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Original Studies |
Dipartimento di Fisiopatologia Clinica, Unita’ di Andrologia, Università di Firenze, I-50139 Florence, Italy
Address all correspondence and requests for reprints to: Michaela Luconi or Elisabetta Baldi, Dipartimento di Fisiopatologia Clinica, Unità di Andrologia, Università di Firenze, viale Pieraccini 6, I-50139 Florence, Italy. E-mail: m.luconi{at}dfc.unifi.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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H222
antibody, which is directed against the steroid binding domain of the
genomic estrogen receptor. The biological effects of estrogen receptor
were investigated by analyzing calcium fluxes, tyrosine
phosphorylation, and acrosome reaction (AR) in response to
17ß-estradiol (17ßE2) and by measuring the steroid
influence on calcium and AR in responses to progesterone (P), a
well-known physiological stimulus for human spermatozoa. Our results
demonstrate that 17ßE2 induces a rapid and sustained
increase of intracellular calcium concentrations
([Ca2+]i). This effect is totally dependent
on the presence of extracellular calcium, because it is completely
abolished in a calcium-depleted medium. The dose-response curve for
calcium increase to 17ßE2 is biphasic with a first
component in the nanomolar range (effective concentration 50 =
0.60 ± 0.12 nmol/L) and a second component in the micromolar
range (EC50 = 3.80 ± 0.26 µmol/L).
17ßE2 stimulates tyrosine phosphorylation of several
sperm proteins, including the 29-kDa protein band, and determines a
reduction of calcium response to P, finally resulting in inhibition of
P-stimulated sperm AR. Conversely, no direct effect of
17ßE2 is observed on AR. 17ßE2 effects on
calcium are clearly mediated by a membrane receptor, because they are
reproduced by the membrane-impermeable conjugate of the hormone
BSA-E2 and reduced by sperm preincubation with H222
antibody. Taken together, our results clearly show the presence of a
functional surface estrogen receptor, of 29 kDa, on human spermatozoa.
This receptor may play a role in the modulation of nongenomic action of
P in these cells during the process of fertilization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, some interactions between P and estrogen at membrane level have been suggested both in spermatozoa and brain tissues. P competes with [3H]17ßE2 binding to intact human spermatozoa (18), and estradiol can displace iodide P binding to a protein of 29 kDa identified on mouse brain membrane lysates (19, 20).
In the present study, we report identification and partial characterization of a novel receptor for estrogen on human sperm membrane, using functional and biochemical approaches similar to those applied by our group to characterize the nongenomic receptor for P on human sperm surface (11). We investigated the biological effects of 17ßE2 on intracellular calcium levels in fura-2-loaded spermatozoa and on AR. In addition, we examined the possible interference exerted by this steroid on calcium and AR in response to P. Finally, by ligand and Western blot analysis of sperm lysates, we initiated the molecular characterization of estrogen receptor and investigated the modulation of tyrosine phosphorylation pattern of this protein in response to administration of the steroid.
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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 from
Irvine (Santa Ana, CA). All free steroids, peroxidase-conjugated
estradiol (E2-POD), 6-(O-carboxymethyl)oxime-estradiol
conjugated with BSA (BSA-E2), secondary conjugated
antibodies, fluorescein isothiocyanate-labeled Arachis hypogea (peanut)
lectin, and all the other chemicals were from Sigma Chemical Co. (St. Louis, MO). Reagents for SDS-PAGE and for protein
measurement were from Bio-Rad Laboratories, Inc.
(Hercules, CA). Monoclonal H222 antibody was a kind gift of Prof.
Geoffrey Greene (The Ben May Institute for Cancer Research, University
of Chicago, Chicago, IL). Peroxidase-conjugated monoclonal (PY20-HRP)
antiphosphotyrosine antibodies were from ICN (Costa Mesa, CA).
Digitonin and Fura-2/AM were obtained from Calbiochem (La
Jolla, CA). The BM enhanced-chemiluminescence system was from
Boehringer (Mannheim, Germany).
Preparation of spermatozoa
Human semen was collected, according to the World Health Organization (WHO)-recommended procedure (21) 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 (22). Briefly, spermatozoa were separated on 40 and 80% Percoll gradients, combined, washed in HTF medium containing 0.3% fatty acid free-BSA, and resuspended in the same medium at the indicated concentration. Alternatively, sperms were separated by swim-up collection, according to the WHO-recommended procedure (21). Spermatozoa were capacitated for 2 h or otherwise indicated in 0.3% BSA-containing HTF and treated as indicated in each experiment.
Preparation of sperm membranes
Sperm membranes were prepared as previously described (11). Briefly, spermatozoa were lysed in lysis buffer [20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 0.25% Nonidet P40, 1 mmol/L Na3VO4, 1 mmol/L phenylmethanesulfonyl flouride] for 1 h on ice. Then the samples were subjected to two subsequent cycles of homogenizing (teflon-glass) and sonicating 3 x 15-sec 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.
Preparation of uterine and myometrial cell lysates
Human uterine samples in the proliferative phase of the menstrual cycles, obtained at surgery, were processed as previously described (23).
Myometrial cells, obtained as previously described (23), were resuspended in lysis buffer (see above). After protein measurement (Biorad kit, Bio-Rad Laboratories, Inc.), aliquots of cell extracts were applied onto SDS-polyacrylamide gels.
Measurement of intracellular calcium concentration
Spermatozoa, prepared as described above, were loaded with 2 µmol/L Fura-2/AM for 45 min at 37 C, washed, 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 Na-lactate, 2.5 mmol/L Na-pyruvate, 2 mmol/L HEPES, 0.3% BSA, pH 7.5), and [Ca2+]i, before and after stimulation with the different agonists, was measured (as described previously) using a spectrofluorimetric method (22), except that, in the present experiments, we used a Perkin-Elmer Corp. (Foster City, CA) LS50B instrument equipped with a fast rotary filter shuttle for alternate 340- and 380-nm excitation. Fluorescence measurements were converted to [Ca2+]i by determining maximal fluorescence with 0.01% digitonin, followed by minimal fluorescence with 10 mmol/L EGTA, pH 10. [Ca2+]i was calculated according to Grynkiewicz (24) using the ratio 340/380 and assuming a dissociation constant (Kd), of Fura-2 for calcium, of 224 nmol/L.
SDS-PAGE
After the different incubations, as indicated, samples were processed for SDS-PAGE as previously described (25). Briefly, sperm samples containing 107 cells/mL were added with 1 mmol/L Na3VO4, centrifuged at 400 x g at 4 C for 10 min, washed in PBS, and resuspended in 20 µL lysis buffer. After protein measurement (Biorad kit, Bio-Rad Laboratories, Inc.), the sperm extracts, containing the same protein amount, were diluted in an equal volume of reducing 2x loading buffer (1x = 62.5 mmol/L Tris (pH 6.8), 10% glycerol, 20% SDS, 2.5% pyronin, and 100 mmol/L dithiotheitrol), incubated at 95 C for 5 min, and loaded onto 10% polyacrylamide-bisacrylamide midi- and minigels. After SDS-PAGE, proteins were transferred to nitrocellulose membranes.
Ligand blot analysis
Nitrocelluloses filters with transferred proteins were treated for ligand blot analysis of sperm proteins, as previously described (11), with slight modification. Briefly, the membranes were incubated for 30 min in 3% NP-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 overnight in 0.3% BSA/0.1% Tween-20/PBS containing peroxidase-conjugated estradiol (E2-POD, 0.5 µmol/L). After several washes in 0.1% Tween-20/PBS, reacted proteins were revealed by a BM chemiluminescence system.
Western blot analysis
Nitrocelluloses filters with transferred proteins were blocked
overnight at 4 C in TTBS (0.1% Tween-20, 20 mmol/L Tris, 150 mmol/L
NaCl) containing 5% BSA, then washed repeatedly in TTBS, and incubated
for 2 h in 2% BSA-TTBS containing PY20-HRP antibody (1:1000).
After several washes in TTBS, reacted proteins were revealed by a BM
chemiluminescence system. In some experiments, blots were washed for 30
min at 50 C in stripping buffer (10 mmol/L Tris (pH 6.8), 1% SDS, 5
mmol/L ß-mercaptoethanol), to remove bound antiphosphotyrosine
antibodies, then immunostaining was performed by 3-h incubation with
H222 antibody (1:400 in 2% BSA-TTBS), followed by 1-h incubation
with antirat IgG-POD (1:4800 in 2% BSA-TTBS). Finally, the bands were
visualized by the BM system. The immunospecificity of PY20 was
determined by preadsorbing the antibody with 40 mmol/L
o-phospho-DL-tyrosine for 1 h at room temperature.
AR assay
Acrosome-reacted spermatozoa were evaluated using the fluorescent probe fluorescein isothiocyanate-labeled Arachis hypogea (peanut) lectin, according to Aitken et al. (26), as previously described (27). Briefly, after 2-h capacitation, spermatozoa (106/mL) were preincubated for 10 min with 17ßE2 at different concentrations and then stimulated with P (10 µmol/L), or appropriate control solvent (dimethyl sulfoxide) for 2 h at 37 C. After staining with fluorescent lectin, fluorescence was observed under a fluorescent microscope (Leitz, Type 307–148.002, Wetzlar, Germany), and AR was evaluated on a total of 100 spermatozoa/slide. According to Aitken et al. (26), only curly-tailed spermatozoa were considered viable and thus scored.
Analysis of experimental results
The computer program ALLFIT (28) was used for the analysis of sigmoidal dose-response curves obtained in calcium studies. Data are expressed as mean ± SEM. Statistical analysis was made with Student’s t test and one-way ANOVA.
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Results |
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Addition of 17ßE2 to fura-loaded spermatozoa
induced a rapid and sustained rise of [Ca2+]i
in a dose-dependent manner. Figure 1
reports the typical calcium waves in response to increasing
concentrations of 17ßE2 (0.1 nmol/L–100 µmol/L). The
dose-response curve for the calcium effect of 17ßE2, as
generated by the simultaneous computer analysis with the program ALLFIT
(28), is biphasic (Fig. 2), showing a
first component with an effective concentration 50 of 0.60
± 0.12 nmol/L and a second component with an EC50 of
3.80 ± 0.26 µmol/L. Also, because P stimulates a rapid calcium
influx in human spermatozoa with a similar biphasic dose-response curve
(11), and interactions between P and estrogen have been reported (19, 20), we tested the hypothesis of an eventual interference between the
effects of the two steroids. Interestingly, the shapes of P- and
17ßE2-induced calcium waves were different: P induced
first a rapid peak, followed by a long sustained plateau, whereas
17ßE2 induced a slow sustained response (Fig. 1). The
typical [Ca2+]i transient in response to P
(10 µmol/L) was reduced in a dose-dependent manner by a previous
administration of 17ßE2, both in the peak and plateau
components (Fig. 1, also see Table 1).
Table 1 reports the percentage peak and plateau
[Ca2+]i increases in response to P (10
µmol/L) alone or after previous administration of increasing
concentrations of 17ßE2. Inhibition of the plateau phase
was statistically significant for all the tested doses of estradiol,
whereas peak inhibition was statistically significant only for high
concentrations (Table 1). The effect of 17ßE2 was
specific for P-response, because the steroid did not affect
[Ca2+]i increase obtained after stimulation
with the endoplasmic Ca2+-ATPase inhibitor thapsigargin (10
µmol/L, Fig. 3), previously shown to
increase calcium levels (29, 30) and AR (31) in human spermatozoa.
Effects of 17ßE2, both on calcium levels and on calcium
response to P, were not antagonized by the cytosolic estrogen receptor
antagonist tamoxifen (not shown), suggesting that the classical
estrogen receptors are not involved. The effect of 17ßE2
seemed to be specific, because comparable concentrations of
17-estradiol (17E2), even at 10 µmol/L
concentration, neither stimulated [Ca2+]i
rise nor interfered with P-induced response (Fig. 4). To further demonstrate that the
effect of 17ßE2 on [Ca2+]i was
mediated by a receptor present on sperm membrane, we used the
membrane-impermeable estradiol conjugate BSA-E2. This
compound induced an [Ca2+]i increase similar
to that of 17ßE2 (Fig. 5),
whereas the addition of BSA alone, the macromolecular component of the
conjugate, was ineffective (not shown). BSA-E2 was also
able to mimic the inhibitory effect exerted by the free steroid on
P-induced calcium waves (Fig. 5). The biological effects of
BSA-E2 were observed until 0.1 µmol/L concentration was
achieved, which elicited an increase of basal
[Ca2+]i of about 1.16-fold (data not shown).
Taken together, all these data demonstrate that 17ßE2
acts through interaction with a surface receptor. The increase in
[Ca2+]i after addition of 17ßE2
was totally dependent on the presence of extracellular calcium, because
the response was absent when spermatozoa was stimulated in
calcium-depleted medium in the presence of 2 mmol/L EGTA, and it was
restored by subsequent replacement of external calcium to normal levels
(Fig. 6B). Similarly, the calcium wave
induced by BSA-E2 (1 µmol/L) was blunted in the absence
of extracellular calcium and was restored when
[Ca2+]e was replaced (Fig. 6C).
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Because a [Ca2+]i rise, induced by P,
leads to an increase in AR of human spermatozoa, we next investigated
whether 17ßE2 effects on calcium were also involved in
regulation of AR. As shown in Fig. 7, 2
-h incubation of capacitated
spermatozoa with increasing concentrations of 17ßE2
induced only a slight stimulation of AR at the highest dose used (10
µmol/L). Interestingly, all the three doses of 17ßE2
blunted AR in response to P (Fig. 7).
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To characterize 17ßE2-binding proteins in human
spermatozoa, we performed, on total sperm lysates, ligand blot
experiments using E2-POD as probe, and Western analysis
with the monoclonal antibody H222, directed against the
steroid-binding domain of the genomic receptor (32). E2-POD
has been shown to bind to a membrane estrogen receptor in pancreatic
islet cells (33), indicating that such molecule is a good tool to
investigate this type of receptor. H222 antibody has been shown to
recognize a membrane estrogen receptor in rat pituitary tumor cells
(34). Moreover, the approach of using an antibody produced against the
steroid binding sequence of the genomic receptor was applied by our
(11) and other groups (35, 36) to identify the putative membrane
receptors for P in human spermatozoa. In addition, preincubation of
sperm samples with H222 antibody (1:20, Fig. 8B), but not with normal rat serum (1:20,
Fig. 8C), reduced 17ßE2 stimulation of calcium influx
(Fig. 8A), suggesting that the sperm membrane receptor for estradiol is
recognized by this antibody. A single band, of approximately 29-kDa
molecular mass, is revealed both by E2-POD (0.5 µmol/L,
Fig. 9A) and H222 antibody (1:400,
Fig. 9B). An estrogen-binding protein of similar molecular mass has
been described also in other cell types (19, 20, 37). The same protein
band of 29 kDa was detected on purified sperm membranes stained with
H222 antibody (Fig. 9D). Longer exposures of the H222-stained
blots revealed the presence of two additional bands, of about 42–45
kDa and 54–58 kDa (Fig. 9E). A protein band, at the expected 54–58
kDa molecular mass range, probably corresponding to one of the known
isoforms of the genomic estrogen receptor (38, 39), was detected by
H222 both on myometrial cell and total uterus lysates, used as
control for genomic estrogen receptors (Fig. 10). Interestingly, myometrial cells
also show the presence of a 29-kDa protein band (Fig. 10).
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, A and B, with PY20
antibody revealed that this protein was phosphorylated on tyrosine
residues (Fig. 9C). Moreover, a rapid (10 min) stimulation of
capacitated spermatozoa with 17ßE2 induced an increase of
phosphorylation in tyrosine residues of several protein bands,
including the p29 kDa one (Fig. 11A). A
comigration between this tyrosine phosphorylated band (Fig. 11A) and
the putative estrogen receptor was observed when the blot was washed
thoroughly and reprobed with H222 antibody (Fig. 11B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H222 antibody as probes, reveal the
presence of a protein band with an apparent molecular mass of 29 kDa.
Location of this receptor on sperm surface is demonstrated both by the
ability of the impermeable conjugate E2-BSA to induce
similar calcium waves, as 17ßE2, as well as by the
detection of the 29-kDa protein band in purified sperm membranes by
Western analysis. The possible involvement of such a protein in the
biological effects of 17ßE2 is suggested by the
inhibition of 17ßE2-stimulated
[Ca2+]i increase by H222 antibody, which
probably competes with the steroid for the binding domain of the
membrane receptor. Similarly, Morey et al. (42) showed that
H222 antibody reverted the biological effects of estrogen in
vascular smooth muscle cells. A protein with a molecular mass of about
29 kDa, identified by photoaffinity labeling with
progesterone-11-hemisuccinate-(2-[125I)iodo-histamine),
and specifically displaced by incubation with estradiol, has been
detected in mouse brain membranes (19, 20) and has been suggested as
the putative membrane binding site for estrogen (20). Moreover, Monje
and Boland (37), using monoclonal antibodies against different domains
of the intracellular estrogen receptor, identified on uterine membranes
a 28- to 32-kDa protein, besides the expected 65-kDa band representing
one of the genomic receptors. Such molecular mass (29 kDa) is quite
different from the known classical and ß estrogen receptors (38, 39). Higher exposures of films in our Western blot analysis of sperm
lysates reveal that H222 antibody faintly detects two additional
sperm protein bands, the higher of which shows a molecular weight
similar to one of the classical genomic receptors. Although we have
used all the necessary precautions to minimize eventual protein
cleavage, the possibility that the 29-kDa protein band is a proteolytic
fragment of the full-length estrogen receptor cannot be excluded. Also,
it possible that a specific regulatory protein cleavage is involved in
synthesis of the functional estrogen receptor in spermatozoa. The fact
that other bands are seen with H222 antibody suggests this
possibility. On the other hand, a 66-kDa estrogen receptor that
comigrates with a similar protein in the endometrial tissue has been
detected, with a different antibody in human spermatozoa, by Durkee
et al. (15). However, these authors could not discriminate
whether this form represented the genomic receptor or not.
Interestingly, the same authors detected, by RT-PCR analysis of sperm
RNA, two different amplified nucleotidic bands (15), suggesting the
presence of different messenger RNAs for estrogen receptors in human
spermatozoa, as also described in other cell types (43, 44). So far,
the question of whether classical genomic estrogen receptors are
present in human spermatozoa still remains open. On the other hand, it
is unlikely that the genomic estrogen receptor, if present, could be
functional, because mature spermatozoa are transcriptionally silent.
Moreover, our experiments clearly show that the 29-kDa protein band is
the only one detected by both ligand and Western blot analyses,
strongly indicating that this protein represents the membrane receptor
for estrogen in human spermatozoa. The rapid increase of [Ca2+]i and phosphorylation induced by 17ßE2 in human spermatozoa confirms the findings in other cell types for nongenomic/rapid actions of estrogens (37, 45, 46, 47, 48, 49). As in the case of P (50), both 17ßE2- and BSA-E2-stimulated [Ca2+]i increases in spermatozoa are strictly dependent on the presence of extracellular calcium. Because this steroid is present in the follicular fluid (6, 7, 8) and in the male genital tract (12) at concentrations similar to those inducing the biological effects observed in vitro in human spermatozoa, it is conceivable that these effects may be physiologically relevant. The increase of calcium and tyrosine phosphorylation of proteins stimulated by 17ßE2 in human spermatozoa is not followed by induction of AR; rather, these effects interfere with those exerted by P. Indeed, a previous addition of 17ßE2 inhibits, in a dose-dependent manner, the subsequent calcium and AR responses to P. In particular, the plateau phase of P calcium response is significantly reduced after a first priming with very low concentrations of 17ßE2. Because the plateau phase of P-induced [Ca2+]i increase has been associated with induction of AR (50), it is conceivable that inhibition of P-stimulated AR by 17ßE2 is attributable to inhibition of the plateau phase. Stimulation of tyrosine phosphorylation of its own receptor may be involved in the modulation of receptor binding activity. Indeed, modulation of the phosphorylation state of the estrogen receptor by the steroid itself or other substances has been associated with transactivation of the classical genomic estrogen receptor (40, 41). In particular, tyrosine phosphorylation occurs in the ligand binding domain of the genomic receptor (52).
The precise mechanism involved in 17ßE2 inhibition of
calcium and AR response to P in human spermatozoa is still unclear.
Other groups reported rapid inhibitory effects of 17ßE2
on vascular smooth muscle contraction (53, 54, 55, 56) and on neuron
hyperpolarization (57). In particular, the rapid inhibitions of
coronary artery contraction [either basal (56) or induced by PG
F2, extracellular potassium (54), and endothelin (55)]
seem to be mediated by reduction of cellular calcium influx via
blockage of L-type Ca2+ channels (53). Lagrange et
al. (57) reported 17ßE2 reduction of µ-opioids’
ability to hyperpolarize guinea pig hypotalamic neurons via G
protein-coupled receptors. However, in all these cases, the inhibitory
effects of 17ßE2 are never associated with an increase of
calcium influx induced by the steroid itself, as in our experiments. It
is possible that the partial stimulation by 17ßE2 of the
same signal transduction pathways of P interferes with the biological
response to the latter, leading to inhibition of AR. However, the
possibility that 17ßE2 and P compete for the same
receptors cannot be excluded.
Interestingly, the sperm calcium curve, in response to 17ßE2, shows a biphasic behavior, with two components (one in the nanomolar and the other in the micromolar range), similar to the calcium curve obtained for P (11). Although this result may suggest the presence of two different binding sites for estradiol, we have constantly observed the presence of a single 29-kDa protein band in ligand blot experiments with E2-POD. On the other hand, binding of 3H-17ßE2 to intact human spermatozoa revealed the presence of a single binding site, with a an apparent Kd of 0.6 nmol/L (13), consistent with the first component of our curve. Similarly, the effect of 17ßE2 on P-induced AR and plateau phase of calcium increases was observed at nanomolar concentrations.
Inhibition of rapid responses to 17ßE2 by tamoxifen is controversial (45, 49, 58). Indeed, whereas Lantin-Hermoso et al. (58) described a complete inhibition by tamoxifen on estradiol acute stimulation of nitric oxide synthase activity in artery endothelium, Watters et al. (49) found no effect of tamoxifen on rapid membrane effects of estrogen in neuroblastoma cells. Moreover, Morley et al. (45) showed that tamoxifen could not affect the rapid estrogen-triggered [Ca2+]i increase in chicken granulosa cells. In our hands, this cytosolic estrogen receptor antagonist was ineffective in counteracting estradiol action on intracellular calcium, further suggesting that the estrogen receptor in spermatozoa differs from the genomic one.
In conclusion, our results demonstrate the presence of a biologically active sperm receptor for estrogen in human spermatozoa, suggesting a novel role for estradiol, in the process of fertilization, as a possible physiological modulator of P action on spermatozoa. Because levels of estradiol in the follicular fluid are similar to those inducing the observed nongenomic effects, the strict cross-talk between sperm membrane receptors for 17ßE2 and P may be important for an appropriate timing of capacitation and AR in the female genital tract. Further studies are required to elucidate whether environmental chemicals with estrogen action might have similar effects on human sperm and to evaluate whether the absence of sperm response to P in several cases of idiopathic male infertility (27, 59) may be attributable to alteration in the interactions between these two steroids.
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Acknowledgments |
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H222 and for helpful suggestions. We are indebted to Prof.
Mario Serio (Unità di Endocrinologia, Università di
Firenze) and Dr. M. Maggi (Unità di Andrologia, Università
di Firenze) for critical reading of the manuscript. We thank Prof.
Gianna Fiorelli (Unità Endocrinologia, Università di
Firenze) for helpful advice. Received December 1, 1998.
Revised January 19, 1999.
Accepted February 4, 1999.
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identifies four protein bands in mouse brain membranes. J
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in mouse brain membranes. J Steroid Biochem Mol Biol. 58:89–94.[CrossRef][Medline]
in vivo and in
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