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Original Studies |
Institute of Reproductive Medicine of the University (M.S., J.G., A.K., T.K., D.S., E.N.), D-48129 Münster; and Institute of Hormone and Fertility Research, University of Hamburg (W.H.), D-22529 Hamburg, Germany
Address all correspondence and requests for reprints to: Prof. Dr. E. Nieschlag, F.R.C.P., Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Munster, Germany. E-mail: nieschl{at}uni-muenster.de
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Study 1: mutational analysis of the FSH receptor in male
infertility. Mutational analysis of the FSH receptor was performed
in 48 patients consulting our infertility clinic. All patients gave
informed consent to participate in the study. The clinical parameters
of the patients are shown in Table 1
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This group includes 22 patients with nonobstructive azoospermia, 24
patients with severe oligozoospermia (sperm concentration, <10 x
106/mL; median value, 0.4 x 106/mL), and
2 patients with slightly reduced sperm concentration. Serum FSH levels
were above the upper normal limit (>7 IU/L) in 17 azoospermic
patients, 14 oligozoospermic patients, and 2 infertile men with
slightly reduced sperm concentrations. The remaining subjects had
normal FSH levels, and obstruction was excluded by testicular biopsy,
sonography, and chemical markers of ductal patency in the seminal
plasma. The median FSH concentration values were 22.3 and 9.6 IU/L in
the azoospermic and severely oligozoospermic groups, respectively.
Testicular histology was available in 16 azoospermic men and showed
complete SCO in 12 cases and spermatogenic arrest at the stage
of primary spermatocytes in 4 cases. In 11 oligozoospermic patients,
testicular histology showed monolateral or focal SCO, in 2
patients spermatogenic arrest, and in 1 decreased spermatogenesis.
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Clinical parameters
All serum hormones were measured in duplicate. Serum FSH and LH were assayed by immunofluorimetric assay using the Autodelfia system (Wallac, Freiburg, Germany). The sensitivities were 0.05 and 0.025 IU/L for FSH and LH, respectively. The intra- and interassay coefficients of variation (CVs) were less than 3% for both hormones. Serum testosterone was measured by RIA using a direct method (DSL, Sinsheim, Germany). The sensitivity and intra- and interassay CVs were 0.4 nmol/L, 5.5%, and 9.2%, respectively. Serum inhibin B was measured by enzyme-linked immunosorbent assay (9) using the Serotec inhibin B dimer assay kit (Camon, Wiesbaden, Germany) according to the instructions of the manufacturer. The sensitivity was 7.8 pg/mL. Intra- and interassay CVs were 2.5% and 6.7%, respectively. Semen analysis was performed according to the WHO guidelines (10). Testicular volume was determined by ultrasonography, using Sonoline SL2 equipment (Siemens, Erlangen, Germany) (11).
DNA isolation and analysis
Genomic DNA was obtained from peripheral blood leukocytes as previously described (12). The entire FSH receptor gene was screened for mutations by single stranded conformation polymorphism (SSCP) gel electrophoresis. Exons 1–9, encoding the extracellular domain of the FSH receptor, were amplified by PCR using primers flanking each exon as previously described (13). The PCR amplification of exon 10, encoding the transmembrane and intracellular domains, SSCP and sequence analysis were performed as described previously (12). The SSCP analysis was employed for the characterization of the polymorphism at position 307. The SSCP results were confirmed by direct sequencing of about 10% of randomly chosen DNA samples .
Restriction fragment length polymorphism (RFLP) of the Asn680Ser variant
The presence of the Asn680Ser variant introduces a restriction site that can be exploited in the RFLP technique. A fragment of exon 10, 10E to 10G (12), was amplified from genomic DNA. The PCR fragment was purified by purification columns (Qiagen, Hilden, Germany) and further subjected to restriction digestion by Bsr1. After digestion, the fragments were run on a 2% agarose gel electrophoresis and analyzed. The uncleaved fragment, homozygous for Asn, has a size of 579 bp, whereas the cleaved fragment, homozygous for Ser, gives rise to 443- and 136-bp fragments. The presence of all three fragments indicated a heterozygous state.
Mutagenesis, transfection of COS-7 cells, and cAMP assay
The human FSH receptor complementary DNA originally cloned in the EcoRI restriction site of pSG5 (Stratagene, Heidelberg, Germany) carries ACT (Thr) in codon 307 and AAT (Asn) in codon 680. These two sites were mutagenized to GCT (Ala) and AGT (Ser), respectively, by oligonucleotide-directed mutagenesis using the Transformer Site-Directed Mutagenesis Kit (Clontech, Heidelberg, Germany).
Transfection experiments were carried out in COS-7 cells essentially as previously described (12). In preliminary experiments the transfection conditions were optimized to obtain about a 50% transfection rate using 7.5 µg/mL Lipofectamine reagent (Life Technologies, Eggenstein, Germany) and 2 µg/mL plasmid DNA. Transfection efficiency was checked by transfecting the cells with the pcDNA3.1/His/lacZ plasmid. Twenty-four hours after transfection, cells were washed and stimulated with human recombinant FSH (Serono Laboratories, Inc., Aubonne, Switzerland) as previously described (12). cAMP in the medium was measured by a cAMP enzyme-linked immunosorbent assay kit (IHF, Hamburg, Germany).
Binding studies
The binding characteristics of the two allelic variants of the FSH receptor were studied in transiently transfected COS-7 cells as described by van Loenen et al. (14). COS-7 cells seeded in 24-well plates were transfected as described above. Twenty-four hours after transfection, cells were washed twice with HEPES-buffered Krebs-Ringer buffer without NaCl, with sucrose (200 mmol/L sucrose, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2.5 nmol/L CaCl2, 5 mmol/L MgCl2, and 25 mmol/L HEPES, pH 7.5) containing 0.1% BSA. Cells were incubated with increasing concentrations of [125I]human FSH (NEX-173, New England Nuclear-DuPont, Bad Homburg, Germany; SA, 127 µCi/µg) in the dose range 9.4–150 pmol/L, in the presence and absence of an excess (2 IU) of unlabeled FSH (Fertinorm, Serono, Unterschleissheim, Germany). After 3 h at 37 C, cells were washed twice with ice-cold buffer, dissolved in 1 N NaOH, and total cell-associated radioactivity was counted. The binding experiments were performed twice. Binding data were calculated with the GraphPad Prism program (San Diego, CA).
Statistical analysis
All clinical variables were checked for normal distribution in
the Kolmogorov-Smirnov test. Statistical analysis between the groups
was then performed by t test. Differences in the
distribution of proportions between the groups were tested by
2 test. Two-sided P < 0.05 were
considered significant. These analyses were performed using the
statistical software SigmaStat for Windows, version 2.0 (SPSS, San
Rafael, CA). Statistical analysis of binding and functional parameters
of transiently transfected COS-7 cells was performed on log-transformed
data using the GraphPad Prism program.
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Results |
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No aberrant SSCP migration pattern was detected in exons 1–9 in any subject. In several patients, the SSCP analysis showed two possible patterns of migration on gel electrophoresis of the amplified fragments A and G of exon 10. Sequencing revealed that codon 307, in the extracellular domain, could be occupied by either ACT (Thr) or GCT (Ala) and that codon 680 could be occupied by either AGT (Ser) or AAT (Asn). These data confirm two previously reported polymorphisms (3, 4, 15, 16, 17).
A nonpolymorphic heterozygous point mutation was found in one patient, exchanging GTG (Val) in codon 341 to GCG (Ala). This patient had only one testis (volume, 17 mL) because of a possible testicular malformation on the left side and was azoospermic. His serum FSH level was 34 IU/L, and his inhibin B concentration was 12.4 pg/mL. The histology of the right testis showed spermatogenic arrest at the stage of primary spermatocytes. In vitro studies in transiently transfected COS-7 cells showed that the mutated receptor was normally responsive to FSH stimulation (not shown). Therefore, this heterozygous mutation is not expected to result in any impairment of receptor function. No other mutations were identified.
Study 2: frequency of the allelic variants of the FSH receptor in proven fathers and infertile men
The finding that the FSH receptor can be polymorphic at two
positions in infertile men prompted us to analyze the frequency and
distribution of the polymorphic variants in proven fathers and in
infertile patients. To this end, analysis of codon 307 was carried out
by SSCP, whereas codon 680 was analyzed by RFLP (Fig. 1). This investigation revealed that at
the genomic level, there is a tight linkage between the two polymorphic
sites and the two polymorphisms occur as two discrete allelic variants,
i.e. Thr307-Asn680 and
Ala307-Ser680, giving rise to two receptor
isoforms at the protein level. The frequency and distribution of these
two allelic variants in proven fathers and infertile patients are shown
in Table 2. Of 172 alleles in the 86
fathers, 103 were Thr307-Asn680 (60%) and 69
were Ala307-Ser680 (40%). Similarly, in the 75
infertile men, 84 of 150 alleles were
Thr307-Asn680 (56%), and 66 were
Ala307-Ser680 (44%). No significant difference
in the distribution of the two variants between the groups could be
found.
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, the cAMP production in
response to increasing concentrations of FSH was similar for the two
receptor isoforms. In repeated experiments, the FSH ED50
were 0.21 ± 0.07 and 0.24 ± 0.09 IU/L for the
Thr307-Asn680 and
Ala307-Ser680 variant, respectively
(P = 0.99; n = 3). In binding experiments, the
Kd of the two receptor variants did not differ
significantly and were 261.8 ± 114.9 and 149.5 ± 52.54
pmol/L for the Thr307-Asn680 and
Ala307-Ser680 variant, respectively
(P = 0.42; n = 3), suggesting similar binding
affinities. Although the Ala307-Ser680 variants
were always less expressed than the
Thr307-Asn680 receptor (binding capacity,
27.86 ± 11.05 vs. 10.46 ± 3.34 cpm/100 cells
corresponding to 12.59 ± 6.19 vs. 4.53 ± 2.15
pmol/L for the Thr307-Asn680 and
Ala307-Ser680 variants, respectively), the
difference was not statistically significant (n = 3;
P = 0.39). These data suggest similar functional
characteristics for the two receptor isoforms.
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). These three parameters differed
significantly between controls and patients, but no significant
differences could be observed within the groups and between the
subjects with different allelic variants, even when only the homozygous
subjects were considered.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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FSH receptor analysis in normal and infertile men confirmed our previous finding of two polymorphisms at positions 680 and 307 (3, 4). The present investigation revealed for the first time that these polymorphisms occur in two possible arrangements, suggesting the existence of two discrete allelic variants of a gonadotropin receptor in the Caucasian population. In contrast, the two polymorphic variants were reported to occur in linkage disequilibrium in Brazilian subjects, revealing ethnic differences (16).
Comparison with the reported amino acid sequence of the FSH receptor cloned from other animal species shows that the position corresponding to the human FSH receptor position 307 is occupied by Thr in the cynomolgus monkey (18); by Ala in the bovine, equine, and ovine receptors (19, 20, 21); and by Ile in the rat (22), whereas position 680 is invariably occupied by Asn in all these species. As the occurrence of polymorphisms of the FSH receptor in animal species has not been investigated, it is difficult to assess whether and which of the two human alleles is phylogenetically more closely related to other species.
The presence of an Asn residue at position 680 introduces a potential glycosylation site, which might be important for posttranslational receptor processing and expression at the cell surface (23), whereas a Ser residue could contribute to a potential phosphorylation site involved in receptor function. When transiently transfected in COS-7 cells, the Asn680 receptor isoform showed only a slightly, not significantly higher expression at the cell surface. Common polymorphisms of G protein-coupled receptors are known to influence pathophysiological functions. For example, a polymorphism in the opsin gene accounts for genetic variations in sensitivity to long wavelength light (24). A polymorphism in the glucagon receptor was more common in subjects with noninsulin-dependent diabetes mellitus (25). Variants of the MSH receptor gene are associated with red hair and fair skin (26). Moreover, a TSH receptor variant has been found to have enhanced sensitivity to TSH stimulation in vitro (27). The two FSH receptor isoforms described in this paper show similar hormone affinity and cAMP production in vitro and in parameters of FSH action in vivo, at least in adult males. These data suggest that FSH receptor isoforms are not functionally different in normal and infertile men. However, we cannot exclude the possibility that different activities of the two receptor isoforms might become evident in other pathophysiological conditions. The existence of several FSH isoforms with different specific activities is well known (28). It has been speculated that, depending on FSH isoform interaction, FSH receptors can couple to different signal transduction pathways and elicit different physiological responses (28). Our finding that FSH receptor isoforms exist as well renders the pleiotropism of FSH action even more multifaceted. Conversely, receptor isoforms could be relevant to the action of synthetic hormone analogs (29, 30) and may have an important impact on drug development (31). These possibilities should be analyzed in future studies.
The roughly Mendelian distributions of the two allelic variants were similar in fertile and infertile men, excluding a role of the FSH receptor genotype in male fertility determination. Likewise, mutations of the FSH receptor were not found to play a pathogenetic role in the spermatogenetic failure of the patients analyzed in this study, extending previous observations (7, 17). Previous studies had shown that serum FSH is bioactive (32) and excluded the occurrence of circulating inhibitors of the FSH receptor in infertile men (33). Thus, FSH action is not impaired in patients with idiopathic azoospermia or severe oligozoospermia, and the cause of their infertility remains to be determined. Compared to the other glycoprotein hormone receptors, mutations of the FSH receptor appear to be extremely rare (16, 34, 35). This is compatible with an irreplaceable role of FSH in human reproduction so that mutations affecting its action are self-eliminating. Finally, the coexistence of both FSH and FSH receptor isoforms supports the idea of an ongoing process of coevolution of ligand-receptor pairs that might be necessary for improving the reproductive function of the species (36).
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Acknowledgments |
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Footnotes |
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Received February 25, 1998.
Revised November 4, 1998.
Accepted November 10, 1998.
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