by Roussel-UCLAF (Romainville, France). Steroids,
peroxidase-conjugated P (P-POD), antimouse IgG-POD, antirabbit IgG-POD,
GABA, and all other chemicals were obtained from Sigma Chemical Co.
(St. Louis, MO). Reagents for SDS-PAGE were purchased from Bio-Rad
Laboratories (Hercules, CA). Monoclonal c262 antibody was obtained
from StressGen (Victoria, Canada). Polyclonal anti-DNA-binding domain P
receptor antibody was purchased from Dako (Milan, Italy). Monoclonal
PR anti-P receptor was obtained from Biogenex (San Ramon, CA).
Ionomycin and fura-2/AM were obtained from Calbiochem (La Jolla,
CA).
Preparation of spermatozoa
Human semen was collected, according to the WHO recommended procedure (25), by masturbation from normozoospermic men undergoing semen analysis for couple infertility. Samples with a linear progressive motility of less than 50% and with leukocytes and/or immature germ cell concentration greater than 106/mL were not included in the study. Semen samples were processed as previously described (11). Briefly, spermatozoa were separated on 40% and 80% Percoll gradients, combined, washed in HTF medium containing 0.3% fatty acid-free BSA, and finally resuspended in the same medium at the indicated concentration. Spermatozoa were capacitated for 2 h or otherwise indicated in 0.3% BSA-containing HTF.
Equilibrium binding studies
Binding studies were performed on human intact spermatozoa.
After 2 h of capacitation, spermatozoa were washed in
phosphate-buffered saline (PBS) and resuspended in 0.3% BSA-PBS. One
hundred-microliter aliquots of spermatozoa (8 x
107/mL) were incubated for 1 h at 4 C with 77 pmol/L
progesterone-11-glucuronide-[125I]iodotyramine in
0.3% BSA-PBS in the presence of increasing concentrations (1E-11 to
1E-3 mol/L) of unlabeled compounds: P, 17-hydroxyprogesterone
(17OHP), 11ß-hydroxyprogesterone (11ßOHP), RU486, GABA,
dihydrotestosterone (DHT), and Norgestrel (Sigma). After incubation, 1
mL iced Tris (50 mmol/L) was added to each tube. Samples were
centrifuged at 3000 rpm for 10 min at 4 C, and radioactivity associated
with the obtained pellets was measured in a -counter at 70%
efficiency. In some experiments, pellets were resuspended in 1 mL
glycine acid buffer (pH 2), incubated for 10 min on ice, and then
centrifuged, and the obtained pellets and supernatants were counted in
a -counter to determine the loss of radioactivity after the acid
wash.
Measurement of [Ca2+]i
Spermatozoa, prepared as described above, were loaded with 2 µmol/L fura-2/AM for 45 min at 37 C, washed, and resuspended in FM medium (125 mmol/L NaCl, 10 mmol/L KCl, 2.5 mmol/L CaCl2, 0.25 mmol/L MgCl2, 19 mmol/L sodium lactate, 2.5 mmol/L sodium pyruvate, 2 mmol/L HEPES, and 0.3% BSA, pH 7.5), and [Ca2+]i before and after stimulation with the different agonists was measured as described previously using the spectrofluorometric method (11). Fluorescence measurements were converted to [Ca2+]i by determining maximal fluorescence (Fmax) with ionomycin (8 µmol/L, final concentration) followed by minimal fluorescence (Fmin) with 10 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, pH 810. [Ca2+]i was calculated according to the method of Grynkiewicz (26), assuming a dissociation constant of fura-2 for calcium of 224 nmol/L.
Preparation of uterine lysate
Human uterine samples in the proliferative phase of the menstrual cycle obtained at surgery were minced with sharp scissors, suspended in lysis buffer (20 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 0.25% Nonidet P-40; 1 mmol/L Na3VO4; and 1 mmol/L phenylmethylsulfonylfluoride), and homogenized (Teflon-glass). The homogenates were centrifuged at 1500 rpm for 10 min at 4 C, and supernatants corresponding to total lysates were subjected to protein measurement.
Sperm membrane preparation
Spermatozoa stored in liquid nitrogen were lysed in lysis buffer for 1 h on ice. Then, the samples were subjected to two subsequent cycles of homogenizing (Teflon-glass) and sonicating (three times, 15-s 8 burst). The homogenates were centrifuged at 1,500 rpm for 10 min at 4 C, and supernatants were ultracentrifuged at 48,000 rpm for 45 min at 4 C. The resulting pellets (cellular membranes) were resuspended in lysis buffer and homogenized. The supernatants (cytosolic fractions) were dried in a speed vacuum and resuspended in lysis buffer. Proteins were measured in cell lysates using a Bio-Rad kit (Bio-Rad, Hercules, CA).
SDS-PAGE
After 5-h capacitation in 0.3% BSA-containing HTF, sperm samples were processed for SDS-PAGE as previously described (22, 27). Briefly, they were centrifuged at 400 x g at 4 C for 10 min, washed twice in PBS, and resuspended in 10 µL lysis buffer. After measurement of proteins, the sperm extracts, containing approximately 30–50 µg proteins, were diluted in equal volume of 2-fold concentrated Laemmli sample buffer, vortexed, incubated at 95 C for 5 min, and then loaded onto 10% polyacrylamide-bisacrylamide gels. After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Sigma Chemical Co.).
Ligand blot analysis
The nitrocellulose was incubated for 30 min in 3% Nonidet P-40/PBS, then for 2 h in 0.3% BSA/0.1% Tween-20/PBS, for 10 min in 0.1% Tween-20/PBS, and finally overnight in 0.3% BSA/0.1% Tween-20/PBS containing P-POD (0.5 µmol/L) in the presence or absence of a high concentration of free P (10 µmol/L). After several washes in 0.1% Tween-20/PBS, reacted proteins were simultaneously revealed by an ECL system (Amersham). Particular attention was paid in these experiments to treat the samples (with or without free P) simultaneously and for exactly the same incubation times.
Western blot analysis
Transferred nitrocellulose was blocked for 3 h at room
temperature in TTBS (0.1 Tween-20, 20 mmol/L Tris, and 150 mmol/L NaCl)
containing 5% BSA, then washed repeatedly in TTBS and incubated for
2 h in 2% BSA-TTBS containing c262 (1:400). After washing,
nitrocellulose were incubated with goat antimouse IgG-POD (1:5000 in
2% BSA-TTBS). In other experiments, different primary antibodies were
used (PR and DNAbd-PR, 1:100 dilution). After several washes in
TTBS, reacted proteins were revealed by the ECL system (Amersham).
AR assay
Acrosome-reacted spermatozoa were evaluated using the
fluorescent probe FITC-labeled Arachis hypogea (peanut)
lectin according to the method of Aitken (28) as previously described
(29). Briefly, after 2-h capacitation, spermatozoa (106/mL)
were preincubated for 30 min with c262 antibody or PR (1:100) and
then stimulated with P (10 µmol/L), A23187 (10 µmol/L), or the
appropriate control solvent (dimethylsulfoxide) for 2 h at 37 C.
After staining with fluorescent lectin, fluorescence was observed under
a fluorescent microscope (type 307–148.002, Leitz, Wetzlar, Germany),
and the AR was evaluated on a total of 100 spermatozoa/slide. Using the
method of Aitken (28), only curly-tailed spermatozoa were considered
viable and thus scored.
Analysis of experimental results
The binding data were evaluated quantitatively with nonlinear least squares curve fitting using the computer program Ligand (30). The program provides objective measures of goodness of fit in terms of both magnitude and randomness of residuals. The computer program Allfit (31) was used for the analysis of sigmoidal dose-response curves obtained in binding and calcium studies. AR data were analyzed by Student’s t test and one-way ANOVA after data transformation in arcsins of the root square. Each data point represents the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify and pharmacologically characterize P-binding sites on
human spermatozoa, we performed binding studies in pooled capacitated
human spermatozoa using
progesterone-11-glucuronide-[125I]iodotyramine as
tracer (Fig. 1
, inset).
Preliminary time-course experiments indicated that
progesterone-11-glucuronide-[125I]iodotyramine binding
was time dependent and reached apparent equilibrium within 60 min at 4
C (Fig. 1). Accordingly, all subsequent binding experiments were
conducted using these experimental conditions. The steric compliance
and the hydrophilic properties of the substitution group should prevent
11-glucuronide-[125I]iodotyramine from crossing the
plasmalemma, as indicated by the dramatic decrease in radioactivity
(73.96 ± 0.74%; n = 10) after acid washing of samples. To
exclude the possibility that binding of
progesterone-11-glucuronide-[125I]iodotyramine to BSA
could be responsible for the observed binding, these experiments were
also conducted in the absence of BSA with results similar to those
obtained in its presence (results not shown).
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shows a typical Scatchard plot
for progesterone-11-glucuronide-[125I]iodotyramine
binding to human spermatozoa. The curvilinear relationship indicates
heterogeneity of P binding in human spermatozoa. To further
characterize P-binding sites, we performed 10 experiments of
competition curves for
progesterone-11-glucuronide-[125I]iodotyramine binding
using increasing concentrations of P, 17OHP, and 11ßOHP as
competitors for the radioactive tracer. Competition experiments were
conducted in sperm derived from different subjects or in pooled sperm
from different donors (n = 10). Simultaneous mathematical analysis
of experimental results strongly indicated the presence of at least two
distinct classes of sites. The introduction of a second independent
class of sites dramatically improved the fitting of experimental
results (P < 0.0001). A typical family of competition
curves is shown in Fig. 3. Affinity
constants and concentrations of receptors are reported in Table 1. We found a high affinity, low capacity
binding site that is apparently selective for P and a low affinity,
high capacity site that binds with virtually equal affinity all three
progestins. Note that GABA, DHT, and Norgestrel did not displace
binding even at micromolar concentrations. To exclude the possibility
that displacement of radioactive tracer by P might be due to induction
of the AR and, thus, loss of acrosome in a high percentage of cells, we
evaluated the AR in spermatozoa treated with P (1 nmol/L, 0.1 µmol/L,
and 10 µmol/L) for 60 min on ice (same experimental conditions as
those used for binding experiments). We found that in these conditions,
P was unable to stimulate the AR (mean ± SEM
acrosome-reacted sperm, 15.5 ± 2.0 in controls, 13.2 ± 2.7
with 1 nmol/L P, 12.5 ± 3.5 with 0.1 µmol/L P, and 17.5 ±
4.4 with 10 µmol/L P; n = 2). Similar results were obtained by
Tesarik and Mendoza (32) using FITC-BSA-conjugated P.
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To investigate the biological function of the two binding sites
identified with equilibrium binding studies, we evaluated the effects
of increasing concentrations of P, 17OHP, and 11ßOHP on
[Ca2+]i of fura-2-loaded spermatozoa. Figure 4 reports the percent increase in
[Ca2+]i in response to increasing
concentrations of P, 17OHP, and 11ßOHP, as generated by the
computer program Allfit after simultaneous analysis of the different
curves. The dose-response curve for P was clearly biphasic, with a
first component in the nanomolar range (EC50 = 55.6 ±
15.4 nmol/L; n = 12) and a second component in the micromolar
range (EC50 = 40.1 ± 20.1 µmol/L; n = 12). On
the contrary, dose-response curves for 17OHP and 11ßOHP were
monophasic, with EC50 values, respectively, of 53.4 ±
41.4 µmol/L (n = 7) and 5.8 ± 4.4 µmol/L (n = 8). A
significant correlation was observed between pK and logEC50
(r = 0.946; P = 0.05). Interestingly, at low
concentrations (10–50 nmol/L), the intracellular calcium increase in
response to P was significantly higher (P < 0.05) than
that of the other steroids, whereas at micromolar concentrations, the
three progestins induced similar responses, as previously reported (11, 33). The addition of different concentrations of solvent control
(0.01–1% dimethylsulfoxide) did not modify sperm
[Ca2+]i (not shown). In Table 2, the percent stimulation of
[Ca2+]i vs. basal in response to
several agonists at two different concentrations is reported. At the
concentration of 50 nmol/L, only P induced a significant response,
whereas at 50 µmol/L P, 11ßOHP and 17OHP stimulated a similar
influx of calcium. The other tested molecules did not stimulate an
appreciable increase in sperm [Ca2+]i (Table 2).
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, a first dose of 50 nmol/L P induced an
increase in [Ca2+]i that could not be
reproduced by a subsequent equimolar administration of the same agonist
(Fig. 5a), indicating the occurrence of homologous desensitization
(34). No response and no modification of the response to subsequent
administration of P were observed with 50 nmol/L 11ßOHP or 17OHP
(not shown). At the concentration of 50 µmol/L, homologous and
heterologous (34) desensitization occurred with all three steroids. At
this concentration, a first administration of P (Fig. 5b) or 11ßOHP
(Fig. 5c) induced similar intracellular calcium transients (see also
Table 2
), which was not followed by a response to a subsequent
equimolar dose of P (compared with the response to P in the same
subject; Fig. 5d). Similar results were obtained with 17OHP (not
shown).
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, GABA did not stimulate an increase in
[Ca2+]i at either low (50 nmol/L; Fig. 6a) or
high (50 µmol/L; Fig. 6c) concentrations. Moreover, GABA did not
affect calcium transient in response to a subsequent equimolar
administration of P at both low (Fig. 6, a vs. b) and high
(Fig. 6, c vs. d) concentrations compared to the respective
control values. In addition, in our hands, GABA at concentrations
ranging from 50 nmol/L to 50 µmol/L did not induce acrosome reaction
of capacitated human spermatozoa or modify the response to P (percent
AR; mean ± SEM of three experiments; control, 8
± 1.1; 10 µmol/L P, 14 ± 1.1; 50 nmol/L GABA, 8 ± 1.0;
500 nmol/L GABA, 8 ± 1.0; 5 µmol/L GABA, 7.5 ± 0.5; 50
µmol/L GABA, 8.6 ± 0.3; 50 nmol/L GABA plus 10 µmol/L P,
16 ± 4; 50 µmol/L GABA plus 10 µmol/L P, 14.6 ±
2.7).
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To identify the molecular mass of P-binding proteins in
capacitated human spermatozoa, we performed ligand blot experiments
using P-POD (0.5 µmol/L) as probe. This molecule has been shown to be
effective in inducing AR in human spermatozoa (13) and thus is an
interesting tool to perform this type of experiment. As shown in Fig. 7A, P-POD identifies two protein bands on
total sperm lysate (right panel) of 54 and 57 kDa, which
totally disappear when P-POD is added in the presence of a high
concentration (10 µmol/L) of free P (left panel),
suggesting that these proteins bind P in a specific manner. These
results have been consistently reproduced in six different sperm
samples.
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c262, which is directed against the C-terminal tail of
genomic P receptor (35). Recent data demonstrated that this antibody is
able to counteract the effect of P in human spermatozoa and stains some
protein bands in sperm lysates (19). As shown in Fig. 7B (left
panel), Western blot immunostaining of sperm lysates with c262
identified two protein bands of similar molecular mass (57 and 54 kDa)
as those revealed by ligand blot with P-POD (Fig. 7A) and an additional
band at 66 kDa. In uterine lysates, used as a control for P genomic
receptor, the antibody detected several bands. Among these, two bands
of about 100–110 and 60 kDa, which correspond to known isoforms of
genomic P receptor (35, 36), are not present in spermatozoa, confirming
the absence of genomic P receptors in these cells (37).
The same sperm and uterine total lysates as those in Fig. 7B
(left panel) were stained with antibodies directed against
different regions of the genomic P receptor, respectively the
N-terminal (PR) and the DNA-binding domains of the genomic receptor.
Interestingly, PR detected only the 66-kDa band (Fig. 7B, right panel), whereas no protein bands were revealed by the
antibody directed against the DNA-binding domain (data not shown),
indicating that the putative 57- and 54-kDa P-binding proteins do not
show homology with the classical nuclear receptor family at the
N-terminal and DNA-binding sequences. To exclude the possibility that
the 66-kDa protein band detected with -c262 and PR is due to the
residual presence of BSA, we performed Western blot analysis of BSA
standard with the two antibodies. In this case, no protein bands were
detected (not shown), indicating that the 66-kDa band revealed in sperm
lysates is indeed a sperm protein.
The possible sperm surface localization of 54- and 57-kDa P-binding
proteins was demonstrated by Western blot analysis with c262 of
purified sperm membrane and cytosol shown in Fig. 7C. The monoclonal
antibody detected the same three bands of 66, 57, and 54 kDa as in
total lysate (Fig. 7B, left panel), whereas in the cytosolic
fraction, only the 66-kDa band was observed.
In Table 3, we summarize the results of
Western and ligand blot experiments on sperm P-binding proteins (six
experiments for ligand and four experiments for Western blot). We
believe that only the 54- and 57-kDa proteins are specific P-binding
proteins, as only these two proteins were detected by both c262 and
P-POD and disappeared when P-POD was used in the presence of a high
concentration of free P (Table 3). Moreover, our results confirm that
genomic P receptors are not present in spermatozoa (37), as no protein
bands are detected by the antibody directed against the DNA-binding
domain of genomic receptor.
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c262 antibody, but the heavy chain of the antibody
(
60 kDa) used to immunoprecipitate masks the proteins of interest as
they comigrate at the same molecular mass (data not shown).
Effects of c262 and PR antibodies on AR
As P is a well known stimulus for acrosome reaction (5), we
investigated whether c262 antibody affected this effect. Human
capacitated spermatozoa were stimulated with P (10 µmol/L) in the
presence or absence of monoclonal antibody c262 or PR antibody
(1:100 dilution). As shown in Table 4,
c262 antibody did not alter the spontaneous AR, whereas it totally
blunted the effect of P. No effects of the antibody were observed on
A23187-induced AR (data not shown). These results strongly indicate
that the membrane p54 and p57 proteins revealed by c262 are involved
in transducing P biological effects in human spermatozoa. Conversely,
the PR antibody, which did not detect the 57- and 54-kDa protein
bands, but only the 66-kDa one, in Western blot analysis of sperm
lysates, did not inhibit the P-stimulated AR (Table 4), suggesting that
the 66-kDa band is not involved in mediating P activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Computerized analysis of binding displacement curves with the program
Ligand strongly indicates the presence of two distinct P-binding sites
on human spermatozoa. The high affinity, low capacity site is
displaced, among the different molecules used in our experiments, only
by P, whereas the low affinity, high capacity site is displaced by P,
11ßOHP, 17OHP, and RU486. Both binding sites appear to be relevant
for P-induced calcium fluxes in human spermatozoa. Indeed, we found
that the dose-response curve of P-induced
[Ca2+]i increase was characterized by two
distinct components, with EC50 values, respectively, in the
nanomolar and in the micromolar ranges, whereas dose-response curves
for 11ßOHP and 17OHP were characterized by a single component with
an EC50 in the micromolar range. Most relevant,
EC50 values for [Ca2+]i
dose-response curves and binding affinity constants for the three
progestins were correlated, indicating that the two surface receptors
identified with equilibrium binding studies are involved in P-induced
calcium influx. As P is present at micromolar levels in the cumulus
oophorus surrounding the oocyte (3), it can virtually bind both the
high and low affinity receptors present on spermatozoa; hence, it can
be hypothesized that both sites are physiologically involved in the
action of the steroid in human spermatozoa. Moreover, the ability of
other molecules sharing a progestin structure, such as 17OHP and
11ßOHP, to displace the low affinity site suggests that 17OHP,
which is also present in high concentrations in follicular fluid and
cumulus matrix (3), might be physiologically relevant during the
process of fertilization. The low affinity site is also displaced by
RU486, a potent antagonist of the genomic P receptor. This finding is
consistent with its reported inhibition of P biological effects in
human spermatozoa when used at micromolar doses (38). In a previous
report, Neulen et al. (39), using [3H]P as
ligand, found a single site for P with a Kd of about 10
nmol/L in human seminal plasma and spermatozoa. However, a comparison
of these results with the present data is difficult, because these
researchers used as tracer [3H]P, which can easily cross
the plasma membrane, and performed most of the experiments in seminal
plasma (39).
Ligand blot analysis of sperm lysates using P-POD identified two
protein bands of about 57 and 54 kDa that were displaced in the
presence of unconjugated P. Two proteins with similar molecular masses
were also identified by Western blot analysis of sperm proteins with an
antibody directed against the C-terminal domain of the P genomic
receptor (-c262). At present, we do not have any evidence that these
two proteins are related to the two receptors evidentiated with
equilibrium binding studies. On the other hand, the inhibitory effect
of the -c262 antibody of P-induced AR (Ref. 19 and the present
study) and the increase in [Ca2+]i (19)
strongly suggests that the proteins detected with this antibody are
involved in the biological effects of P. P-POD has been used as a probe
in ligand blot analysis of sperm proteins by Benoff et al.
(18), who identified two proteins of 70 and 58 kDa present on sperm
crude membranes. It must be also mentioned that in Western blot
analysis of sperm proteins with -c262 antibody, Sauber et
al. (19) detected two major proteins with molecular masses of
50–52 and 46–48 kDa and additional bands at lower molecular masses.
The discrepancies in apparent molecular mass of the two P-binding
proteins identified in these studies may be due to covalent
modifications (glycosylation, phosphorylation) of the proteins that
could be altered by the different methods of protein preparation. Among
proteins capable of binding P, cortisol-binding globulin (40) and
albumin (41) show molecular masses similar to those of p54 and p57.
Using pure BSA as a positive control, we excluded c262
cross-reaction with albumin, confirming data reported by Benoff
et al. (18). In addition, Sauber et al. (19) did
not detect any immunoreactivity with antibody against cortisol-binding
globulin in washed spermatozoa. In our study we detected an additional
protein band at 66 kDa, which is also present in Western blot performed
with an antibody directed against a different domain of the P genomic
receptor (PR). However, the lack of effect of this antibody on
P-stimulated AR suggests that this band is not specific and is not
involved in the biological effect of the steroid. Using proteins
lysates from partially purified sperm membranes, we demonstrated that
the two binding proteins of 57 and 54 kDa are selectively localized in
the membranes and absent in the cytosol, in agreement with
immunofluorescence studies demonstrating surface localization of the
staining with -c262 (19). Recently, high affinity binding sites for
P have been characterized and purified in porcine liver membranes (42).
Interestingly, binding studies using [3H]P as ligand
identified two binding sites for P with apparent
Kd values of 11 and 286 nmol/L, while
purification of P-binding proteins revealed the presence of two
proteins of 56 and 28 kDa (42). From these studies it appears that
nongenomic P receptors show profound structural and functional
differences compared to the genomic receptor family. In keeping with
this conclusion, spermatozoa from transgenic mice lacking the nuclear
genomic P receptor show normal surface P receptors (43), suggesting the
presence of two different genes encoding for genomic and nongenomic
receptors for the steroid.
As stated above, the possible involvement of GABA receptors in the mechanism of action of P in mammalian sperm is still a matter of discussion, as both positive (9, 10, 14) and negative (11, 12, 13) results have been reported. Binding sites for GABA have been shown in spermatozoa (9, 44). However, competitive displacement of [3H]GABA tracer by nipecotic acid, a blocker of GABA transport protein, suggests the presence of GABA transport proteins more than true GABA receptors (44). [3H]GABA transport has been demonstrated in human spermatozoa (45). By immunofluorescence studies, a GABAA receptor/Cl- channel complex has been localized on the surface of the sperm head at the equatorial segment (9). It has been proposed that the effects of P may be mediated by such a channel on the surface of human sperm (8). However, despite different results obtained in mice (10), GABA has been reported to only weakly stimulate the AR in human spermatozoa (9) and, in contrast to the effects of P, to be devoid of stimulatory effects on sperm-oocyte fusion (13). At least in the mouse, the effect of GABA appears to be mediated by the influx of calcium (10). We were unable to demonstrate any effect of GABA on AR in either the absence or presence of P. Moreover, our data confirm that GABA does not stimulate an influx of calcium in human spermatozoa or modify P-induced calcium influx, as reported previously (11, 12, 13). In addition, competitive binding studies demonstrate no interaction of GABA with either the high or the low affinity P-binding sites. Taken together, our results seem to exclude the involvement of GABA receptors in calcium signaling stimulated by P in human spermatozoa. In a recent report, Meizel et al. (14) demonstrated, by image analysis of single sperm cells loaded with fura-2, that the effect of P could be partially reverted by picrotoxin, suggesting the involvement of GABAa receptors. However, direct effects of GABA on calcium influx were not investigated in this study (14), and using an identical experimental approach, Aitken et al. (13) did not demonstrate any effect of GABA on sperm calcium. As the P-induced AR clearly requires chloride efflux and activation of GABAA receptor/Cl- channel complex (8), it is possible that P receptors and GABAA receptor/Cl- channel complex are distinct and cooperate to induce the AR.
In conclusion, our results demonstrated the presence of two functional membrane P receptors in human spermatozoa. The p54 and p57 P-binding molecules detected in our study may represent novel nongenomic P receptors on human sperm membrane that can mediate the steroid effects. Isolation, sequencing, and further functional characterization of these two protein candidates of nongenomic P receptors are presently under investigation in our laboratory. As we have recently shown that a correlation exists between sperm responsiveness to P and sperm fertilizing ability (29, 46), identification and characterization of nongenomic P receptor(s) may be clinically relevant not only in future research in male infertility, but also in the development of new contraceptive tools.
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Acknowledgments |
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Footnotes |
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Received August 8, 1997.
Revised December 1, 1997.
Accepted December 8, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyprogesterone: novel stimulators of
calcium influx in human sperm. J Biol Chem. 265:1367–1380.
-aminobutyric acid specific binding sites on human
spermatozoa. Mol Hum Reprod. 1:1885–1890.
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