Original Article
Brief Report
Normal Spermatogenesis in a Man with Mutant Luteinizing Hormone
N Engl J Med 2009; 361:1856-1863November 5, 2009
- Abstract
Men with mutations in LHB, the gene encoding the beta subunit of luteinizing hormone (LHB), have azoospermia with absent or few fetal Leydig cells. We report a mutation in LHB in a man and his sister. The man presented with absence of virilization, undetectable luteinizing hormone, and a low serum testosterone level. He had complete spermatogenesis with a normal sperm count. The mutant luteinizing hormone had a low level of partial activity in vitro. We concluded that the residual luteinizing hormone activity, resulting in the expression of steroidogenic enzymes in few mature Leydig cells producing small amounts of intratesticular testosterone (20.2 ng per gram), was sufficient for complete and quantitatively normal spermatogenesis.
Media in This Article
Figure 1
Pedigree of the Patient with Hypogonadism and LHB Mutation.Figure 2
Histologic and Immunocytochemical Studies of the Patient's Testis.Article Activity
- Article
Mutations that abolish the activity of luteinizing hormone are rare; they have been reported in five men and one woman.1-5 The phenotypes of these persons suggest that luteinizing hormone is not required for male sexual differentiation but is critical to the proliferation and function of Leydig cells and to the induction of puberty. Infertility and very low levels of spermatogenesis persist in the affected men, despite long-term exposure to human chorionic gonadotropin, suggesting that the absence of perinatal exposure to luteinizing hormone alters Leydig cells' proliferation and maturation, impairing the onset of normal spermatogenesis, which is thought to be critically dependent on a high level of intratesticular testosterone.6-9
We report a case of familial hypogonadotropic hypogonadism involving a partial loss of luteinizing hormone function. The proband had an undetectable circulating luteinizing hormone level and a low serum testosterone level. He had a markedly small population of mature Leydig cells expressing the steroidogenic enzymes necessary for androgen synthesis and producing low levels of intratesticular testosterone. He had a mutant form of luteinizing hormone beta that showed low-level residual function in vitro, indicating the presence of low levels of luteinizing hormone activity from birth to adulthood, which permitted the maturation and function of a small number of Leydig cells. This was nevertheless sufficient to trigger and maintain complete and quantitatively normal spermatogenesis.
Case Report
This study was approved by the review boards of the institutions involved, and written informed consent was obtained from the patient and his family members. The 43-year-old proband (Subject IV-3, with a 46,XY karyotype) (Figure 1Figure 1
Pedigree of the Patient with Hypogonadism and LHB Mutation.) had been born in Morocco to consanguineous parents and was previously treated there for hypogonadism at 28 years of age. Treatment with intramuscular testosterone resulted in masculinization and penile growth. The treatment was irregular because of poor adherence; it had been discontinued for 3 months before the patient was referred to our department. Examination showed that the patient was virilized (185 cm in height, 75 kg in weight) with normal masculine features: pubic hair of Tanner stage 5, axillary hair of Tanner stage 3, penile length of 12 cm, normal testicular volume, and absence of gynecomastia. Initial and subsequent laboratory tests (Table 1Table 1
Serum Hormone Levels in the Proband with LHB Mutation, His Affected Sister, and Members of Their Family.) showed undetectable luteinizing hormone levels (i.e., <0.05 IU per liter), high follicle-stimulating hormone and anti-Müllerian hormone levels, normal inhibin B levels, and very low serum testosterone levels. Magnetic resonance imaging of the brain and pituitary was normal. Ultrasonography revealed bilateral testicular microlithiasis and testis volume in the lower normal range (right testis, 14.5 cm3; left testis, 12.0 cm3).Hypogonadism due to isolated deficiency of luteinizing hormone beta was suspected. Surprisingly, several spermiograms performed over a 10-month period consistently revealed normal numbers of spermatozoa (76.4×106 to 127.0×106 spermatozoa per milliliter) and qualitatively normal-to-subnormal spermatogenesis (5 to 23% of sperm with normal morphology and 25 to 35% with motility), with low semen volume (0.8 cm3).10
Testicular biopsy showed heterogeneous seminiferous tubules separated by fibrous tissue (Figure 2AFigure 2
Histologic and Immunocytochemical Studies of the Patient's Testis.), containing few mature, vacuolated Leydig cells (Figure 2B and 2C). Half the tubuli were hyalinized or hypoplastic, with a thickened lamina, immature Sertoli cells, and spermatogonia. The other half were composed of mature Sertoli cells and germ cells at all stages of differentiation (Figure 2A, 2B, and 2C).Immunohistochemical analysis showed that the small number of typical mature Leydig cells expressed enzymes necessary for androgen synthesis: cytochrome P-450 (CYP) 17α-hydroxylase (Figure 2D), 3β-hydroxysteroid dehydrogenase (Figure 2F), and CYP cholesterol-side-chain cleavage enzyme (not shown). We also detected fibroblast-like fetal Leydig precursors, located outside the tubular basal lamina, expressing 3β-hydroxysteroid dehydrogenase (Figure 2G) but devoid of CYP 17α-hydroxylase (not shown).9,11 There were markedly fewer Leydig cells in the patient's testis-tissue sample than in a sample from an age-matched control (Figure 2E). The patient's Sertoli cells expressed anti-Müllerian hormone (Figure 2H), the persistence of which indicates low levels of intratesticular testosterone,12 whereas control cells had an absence of anti-Müllerian hormone (Figure 2I). The expression patterns of the androgen receptor (Figure 2J and 2K) and of both markers of germ-cell-maturation histone H1 (Figure 2L and 2M) and proacrosin (Figure 2N and 2O) were similar in samples from the patient's testis and the control testis.
Testosterone measurements were obtained with the use of gas chromatography–mass spectrometry.13 The level of intratesticular testosterone in the patient's biopsy sample (20.2 ng per gram of tissue) was approximately one eighteenth that of a specimen from an age-matched control (356.6 ng per gram of tissue); the control value matches values in previous reports.14 Similarly, the level of intratesticular 5α-dihydrotestosterone was much less in the patient's sample (0.59 ng per gram of tissue) than in the control sample (12.5 ng per gram of tissue) (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). The serum testosterone level was 0.4 ng per milliliter (1.4 nmol per liter), confirming the immunoassay results.
The proband's siblings (Figure 1A) underwent normal, spontaneous puberty. One sister, Subject IV-5 (born in 1961), had menarche at 14 years of age and subsequently had oligomenorrhea and secondary amenorrhea. Repeated ultrasonography in the sister, for evaluation of infertility, at 30 years of age revealed bilateral ovarian macrocysts. Recent assays showed undetectable luteinizing hormone and low estradiol and high follicle-stimulating hormone levels (Table 1). A brother, Subject IV-2 (born in 1967), was the father of two children, and another sister, Subject IV-4 (born in 1975), had normal hormonal levels (Table 1). Yet another sister, Subject IV-1 (born in 1957), was the mother of two children; she and the other brother, Subject IV-6 (born in 1977), were unavailable for study.
Methods
DNA Sequencing
We sequenced the three exons of LHB, as described in the Supplementary Appendix.
Immunohistochemical Analysis
Immunohistochemical evaluation was performed with the use of a commercial kit (LSAB+ system with 3-amino-9-ethylcarbazole, Dako), as described previously.15
Functional Analysis of Mutant Luteinizing Hormone Beta
Coding sequences of human LHA (encoding the alpha subunit of luteinizing hormone) and LHB were inserted into the pSG5 vector (Stratagene). We then introduced the deletion carried by the proband into LHB to create a mutant luteinizing hormone beta construct; we also introduced the V5 epitope into the C-terminal of the luteinizing hormone alpha subunit to make the construct α-V5. Western blot analyses were performed with the use of an anti–human chorionic gonadotropin β antibody (ab14301, Abcam), which cross-reacts with the beta subunit of human luteinizing hormone; the samples blotted were cell lysates and concentrated medium of human embryonic kidney (HEK) 293T cells transfected with the use of a transfection reagent (SuperFect, Qiagen). For coimmunoprecipitation analysis, cell lysates from transfected COS-7 cells (derived from fibroblasts from kidneys of African green monkeys) were incubated with anti-V5 antibody (Invitrogen), and immunoprecipitates were analyzed by means of Western blotting. The activity of recombinant secreted mutant luteinizing hormone relative to that of wild-type luteinizing hormone was determined by assessing cyclic AMP accumulation in HEK 293T cells transiently expressing the human luteinizing hormone receptor. The level of wild-type luteinizing hormone in the culture medium was determined by means of immunofluorometric assay (LHsp enzyme-linked immunosorbent assay, Biosource). Details are given in the Supplementary Appendix.
Results
Sequence analysis of LHB from the proband (Fig. S1A in the Supplementary Appendix) revealed a homozygous nine-base deletion in exon 2, predicted to result in a deletion of amino acids 10 to 12 (histidine–proline–isoleucine) in the mutant luteinizing hormone beta (Figure 1B). Proline and the hydrophobic residues (isoleucine or valine) are highly conserved in mammalian LHB genes. The affected sister, Subject IV-5, was also homozygous for the deletion; the mother (Subject III-2) and asymptomatic siblings (Subjects IV-2 and IV-4) were heterozygous for the deletion (Fig. S1B in the Supplementary Appendix).
Both the mutant and wild-type luteinizing hormone beta (both approximately 15 kD in size)16 were synthesized in HEK 293T cells (Figure 3AFigure 3
Production and Heterodimerization in Mutant and Wild-Type Luteinizing Hormone (LH) Beta and Bioactivity of Mutant and Wild-Type LH.). However, we observed less mutant protein than wild-type protein in cell lysates (mean [±SD] ratio of wild-type:mutant, 2.3±0.8, from three independent experiments), suggesting that the deletion may result in decreased stability of the mutant protein or affect the stability or translational efficiency of its corresponding messenger RNA. In concentrated culture medium, the level of wild-type protein (expressed as the mean [±SD] of three independent experiments) was 67.9±8.6 times that of the mutant protein (Figure 3A). After normalization of this ratio on the basis of the intracellular expression ratio, we calculated that the secretion level (expressed as the mean [±SD] of three independent experiments) associated with wild-type luteinizing hormone was 32.8±11.7 (mean [±SD] of three independent experiments) times that associated with the mutant luteinizing hormone beta, when coexpressed with the alpha subunit in HEK 293T cells.Coimmunoprecipitation studies with COS-7 cells cotransfected with expression vectors containing the α-V5 construct and the wild-type or mutant luteinizing hormone beta subunit were performed to determine whether the defect in secretion of mutant luteinizing hormone beta resulted from defective subunit heterodimerization. Immunoprecipitates from cell lysates incubated with anti-V5 antibody, as previously described,2 contained both subunits, indicating the occurrence of heterodimerization of the alpha and beta subunits (Figure 3B). However, immunoprecipitates contained less mutant luteinizing hormone beta than the wild type (ratios in two independent experiments, 1:8 to 1:10).
Cyclic AMP accumulation was markedly depressed in association with the mutant luteinizing hormone as compared with the wild-type hormone (Figure 3C). Assuming that most of the secreted mutant luteinizing hormone is dimerized, as is wild-type luteinizing hormone, the mean (±SD) residual function (measured in three independent experiments) of the mutant hormone was 0.73±0.32% of that of the wild-type hormone.
Discussion
The patient described here had an absence of spontaneous virilization, very low serum testosterone levels, and minimal luteinizing hormone activity — but complete, quantitatively normal spermatogenesis.
In humans, there are three waves of Leydig-cell growth: the first, during the antenatal period, when growth is dependent on human chorionic gonadotropin; and the second and third, during the perinatal period and puberty, respectively, when growth is strictly under the control of luteinizing hormone.9-11 Male infants display transient postnatal activation of the gonadotropin-releasing hormone pulse generator, inducing a surge in follicle-stimulating hormone, luteinizing hormone, and testosterone that is correlated with normal adult spermatogenesis and fertility.17 Partial stimulation of Leydig-cell proliferation, maturation, and function by luteinizing hormone may have occurred in the testes of our patient, both perinatally and during puberty, to induce spermatogenesis. The mutant luteinizing hormone in our patient is known to retain partial activity, for two reasons. First, his testicular biopsy specimen contained mature Leydig cells expressing the steroidogenic enzymes CYP cholesterol-side-chain cleavage enzyme and CYP 17α-hydroxylase, which are required for testosterone synthesis — the expression of these enzymes is strictly dependent on luteinizing hormone.9,11 Second, the intratesticular testosterone level in the patient (20.2 ng per gram) greatly exceeded (by a factor of 40) the serum testosterone level, a discrepancy that is consistent with testosterone production within the testes.
The low production of intratesticular testosterone and consequent weak testosterone secretion into the serum (Table 1, and Table S1 in the Supplementary Appendix) were insufficient to induce virilization but were nevertheless capable, in cooperation with a functional follicle-stimulating hormone–gonadal axis, of exerting a local paracrine effect on contiguous Sertoli cells and inducing the development and maturation of seminiferous tubules that are required for complete spermatogenesis.18 Our findings support the recent proposal that congenital hypogonadotropic hypogonadism be managed both neonatally and at puberty through the administration of exogenous gonadotropins mimicking the physiologic gonadotropin surges.17,19 As has been described in men with other LHB mutations, our patient had inappropriately normal levels of inhibin B, given his high plasma level of follicle-stimulating hormone.2,3 This may have been due to the insufficient development of the follicle-stimulating hormone–inhibin feedback loop.20
The homozygous LHB deletion of the patient and two of his heterozygous, unaffected family members was found to occur near a disulfide bridge forming the cystine-knot folding motif. This region may tolerate conformational changes.16
In male mice deficient in the luteinizing hormone receptor, spermatogenesis proceeds until the elongated spermatid stage, and the mice are infertile.21 However, major differences in the hormonal regulation of spermatogenesis are now known to exist between primates and rodents. Thus, to obtain complementary information on the maintenance of spermatogenesis in normal men undergoing gonadotropin suppression, studies have been performed in men with the use of hormonal contraception protocols. On administration of testosterone, both luteinizing hormone and follicle-stimulating hormone are decreased to minimal levels, suppressing intratesticular testosterone production in these men.22,23 Intratesticular testosterone levels similar to the serum levels (3.8±1.3 ng per milliliter [13.2±4.5 nmol per liter])24 cannot maintain quantitatively normal sperm production. However, low intratesticular testosterone levels have been found to be sufficient for the maintenance of low levels of spermatogenesis in some men with previously normal testicular development and normal sperm maturation.22 Currently, the minimal intratesticular testosterone level required to trigger and maintain normal sperm production in men is not known.6,22
This study goes against current wisdom in that it shows that complete and quantitatively normal spermatogenesis may be triggered and maintained by low levels of luteinizing hormone activity postnatally and at puberty. A better understanding of the intratesticular hormonal microenvironment required throughout life (and especially during the perinatal and peripubertal periods) for adult spermatogenesis could result in better strategies to treat certain patients with infertility and to develop hormonal contraception methods for men.22,23
Supported by grants from INSERM, the Société Française d'Endocrinologie, Assistance Publique–Hôpitaux de Paris, and the French Ministry of Health (DHOS).
No potential conflict of interest relevant to this article was reported.
Drs. Achard, Courtillot, and Lahuna contributed equally to this article, as did Drs. Touraine and Misrahi.
We thank G. Schaison, P. Roger, N. Lahlou, and E. Pussard for helpful discussions; N. Josso (INSERM Unité 293, Montrouge, France) and D. Escalier (Centre Hospitalier Universitaire Bicêtre, Le Kremlin Bicêtre, France) for kindly providing the anti-Müllerian hormone and anti-proacrosin antibodies, respectively; Prof. N. Suganuma (Nagoya University School of Medicine, Handa, Japan) for kindly providing the pM2α and pM2β vectors; S. Maillet for help with sequencing analyses and polymerase-chain-reaction–restriction-fragment–length polymorphism experiments; C. Coussieu, S. Brailly, and N. Lahlou for performing hormonal assays and S. Bart for performing the testicular biopsy; and A. Pianos, B. Eychenne, and A. Cambourg for technical assistance with steroid determinations by means of gas chromatography–mass spectrometry.
Source Information
From INSERM Unité 854, University Paris South 11 (C.A., O.L., M.M.); and the Laboratories of Molecular Genetics, Pharmacology, and Hormonology (G.M., M.M.) and Biological Andrology (J.-C.S.), Assistance Publique–Hôpitaux de Paris; and INSERM Unité 788 (P.L., M.S.) — all at Bicêtre Hospital, Le Kremlin Bicêtre, France; Université Paris VI, Department of Endocrinology and Reproductive Medicine, Pitié–Salpêtrière Hospital, and Centre des Maladies Endocriniennes Rares de la Croissance (C.C., A.B., F.K., P.T.) and University Paris Descartes (O.L., J.-C.S., M.M.) — all in Paris; and Abdou Hay Al Mahatta, Oujda, Morocco (H.B.).
Address reprint requests to Dr. Misrahi at INSERM Unité 854, Laboratory of Molecular Genetics, Pharmacology, and Hormonology, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, 94275 Le Kremlin Bicêtre, France, or at micheline.misrahi@bct.aphp.fr.
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- Citing Articles (3)
Citing Articles
1
Stephanie T Page. (2011) Physiologic role and regulation of intratesticular sex steroids. Current Opinion in Endocrinology, Diabetes and Obesity 18:3, 217-223
CrossRef2
N. Ansari. (2011) Les hypogonadismes hypogonadotrophiques congénitaux masculins, quelles données récentes ?. Andrologie 21:2, 68-74
CrossRef3
Ursula B. Kaiser. 2011. Gonadotropin Hormones. , 205-260.
CrossRef
