Immunolocalization of Inhibin and Activin and B Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis

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1 BIOLOGY OF REPRODUCTION 68, (2003) Published online before print 14 October DOI /biolreprod Immunolocalization of Inhibin and Activin and B Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis C. Marchetti, 2,3 M. Hamdane, 2 V. Mitchell, 2,3 K. Mayo, 4 L. Devisme, 5 J.M. Rigot, 6 J.C. Beauvillain, 3 E. Hermand, 3 and A. Defossez 1,2,3 Inserm U422, Lille cedex, France Laboratoire de Biologie de la Reproduction-Histologie, 3 CHRU-Faculté demédecine, Lille cedex, France Department of Biochemistry, 4 Northwestern University, Evanston, Illinois Laboratoire d Anatomo-pathologie, 5 CHRU, Lille cedex, France Service d Andrologie, 6 CHRU, Lille cedex, France ABSTRACT Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an and a B subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mrna probes, the mrna expression patterns of inhibin and inhibin/activin B subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin subunit and expressed inhibin subunit mrna. Using inhibin B subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin B subunit mrna expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin B subunit mrna labeling density was observed in both Sertoli cells and Leydig cells, whereas B subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells. inhibin, Sertoli cells, spermatogenesis, testis INTRODUCTION Inhibins/activins were first recognized as gonadal hormones that regulate the production and release of FSH from the anterior pituitary gland in a classic feedback loop [1]. Studies now show that inhibins are composed of a dimer of a common subunit and either a A (inhibin A) or B (inhibin B) subunit while the activins are subunit dimers (activin A, activin B, and activin AB). These proteins, which are members of the transforming growth factor beta (TGF beta) superfamily, are produced in the testis and are 1 Correspondence: André Defossez, Laboratoire de Biologie de la Reproduction-Histologie, CHRU, av. Oscar Lambret, Lille cedex, France. FAX: ; a-defossez@chru-lille.fr Received: 8 February First decision: 26 February Accepted: 7 August by the Society for the Study of Reproduction, Inc. ISSN: considered as having paracrine and autocrine actions in the regulation of steroidogenesis and spermatogenesis [2]. The development of immunoassays specific for the dimeric forms has allowed the demonstration that inhibin B is the relevant circulating form produced in the fetal and adult human male [3 6]. Prior to puberty, Sertoli cells appear to be the most likely cellular source of inhibin B production, while after puberty, serum inhibin B levels are closely related to spermatogenic status, as shown by the direct correlation between serum inhibin B and sperm count [7]. Thus, in the adult, the cellular site of intratesticular production of both the and B subunits of inhibin is still controversial. Inhibin/activin subunit (, A, and B ) immunoreactive protein localization patterns have been examined in fetal and adult human testes. In the fetal human testes, and B but not A subunits were immunolocalized in Leydig and Sertoli cells [8, 9]. Inhibin subunit mrna signal is present in Sertoli cells and interstitial cells, whereas B subunit mrna is expressed predominantly in seminiferous tubules and A subunit mrna was detected in the interstitial cells [10]. In normal adult testes, subunit immunostaining is present in both Leydig and Sertoli cells [11] and the A subunit is localized in both Leydig and Sertoli cells [12]. Anderson et al. [13] show a specific immunoreactivity for both the and B subunits in Sertoli cells and in Leydig cells. Andersson et al. [14, 15] obtain also in adult testes a positive immunostaining for inhibin subunit in both Sertoli cells and Leydig cells. But these authors, using two different monoclonal anti- B subunit antibodies, observe an intense immunostaining located in germ cells from pachytene spermatocytes to round spermatids. No staining for the inhibin B subunit was observed in Sertoli cells, spermatogonia, preleptotene, leptotene, or zygotene spermatocytes or late spermatids. However, Leydig cells were also positive for B subunit immunostaining. Thus, while it seems clear that the subunit is contained in the Sertoli cells, the B subunit is present in the Sertoli cells and in spermatocytes. The distribution of inhibin/activin subunit (, A, and B ) mrna in adult human testes has not been described to our knowledge. Taking into account these divergent results, the aim of the present study was to determine the immunocytochemical localization of the and B subunits of inhibin/activin in testicular biopsies from adult men with normal or abnormal spermatogenesis. In addition, we report subunit mrna and B subunit mrna expression observed by in

2 INHIBIN B AND GERM CELLS 231 situ hybridization in seminiferous tubules and in the interstitial cells. MATERIALS AND METHODS Subjects Testicular biopsies were obtained from men referred to our clinic for infertility and were performed as part of a clinical routine evaluation. Based on histologic examination, men were grouped according to the presence of normal spermatogenesis (n 6), spermatogenic arrest (n 5), or Sertoli cell only (SCO) syndrome (n 8). The men with normal spermatogenesis present a bilateral agenesis of the vas deferens. Men with spermatogenic arrest were defined as having bilateral spermatogenic arrest and azoospermia. The SCO men were defined as having SCO and azoospermia. This study was conducted according to guidelines established for research on human subjects (Committee for the Protection of People in Biomedical Research, CHU Lille). Testicular Biopsies Testicular biopsies of approximately 2 2 mm were obtained by open surgery and fixed in modified Bouin Hollande Sublime solution (paraformaldehyde, copper acetate, mercuric chloride, and picric acid (Sigma Chemical Co., St. Louis, MO). After embedding in paraffin, 5- m-thick sections were mounted onto gelatin-coated slides and stored at room temperature until used for immunohistochemistry or in situ hybridization. For evaluation of spermatogenetic status, slides were stained with hematoxylin and eosin before evaluation by light microscopy. Immunohistochemistry Before incubation with primary antibody, sections were dewaxed, rehydrated in graded ethanol, washed in water and 0.1 M PBS. Sections were subjected to antigen retrieval by microwaving in 0.01 M citrate buffer (ph 6) on full power (800 watts) for 5 min and on half power (400 watts) for 8 min. Sections were allowed to cool at room temperature for 30 min before being washed for 10 min in PBS and subsequently incubated in 1% H 2 O 2 in PBS for 30 min to block endogenous peroxidase. After washing in PBS (10 min), sections were preincubated in 0.3% triton-3% normal goat serum in PBS for 90 min to prevent nonspecific immunostaining with secondary antibodies. Sections were washed in PBS (10 min) and incubated with primary antibodies in a humid chamber overnight at 4 C. Two monoclonal antibodies (Serotec Ltd., Oxford, England), respectively directed against and B subunits of human inhibin, were used. The first, used against a synthetic peptide corresponding to the 1 32 peptide of the subunit of human inhibin was used at a concentration of 1:200 in PBS. The second, used against a synthetic peptide corresponding to residue of the inhibin B subunit was used at a concentration of 1:5 in PBS. After washing in PBS for 10 min, the sections were incubated with biotinylated goat antimouse immunoserum (1:200) for 90 min. The sections were washed again in PBS and then exposed to the streptavidin-biotinhorseradish peroxidase complex (AbCys, Paris, France) for 90 min. The tissue sections were washed and then the reaction products were visualized by treating them with 0.025% (w/v) 3.3 -diaminobenzidine tetrachloride (DAB; Sigma Chemical Co.) in 100 mm Tris-buffered saline containing 0.01% H 2 O 2 for 5 10 min. After washing in water, sections were counterstaining with hematoxylin for cell nuclear visualization and mounted with Eukitt. The specificities of the primary antibodies were previously tested in an ELISA and reported elsewhere [16]. Different authors obtained, with the same antibodies, an immunohistochemical-specific labeling in testis and ovary [17 19]. In negative control sections (representative sections where the primary antibody was substituted with mouse immunoglobulin), we obtained no specific positive immunoreactivity (Fig. 1, f and g). Positive controls that comprised ovaries containing follicular cysts or corpora lutea were also routinely included. In Situ Hybridization Generation of RNA probes. 35 S-Labeled RNA transcripts were used in radioactive in situ hybridization to localize mrnas of inhibin and B subunits. Human inhibin cdna [20] (with accession number M13981 in GenBank in 01/06/1995) was cloned into PGEM-1. Rat inhibin B subunit cdna [21, 22] (with accession numbers M32756 and M32757 in GenBank in 04/27/1993) was cloned into PGEM-4. Antisense and sense crna probe were transcribed using T7 and SP6 RNA polymerases, respectively. There is 93% homology between rat and human inhibin B subunit cdna sequences for M32756 and 95% homology for M32757 (refer to GenBank DATAbase). In situ hybridization. The hybridization protocol was that described by Mitchell et al. [23]. Briefly, sections of testis tissue were placed into 0.1 M glycine-0.2 M Tris HCl (ph 7,4) for 10 min before treatment with proteinase K (1 mg/ml in 100 mm Tris [ph 8] and 50 mm EDTA) for 15 min at 37 C. Slides were immersed in 4% paraformaldehyde/0.1 M phosphate buffer saline for 15 min and treated with 0.1 M triethanolamine (ph 8)-0.25% acetic anhydride for 10 min. The sections were dehydrated in graded concentrations of ethanol and hybridized during 16 h in a 60 C oven in diluted probes/hybridization buffer containing 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 20 mm Tris-HCl (ph 8), 5 mm EDTA, 1 Denhart solution, 0.5 mg/ml Escherichia coli transfer RNA, 100 mm dithiothreitol (DTT). The slides were then washed twice with 4 sodium saline citrate (SSC) and 10 mm DTT for 30 min and for 1 h, then in 0.3 M NaCl, 20 mm Tris-HCl (ph 8), 5 mm EDTA, and 50% formamide for 30 min. After treatment with ribonuclease A (20 g/ml in 0.1 M Tris [ph 8], 0.5 M NaCl, and 0.5 M EDTA) for 30 min at 37 C, the slides were rinsed in 2 SSC for 15 min at 60 C and in 0.1 SSC for 15 min at 60 C. The slides were dehydrated in 70% ethanol in ammonium acetate and 100% ethanol and dipped in Kodak NTB-2 emulsion (Kodak, Rochester, NY). The slides were exposed for 4 wk prior to development and counterstaining with 0.1% blue Azur. Sense RNA probes specific for each subunit were used for control hybridization (Fig. 2, e and g). RESULTS Immunohistochemistry In all testes (normal spermatogenesis, spermatogenic arrest, SCO), positive immunostaining for inhibin subunit was observed in Sertoli cells and Leydig cells (Fig. 1, a and b). In testes with complete spermatogenesis and with spermatogenic arrest, no germ cells were immunolabeled (Fig. 1a). In testes with SCO, intense immunostaining was observed in Sertoli cells and Leydig cells, particularly when interstitial tissue show a Leydig hyperplasia (Fig. 1b). Using inhibin B subunit immune serum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids (Fig. 1, c and e). In contrast, other germ cells (spermatogonia; preleptotene, leptotene, or zygotene spermatocytes; late spermatids) and Sertoli cells were not immunolabeled. Staining of Leydig cells was weakly positive (Fig. 1e). On testes with SCO, B subunit immunostaining was negative for Sertoli cells but faintly positive for Leydig cells (Fig. 1d). In Situ Hybridization In testes with normal spermatogenesis and with spermatogenic arrest, both Sertoli cells and Leydig cells expressed inhibin subunit mrna. Intratubular high levels of hybridization signal were observed in Sertoli cells, between germ cells, whereas levels of inhibin subunit mrna in Leydig cells were heterogeneous (Fig. 2a). In testes with SCO, high levels of inhibin subunit mrna were observed in both Sertoli cells and Leydig cells (Fig. 2, b and f). Inhibin B subunit mrna expression was intense in germ cells and in Sertoli cells in testes with normal spermatogenesis and with spermatogenic arrest. High levels of mrna hybridization signal were observed in spermatogonia, spermatocytes, and round spermatids. Most mature germ cells showed no labeling whereas Leydig cells were faintly labeled (Fig. 2, c and d). In testes with SCO, high inhibin B subunit mrna la-

3 232 MARCHETTI ET AL. FIG. 1. Immunolocalization of inhibin and B subunits in human adult testes. a, b) Immunostaining for subunit in testes with normal spermatogenesis (a) or Sertoli cell only (b). c e) Immunostaining for B subunit in testes with normal spermatogenesis (c), Sertoli cell only (d), and spermatogenic arrest (e). f, g) Negative controls in testes with spermatogenic arrest (f) or with normal spermatogenesis (g). The primary antibody (anti- in f and anti- B in g) was substituted with mouse immunoglobulin. a, b) Bar 40 m; c, d) bar 15 m; e) bar 20 m; f, g) bar 50 m. C, Spermatocyte I; G, spermatogonia; L, Leydig cell; S, Sertoli cell; T, spermatid.

4 INHIBIN B AND GERM CELLS 233 FIG. 2. Expression of inhibin and B subunit mrnas in human adult testes. a, b, f) Expression for subunit mrna in testes with normal spermatogenesis (a) or Sertoli cell only (b f). c, d, h) Expression for B subunit mrna in testes with normal spermatogenesis (c) or Sertoli cell only (d h). e g) Controls with sense RNA probes for subunit (e) and for B subunit (g). a d) Bar 15 m; e h) bar 50 m. Compare the signal obtained for the subunit at the periphery of tubules, in Sertoli cells (a, b, f), and the signal obtained for the B subunit around the nuclei of round germ cells (c, d). Note that in SCO, subunit mrna (b f) and B subunit mrna (h) are expressed in Sertoli and Leydig cells. In testes with normal spermatogenesis, Leydig cells and Sertoli cells express and B subunits (a, c, d). B subunit mrna signal is observed in spermatogonia and spermatocytes but is absent in elongated spermatids. C, Spermatocyte I; G, spermatogonia; L, Leydig cell; S, Sertoli cell; T, spermatid.

5 234 MARCHETTI ET AL. beling density was observed in both Sertoli cells and Leydig cells (Fig. 2, d and h). There were no positive signals for the or B subunits in sections treated with sense probes (Fig. 2, e and g). DISCUSSION The immunocytochemical results obtained from the present investigation demonstrate the presence of inhibin subunit in both Sertoli cells and Leydig cells but not in the germ cells of adult human testes with normal or altered spermatogenesis. Similar findings were previously reported in fetal or adult testes [8, 9, 11, 12]. In agreement with these results, inhibin subunit mrna was also localized using in situ hybridization in both Sertoli cells and Leydig cells but not in the germ cells. These observations are consistent with data previously obtained in the human fetus [10]. Moreover, we found that the B subunit was immunolocalized in pachytene spermatocytes to round spermatids as well as in Leydig cells but not in Sertoli cells in testes with normal spermatogenesis and with spermatogenic arrest. In testes with SCO, only Leydig cells were immunostained. These immunocytochemical results corroborate the findings of Andersson et al. [14, 15] but are inconsistent with the observations of Anderson et al. [13], who observed B subunit immunolocalization in Leydig cells and Sertoli cells but not in germ cells. These two teams used the same antibodies we used, and the discrepancy between their results may be due to differences in the fixation in the immunohistochemical procedure. Anyway, the specificity of the primary antibody anti- B subunit was tested, the staining pattern was highly reproducible in all testis sections from different men and immunostaining was restricted to germ cell types with no background staining of other cell types. However, we cannot rule out that the anti- B subunit antibodies recognize subunits destined to become part of activins that play intratesticular autocrine/paracrine roles [15]. We also report the first examination of inhibin/activin B subunit mrna signals in the adult human testis. In agreement with our results obtained with immunohistochemistry, B subunit mrna is expressed in germ cells from pachytene spermatocytes to round spermatids in testes with normal or altered spermatogenesis. Surprisingly, in contrast with our immunohistochemistry results, an intense mrna signal for B subunit was detected in Sertoli cells and Leydig cells of all testes, particularly in testes with SCO. In SCO, inhibin B subunit mrna expression was intense in both Sertoli cells and Leydig cells, whereas B subunit immunostaining was negative in Sertoli cells and faintly positive in Leydig cells. Discrepancies between presence of mrna and of the corresponding peptides have been described in other systems: in the human ovary, no inhibin subunit immunoreactivity was detected in thecal cells in spite of the presence of an subunit mrna signal there [24]. This may be explained by differences in the sensitivities of the antisera and probes used to detect subunit proteins and messages and/or to relatively low levels of protein in these cells. Alternatively, the rapid release or the rapid degradation of inhibin subunits from the cells may occur. It must be added that inhibin B was undetectable in subjects with SCO tubules [14, 25]. In the testes with presence of germ cells, B subunit is present in all germ cells prior to the spermatid stage and at a lower level in Leydig cells, whereas subunit is confined to Sertoli cells and Leydig cells. These results agree with the suggestion that testicular production of the - B dimer could occur from a combination of subunit peptides from different cell types [26]. Thus, germ cells might directly contribute to inhibin production. This view is supported by the observations that inhibin B concentrations fall to undetectable levels following loss of all germ cells in men after chemotherapy or radiotherapy [27, 28]. Identical results were obtained by Foppiani et al. [29] in the irradiated nonhuman primate model. Moreover, a direct positive correlation between inhibin B and sperm concentration in normal men is noted [6, 7, 13, 30, 31]. Finally, studies in which serum inhibin B levels were related to the histologic pattern of testicular biopsies have confirmed that inhibin B levels are reduced in men with more severely affected spermatogenesis [14, 21, 32 34]. Inhibin B would be secreted into the seminiferous tubular fluid and would reach the bloodstream very rapidly [35]. This hypothesis is supported by the fact that inhibin B is detected in seminal plasma of normal men at variable levels but is undetectable in vasectomized patients [13, 36]. However, the pathways via which inhibin reaches seminal plasma and blood may be differentially regulated [26]. Thus, different authors agree with the occurrence of a major switch in inhibin regulation around puberty: while FSH governs directly inhibin secretion by Sertoli cells in childhood, germ cells are a major determinant of inhibin B production in adulthood that depend only secondarily on FSH [14, 26, 37]. The localization of and B subunits and their mrna in the Leydig cells of all examined testes whether spermatogenesis was present or absent led to the suggestion that Leydig cells could contribute to inhibin B production. Thus, Jin et al. [38] suggest that Leydig cells are the main source of inhibin B in male Göttingen pigs. However, in hypogonadal or normal men, recombinant LH or hcg do not stimulate the production of inhibin B [39, 40]. Therefore, in man, Leydig cells do not appear to contribute to the pool of circulating inhibin B but might be a site of action of inhibin and activin, e.g., in the regulation of steroidogenesis, and thus might contribute to the regulation of inhibin production by the Sertoli cells [37]. In conclusion, the immunolocalization of inhibin/activin B subunits and the detection of their mrna in germ cells in human adult testes corroborate the view that, in adult man, inhibin B may be a joint product, with Sertoli cells providing the subunit and certain stages of germ cells providing the B subunit. This hypothesis might explain the molecular basis of the close link between inhibin B production and spermatogenesis in adult men [15]. REFERENCES 1. Burger HG, Igarashi M. Inhibin: definition and nomenclature, including related substances. 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