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Unité de Recherches, INSERM, U-135, Hormones, Gènes, et Reproduction, Hôpital Bicêtre (G.M., N.C., B.V., H.L., E.M.), 94275 Le Kremlin-Bicêtre, France; INRA, Center de Recherches de Tours (M.-A.D.), 37380 Nouzilly, France; Unité de Recherches, INSERM, U-274, Université Paris-Sud (L.C.), 91400 Orsay, France; and Institut Mutualiste de Montsouris, Laboratoire de Procréation Médicalement Assistée (P.G.), 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. Edwin Milgrom, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. E-mail: . u135{at}kb.inserm.fr
Abstract
The regulatory mechanisms of oocyte maturation remain poorly understood. Although gonadotropins play a major role in these processes, they have generally been considered to act on somatic supportive cells, but not directly on germ cells. We have raised high affinity monoclonal antibodies against LH and FSH receptors. When using the latter to study receptor distribution in human and pig ovaries we have observed the presence of FSH (but not LH) receptors in the oocytes. FSH receptors appeared in the oocytes of primary follicles during follicular development and persisted up to the preovulatory stage. In denuded human preovulatory oocytes, FSH receptor mRNA was detected at a concentration per cell exceeding by about 20-fold that present in granulosa cells. Saturable binding of [125I]FSH to the membrane of oocytes was demonstrated by autoradiography. When incubated with FSH, denuded oocytes responded by a mobilization of Ca2+. These observations concur to demonstrate the presence of functional FSH receptors in oocytes and raise the possibility of direct control of oocyte development by FSH.
THE MECHANISMS of maturation of germinal cells have always been the subject of great scientific interest. In the case of the oocyte, arrest and resumption of meiosis as well as acquisition of the competence to become fertilized have been specially investigated (1, 2, 3). Recent progress in assisted reproduction has increased interest in these mechanisms. Cloning by nuclear transfer objectifies the ability of the oocytic cytoplasm to set off a program of embryonic development even in differentiated nuclei (4). Culture of oocytes to maturity is an emerging technology that may transform the practice of in vitro fertilization (IVF) (5, 6).
In all cases the regulatory mechanisms of oocyte maturation remain poorly understood. Gonadotropins play a very important role and are supposed to act on somatic supportive cells and not directly on germ cells (7, 8). The oocyte develops inside the ovarian follicle, enclosed by granulosa cells. The latter are separated by a basement membrane from layers of thecal cells. FSH is known to act on granulosa cells, whereas LH has mainly a steroidogenic activity in thecal cells. LH receptors (LHR) are present in granulosa cells only at later stages of development, in large antral follicles. These conclusions derive mainly from experiments involving stimulation by gonadotropins of isolated cells or from analysis of receptor distribution using radioactive gonadotropins and autoradiography (9, 10). Recently, RT-PCR and in situ hybridization methods allowed the study of gonadotropin receptor mRNAs (11, 12, 13, 14, 15). However, several of these methods did not allow very precise localization or were at the limit of their sensitivity due to the low concentration of receptors. We have raised high affinity monoclonal antibodies against LHR and FSH receptors (FSHR) (16, 17, 18). Using the latter to study receptor distribution in human and pig ovaries, we have observed the presence of FSH (but not LH) receptors in the oocytes. This observation, which was confirmed by the presence of receptor mRNA in denuded human preovulatory oocytes as well as autoradiography and Ca2+ mobilization studies, raises the possibility of direct control of oocyte development by FSH.
Materials and Methods
Ovaries and oocytes
Porcine ovaries were obtained from 20 adult sows. Human ovaries originated from tissues discarded after hysterectomy for benign uterine pathology of 9 cycling women (37–43 yr old). Tissue specimens were frozen in liquid nitrogen. Oocytes were retrieved from 6 preovulatory sows. For RNA studies porcine oocytes were mechanically denuded of granulosa cells. Human oocytes were obtained from an IVF and an intracytoplasmic sperm injection (ICSI) program. For ethical reasons only the oocytes that failed to fertilize were used in this study. IVF oocytes were denuded by repeated passages through a fine needle pipette; ICSI oocytes were denuded by a 25-sec hyaluronidase treatment (80 IU/ml). Microscopic examination allowed the retention of only oocytes devoid of granulosa cell contamination. Oocytes were pooled and frozen in liquid nitrogen for RNA extraction. For immunohistochemistry, autoradiography, or calcium measurements, oocytes were placed in 1 ml IVF medium (Medi-Cult, Jyllinge, Denmark) under 5% CO2.
Human granulosa cells were collected during oocyte retrieval. After gradient Percoll centrifugation and washing, the cells were counted and frozen as aliquots.
Immunocytochemistry
Monoclonal antihuman FSHR 323 and 156 and anti-LHR 38 antibodies have been previously described, and their specificities were extensively studied (17, 18, 19). They recognize the extracellular domain of FSHR and LHR, respectively. These antibodies have also been used in a variety of immunocytochemical, immunopurification, and immunoprecipitation studies (17, 18, 20, 21, 22, 23, 24, 25). Frozen sections and whole fixed oocytes were immunolabeled as previously described (17, 18, 20). In the present experiments controls included replacement of the specific antibodies with nonimmune mouse Igs (Sigma, St. Louis, MO) or with anti-FSHR antibodies preincubated overnight at 4 C with a 100-fold molar excess of FSHR ectodomain expressed in Escherichia coli (17). Unfixed oocytes were immunolabeled by successive micropipetting onto petri dishes filled with 200 µl of the same reagents as those used for immunolabeling of sections. The oocytes were deposed on sylanized slides.
Autoradiography
Human denuded oocytes were washed in DMEM and incubated at 37 C for 90 min at room temperature in 0.2 ml 0.05 M sodium phosphate, 0.15 M NaCl, 0.01 M CaCl2, and 0.01 M EDTA buffer, pH 7.4, containing 0.1% sodium azide and 2.25% fatty acid-free BSA (Sigma). Oocytes were incubated with 10-8 M [125I]hFSH (SA, 80 µCi/µg) or 10-8 M [125I]hCG (SA, 80 µCi/µg; NEN Life Science Products, Boston, MA). Other oocytes were incubated with [125I]hFSH or [125I]hCG in the presence of an excess of unlabeled hormone [10-6 M Metrodin HP (Serono, Braintree, MA) or 10-6 M hCG (Endo, Organon, West Orange, NJ)]. After five washes the oocytes were placed on slides precoated with an emulsion for microautoradiography (LM1, Amersham Pharmacia Biotech, Arlington Heights, IL). The slides were revealed (9) after 1 wk and examined by Nomarski differential interference contrast microscopy. Similar experiments were performed on granulosa cells.
FSHR and LHR mRNA
The absence of granulosa cells was verified individually for every denuded oocyte by microscopic examination. RNA was extracted from the pooled oocytes (and in a parallel experiment from varying numbers of granulosa cells) with the RNAeasy mini kit (QIAGEN, Chatsworth, CA). Half of the extract was used for RT with random primers (Life Technologies, Inc., Gaithersburg, MD; 20 pmol/20 µl reaction) and Superscript RNase H- reverse transcriptase (Life Technologies, Inc.; 100 IU/reaction). Ten microliters of the cDNA mixture were used for PCR (total volume, 50 µl). The following synthetic FSHR primers were used: either nucleotides 202–223 (exon 2) and 559–583 (exon 7) or nucleotides 568–592 (exon 7) and 909–933 (exon 10; nucleotides are numbered from the initiation ATG codon). The following LHR primers were used: nucleotides 170–206 (exon 2) and 336–360 (exon 4). After 31 cycles of amplification, 30 µl were electrophoresed in 3% agarose and analyzed by Southern blot using an internal 32P-labeled oligonucleotide probe (105 cpm/ml). The probe corresponded to either nucleotides 343–366 (exon 4) or nucleotides 790–813 (exon 9) for the FSHR and to nucleotides 273–298 (exon 4) for the LHR. Human muscle RNA was used as a negative control.
FSH effect on intracellular Ca2+
Denuded human oocytes were incubated in IVF medium (Medicult) containing 3 µM fura-2/AM, then plated onto polylysine-coated glass coverslips and incubated for 30 min at 37 C under an atmosphere containing 5% CO2. In some experiments granulosa cells collected from patients in an IVF program were harvested from the follicular aspirate, seeded onto circular glass polylysine-coated coverslips, and challenged with fura-2 for 30 min at 37 C. In all cases the coverslips were then washed with saline solution. They were put onto a thermostated holder (37 C) on the stage of a Axiovert 35 microscope (Carl Zeiss, New York, NY). Ca2+ imaging was performed as described by Combettes et al. (26). Briefly, fluorescence images were collected by a low light level ISIT camera (Lhesa, France), digitized, and integrated in real time by an image processor (Metafluor, Princeton, NJ).
Calibration of fura-2 fluorescence in terms of the Ca2+ concentration was performed in individual oocytes and calculated from the ratio of 340/380 nm fluorescence as described by Grynkiewicz et al. (27) using a Kd (Ca2+-dye) of 250 nM. The ratios Rmax and Rmin were determined as previously described (26). The oocytes or granulosa cells were continuously superfused at a rate or 1.5 ml/min with the test solution or with the control buffer.
For experiments using gap junction inhibitors, oocytes were incubated with 1-octanol (1 mM) or 18
-glycyrrhetinic acid (250 µM) for 10 min and then with recombinant FSH (100 mIU/ml).
Results
Immunostaining of FSHR in pig oocytes
During a study of FSHR distribution in pig ovaries we observed strong immunostaining in oocytes. Figure 1
, a and b, shows sections of a preantral follicle reacted with anti-FSHR 156 monoclonal antibody. The zona pellucida clearly separates the oocyte from the granulosa cells; both are strongly labeled. Immunostaining is present on the cell membrane and also inside the cells, as previously observed for gonadotropin receptors. This probably corresponds to the receptor precursor that accumulates in the cells (17, 19). A similar labeling was observed when using another monoclonal anti-FSHR antibody (antibody 323) recognizing a different epitope on the ectodomain of the receptor (not shown). As a control for staining specificity by FSHR 156 antibody, adjacent sections were reacted with a nonreceptor-related monoclonal antibody of the same class (IgG1) and at the same concentration. No labeling was observed (Fig. 1c). Another control involved preincubation of the antireceptor monoclonal antibody with purified FSH receptor ectodomain expressed in Escherichia coli (Fig. 1e). The immunostaining was suppressed (compared with control in Fig. 1d). When the anti-FSH receptor antibody was incubated with LHR ectodomain expressed in E. coli, there was no decrease in immunostaining (not shown). As is often the case for very rare proteins, immunostaining for gonadotropin receptors could only be studied on frozen sections (28, 29). For this reason the morphology of the oocytes was not as good as in Formol-fixed paraffin-embedded sections. However, the immunolabeling of oocyte sections or of the surface of intact oocytes was very clear-cut.
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Forty pig ovaries were analyzed, and 219 different follicles containing oocytes were observed. Primordial follicles, showing an oocyte surrounded by a squamous layer of pregranulosa cells, were never labeled by anti-FSHR antibodies (Fig. 2a). Follicles intermediary between primordial and primary stages with both squamous and cubical granulosa cells showed faint immunostaining. The boundaries between the oolemma and the surrounding cells were not clearly distinguishable. In most follicles immunostaining was present at this stage, but it was difficult to determine whether it was associated with the oocyte or the granulosa cells. In most cases granulosa cells were not labeled on the membrane regions outside those contacting the oocyte. The majority (91%) of primary follicles (Fig. 2b) were labeled with the anti-FSHR antibody. The immunostaining was moderately intense. The immunolabeled oocyte was surrounded by cubical granulosa cells whose immunostaining was no longer restricted to the areas in contact with the oolemma, but included the whole cellular membrane. All secondary follicles were immunolabeled, most of them strongly. With increasing size of the follicle, the development of the zona pellucida allowed clear delineation of immunostaining of the oocyte from that of granulosa cells (Fig. 2c). The number of antral follicles containing oocytes that could be observed was smaller. Indeed, at this stage the oocytes that were weakly attached to the follicle were frequently washed out during the various incubation and washing procedures. Twelve oocytes could be examined, present in apparently healthy follicles. Ten were stained with anti-FSHR antibodies (Fig. 2d); two were not labeled. The LHR antigen was absent in the oocytes at all stages of follicular development. Figure 2e shows an antral follicle in which only the theca interna was labeled by anti-LHR antibodies and the oocyte was not immunostained.
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The study of human ovaries was less extensive due to the limited availability of tissues from normal cycling women. However, the observed patterns were very similar to those seen in pig ovaries. Primordial follicles were never stained (not shown), whereas primary follicles were immunolabeled by anti-FSHR antibody (Fig. 3a). The aspect was very similar to that observed at the same stage in the pig: overall oocyte staining contrasted with granulosa cells displaying staining limited to the membrane bordering the oocyte. Secondary follicles of all sizes were also labeled (Fig. 3b). The presence of the pellucida allowed us to clearly delineate the immunostaining of the oocyte from that of the granulosa cells. Preovulatory oocytes obtained during IVF procedures also reacted with anti-FSHR antibodies. Figure 3c shows a section of such an oocyte stained with anti-FSH receptor antibody 323, whereas Fig. 3e shows the surface labeling of an intact nonsectioned oocyte. Control experiments involved the absence of labeling with a nonreceptor-related IgG2a monoclonal antibody (Fig. 3d) and with antibody 323 previously preincubated with an excess of FSH receptor ectodomain synthesized in E. coli (not shown). The preovulatory oocytes were not stained with anti-LHR antibodies (Fig. 3f).
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Several oocytes were observed inside atretic follicles in both pig and human ovaries (Fig. 4). A variety of situations were observed. In some cases oocyte staining disappeared, whereas granulosa cells remained labeled. In other cases the opposite situation was seen; oocytes were labeled, but there was no more FSHR staining in the granulosa cells. In some follicles FSHR immunostaining was suppressed in both oocyte and granulosa. Finally, in the rare cases where FSH immunolabeling persisted in both granulosa and oocyte, it was usually diminished in either one of these structures.
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To confirm the presence of FSHR in the oocytes we used RT-PCR to detect the mRNA. We obtained human oocytes from an IVF program. For ethical reasons only oocytes that failed to develop after IVF or ICSI were used. In all cases they were individually examined and retained only if they were totally devoid of attached granulosa cells. RNA was extracted from 60 oocytes, transcribed into cDNA with random primers, and amplified using primers corresponding to exons 7 and 10 of the receptor gene. A Southern blot was performed using a DNA corresponding to exon 9 as a probe. In parallel, RNA was extracted from various numbers (ranging from 60–5000) of granulosa cells and identically submitted to RT-PCR. As shown in Fig. 5A, the signal given by 60 oocytes corresponded approximately to the signal given by 1200 granulosa cells. Similar results were obtained using primers corresponding to exons 2 and 7 of the human FSHR gene (probe in exon 4). On the contrary, there was no signal for LHR mRNA in oocytes in similar conditions, whereas LHR mRNA was readily detected in granulosa cells (Fig. 5B).
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) was also sequenced and shown to correspond to a splicing variant (addition of 102 intronic nucleotides at the end of exon 8). A parallel experiment using the mRNA of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) (31) showed a similar ratio of mRNA per cell between the oocyte and the granulosa cells (Fig. 5C). FSHR mRNA was also detected in denuded porcine oocytes (data not shown). [125I]FSH binding to human oocytes
The binding of FSH to oocytes could not be studied using biochemical methods, because this would have necessitated using thousands of oocytes. We thus used autoradiography, which allows determination of hormone binding to individual oocytes. Oocytes were incubated with either [125I]FSH or [125I]FSH in the presence of an excess of unlabeled hormone. After extensive washing an autoradiographic film was applied. As shown in Fig. 6A, the granulosa-free oocytes bound [125I]FSH, and this binding was saturable because it was competed out by unlabeled FSH (Fig. 6B). A positive control involved granulosa cells and gave a similar result (Fig. 6D). On the contrary, [125I]hCG did not bind to oocytes (Fig. 6C).
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FSHR are coupled to adenylate cyclase and also to PLC (32, 33, 34, 35). The latter is known to modulate Ca2+ mobilization, which may be analyzed in individual cells. Such studies could be performed even with the limited number of oocytes available. Human oocytes were loaded with fura-2, washed for 10 min with saline, and then challenged with 100 mIU/ml recombinant (Puregon Organon) or purified (Metrodin HP) human FSH. The basal intracellular calcium Ca2+ concentration in the oocytes was 47 ± 4 nM. Addition of Metrodin HP induced a 5-fold increase in the intracellular Ca2+ concentration to 250 ± 10 nM (mean ± SEM of 11 experiments). The concentration returned spontaneously to the basal level within 3–4 min. Delivery of control medium had no effect on Ca2+. The same pattern of response was observed with recombinant FSH, excluding the possibility of contamination with nonrelevant proteins. This Ca2+ increase was observed in 54% of the studied oocytes (n = 48). This may be due to alteration of some of these oocytes that were studied 48 h after recovery and were failures of IVF or ICSI. Oocytes that failed to respond to FSH also gave a minimal response to thimerosal (200 µM), which has been shown to provoke a large Ca2+ increase in oocytes by direct mobilization of Ca2+ stores (36). The studied oocytes were totally devoid of contaminating granulosa cells when examined by a variety of microscopic methods. Further evidence for the absence of granulosa cells was obtained from the following experiment. Human granulosa cells were loaded with fura-2 and challenged with 10 µM ATP. An oscillatory intracellular calcium response was observed (data not shown), confirming previous reports (37). In contrast, FSH-responding oocytes did not respond to ATP (Fig. 7). These oocytes also failed to respond to hCG (500 mIU/ml).
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-glycyrrhetinic acid (39). For each compound three experiments were performed, and in all cases the Ca2+ response was not modified (Fig. 7). These experiments as well as microscopic examination, immunocytochemistry, autoradiography, and RT-PCR showed no evidence of contamination by granulosa cells. Discussion
Immunocytochemical studies showed the presence of FSHR in both pig and human oocytes. These observations contradict the presently accepted dogma. It was thus very important to eliminate the possibility of artifacts. We have very carefully examined the specificity of the antibodies used for these studies and also the specificity of oocyte immunostaining. The monoclonal anti-FSHR antibodies have been prepared by immunization of mice with recombinant FSHR obtained in E. coli. Their characterization has been described in detail (17). That these antibodies did indeed recognize the FSHR was shown by their ability to immunoprecipitate [125I]FSHR complexes and to specifically detect the receptor in Western blot experiments (17). The antibodies allowed immunopurification of the receptor (17) and immunostained cells transfected with expression vectors encoding FSHR (22, 23). No staining was seen on parent nontransfected or mock-transfected cells. The monoclonal antibodies labeled, as expected, Sertoli cells in the testis (17) and granulosa cells in the ovary (24). In addition to these previously published controls of the specificity of the monoclonal antibodies, some further controls were performed in the present work. Immunostaining was similar on adjacent sections of oocytes incubated with two monoclonal antibodies recognizing two different epitopes on the FSHR. There was no immunostaining with control nonrelated monoclonal antibodies of the same class and at the same concentration. The immunostaining of the oocytes was extinguished after preincubation with recombinant FSHR, but not with recombinant LHR.
The presence of FSHR and its mRNA in oocytes was confirmed by autoradiography, RT-PCR, and Ca2+ mobilization studies. These results were not due to oocyte contamination by granulosa cells, as all of the oocytes were denuded and verified individually by light microscopy. In some experiments the absence of contamination was confirmed by using semithin sections and electron microscopy. RT-PCR showed a 20-fold excess of FSHR mRNA per cell in oocytes compared with granulosa cells. The contamination by granulosa cells should thus have been massive (but invisible with microscopy) to explain our results. Autoradiography showed saturable binding of [125I]FSH to the entire surface of the oocytes and was not restricted to some regions, as would have been the case if the binding was due to contaminating adherent granulosa cells. In Ca2+ mobilization studies the oocytes were loaded with fura-2 and examined for fluorescence. Fluorescent granulosa cells would have been obvious in these conditions. Finally, the Ca2+ response to ATP was different in oocytes and granulosa cells, and gap junction inhibitors did not prevent FSH-induced Ca2+ mobilization in the oocytes.
Ovarian follicular development is known to proceed to primordial and primary stages independently of the action of FSH. This has been observed in mice carrying invalidations of the FSHß and FSHR genes (40, 41) and also in patients with mutations suppressing the function of the FSHR (24, 25, 42). Increasing FSH concentrations seem, on the contrary, necessary for further stages of development (43, 44). Although a minority of previous studies have given some hints of the existence of FSH-binding sites in the oocytes (45, 46) and also in spermatogonia (47, 48), their methodology could not demonstrate this unequivocally. Furthermore, a majority of studies showed FSH binding or FSHR mRNA only in the granulosa (15, 49, 50, 51). This has led to a consensus that this hormone would act only on granulosa cells (52). Oocyte development was considered secondary to these effects and due to diffusable substances, possibly passing through gap junctions between granulosa cells and the oocyte. However, it is known that follicles enucleated from their oocytes fail to develop even in presence of FSH (53). It is thus possible that two-way exchanges occur and that a direct action of FSH on oocytes produces compounds whose diffusion into the granulosa cells is necessary for their proliferation and maturation. In atretic follicles we observed that FSHR are lost (or markedly decreased) in the oocyte, the granulosa layers, or both. This may be a phenomenon secondary to atresia. Alternatively, it may be its primum movens, with the lack of activity of FSH in either the oocyte or the granulosa inducing atresia. It is thus possible that FSH must act in both cell types to promote follicular growth and development. During development of the human ovarian follicle, the oocyte undergoes a 300-fold size increase. The number of organelles (mitochondria, Golgi, ribosomes, cortical granules, etc.) increases dramatically (54). A variety of RNA and protein molecules are synthesized and stored (55). The oocyte is blocked in the diplotene stage of prophase I of the first meiotic division. The block will only be relieved at a later stage, secondarily to the LH peak (6). It is during this growth period that oocytes acquire the ability to undergo meiosis (meiotic competence). It is still largely unknown which of these events is FSH dependent and what are the underlying molecular mechanisms occurring in granulosa cells, oocytes, or both.
Acknowledgments
We thank Dr. D. Pathier, C. Cheramy, and C. Pierre for their help. V. Coquendeau and A. D. Dakhlia typed the manuscript.
Footnotes
This work was supported by INSERM, the Faculté de Medecine Paris Sud, the Assistance Publique-Hôpitaux de Paris, and the Association pour la Recherche sur le Cancer.
1 G.M. and N.C. contributed equally to the work. 
Abbreviations: FSHR, FSH receptor; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; LHR, LH receptor.
Received December 7, 2001.
Accepted January 17, 2002.
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