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Original Studies |
Institute of Reproductive Medicine of the University, D-48129 Münster, Germany; and Growth, Puberty, and Adolescence Foundation (U.E.), CH-8006 Zürich, Switzerland
Address all correspondence and requests for reprints to: Prof. Dr. Eberhard Nieschlag, 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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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Aberrant hormone binding/signal transduction of LH/hCG through the LH receptor (LHR) directly affects androgen production, leading to disturbances of fetal as well as pubertal male development. The clinical phenotypes resulting from altered LH/hCG-LHR interaction in genetically male subjects range from male pseudohermaphroditism, characterized by female external phenotype with a blind-ending vagina, lack of breast development, and primary amenorrhea, to incomplete virilization of the external genitalia, with micropenis and/or hypospadia (2). In severe cases the responsiveness of the Leydig cells to hCG is abolished, resulting in Leydig cell hypoplasia (LCH). Two types of LCH have been described (3). Type I LCH is the most severe form, resulting in female phenotype and is caused by inactivating mutations in the LHR, which completely prevent hCG/LH signal transduction. Patients with type II LCH are characterized by milder signs of androgen deficiency and are generally hypogonadal. This milder form derives from mutations of the LHR, which only partially inactivate the LHR. Patients with type II LCH might retain partial responsiveness to hCG (4).
In the present paper we report the unique case of a patient with LCH type II caused by a genomic deletion resulting in the complete absence of exon 10 of the LHR. The patient displayed a normal male phenotype and came to clinical attention because of lack of pubertal development and hypogonadism. This patient represents the clinical counterpart of the normal male marmoset monkey (Callithrix jacchus) in which the expressed LHR lacks exon 10 in toto. In this patient hypogonadism was treated, and spermatogenesis was induced by hCG administration. The successful induction of normal testosterone production and complete spermatogenesis by hCG administration not only provides important clinical insights about the possible role of exon 10 of the LHR in discriminating between LH and hCG action, but also is the first description of a gonadotropin-based therapy in a LCH type II patient.
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Subjects and Methods |
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The patient was the second of three sons of consanguineous Turkish parents (first degree cousins). Family history revealed no delayed or incomplete puberty and no infertility. The patient was first presented at the out-patient department of a pediatric clinic at the age of 18.2 yr because of retarded pubertal development. Body proportions were eunuchoid [height, 177.8 cm (+0.2 SD); target height, 176 cm; father, 175 cm; mother, 165 cm; sitting height, 85 cm (-2.3 SD)]. He showed Tanner stage 2 for pubic hair and a pubertal testicular volume of 8 mL bilaterally. The bone age was 14.0 yr, FSH was normal (3.4 U/L), but LH was elevated (17.1 U/L) with low plasma testosterone for the bone age (1.8 nmol/L). Without further diagnostics, probative treatment with 250 mg testosterone enanthate, im, every 4 weeks was instituted. After 6 months, testicular volume had increased to 12 and 15 mL, pubic hair corresponded to Tanner stage P4–5, and treatment with testosterone was discontinued. At 19.3 yr the patient was referred to the out-patient department of the Growth, Puberty, and Adolescence Foundation in Zürich because of arrest of pubertal development and hypogonadism. His height was 186.6 cm (+1.2 SD), body proportions were still eunuchoid, bone age was 14.5 yr, and pubic hair was at Tanner stage 4–5. Testicular volume had regressed to 10 mL. Basal LH was elevated (25 IU/L) with a further increase to 55 IU/L after GnRH (25 µg/m2) in the presence of testosterone levels in the castrate range that did not increase after GnRH stimulation. Basal FSH was normal without increase after GnRH (2.5 IU/L). A LH receptor defect was suspected. At the age of 22 yr the patient consulted another medical institution in Zürich for a second opinion. Eight weeks after withdrawal of testosterone substitution, serum LH was 29.2 IU/L, testosterone was 3.9 nmol/L, and FSH was 5.8 IU/L. Eleven weeks after withdrawal, serum testosterone was 1.8 nmol/L, LH rose further to 43.2 IU/L, and FSH was unchanged (5.5 IU/L). LH bioactivity was tested using a mouse Leydig cell bioassay and was normal, suggesting a complete resistance to LH action. Semen analysis revealed azoospermia. Treatment with testosterone enanthate (250 mg every 3 weeks) was instituted again and then stopped upon demand by the patient at the age of 24 yr.
DNA isolation and PCR
DNA was isolated from blood samples obtained from the patient as well as from the father, mother, and one brother. In addition, two blood samples from male volunteers with normal hormonal levels and normal sperm counts according to WHO criteria were taken. Genomic DNA was purified using the Nucleon Kit (Herolab, Braunschweig, Germany). Exons 1–11 of the LHR gene were amplified using the primers and cycling conditions described by Atger et al. (5). Each PCR sample (25 µL) contained 10 nmol/L Tris-HCl (pH 8.3), 50 nmol/L KCl, 0.01% gelatin, 2 nmol/L MgCl2, 0.2 mmol/L deoxy-NTPs, 2 U Taq polymerase (Promega Corp., Heidelberg, Germany), 100 mmol/L primer, and 200 ng DNA. The amplified products were subjected to 1–2% agarose gel electrophoresis for further analysis. Exons 1–9 and 11 of the patient were sequenced using the Licor-System (MWG-Biotech, Ebersberg, Germany), and no sequence abnormalities were noted.
Long template-PCR
For amplification of the genomic region of exon 9–11 from the LHR gene, the Expand Long Template PCR System (Roche Molecular Biochemicals, Mannheim, Germany) was used. The following primers, designed on the basis of the published LHR complementary DNA (cDNA) (6), were used to amplify genomic regions from DNA of either the control persons or the patient and his family members: exon 9, forward, 5'-GGCCCTGCCGAGCTATGGCCTAG-3'; exon 10, reverse, 5'-CCTTACTGTGCTTTCACATTGTTTGG-3'; exon 10, forward, 5'-CCAAACAATGTGAAAGCACAGTAAGG-3'; and exon 11, reverse, 5'-AGTCCCAGCCACTCAGTTCACTCTC-3'.
Each PCR sample contained the PCR buffer 3 contained in the kit, 10 mmol/L deoxy-NTPs, 100 mmol/L primer, and 200–300 ng template DNA. The cycling conditions were as follows: denaturation at 92 C for 2 min, followed by 10 cycles at 92 C for 10 s, at 58 C for 30 s, and at 68 C for 8 min; the program was continued by 25 cycles at 92 C for 10 s, at 58 C for 30 s, and at 68 C for 8 min, including a time increment of 20 s/cycle. The PCR program was completed after a final elongation step at 68 C for 7 min. The different amplicons were analyzed by 0.8% agarose gel electrophoresis.
Sequence analysis of the genomic region covering exons 9–11 of the LHR gene
The amplified DNA fragments were cloned into the pGEM-T easy vector (Invitrogen, Heidelberg, Germany), and the clones obtained were further analyzed by DNA sequencing using the primer walk method. The sequences obtained were aligned using the Sequencher DNA software (Gene Code Corp., Ann Arbor, MI).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Each of the 11 exons of the LHR gene were amplified by PCR, and
the size of the amplicons was determined by agarose gel
electrophoresis. All exons of the patient except exon 10 were
sequenced, and no nucleotide abnormalities could be observed compared
to the wild-type LHR (data not shown). In the patient, amplification of
exon 10 consistently failed in several independent experiments using
different cycling programs and altered annealing temperatures.
Amplification of exon 10 using DNA obtained from a healthy control
person, the father, the mother, and the brother of the patient resulted
in the expected 174-bp amplicon with 62 bp of 5'-intronic sequences, 81
bp encoding exon 10, and 31 bp of 3'-intronic sequences (Fig. 1
). These results indicated that exon 10
of the LHR gene was deleted in the patient, but did not give
information about the extent of the deletion.
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, lanes 1 and 2). This long template-PCR
resulted in amplicons with approximately 4 kbp for a fragment covering
exons 9 to 10 and approximately 5 kbp for the fragment covering exons
10 to 11, respectively. In a second step the setting of the long
template-PCR was adjusted to amplify the region covering exons 9–11 in
one reaction step (Fig. 2, lane 3), resulting in the expected 9-kbp
fragment. Using exactly the same long template-PCR conditions, the
corresponding region of exons 9–11 was amplified in the patient. The
amplicon obtained had a size of approximately 4 kbp, indicating the
loss of about 5 kbp compared to the control DNA.
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). In total, the region covers
9770 bp, including exon 10. The amplicon spanning exons 9–11 of the
patient displayed 3691 bp. Alignment of the control and patient
sequences revealed one breakpoint at position 1747 in intron 9 and the
second breakpoint at position 7834, within intron 10. Thus, the
deletion completely removes exon 10. Analysis of the two breakpoints
gives no insight about the mechanism possibly causing the deletion, as
no homologous sequences, potentially involved in aberrant
recombination, could be identified (Fig. 4).
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Treatment of the patient with hCG
Treatment with testosterone enanthate (250 mg/3 weeks) was stopped
(upon demand by the patient) at the age of 24 yr. Several months later
LH was again elevated (41 IU/L), and testosterone was undetectable
(Table 1). A hCG test (5000
U/m2; Profasi, Serono, Milan, Italy) was
performed, resulting in nearly normal testosterone production (37
nmol/L on day 4; Table 1). hCG treatment continued at 5000 IU/week for
2 months and later was reduced to 3000 IU/week. By this treatment
testosterone levels were maintained within the normal range, and
testicular volume increased from 20 to 35 mL on each side. During hCG
treatment inhibin B levels decreased from 427 to 120 pg/mL, whereas FSH
levels initially increased from 3.4 to 25 IU/L and decreased thereafter
(7.4 IU/L at the last examination). Although the patient was
azoospermic previously, after 4 months of hCG treatment semen analysis
revealed a sperm concentration of 5.3 million/mL and total sperm counts
of 24 million/ejaculate.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The parents and one brother of the patient were heterozygous carriers of the same deletion. Indeed, in further long template-PCR experiments with genomic DNA we were able to amplify both the deleted form and the wild-type sequences of the region covering exons 9–11 (data not shown). The heterozygous family members were not clinically affected, confirming that one functional allele is sufficient for normal receptor activity, normal phenotype, and fertility.
Recently, the LHR cDNA of the marmoset monkey was cloned and characterized (10). Interestingly, in this species, the wild-type LHR cDNA completely lacks exon 10. When expressed in vitro, the marmoset monkey LHR cDNA showed normal hCG binding and normal hCG-induced cAMP and inositol trisphosphate signal transduction compared to the human wild-type LHR. In further experiments Zhang et al. (11) removed exon 10 from the human LHR cDNA and studied the binding and signal transduction properties of this mutant. The transport of the mutant receptor to the cell membrane was severely affected, resulting in decreased receptor expression at the cell surface. These experiments revealed that lack of exon 10 in the human LHR trapped most of the receptor inside the cell, and only one fourth of the receptor protein is integrated into the cell membrane. Stimulation with hCG, however, induced a similar dose response of cAMP production between wild-type human LHR and human LHR lacking exon 10 (11).
To date only a few naturally occurring mutations of the LHR causing LCH type I and II have been described (3). In most cases point mutations led to substitution of an amino acid crucial for receptor function. The phenotypes range from male pseudohermaphroditism to incomplete virilization depending on whether the mutation inactivates the LHR completely or only partially. In the cases of male hypogonadism some residual activity of the LHR has always been shown by the increase in serum testosterone concentration upon a GnRH or hCG stimulation test (4). Furthermore, in vitro studies confirmed these findings, as high doses of hCG could increase cAMP or inositol trisphosphate production of the mutagenized receptor (12). The residual reactivity of the LHR to hCG explains the development of a male phenotype. There is obviously a clear correlation between the severity of the clinical phenotype and overall receptor signal capacity, consisting of hormone binding, cell surface expression, and coupling efficiency (13). In a case of compound heterozygosity leading to male pseudohermaphroditism, a deletion of exon 8 was observed. Functional studies of the LHR lacking exon 8 showed its complete inactivation (14). To our knowledge, this is the only other description of a complete deletion of a LHR exon besides the case described here.
Compared to other LCH type II patients our patient shows some clear discrepancies. The residual activities of the LHR described in these patients led to low, albeit measurable, testosterone levels, whereas our patient showed testosterone levels below the detection limit (<0.7 nmol/L). These hormonal differences are also reflected by the testicular volumes, with LCH type II patients having testicular volumes of approximately 20 mL vs. 8 mL in our patient at the first presentation (4). Testicular biopsies of patients with LCH type II showed spermatogenetic arrest at the stage of spermatid elongation. Complete spermiation failed because of the necessity for high intratesticular testosterone levels for sperm release (1, 2). Testosterone treatment of our patient led to an increase in testicular volume from 8 to 25 mL, indicating the initiation of spermatogenesis. However, semen analysis revealed azoospermia, which might be caused by spermatogenetic arrest at the stage of spermiation, similar to that observed in patients with residual LHR activity. Complete spermatogenesis could only be obtained by treatment with hCG, presumably leading to the high intratesticular testosterone levels required.
The fact that FSH levels in some LCH type II patients remain in the normal range is noteworthy and might be explained by a spermatogenic failure beyond the stages of spermatogenesis regulated by FSH (15). In our patient FSH was normal before treatment, increased markedly during the first weeks of hCG administration, and decreased thereafter. Instead, serum inhibin B concentrations decreased progressively. This suggests that the initial increase in FSH levels was probably secondary to the inhibin B decrease, which, in turn, might have been induced, directly or indirectly, by the increase in intratesticular testosterone. This peculiar hormonal situation has not been described previously and might reflect a maturational switch in inhibin B production taking place at puberty (16). In fact, before puberty inhibin B is a pure Sertoli cell marker, but is obviously influenced by the spermatogenic state and becomes a more general spermatogenesis marker in adulthood. Testosterone might be the factor inducing this pubertal switch. On the other hand the increase in serum FSH during the first weeks of treatment observed in our patient is reminiscent of a compensatory rise of FSH after hemicastration in monkeys, leading to a decrease in inhibin B secretion and accompanied by an increase in intratesticular testosterone (17). The completion of spermatogenesis in this patient is now accompanied by normal gonadotropin and inhibin B levels, suggesting that the adult feedback regulation is fully established.
The deletion of exon 10 probably affects the extracellular domain involved in hormone binding, but not necessarily signal transduction. Considering that serum hCG levels during treatment were similar to LH concentrations in the absence of therapy and that LH serum levels decreased progressively during treatment, the total amounts of circulating hCG and LH alone cannot explain the induction and maintenance of testosterone biosynthesis. In fact, even assuming that the trafficking of the deleted receptor to the cell membrane is probably hampered (11), the high levels of biologically active LH should have supported at least low testosterone levels. Rather, our data suggest that exon 10 might be able to discriminate between LH and hCG. Recent studies of hCG binding to the extracellular domain have demonstrated that the ß-subunit of hCG makes direct contact with the extracellular domain (18). The studies of Zhang et al. (11) have shown that hCG is able to bind to and activate the human LHR lacking exon 10. Therefore, it is tempting to speculate that the ß-subunit of hCG may be due to its C-terminal elongation is more flexible compared to the LH ß-subunit and that hCG binds to the exon 10-deleted LHR, whereas LH does not.
The importance of the glycosylation sites and the cystein residue of exon 10 for hormone binding and/or signal transduction is still matter of investigation. Modeling of the extracellular domain of the LHR revealed that the C-terminus, including exon 10, displays a chemokine-like structure putatively forming another ß-sheet, which might represent a contact site for hormone interaction (19). Moreover, recent studies from the group of Moyle have shown that the extracellular domain possesses, beside the high affinity binding sites in the N-terminal part, a hormone discrimination site at the C-terminus (20). This site obviously prevents LH binding in the presence of hCG. Thus, the C-terminus is a crucial part of the extracellular domain involved in hormone binding and signal transduction.
The in vitro experiments using the human LHR cDNA lacking exon 10 performed by Zhang et al. (10) help to interpret the clinical picture of the patient presented in this report. Obviously, the lack of exon 10 in the propositus did not prevent masculinization during fetal life, confirming in vivo the in vitro data published by Zhang et al. (11). However, with the switch to LH after birth, Leydig cell signal transduction must have been impaired, resulting in the observed picture of LH insensitivity. Maternal hCG synthesized during pregnancy led to the development of a normal male phenotype, whereas LH was unable to stimulate the mutant receptor at the time of puberty. This assumption is further supported by the fact that hCG treatment with concentrations comparable to the LH levels observed in the patient is capable of inducing testosterone biosynthesis and complete spermatogenesis. This suggests a possible dual mechanism of hormone binding and signal transduction for hCG and LH.
Future studies may reveal which parts of exon 10 are involved in hormone selectivity of LH and hCG and whether the C-terminal part of the extracellular domain from the LHR can be used in the design and development of hormone-specific analogs.
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Acknowledgments |
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Received September 2, 1999.
Revised February 1, 2000.
Accepted February 5, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit. Mol Endocrinol. 13:1285–1294.This article has been cited by other articles:
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K. Nakamura, S. Yamashita, Y. Omori, and T. Minegishi A Splice Variant of the Human Luteinizing Hormone (LH) Receptor Modulates the Expression of Wild-Type Human LH Receptor Mol. Endocrinol., June 1, 2004; 18(6): 1461 - 1470. [Abstract] [Full Text] [PDF]
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HindIII digest was used
(lane M), and its corresponding sizes are outlined.
