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Department of Pediatric Endocrinology (L.S.-G., J.-C.C.), Laboratory of Pathology (P.B.), and the Laboratory for Hormone Biology (M.R., N.L.), Centre Hospitalier Universitaire Cochin, Saint Vincent de Paul, 75014 Paris, France; Department of Endocrinology (L.S.-G.), Hospital Infantil Nino Jesus, 28009 Madrid, Spain; and Laboratory of Spermiology and Histology (V.M.), Centre Hospitalier Régional Universitaire, Faculty of Medicine, 59037 Lille, France
Address all correspondence and requests for reprints to: Dr. Najiba Lahlou, Biologie Hormonale, Hôpital Saint Vincent de Paul, 82 avenue Denfert-Rochereau, 75014 Paris, France. E-mail: najiba.lahlou{at}svp.aphp.fr.
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Abstract |
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Objectives: The objective of the study was to assess the cellular origin of serum inhibin thanks to testis section immunostaining.
Main Outcome Measures: Serum testosterone, gonadotropin, inhibin B, pan-
C-inhibin, and anti-Mullerian hormone levels were measured. Immunostaining was performed using specific anti-- and anti-ß-subunit antibodies.
Subjects and Methods: Five boys from three families (mutation M398T or I542L) were investigated at onset (2–6 yr), on ketoconazole treatment, and at adolescence. Testis biopsies were performed in three subjects before the disease was fully characterized.
Results: The high testosterone levels were suppressed by ketoconazole. Anti-Mullerian hormone levels were inversely related to testosterone: low at diagnosis, elevated after testosterone suppression. Despite FSH suppression, inhibin B and pan-C-inhibin levels were high from clinical onset to adolescence. Biopsy specimens showed normal Sertoli cell complement and germ cell maturation until the spermatocyte II stage. Sertoli and Leydig cells displayed positive inhibin -subunit immunostaining. Only Leydig cells and spermatogonia stained positively for the inhibin ßB-subunit.
Conclusions: Familial male-limited precocious puberty is a unique model of inhibin B secretion, demonstrating that Leydig cells can produce significant amounts of the dimeric molecule. Our results also suggest that the pubertal FSH rise is not required for full expression of the two inhibin B genes and for the initiation of germ cell maturation.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit and a specific ß-subunit, ßA and ßB, respectively. Inhibin B is the only circulating bioactive form in males and mainly reflects Sertoli cell function (1, 2). FSH is thought to be the main regulator of inhibin B secretion. Inhibin B and FSH levels are positively correlated during the dynamic phases of Sertoli cell proliferation (reviewed in Ref. 3). By contrast, they are inversely correlated in adult males, a model consistent with a classical negative feedback loop, as demonstrated in several clinical studies (reviewed in Ref. 4). Indeed, inhibin was primarily hypothesized, more than 70 yr ago, as a nonsteroidal testicular factor responsible for the inhibition of FSH secretion. This effect seems to result from its competition with the stimulatory peptide activin, a gonadal and extragonadal factor, another member of the TGFß family. Inhibin can prevent the activin binding to its receptors at the cell membrane. One proposed mechanism is to use ß-glycan to occupy the activin receptor II binding subunit of the activin receptor complex, thus preventing activin from exerting its stimulatory effect on the pituitary gonadotrophs and GnRH neurons (reviewed in Refs. 5 and 6). During infancy and childhood, inhibin B levels are significantly higher in males than females, which may account for the significantly lower FSH levels in males than females. In boys aged 2–6 yr, inhibin B levels are much lower than in adult males; they progressively increase after the age of 8 yr simultaneously with the pubertal increase in FSH secretion (reviewed in Ref. 3). Familial male-limited precocious puberty (FMPP) is due to the gonadotropin-independent precocious secretion of testosterone by Leydig cells resulting from activating mutations in the LH receptor gene. This disorder is dominantly transmitted among boys, whereas girls are only mutation carriers. Affected boys generally undergo rapid growth, bone age maturation, and progressive virilization as early as 2–4 yr of age. Testosterone levels are in the pubertal range, gonadotropin secretion is markedly suppressed, and GnRH agonist treatment is ineffective (7). Administration of the steroidogenic enzyme inhibitor ketoconazole lowers testosterone levels, stops the progression of pubertal symptoms, and allows the patient to reach normal adult height (8).
In five boys with FMPP, we observed continually elevated inhibin B levels long before the physiological age of puberty. This raises the question of the cellular origin of inhibin in these boys. In this study, we aimed to elucidate the significance of this non-FSH-dependent inhibin overproduction. Testis biopsy samples from three boys, taken to rule out a diagnosis of Leydig cell tumor before full characterization of the genetic disorder in 1993 (9), gave us a unique opportunity to compare immunostaining data with extensive hormonal investigations.
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Subjects and Methods |
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Five patients from three families with FMPP (F, P1, P2, M1, M2) were followed up from clinical onset to adulthood. Subjects M1 and M2 and subjects P1 and P2 are brothers. Their clinical characteristics have been described in detail elsewhere (8). The main clinical features are given in Table 1
. All harbored either the M398T or I542L mutation.
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Three boys, F, P1, and M1, underwent testis biopsies between 1984 and 1990 at the age of 3.5, 6.0, and 3.6 yr, respectively. Testis biopsy samples from adults with congenital obstructive azoospermia but normal spermatogenesis were used as controls (10).
Hormone assays
Inhibin B was measured by ELISA as previously described (11), using Oxford BioInnovation reagents (Diagnostic Systems Laboratories-France, Cergy-Pontoise, France). Inhibin A exhibited a 1% cross-reactivity in this assay. The intra- and interassay coefficients of variation were 5.7 and 12%, respectively, at the level of 112 pg/ml and 4.1 and 8.5%, respectively, at the level of 225 pg/ml. The sensitivity was 6 pg/ml.
Total immunoreactive -inhibin or pan-C-inhibin was measured by RIA as previously described (12) using antiserum 1989 kindly donated by D. M. de Kretser (Monash University, Melbourne, Australia). The tracer was purified 31-kDa bovine inhibin iodinated by the lactoperoxidase method. A pool of human follicular fluid was used as a standard. Its bioactivity was 280 U/ml in an in vitro bioassay with dispersed rat pituitary cells in monolayer culture. The intra- and interassay coefficients of variations were 3.8 and 12%, respectively, at the level of 270 U/ml and 4.6 and 15%, respectively, at the level of 518 U/ml. The sensitivity was 28 U/ml.
Anti-Mullerian hormone (AMH), or Mullerian-inhibiting substance, was measured by ELISA using reagents from Diagnostic Systems Laboratories. A recombinant AMH preparation supplied by the manufacturer was used as a standard. The intra- and interassay coefficients of variation were 2.3 and 3.1%, respectively, at the level of 15 ng/ml and 1.4 and 2.5%, respectively, at the level of 78 ng/ml. The sensitivity was 0.2 ng/ml.
Testosterone was measured by a direct RIA as previously described (13) using Orion reagents (CIS Biointernational, Gif-sur-Yvette, France). Dihydrotestosterone was the only cross-reacting steroid: 4.5%. The intra- and interassay coefficients of variation were 3.8 and 5.4%, respectively, at the level of 1.2 ng/ml and 4.6 and 6.5%, respectively, at the level of 8.6 ng/ml. The sensitivity was 0.017 ng/ml.
FSH and LH were measured by time-resolved fluoroimmunometry as previously described (13), using Wallac reagents (PerkinElmer, Courtaboeuf, France). In the FSH assay, the intra- and interassay coefficients of variation were 1.2 and 3.9%, respectively, at the level of 3.1 IU/liter and 1.5 and 2.8%, respectively, at the level of 16.6 IU/liter. In the LH assay, the intra- and interassay coefficients of variation were 1.4 and 2.6%, respectively, at the level of 0.3 IU/liter and 1.7 and 2%, respectively, at the level of 6.9 IU/liter. The sensitivity was 0.01 IU/liter for both assays.
Reference values for all these parameters were established in our laboratory on the basis of data obtained from 651 normal boys (Table 2). They were sampled in the course of school population-based studies or for systematic investigation in siblings of children with allergic disease. All subjects were selected on a clinical basis: height, weight, bone age, and pubertal development normal for age and no clinical symptom of either endocrine or metabolic disorder.
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Testis biopsy samples of approximately 2 x 2 mm were obtained by open surgery. They were fixed in a modified Bouin Hollande Sublime solution (paraformaldehyde, copper acetate, mercuric chloride, picric acid) for 48 h, embedded in paraffin, and stored at room temperature. Five-micrometer-thick paraffin sections were processed for classic histology. The immunochemical detection of inhibins was performed on the archival biopsy samples only available from P1 and M1 patients. No paraffin-embedded piece was left for patient F.
Testicular paraffin sections were processed for inhibin -, ßA-, and ßB-subunits immunohistochemistry by using immunoperoxidase and immunofluorescent method with tyramide amplification system as described by Csaba et al. (14).
Sections were dewaxed, rehydrated in graded ethanol, washed in water, and then subjected to antigen retrieval by microwaving in 0.01 M citrate buffer (pH 5.2). After cooling for 30 min, sections were washed in 0.02 M potassium PBS (KPBS) (pH 7.4) and preincubated in 0.3% Triton X-100–5% normal sheep serum in KPBS for 60 min. They were then incubated with monoclonal primary antibodies directed against -, ßA-, and ßB-subunits of inhibins obtained from Oxford BioInnovation (Diagnostic Systems Laboratories) for 72 h at 4 C at a concentration of 1:100, 1:5, and 1:5, respectively, in 0.3% Triton X-100–0.02 M KPBS. The anti--subunit antibody MCA951S clone R1 was raised against a synthetic peptide corresponding to the 1–32 peptide of the -subunit. The anti-ßA-subunit antibody MCA950S clone E4 was raised against a synthetic peptide corresponding to the 82–114 peptide of the ßA-subunit. The anti-ßB subunit antibody MCA1661 clone C5 was raised against a synthetic peptide corresponding to a 33-amino acid peptide near the N terminus of the inhibin ßB-subunit. After washing in KPBS for 10 min, the sections were sequentially incubated for 90 min in 1:150 biotinylated sheep antimouse IgG diluted in KPBS and for 90 min in avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, AbCys, Paris, France). After washing in KPBS, the sections were subsequently incubated for 30 min in a 1:100 biotinyl tyramide solution (PerkinElmer, Boston, MA) at room temperature. Sections were exposed for 90 min to 1:200 streptavidin-peroxidase or 1:400 streptavidin-alexa 488 (Amersham Biosciences, Orsay, France). Peroxidase activity was revealed with 0.05% of 3,3'-diaminobenzidine (Sigma-Aldrich, St.-Quentin-Fallavier, France) in 0.05 M Tris-buffered saline (pH 7.6), in the presence of hydrogen peroxide (0.008%). The reaction was stopped in Tris-buffered saline. Sections were counterstained with hematoxylin for cell nuclear visualization and mounted with Vectashield (Vector Laboratories, Burlingame, CA).
The specificity of the primary antibodies against inhibin - and ßB-subunits was previously established (10, 15). In the present study, the specificity of the antibodies was confirmed by using normal mouse serum instead of primary antibodies, by preabsorbing the antibodies with the corresponding peptide, including for the anti-ßA-subunit antibody. These control sections displayed no labeling (data not shown).
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Results |
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At the onset of sexual precocity, at the age of 2.3–6 yr, both testosterone and AMH levels were in the pubertal range (Fig. 1, A and B).
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C-inhibin and inhibin B levels were also in the pubertal range and remained elevated throughout the follow-up period (Fig. 2, A and B). Before treatment, a positive correlation was found between inhibin B and testosterone levels (r = 0.58, P = 0.02, three successive samples per boy; Fig. 3).
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Before the correct diagnosis of the disorder was established, patients F and P1 were treated with triptorelin (a GnRH agonist) at the age of 3.9 and 5.8 yr, respectively, for 13 and 4 months, respectively. A further decrease in the gonadotropin response to the LHRH test was observed without any decrease in testosterone and inhibin levels.
Treatment with ketoconazole for several years decreased testosterone levels to less than 0.4 ng/ml, except in three samples from patients F and M1 (Fig. 1A
). The blockade of testosterone production stopped pubertal maturation but had no effect on inhibin levels. AMH levels increased to prepubertal range and remained elevated as long as ketoconazole was administered (Fig. 1B). Secondarily, these patients displayed spontaneous maturation of gonadotropic secretions at the age of 10.25–12 yr, with FSH peak: 1.22 ± 0.97, LH peak: 9 ± 0.6 IU/liter. Testosterone levels increased despite continued ketoconazole treatment.
Finally, ketoconazole treatment was discontinued at the age of 10.25–13.3 yr. Testosterone levels increased, and AMH levels decreased. Inhibin B and pan-C-inhibin levels remained in the pubertal range (Fig. 2).
In summary, inhibin B and pan-C-inhibin levels were high throughout the observation period, regardless of testosterone levels and the degree of gonadotropin suppression.
Testis histology
Almost identical histological patterns were obtained from testis sections from patients F, P1, and M1.
There were numerous tightly packed seminiferous tubules containing Sertoli cells and spermatocytes. According to references for normal testicular development (16), the Sertoli cells were normal for age, in both size and number. About 30 Sertoli cells per tube section were counted. The shape of the nucleus and the presence of some nuclear indentations corresponded to normal morphology for age. Sertoli cells from the two younger patients, F and M1, who where younger than 4 yr of age at the time of the biopsy, had the morphology of prepubertal Sertoli cells, without any sign of the active growth typical of pubertal maturation. By contrast, Sertoli cells from patient P1, who was 6 yr old at the time of the biopsy, exhibited some initial signs of active growth, with a few lipid vacuoles, appearance that corresponded to his chronological age (Fig. 4). In none of the three boys did Sertoli cells exhibit the nuclear changes characteristic of puberty (16).
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Leydig cells were scarce and nonhyperplastic and did not contain any Reinke crystalloids.
Immunohistochemistry
Immunostaining for the inhibin -subunit was observed in the Sertoli and Leydig cells of all testis samples analyzed: normal adult and FMPP testes (Fig. 5, A and D).
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) but not FMPP testes.
In control adult testes (Fig. 5C), immunostaining for the inhibin ßB-subunit was observed in germ cells from pachytene spermatocytes to spermatocytes II and in some round spermatids. Leydig cells were also stained. Sertoli cells showed no staining. In patients P1 and M1, faint immunostaining for the inhibin ßB-subunit was observed in Leydig cells (Fig. 5E). In the seminiferous tubules of patient M1, some immunostained cells were identified as spermatogonia (Fig. 5F). Spermatocytes, spermatids (P1), and Sertoli cells were not labeled.
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Discussion |
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In prepubertal human testes, both the - and ßB-subunits of inhibin are colocalized in Sertoli cells (15). In pubertal and adult human testes, ßB-subunits are mostly found in spermatocytes and round spermatids, whereas -subunits are found in Sertoli and Leydig cells (10, 15), suggesting that inhibin B may be produced by cooperation between cells in the seminiferous tubules. Our findings, and the known constitutive activation of Leydig cells in FMPP, are consistent with the production of significant amounts of inhibin B by Leydig cells.
Leydig cells are not thought to produce significant amounts of inhibin B in adult males because human chorionic gonadotropin (hCG) stimulation does not increase inhibin B levels (17, 18). However, prolonged hCG administration in prepubertal boys has been shown to increase inhibin B secretion significantly (19, 20). Leydig cells are stained with an anti-ßB-subunit antibody in the testes of pubertal boys and normal men (10, 15). In FMPP testes, only Leydig cells produce both the - and ßB-subunits. This finding supports the assumption that Leydig cells make a significant contribution to serum inhibin B levels. The positive correlation found between testosterone and circulating inhibin B levels at the onset of clinical symptoms is also consistent with the assumption that LH receptor activation is responsible for premature secretion of both testosterone and inhibin.
The mutations in the FSH ß-subunit gene (21, 22) and the inactivating mutations in the FSH receptor gene (23, 24) are also interesting models of testis development in the absence of FSH action. In the very few reported males harboring either of these mutations, inhibin B levels, when measured, were low but detectable: 6–62 pg/ml. These observations could also support the assumption of a significant production of inhibin B from other sources than Sertoli cells. Unfortunately, because there is no report of the effect of hCG administration on inhibin B levels, the possible role of Leydig cells is not ascertained in these models.
It could also have been hypothesized that testosterone itself or a Leydig cell-produced paracrine factor distinct from testosterone is capable of stimulating inhibin production by Sertoli cell or another cell type. It was reported in 1989 (25) that peritubular cell protein modulating Sertoli cell activity (PModS), a paracrine factor produced by peritubular myoid cells under androgen control, stimulates inhibin secretion by Sertoli cells in culture. But it should be noticed that at that time, no immunoassay specific for the ßB-inhibin was available. Therefore, the possible effect of PModS on inhibin B production remains hypothetical. On the other hand, it is well known that Sertoli cells express the androgen receptor and testosterone was shown to increase intracellular calcium ion levels and activate the MAPK pathway in Sertoli cells (reviewed in Ref. 26). However, no evidence has been given of any effect of testosterone on inhibin gene expression in Sertoli cells. Moreover, in our patients, the levels of inhibin B and pan-C-inhibin were not affected during serum testosterone suppression by ketoconazole. In addition, the maintenance of high levels of intratesticular testosterone during ketoconazole treatment is unlikely because the pretreatment suppression of AMH production was removed when on ketoconazole. That suggests that testosterone is not required for the maintenance of high inhibin production and that inhibin secretion is not driven by testosterone in FMPP.
The absence of Sertoli cell immunostaining for the ßB-subunit suggests that these cells do not produce significant amounts of the dimeric inhibin B in FMPP patients. That does not preclude the possibility that Sertoli cells contribute to pan-C-inhibin levels and even to inhibin B production by Sertoli-germ cell cooperation as proposed by Andersson et al. (15), given the ßB-inhibin immunostaining in some spermatogonia from patient M1. It is well documented that Sertoli cells may play a role in inhibin B production in humans (reviewed in Refs. 3, 4) and other mammals. In the rhesus monkey, the postnatal proliferation of Sertoli cells is maximal during infancy when FSH secretion is high (27), inhibin B secretion peaks during the first trimester of postnatal life (28), and inhibin B secretion is positively regulated by FSH (29). In the male rat, the Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (30). The FSH receptor gene is expressed on Sertoli cell membranes, and inhibin secretion responds to FSH administration in men with hypogonadotropic hypogonadism (17). However, because in our patients FSH levels were in the low prepubertal range or below, additional suppression of FSH by triptorelin in two patients did not alter inhibin production, and the Sertoli cell morphology was prepubertal, a significant contribution by Sertoli cells to inhibin production seems unlikely.
The contrast between germ cell maturation until the spermatocyte II stage and low prepubertal FSH levels raises the question of the relative roles of FSH and testosterone in initiating spermatogenesis. Experiments in rodents have shown that FSH and testosterone act in synergy in germ cell maturation (31) and that androgens are required for normal Sertoli cell number and germ cell development (32). However, testosterone alone can induce significant germ cell maturation, independently of FSH activity (33). Similarly, in the rhesus monkey, mitotic proliferation of type A spermatogonia is relatively gonadotropin independent (34), and differentiation of spermatogonia from type A to type B may be driven by either testosterone or FSH (35). These experimental data support the hypothesis that in FMPP the maturation of germ cells to the spermatocyte I and II stages was gonadotropin independent and probably results from prolonged exposure to testosterone.
In addition, it should be noted that in our FMPP patients, the neonatal Sertoli cell proliferation occurred normally because their Sertoli cells are normal in size and number relative to age as shown by our biopsy specimens and the pattern of AMH secretion after testosterone suppression by ketoconazole was similar to that observed in normal prepubertal boys. These findings also suggest that AMH secretion does not depend directly on FSH levels.
Concluding remarks: FMPP as a model of inhibin B secretion
FMPP provides a unique model of inhibin B overproduction: Leydig cells are constitutively activated, whereas gonadotropins are in the low prepubertal range or below. Leydig cells express the genes encoding both the inhibin - and ßB-subunits, strongly suggesting that the premature secretion of inhibins in this disorder originates from Leydig cells.
According to this model, the pubertal FSH rise is required neither for the expression of inhibin - and ßB-subunit genes nor the initial maturation of germ cells. These findings are consistent with the experimental data both in rodents and in monkeys, demonstrating that testosterone alone or LH receptor activation can initiate germ cell maturation (33) and that differentiation of spermatogonia may be initiated by testosterone alone (35).
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Acknowledgments |
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C-inhibin RIA, the World Health Organization International Laboratory for Biological Standards (Potters Bar, UK) for the supply of gonadotropin standards, and Sharon Bowman for the revision of the manuscript. The skillful assistance of Nathalie Robert, Yvette Le Bihan, Catherine Gaillard, and Aurélie Canicio is gratefully acknowledged. |
Footnotes |
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Disclosure of potential conflicts of interest: L.S.-G., V.M., J.-C.C., P.B., M.R., and N.L. have nothing to declare.
First Published Online May 9, 2006
1 L.S.-G. and V.M. contributed equally to this work. 
Abbreviations: AMH, Anti-Mullerian hormone; FMPP, familial male-limited precocious puberty; hCG, human chorionic gonadotropin; KPBS, potassium PBS.
Received November 28, 2005.
Accepted May 2, 2006.
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References |
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and ßB subunits and expression of corresponding messenger RNAs in the human adult testis. Biol Reprod 68:230–235C levels to gonadotrophic stimulation in normal men before and after steroidal contraceptive treatment. Hum Reprod 18:734–743C during development in the male rhesus monkey. Endocrinology 140:5497–5504This article has been cited by other articles:
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