0021-972X/99/$03.00/0
2
Journal of Clinical Endocrinology and Metabolism
Copyright © 1999 by The Endocrine Society
Mutational Analysis of the Follicle-Stimulating Hormone
(FSH) Receptor in Normal and Infertile Men:
Identification and Characterization of Two Discrete
FSH Receptor Isoforms*
MANUELA SIMONI, JÖRG GROMOLL, WOLFGANG HÖPPNER, AXEL KAMISCHKE,
THORSTEN KRAFFT, DANIEL STÄHLE, AND EBERHARD NIESCHLAG
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
ABSTRACT
In a search for pathophysiological causes of idiopathic male infer-
tility we investigated the occurrence of mutations of the FSH receptor
in 48 men with this disorder. The entire FSH receptor gene was
analyzed by single stranded conformation polymorphism analysis
(SSCP). A heterozygous point mutation without functional conse-
quences, exchanging Val to Ala in codon 341, was found in one patient.
SSCP analysis led to the identification of 2 polymorphisms in exon 10
associated in 2 discrete FSH receptor allelic variants, i.e. Thr307-
Asn680 and Ala307-Ser680. The frequency and distribution of the two
allelic variants was further analyzed in 86 proven fathers and 75
infertile men by SSCP (codon 307) and restriction fragment length
polymorphism (codon 680). The 2 receptor isoforms showed similar
T HE PRODUCTION of gametes depends on the con-
certed action of the two gonadotropins FSH and LH on
the gonads. In the male, FSH is required for the determina-
tion of Sertoli cell number and for the induction and main-
tenance of qualitatively and quantitatively normal spermat-
ogenesis (1, 2). FSH acts through binding to its specific
receptor, a member of the G protein-coupled receptor family
(3, 4). Men with a homozygous inactivating mutation of the
FSH receptor have reduced testicular volume, increased se-
rum FSH concentration, and variable degrees of spermato-
genic damage (5). The crucial importance of FSH for male
gonadal function is demonstrated by the recent description
of infertility in a man with a mutation in the FSH b-subunit
gene (6) and in male mice in which the FSH receptor has been
knocked out (7). These findings suggest that mutations of the
FSH receptor could play a pathogenetic role in male infer-
tility. In a previous study, we did not find mutations of the
FSH receptor in a group of 19 infertile men with histologi-
cally documented focal or complete Sertoli cell only syn-
Received February 25, 1998. Revision received November 4, 1998.
Accepted November 10, 1998.
Address all correspondence and requests for reprints to: Prof. Dr. E.
Nieschlag, F.R.C.P., Institute of Reproductive Medicine of the Univer-
sity, Domagkstrasse 11, D-48129 Munster, Germany. E-mail:
nieschl@uni-muenster.de.
* This work was supported by the Deutsche Forschungsgemeinschaft,
the Confocal Research Group “The Male Gamete: Production, Matura-
tion Function” (Grant Ni-130/15), and the German Federal Ministry of
Health, Bonn (to A.K.).
751
Vol. 84, No.
Printed in U.S.A.
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Mendelian distribution in proven fathers and in infertile men. Serum
FSH, inhibin B, and combined testicular volume did not differ be-
tween subjects with different receptor isoforms. Binding studies in
transiently transfected COS-7 cells showed similar binding affinity
for the two receptor variants. Moreover, the Ala307-Ser680 and the
Thr307-Asn680 FSH receptors responded in vitro to FSH with compa-
rable cAMP production. These data suggest that different isoforms of
the FSH receptor with similar functional properties exist in normal
and infertile men. We conclude that mutations of the FSH receptor or
the FSH receptor genotype do not play a pathogenic role in male
idiopathic infertility. The possibility that different FSH isoforms
might interact differently with the 2 receptor variants remains to be
investigated. (J Clin Endocrinol Metab 84: 751–755, 1999)
drome (8). The studies described in this paper extend our
previous observation to a larger group of infertile men. As
during this investigation two polymorphisms were found in
exon 10, we proceeded to analyze the FSH receptor poly-
morphism in infertile men and in a large group of men with
proven fertility. Finally, we studied the functional charac-
teristics of the naturally occurring FSH receptor allelic vari-
ants in vitro and in vivo.
Subjects and Methods
Subjects
Study 1: mutational analysis of the FSH receptor in male infertility. Muta-
tional analysis of the FSH receptor was performed in 48 patients con-
sulting our infertility clinic. All patients gave informed consent to par-
ticipate in the study. The clinical parameters of the patients are shown
in Table 1. This group includes 22 patients with nonobstructive
azoospermia, 24 patients with severe oligozoospermia (sperm concen-
tration, ,10 3 106/mL; median value, 0.4 3 106/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 mono-
lateral or focal SCO, in 2 patients spermatogenic arrest, and in 1 de-
creased spermatogenesis.
752 SIMONI ET AL.
TABLE 1. Clinical parameters (means 6 SD) of the subjects in which the entire coding region of the FSH receptor was analyzed by SSCP
Sperm conc. Inhibin B
Group FSH (IU/L)
(106/mL) (pg/mL)
Azoospermia (n 5 22) 0 21.5 6 13.7 37.8 6 59.1
Oligozoospermia (n 5 24) 1.6 6 2.4 11.4 6 7.6 75.1 6 68.4
P . 0.05 between the patient groups (by t-test).
Study 2: frequency of the allelic variants of the FSH receptor in proven fathers
and infertile men. The incidence of the 2 FSH receptor alleles was analyzed
in 86 proven fathers and in 75 infertile men. The proven fathers were
recruited in part through advertisements in local newspapers (n 5 46)
and in part by soliciting male blood donors at the time of blood donation
(n 5 40). Inclusion criteria were paternity achieved within the last 5 yr
and age less than 45 yr. The 75 infertile men consisted of 30 azoospermic
men and 45 men with severe oligozoospermia (median sperm concen-
tration, 0.6 3 106/mL) selected by the same criteria as the patients in
study 1. All subjects gave their informed consent to take part in the
study. Forty-five patients were included in both studies 1 and 2.
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 ac-
cording 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 anal-
ysis 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 un-
cleaved 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
JCE & M • 1999
Vol 84 • No 2
Combined
Testosterone Maldescensus Varicocele
testicular LH (IU/L)
(nmol/L) (n) (n)
vol (mL)
18.7 6 9.6 7.0 6 3.4 16.3 6 5.6 8 6
26.4 6 12.1 5.4 6 2.9 17.6 6 9.8 7 9
sites were mutagenized to GCT (Ala) and AGT (Ser), respectively, by
oligonucleotide-directed mutagenesis using the Transformer Site-Di-
rected 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
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7.5 mg/mL Lipofectamine reagent (Life Technologies, Eggenstein, Ger-
many) and 2 mg/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, Switzer-
land) 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 mCi/
mg) in the dose range 9.4 –150 pmol/L, in the presence and absence of
an excess (2 IU) of unlabeled FSH (Fertinorm, Serono, Unterschleis-
sheim, 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 x2 test. Two-sided P , 0.05 were
considered significant. These analyses were performed using the sta-
tistical software SigmaStat for Windows, version 2.0 (SPSS, San Rafael,
CA). Statistical analysis of binding and functional parameters of tran-
siently transfected COS-7 cells was performed on log-transformed data
using the GraphPad Prism program.
Results
Study 1: mutational analysis of the FSH receptor in male
infertility
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 electro-
phoresis of the amplified fragments A and G of exon 10.
Sequencing revealed that codon 307, in the extracellular do-
main, 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, 15–17).
 FSH RECEPTOR ALLELIC VARIANTS
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 mu-
tations 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
FIG. 1. a, SSCP-gel electrophoresis of exon 10, fragment A (12). b,
RFLP of exon 10, fragments E-G (12). The presence of three bands
indicates heterozygosity. The results shown in a and b were obtained
in two different sets of DNA samples.
753
(56%), and 66 were Ala307-Ser680 (44%). No significant dif-
ference in the distribution of the two variants between the
groups could be found.
The functional characteristics of the two FSH receptor
isoforms were studied in vitro in transiently transfected
COS-7 cells. As shown in Fig. 2, 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 6 0.07 and 0.24 6 0.09 IU/L for the Thr307-
Asn680 and Ala307-Ser680 variant, respectively (P 5 0.99; n 5
3). In binding experiments, the Kd of the two receptor vari-
ants did not differ significantly and were 261.8 6 114.9 and
149.5 6 52.54 pmol/L for the Thr307-Asn680 and Ala307-Ser680
variant, respectively (P 5 0.42; n 5 3), suggesting similar
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binding affinities. Although the Ala307-Ser680 variants were
always less expressed than the Thr307-Asn680 receptor (bind-
ing capacity, 27.86 6 11.05 vs. 10.46 6 3.34 cpm/100 cells
corresponding to 12.59 6 6.19 vs. 4.53 6 2.15 pmol/L for the
Thr307-Asn680 and Ala307-Ser680 variants, respectively), the
difference was not statistically significant (n 5 3; P 5 0.39).
These data suggest similar functional characteristics for the
two receptor isoforms.
As possible parameters of the activities of the two allelic
variants in vivo, we compared the serum levels of FSH and
inhibin B and the combined testicular volumes of proven
fathers and infertile men (Fig. 3). 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.
Discussion
Given the fundamental role of FSH in human spermato-
genesis, we postulated that defects of the FSH receptor might
be involved in some form of male infertility with azoosper-
mia or severe oligozoospermia. The screening of a large
number of infertile men, however, does not support this
hypothesis, and extending our previous observation (8), this
study demonstrates that sporadic mutations of the FSH re-
ceptor are not a common finding in male infertility. To our
knowledge, this is the largest cohort of male patients in
which the FSH receptor has been systematically analyzed in
its entirety. In addition, a recent study by Tuerlings et al. (17),
based on 23 oligozoospermic and 5 azoospermic men un-
dergoing intracytoplasmatic sperm injection, did not detect
mutations of the FSH receptor gene in such patients. There-
fore, the only known mutation of the FSH receptor relevant
to male infertility remains the Ala189Val homozygous sub-
stitution found in some Finnish families with hereditary
primary amenorrhea (6, 15). These results restrict the indi-
cation of mutational analysis of the FSH receptor to infertile
men whose parents are related or with a history of amen-
orrhea/infertility in their family.
FSH receptor analysis in normal and infertile men con-
firmed our previous finding of two polymorphisms at po-
sitions 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 dis-
crete allelic variants of a gonadotropin receptor in the Cau-
754 SIMONI ET AL.
TABLE 2. Frequency and distribution of the two FSH receptor allelic variants in the subjects in which the polymorphism was analyzed
by SSCP and RFLP
Group Thr307-Asn680/Thr307-Asn680
Proven fathers (n 5 86) n 5 32 (37.2)
Infertile men (n 5 75) n 5 24 (32)
Values are given as the number of men, with percentages in parentheses.
a
P . 0.05 between the patient groups (by x2 test).
FIG. 2. FSH-stimulated cAMP production of COS-7 cells transiently
expressing the two allelic variants of the FSH receptor (mean 6 SEM
of triplicate determinations of one representative experiment).
casian 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 po-
sition 307 is occupied by Thr in the cynomolgus monkey (18);
by Ala in the bovine, equine, and ovine receptors (19 –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 re-
ceptor isoform showed only a slightly, not significantly
higher expression at the cell surface. Common polymor-
phisms of G protein-coupled receptors are known to influ-
ence pathophysiological functions. For example, a polymor-
phism 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
JCE & M • 1999
Vol 84 • No 2
Thr307-Asn680/Ala307-Ser680 Ala307-Ser680/Ala307-Ser680
n 5 39 (45.4) n 5 15 (17.4)
n 5 36 (48) n 5 15 (20)
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FIG. 3. Serum FSH and inhibin B values and combined testicular
volume in 86 proven fathers (left panels) and 75 infertile men (right
panels) grouped according to their FSH receptor genotype. TN,
Thr307-Asn680/Thr307-Asn680; AS, Ala307-Ser680/Ala307-Ser680; TN/AS,
Thr307-Asn680/Ala307-Ser680. The three clinical parameters did not
differ significantly between subjects with different FSH receptor ge-
notype in both controls and patients (by Kruskal-Wallis ANOVA).
in parameters of FSH action in vivo, at least in adult males.
These data suggest that FSH receptor isoforms are not func-
tionally 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 patho-
physiological conditions. The existence of several FSH iso-
forms with different specific activities is well known (28). It
has been speculated that, depending on FSH isoform inter-
action, FSH receptors can couple to different signal trans-
duction pathways and elicit different physiological re-
sponses (28). Our finding that FSH receptor isoforms exist as
well renders the pleiotropism of FSH action even more mul-
tifaceted. 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.
 FSH RECEPTOR ALLELIC VARIANTS
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 deter-
mination. 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 pre-
vious 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 id-
iopathic 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).
Acknowledgments
We thank Prof. Dr. C. Carani and Prof. Dr. E. Baldini, University of
Modena (Modena, Italy), for providing DNA samples of blood donors.
We thank J. Esselmann, L. Pekel, and B. Schuhmann for the excellent
technical assistance, and S. Nieschlag, M.A., for language editing of the
manuscript. We thank Ares Advanced Technology (Randolph, MA) for
providing us with recombinant human FSH.
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