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CLINICAL CASE SEMINAR |
INSERM E120 (O.L., A.D., I.B., M.M.) and Laboratoire d’Hormonologie et Biologie Moléculaire (G.M., M.M.), Hôpital Bicêtre, 94275 Le Kremlin Bicêtre, France; and Service d’Endocrinologie et Maladies de la Reproduction (P.T., F.K.), Hôpital Necker; 75015 Paris; Service d’Anatomopathologie (M.C. V.L.), Hôpital Cochin, 75014 Paris, France
Address all correspondence and requests for reprints to: Prof. Micheline Misrahi, INSERM E120, Bat. Gregory Pincus Hôpital Bicêtre, 94275 Le Kremlin-Bicêtre, France. E-mail: micheline.misrahi{at}bct.ap-hop-paris.fr.
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Abstract |
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Introduction |
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The first phenotype of premature ovarian failure (POF) associated with a mutation of the FSHR was described in the Finnish population (4). The patients had a severe phenotype with primary amenorrhea, variable development of secondary sex characteristics, high serum levels of FSH, and streak or hypoplastic ovaries. Histological examination of the ovaries allowed the detection of follicles. However, a block in follicular maturation was observed at early stages (4, 5). The same natural homozygous mutation of the receptor, Ala189Val, was found in all affected women. This mutation markedly impaired FSHR function in vitro.
Until recently this mutation or other mutations of the receptor have not been detected in women presenting the same phenotype in various countries (6, 7). This suggests that other etiologies are much more frequent and that the Ala189Val mutation of the FSHR is particularly prevalent in the Finnish population because of a founder effect and subsequent consanguinity. Very recently, another Finnish patient with POF was found to display two heterozygous mutations of the FSHR; a novel Ala419Thr mutation was found associated with the Ala189Val mutation previously described (8). The patient had primary amenorrhea and normal female secondary sexual characteristics. No histological study of the ovaries of the patient was performed. Therefore, complementary comparative studies, including clinical, histological, immunocytochemical, and molecular analyses, are needed to better understand the role of the FSHR during the early steps of folliculogenesis.
We have subsequently described a second partial phenotype of FSH resistance syndrome in two patients (9, 10). The characteristic feature was the existence of normal-sized ovaries in women presenting high serum levels of FSH. The two patients had, respectively, secondary or primary amenorrhea with normal puberty. Histological and immunocytochemical examination of the ovaries showed a normal follicular development up to the small antral stage and a disruption at further stages. The two patients were compound heterozygotes for mutations of the FSHR. These mutations impaired, but did not abolish, receptor function in vitro for one mutated allele: the Leu601Val receptor mutant for one patient and the Arg573Cys receptor mutant for the second patient, exhibiting, respectively, approximately 12% and 24% residual function. Interestingly, there was a correlation between the residual activity of the mutated receptors and the severity of the clinical, biological, and histological phenotypes of the patients.
We describe here a novel homozygous Pro519Thr mutation of the FSHR in a patient presenting with delayed puberty and primary amenorrhea, high plasma FSH levels, and small ovaries. This mutation, located in the second extracellular loop of the receptor, totally impairs receptor function through an altered expression of the mutated receptor at the cell membrane. This residue is thus critical for cell surface targeting of the FSHR. Furthermore, detailed histological and immunohistochemical studies of the ovary provide an opportunity to further analyze the role of the FSHR in the early steps of folliculogenesis.
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Subjects and Methods |
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The proband is a 26-yr-old Caucasian woman who was referred to Necker Hospital for infertility. This patient reported delayed puberty and primary amenorrhea. At the age of 17 yr she began taking an oral contraceptive pill (0.03 mg ethinyl estradiol and 0.15 mg desogestrel), leading to a complete development of secondary sexual characteristics, but she discontinued this treatment when she was 25 yr old because of her desire for pregnancy. She was referred to our department 18 months later. No data are available concerning her family because she was an adopted child.
The patient’s height is 172 cm, and weight is 54 kg, with a body mass index of 18.3. Physical examination revealed normal breast development and normal axillary and pelvic hair without morphological abnormalities. The hormonal evaluations in our department, performed by RIA, revealed high plasma FSH and LH concentrations: FSH, 67 IU/liter (normal range, 1.0–9.0 IU/liter); LH, 21 IU/liter (normal range, 1.4–12 IU/liter); and very low estradiol levels, less than 10 pg/ml (33 pmol/liter; normal range, 20–300 pg/ml). The plasma testosterone level was 0.36 ng/ml (1.2 nmol/liter; normal range, 0.2–0.6 ng/ml), 
4-androstenedione was 2.8 ng/ml (8.4 nmol/liter; normal range, 1.2–1.6 ng/ml), and dehydroepiandrosterone was 14 ng/ml (48.1 nmol/liter; normal range, 2–12 ng/ml). The plasma SHBG level was 85 nmol/liter (normal range, 30–69 nmol/liter). The plasma concentration of inhibin B was markedly low (<10 pg/ml). Thyroid function and plasma PRL levels were normal. No antithyroid or antiovarian autoantibodies could be detected. The karyotype was normal (46,XX). The bone mineral index showed marked osteoporosis at the vertebral site (-2.56 SD; t-score) and osteopenia at the femoral neck (-1.62 SD; t-score). Transvaginal pelvic ultrasonography showed a small uterus (50 x 27 x 17 mm). The two ovaries appeared hypoplastic (0.22 ml for both ovaries), with no evidence of follicular-like structures. A pelvic laparoscopy was performed, revealing a uterus in normal situation, normal right and left fallopian ducts, and small ovaries. One biopsy was performed on each ovary.
Ovarian stimulation by increasing doses of recombinant FSH (Puregon R, Organon, Puteaux, France) was performed after 1 month of treatment with an oral combined estrogen-progestin pill (50 µg ethinyl estradiol and 500 µg norgestrel). It was monitored by hormone assays and pelvic ultrasonography. The total dose of FSH was 10,200 IU. However, after 21 d of stimulation, no follicular development was observed, and plasma estradiol and inhibin B remained at very low levels (<15 and <10 pg/ml, respectively).
The study was approved by the review boards of the different institutions. Informed consent was obtained from the patient and her family.
DNA sequencing
DNA was extracted from peripheral blood leukocytes. The total coding region of the human FSHR gene was sequenced as previously described (9). Genomic sequencing was performed using a Taq dideoxyterminator cycle sequencing kit and a 373A automated sequencer (PE Applied Biosystems, Foster City, CA).
Construction of an expression vector encoding the mutated FSHR
The human FSHR cDNA cloned into the pSG5 expression vector (pSG5-hFSHR) has been described previously (11). The mutation was introduced into the pSG5-hFSHR plasmid vector by oligonucleotide-mediated mutagenesis using QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
The Pro519Thr substitution was engineered with two mutagenic primers: primer A, 5'-GGTGAGCATCTGCCTGACCATGGATATTGAC-3' starting at position 1539 of the cDNA sequence, +1 corresponding to the first nucleotide of the initiation codon; and the reverse primer B, 5'-GTCAATATCCATGGTCAGGCAGATGCTCACC-3'. The mutated base is underlined. The construct was verified by double-strand sequencing.
Transfection of wild-type and mutated FSHRs in COS-7 cells
Wild-type and mutated FSHRs were transiently transfected in COS-7 cells using Superfect (Qiagen, Chatsworth, CA) as previously described (9). The expression of total wild-type and mutated receptors in transfected cells was quantified with an immunoenzymatic assay of the FSHR using two additive monoclonal antibodies as previously described (11). Antibody FSHR18 was coated onto 96-well plates (Maxisorb, Nunc, Naperville, IL) in 50 mM potassium phosphate (pH 7.4) for 16 h at 4 C. After washing, the plates were saturated with PBS containing 0.1% Tween and 1% BSA for 1 h at room temperature. Cells were pelleted, and a total membrane extract was prepared. The extract (11) containing the receptor was added at various dilutions in PBS containing 0.1% Tween and 1% BSA and incubated for 2 h at room temperature. After three washes with PBS, the plates were incubated with biotinylated antibody FSHR323 (1 µg/ml) at room temperature for 1 h. After washing, the plates were incubated with streptavidin-peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL) at a 1:1000 dilution for 1 h at room temperature. The immunocomplexes were colored with 2,2'-Azine-di[3-ethylbenzthiazoline sulfonate] and the OD was read at 492 nm. The concentration of the receptor was measured by reference to known concentrations of FSHR fusion proteins purified from Escherichia coli (11).
Immunofluorescence and confocal microscopy
Indirect immunofluorescence was performed as described previously (9, 10) on transfected COS-7 cells grown on coverslips using antibody FSHR323. This antibody recognizes an epitope located in the extracellular domain of the FSHR (11) and enables study of receptor expression at the cell membrane. Cells were chilled at 4 C and incubated for 1 h in PBS containing 1% BSA and 5 µg/ml FSHR323. The cells were washed and fixed for 15 min in 3% paraformaldehyde in PBS. After washing, the aldehyde groups were quenched with 50 mM NH4Cl in PBS for 30 min. After 1-h saturation with PBS/1% BSA, cells were incubated for 1 h with Alexa Fluor 488-goat antimouse immunoglobulin G (Molecular Probes, Leiden, The Netherlands). The cells were washed and mounted in PBS/70% glycerol. In some experiments the cells were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.075% saponin in PBS/1% BSA (9). The cells were then incubated at room temperature for 2 h with antibody FSHR323 and further processed as described above. An Axiovert 135M microscope (Carl Zeiss, Thornwood, NY) was used in conjunction with a confocal laser scanning unit (Zeiss LSM410) (9).
Functional assays
COS-7 cells in six-well plates were transfected with 2 µg plasmid as previously described (9). The accumulation of cAMP was measured as previously described (9) after 45-min incubation of the transfected COS-7 cells with increasing concentrations of recombinant FSH (0–3000 IU/liter; Gonal-F, Serono Laboratories, Boulogne-Billancourt, France).
FSH binding was performed as previously described (9, 10). Briefly, cells transfected with wild-type or mutated receptor were incubated for 1 h at 30 C with 400,000 cpm/ml radioiodinated FSH (Perkin-Elmer Life Sciences, Boston, MA; specific activity, 135 µCi/µg) in the absence or presence of increasing concentrations of unlabeled recombinant FSH (0–30 nM). All experiments were reproduced at least twice with triplicate samples.
Histological and immunocytochemical studies of the ovary
The ovarian biopsy of the patient was fixed in buffered formol and embedded in paraffin. Histological examination was performed with a conventional optical microscope (Provis, Olympus Optical Co., Tokyo, Japan) on 5-µm thick tissue sections stained with hematoxylin-eosin and Masson’s Trichrome stains. The numbers of primordial, intermediary, and primary follicles per square surface unit were counted on the whole section with an image analysis system (Microvision Instruments, Evry, France) connected to a conventional optical microscope (Leitz) as previously described (12). Immunohistochemistry was performed as follows. Briefly, serial tissue sections were deparaffinized by successive baths in xylene and alcohol, and antigen retrieval was performed before immunohistochemistry in a commercial microwave oven at full power in pH-6 citrate buffer for 15 min. The sections were then incubated overnight with the primary antibodies (polyclonal antibodies anti-P450 side-chain cleavage (anti-P450scc; dilution, 1:3000) (13), anti-P450c17
(14) (dilution, 1:5000), antiaromatase (P450arom; Hauptman-Woodward Medical Research Institute, Inc., Buffalo, NY; dilution, 1:3000), antisteroidogenic factor 1 (anti-SF1; Santa Cruz Biotechnology, Santa Cruz, CA; dilution, 1:100), anti-c-Kit (Santa Cruz Biotechnology; dilution, 1:400), and the monoclonal antibody antiproliferation cellular nuclear antigen (anti-PCNA; clone PC10, Dako, Carpinteria, CA; dilution, 1:200) at 4 C in a humid chamber. After primary antibody incubation, endogenous peroxidases were quenched with 3% H2O2 in PBS (pH 7.4) for 5 min, and the bound antibodies were revealed with a secondary biotinylated antibody and peroxidase-labeled streptavidin (LSAB2 immunostaining kit, Dako Corp., Carpinteria, CA) according to the manufacturer’s instructions. Aminoethylcarbazol (Sigma-Aldrich Corp., St. Quentin-Fallavier, France) was used as a chromogen. The sections were counterstained with Mayer’s hematoxylin. Sections of a biopsy of a normal ovary from an age-matched woman were used as controls.
Terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL; Oncogene, Cambridge, UK) was performed according to the manufacturer’s instructions. The chromogen used was aminoethylcarbazol. Each experiment was performed at least twice in duplicate on nonadjacent sections.
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Results |
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Sequencing of the complete coding region of the human FSHR gene allowed detection of a homozygous missense transversion located in exon 10, where an adenine (A) is substituted for a cytosine (C), resulting in a change of codon 519 (CCC
ACC) of the FSHR gene from proline to threonine (Pro519Thr; Fig. 1A
). The mutation is located in the second extracellular loop of the FSHR (Fig. 1B). This proline is highly conserved in the FSHRs of the different species cloned to date. Furthermore, it is located within a highly conserved motif (KVSIC*PMD) compared with LH and TSH receptors of different species. The patient was adopted, so no familial study could be performed.
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The mutation was reproduced in an expression vector. COS-7 cells were transfected with either wild-type or mutated receptor. As shown in Fig. 2, although FSH stimulation elicited a dose-dependent increase in cAMP accumulation in COS-7 cells transfected with the wild-type receptor, no FSH-induced adenylate cyclase stimulation was detected in COS-7 cells transfected with the mutant receptor even at high (3000 IU/liter) concentrations of FSH (Fig. 2A). The same result was obtained in four independent experiments. FSH binding to intact cells transfected with the Pro519Thr FSHR mutant was barely detectable (Fig. 2B). The mean ± SEM of specific binding to the mutant receptor were 6.3 ± 1.6% compared with the total specific binding obtained with the wild-type receptor (n = 4). Thus, the mutant receptor seemed to be trapped intracellularly.
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, inset). Cell surface expression of the wild-type and mutated receptors
The distribution of receptor molecules was studied by immunofluorescence and confocal microscopy (Fig. 3) as previously described (9, 10). Cells transfected with the wild-type or mutated FSHRs were incubated with a monoclonal anti-FSHR antibody directed against the extracellular domain of the receptor. When cells were permeabilized with saponin before incubation with the antibody, a strong intracellular staining was observed, which was similar for cells transfected with the wild-type or mutated receptors (Fig. 3, a and c). However, when the cells were incubated with the antibody without prior permeabilization of cells, labeling was observed on the cell surface for cells expressing the wild-type receptor (Fig. 3b), but no labeling was observed for cells expressing the Pro519Thr receptor mutant (Fig. 3d). This indicates that the mutant receptor is not properly targeted to the cell surface.
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Histological examination of the ovarian biopsies revealed the presence of a normal cubical surface epithelium. The cortex of the ovary comprised a superficial band of dense fibrous tissue, approximately 0.4 mm thick, which extended beneath the albuginea and was devoid of follicles. Beneath this fibrous layer, numerous small follicles could be observed (Fig. 4, A and B). The small follicles could be distinguished as primordial (Fig. 4, A and B), composed of an oocyte in the first prophase of meiosis and a single layer of flattened squamous pregranulosa cells; intermediary (Fig. 4, A and B), with a mixture of flattened and cubical cells surrounding the oocyte; and primary (Fig. 4C), with a monolayer of cubical granulosa cells. No follicle beyond the primary stage could be found in any of the sections examined.
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, A and C). Similar labeling of the oocytes was observed in a control biopsy of a normal woman of similar age (Fig. 5B).
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).
Conventional optical microscopy on serial sections of tissue did not enable detection of the presence of steroidogenic interstitial cells. Immunostaining with antibodies directed against the steroidogenic enzymes P450scc, P450c17, P450arom, and against SF1 was also performed. No interstitial cells expressing steroidogenic enzymes or SF1 were found in the cortex (data not shown).
A 3' end nonradioactive labeling technique, TUNEL (Oncogene, Cambridge, UK), was performed to assess apoptosis. None of the granulosa cells or oocytes was stained (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Until now only one mutation of the FSHR has been reported in Finnish women presenting an apparent complete phenotype (4). The Finnish patients had delayed or normal puberty and primary amenorrhea. Ovarian histology showed streak or hypoplastic ovaries, with identifiable ovarian tissue always present. The number of follicles detected in the ovaries was very low and was associated with fibrosis (4, 5). However, no quantification of follicular density was performed. Follicular development until the primary stage was observed to be associated with an arrest of follicular maturation at more advanced stages, except occasionally (5). In three of nine cases, normal preantral or more mature follicles were present.
The homozygous Ala189Val mutation, located in the extracellular domain of the receptor, was shown to markedly alter FSHR function. The mutated receptor was sequestered intracellularly (20). A possible remnant FSHR function in vivo has been suggested (20). Recently, another Finnish patient was reported to carry a novel Ala419Thr mutation, associated with the Ala189Val mutation already described. Although the patient had primary amenorrhea, she showed clear signs of endometrial estrogen stimulation, suggesting that the phenotype was less severe compared with the previous patients (8). However, the precise size of the ovaries was not given, and no histological study of the ovaries of the patient was performed (8).
The patient described in this study has a severe clinical and biological phenotype, with delayed puberty, primary amenorrhea, and hypoplastic ovaries. The hormonal evaluation showed very low plasma levels of estradiol and inhibin B. No residual activity of FSHR was detected in vitro, and the ovaries of the patient did not respond to a treatment of 21 d with human recombinant FSH. Indeed, a total dose of 10,200 IU recombinant FSH was unable to induce either an increase in the estradiol or inhibin B levels or the appearance of follicular structure at ultrasonography.
The histological appearance of the ovaries of our patient is characterized by a complete block in follicular maturation beyond the primary stage: no secondary or antral follicles are present. The proportion of each subtype of small follicles corresponds to that described in normal human ovaries (15, 16, 21, 22), and their histological aspect is also normal.
However, the density of small follicles per surface unit appears very high compared with that in age-matched healthy women (15, 16, 21, 22). This density is similar to what is observed in prepubertal ovaries (16, 23). It contrasts with the low number of follicles described in the Finnish patients (5).
The novel mutation present in our patient is a homozygous missense transversion located in exon 10 of the FSHR gene, yielding a Pro519Thr substitution. This mutation is located in the second extracellular loop of the receptor. This proline is highly conserved in the FSHRs of different species cloned to date, but also in the LH and TSH receptors. Furthermore, it is located in a highly conserved motif in the center of exoloop 2, KVSIC*PMDV.
This mutation alters the cell surface targeting of the receptor in vitro, with receptor molecules being trapped intracellularly. When mutations of the second extracellular loop of the LH receptor have been performed (24), mutation of the corresponding proline of the LH receptor (Pro494) into an alanine within the conserved motif also yielded a mutated receptor trapped inside the cells. Human chorionic gonadotropin binding to intact cells was hardly detectable (24). In the case of the FSHR, the mutation Pro519Thr, although corresponding to a different substituted residue, may alter the conformation of the receptor, and this misfolded receptor is trapped intracellularly.
Animal models have also been developed. The ovaries of FSHR-/- mice with targeted disruption of the FSHR gene (25) contain primordial, primary, and secondary follicles up to the preantral stage. In murine knockout models for FSHß, an accumulation of multilayered secondary follicles is observed. The block in folliculogenesis is located before antral formation (26). No normal follicle beyond the preantral stage is present. A well organized thecal layer is present. This has led to the conclusion that theca recruitment is completed autonomously with respect to FSH (26, 27) Taken together, these observations strongly suggest that FSH is not necessary for the initiation of follicular growth or for early follicle development in rodents. The absence of a functional FSHR seems to impede the initiation of follicular growth beyond the primary stage in this patient. This observation is consistent with the presence of FSHR or FSHR mRNAs in primary follicles (28, 29).
To better characterize the mechanism of this block in the follicular growth, we also analyzed the expression of markers previously studied in normal human ovaries. The protooncogene tyrosine kinase c-Kit receptor is one of the elements of the communication between granulosa and oocyte. An antiapoptotic effect of c-Kit ligand on primordial germ cells and oocytes has been demonstrated (reviewed in Ref. 30). c-Kit intervenes in the recruitment of nongrowing follicles to enter the growth phase (31). In the adult human ovary c-Kit is expressed in both oocytes and thecal cells (32, 33). We found expression of c-Kit in more than two thirds of the oocytes in the ovaries of our patient. This observation is consistent with the survival of a large number of small follicles. The lack of further follicular growth does not seem to be due to an absence of c-Kit expression or to apparent oocyte damage.
PCNA is a nuclear protein necessary for cell cycle DNA replication and DNA repair. PCNA is considered by some researchers to herald the beginning of follicular development (31, 34, 35), but DNA synthesis in granulosa cells of small follicles does not seem to be a reliable indicator of impending growth (36). In the fetal ovary of primates, many granulosa cells are immunostained positively for PCNA, whereas fewer of these cells are stained in the adult ovary (37). Thus, the high number of small follicles in our patient, associated with the labeling observed in the majority of granulosa cells and oocytes, is reminiscent of observations made in fetal ovaries (18, 31, 37).
During adult life, follicular apoptosis does not seem to occur in primordial, primary, and secondary follicles in human ovary (38), but is mainly observed in the granulosa cells of antral follicles (39). In our patient the absence of DNA laddering detected by the TUNEL method, an effective technique to show apoptosis in ovarian follicles (38, 39), did not allow the detection of apoptosis in the small follicles. A similar result was obtained in the ovaries of the Finnish patients bearing the Ala189Val FSHR mutation (40). Thus, the lack of follicles at further stages of development does not seem to be due to increased apoptosis.
The absence of steroidogenic cells in the interstitium of this ovary is explained by the absence of secondary follicles, as the steroidogenic cells in the ovarian cortex are derived from thecal cells, which appear only at the secondary stage of follicular growth (41).
Our findings support the concept that in humans, FSH seems mandatory for the initiation of follicular growth only after the primary stage. FSH resistance is a cause of infertility, which, however, is remarkably associated with the persistence of a high number of small follicles. The study of supplementary patients with loss of function mutations of the receptor is warranted because individual variations might also exist. High doses of recombinant FSH may be used in patients carrying a partial loss of receptor function (9), whereas it may be hoped that in vitro oocyte maturation will be possible in the future for patients with complete loss of FSHR function.
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Acknowledgments |
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was a kind gift from Prof. S. Kominami (Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan). We thank C. Pichon for providing the monoclonal anti-FSHR antibody necessary for this work. We also thank K. Laborde and C. Matuchansky for their help. |
Footnotes |
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G.M. and P.T. contributed equally to this work.
Abbreviations: FSHR, FSH receptor; P450arom, aromatase cytochrome P450; P450c17, 17-hydroxylase cytochrome P450; P450scc, side-chain cleavage; PCNA, proliferation cellular nuclear antigen; POF, premature ovarian failure; SF1, steroidogenic factor 1; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling.
Received February 10, 2003.
Accepted April 30, 2003.
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, mutated Pro519Thr receptor.
