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Laboratory of Fish Reproductive Physiology,3 Ehime University, Ehime 790-8566, Japan
R&D Division,4 Q.P. Corporation, Tokyo 183-0034, Japan
Department of Molecular, Cellular, and Developmental Biology,5 University of Michigan, Ann Arbor, Michigan 48109
Research Institute,6 Tokyo University of Agriculture, Tokyo 156-0054, Japan
ABSTRACT
Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.
androgen, follicle-stimulating hormone, in vitro, spermatogenesis, testis
Follicle-stimulating hormone (FSH) is one of the gonadotropins (GTHs) produced in the pituitary that are members of the pituitary glycoprotein family, including luteinizing hormone (LH) and thyroid-stimulating hormone. These hormones are heterodimers, each consisting of a common 
and a hormone-specific β subunit [1]. The actions of FSH are mediated through a specific transmembrane receptor. The FSH receptor (FSHR) is a G protein-coupled, seven-transmembrane domain receptor that can couple to different signaling pathways [2, 3]. Follicle-stimulating hormone-binding activates its receptor and initiates the cascade of events leading to modifications of cellular activities and receptor desensitization [2, 4].
Spermatogenesis, male gametogenesis, is a complex developmental process. The principal hormones controlling vertebrate spermatogenesis are considered to be GTHs and androgens [5–7]. In mammals, the action of FSH is mediated by the FSHR expressed by Sertoli cells [8, 9] and promotes the production of various endocrine and growth factors [10]. Therefore, FSH signaling plays an important role in the initiation of spermatogenesis. In the recently described Fshb or Fshr gene knockout mice, nonfunctional Fsh or Fshr genes lead to reduced testis size and sperm quality, but spermatozoa are still formed and mutant males are fertile [11–14]. These results suggest that the action of FSH is not essential for spermatogenesis in mammals. In contrast, in amphibians, although FSHR is expressed by Sertoli cells similarly to mammals [15], spermatogenesis is induced by stimulation of FSH alone in vitro [16–18]. This suggests that FSH is an important factor for induction of spermatogenesis, but it is assumed that the action of FSH is not mediated through stimulation of steroid hormone production in amphibians. In some teleosts, Fsh is the only Gth produced and secreted by the pituitary of sexually immature animals [19–21]. Fsh also is able to stimulate steroid production and promotes the synthesis of androgens [22–24]. Therefore, it is possible that Fsh acts on early stages of spermatogenesis via the production and secretion of androgen in teleosts. Thus, the definitive functions of FSH have not been established, because there are only a few systems to analyze FSH effects on spermatogenesis. Therefore, it is essential to analyze the function of FSH using an adequate system for studying the role of FSH in regulating spermatogenesis.
To better clarify the various functions of FSH on spermatogenesis, male Japanese eels (Anguilla japonica) were used as experimental animals. Under cultivated conditions, male Japanese eels have immature testes containing only nonproliferated type A and early type B spermatogonia [25]. All stages of spermatogenesis can be induced by treatment of eel testis explants with hCG or 11-ketotestosterone (11-KT) in vitro [6, 26, 27]. This indicates that the eel testicular culture system is a suitable vertebrate model for analyzing FSH functions on spermatogenesis. Therefore, we investigated the role of FSH in spermatogenesis using in vitro eel testicular culture system in this study.
Male cultivated Japanese eels (1 yr old; 180–200 g body weight) were purchased from a commercial eel supplier and kept in circulating 500-liter capacity freshwater tanks at 23°C.
A single injection of hCG dissolved in saline (150 mM NaCl) was intramuscularly administered at a dose of 5 IU/g body weight. Fish were killed either immediately or 1, 3, 6, 9, 12, 15, or 18 days after hCG injection, which induced complete spermatogenesis from spermatogonial proliferation to spermiogenesis within 18 days. Testes were collected for Western blotting and immunohistochemistry. The experiments were conducted in accordance with the institutional animal ethics guidelines of Ehime University.
Testicular Organ Culture Techniques
Testicular organ culture techniques were performed as described previously [6], with minor modifications. In brief, freshly removed eel testes were cut into pieces of 1 x 1 x 0.5 mm3 and placed on floats of 1.5% agarose covered with a nitrocellulose membrane in 24-well plastic tissue culture dishes. The agarose discs were floated in basal medium for 18 h before the start of testicular culture. For culture, testicular explants were maintained in 1 ml basal medium with or without 1 µg/ml 11-KT for 6 days. The testicular fragments then were collected, and their poly (A)+ RNA was extracted using a Fast Track kit (Invitrogen, Carlsbad, CA).
The eel fshr cDNA clone was obtained as one of the cDNA clones upregulated by 11-KT treatment using cDNA subtraction, cloning, and screening, as described previously [28], to identify genes upregulated or downregulated by 11-KT in an in vitro testicular organ culture.
Production of Polyclonal Antibody Against Eel Fshr
To amplify the cDNA fragment encoding the extracellular domain of eel fshr in an open reading frame by RT-PCR, the following primers with a BamHI site, 5'-GGGATCCGTCATCCCAAAAAACACC-3', and with a HindIII site, 5'-GAAGCTTGTCGCATAGCTCATGCAC-3', were used. The PCR product was inserted into the pQE-30 expression vector (Qiagen, Hilden, Germany), with a 6x histidine tag positioned upstream of the initiation codon. Host bacteria carrying the recombinant plasmids were grown at 37°C until log phase, and then 1 mM isopropyl β-D-thiogalactoside was added to induce protein expression. After 4 h of induction, the bacteria were harvested and homogenized in lysis buffer (8 M urea, 0.1 M sodium phosphate monobasic, 0.01 M Tris-HCl, pH 8.0). Recombinant protein was purified by Ni-NTA agarose affinity chromatography (Qiagen).
Female rabbits were immunized with 1 mg purified recombinant protein mixed with Freund complete adjuvant. The rabbits received four immunizations at 2-wk intervals by s.c. injection, and sera were collected after the fourth injection. The diluted antiserum (1:1000) strongly reacted with purified antigen, as assessed by Western blot.
Antibodies against bacterial proteins in the antiserum were removed by the following method. Host bacteria carrying the pQE-30 expression vector were grown, harvested, homogenized in lysis buffer, and centrifuged at 10 000 x g for 1 h at 20°C to obtain bacterial proteins that were dialyzed against 0.01 M PBS. Rabbit antiserum was mixed with bacterial protein in PBS, the mixture was shaken overnight at 4°C, and it was centrifuged at 10 000 x g for 1 h at 4°C to remove antibodies against bacterial proteins.
Plasma membrane fractions of eel testis were obtained by the following method. Fresh testicular tissue samples were homogenized with 0.25 M sucrose buffer at 4°C and centrifuged at 8000 x g for 10 min to remove any remaining nuclear material. The supernatant was centrifuged at 100 000 x g for 1 h to obtain a pellet containing the majority of the plasma membrane fraction.
SDS-PAGE and Western Blot Analysis
Fresh testicular tissue samples were homogenized in physiologic saline solution for eel [25] at 4°C. The homogenate was mixed with an equal volume of sample buffer (125 mM Tris-HCl, 4% [w/v] SDS, 20% [v/v] glycerol and 0.05% [w/v] bromophenol blue, and 10% [v/v] 2-mercaptoethanol). The mixture was heated in a boiling water bath for 5 min and centrifuged at 9000 x g for 5 min, and then the supernatant of the tissue extracts was collected. Samples were separated using SDS-PAGE on 7% and 10% gels. The separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blotting, the membranes were incubated and shaken for 30 min in 5% skimmed milk in 20 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl (TBS) to block nonspecific binding sites. The blocked membranes were immersed overnight in 5% skimmed milk containing the primary antibody against eel Fshr diluted 1:1000. After washing twice with TBS containing 0.025% Tween-20 (TTBS) and then with TBS, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA) diluted 1:2000 in TBS for 1 h at room temperature. After washing, the membranes were analyzed using a LAS-1000mini (Fujifirm, Tokyo, Japan). To demonstrate the specificity of the eel Fshr antibody, the same procedures of Western blot analysis were carried out without primary antibody as a negative control.
Eel testicular fragments were fixed in Bouin solution, embedded in paraffin wax, and cut into 5-µm serial sections. Sections were deparaffinized in xylene and hydrated in a graded ethanol series. Immunohistochemical analysis was performed using a Histofine SAB-AP (R) kit (Nichirei Biosciences, Tokyo, Japan).
Preparation of Recombinant Eel Fsh (r-eFsh) and Its Effect in Japanese Eel Spermatogenesis
R-eFsh was prepared using the yeast synthesis system according to Kamei et al. [29]. In this study, the dosage of r-eFsh was indicated as a unit (U) defined in an eel testis bioassay in comparison with hCG [29, 30]. To investigate the mechanism of FSH action in spermatogenesis, testicular fragments from five eels were cultured for 15 and 36 days at 20°C with either r-eFsh (0.0005, 0.005, 0.05 U/ml) or 11-KT (10 ng/ml) or hCG (0.005, 0.05, 0.5 IU/ml). The medium was changed on Day 7. After cultivation, testicular fragments were fixed in Bouin solution for histologic examinations.
Effects of Trilostane (Steroid Hormone Synthesis Inhibitor) on the Action of r-eFsh and hCG in Eel Spermatogenesis
To investigate whether FSH acts directly or via stimulating steroid hormone production, testicular fragments from five eels were cultured with or without various concentrations of either trilostane (1, 10, and 100 mg/ml), which inhibits 3β-hydroxysteroid dehydrogenase (3β-HSD), or r-eFsh (0.05 U/ml) or hCG (0.05 IU/ml) or 11-KT (10 ng/ml) for 15 days. The medium was changed on Day 7. After cultivation, testicular fragments were fixed in Bouin solution for histologic examinations.
After fixation, testicular fragments were dehydrated by ethanol and lemosol (Wako Pure Chemical Industries Ltd., Osaka, Japan) and embedded in paraffin according to standard procedures. Five-micrometer sections were cut and stained with Delafield hematoxylin and eosin.
Morphometric analysis was carried out on five random sections per testicular fragment for each treatment, and the number of cysts containing each germ cell type was counted using paraffinic sections. The results were expressed in terms of percentage of cysts of a particular germ cell type per total number of cysts observed. According to a previous report [25], the cysts of the following five germ cell types were distinguished and counted: 1) type A spermatogonia and early type B spermatogonia; 2) late type B spermatogonia; 3) primary and secondary spermatocytes; 4) spermatids; and 5) spermatozoa. Isolated type A spermatogonia or groups of two cells surrounded by Sertoli cells and spermatozoa present in the lumen were counted as cysts.
Data analyses were carried out by the Sceirer, Ray, and Hare extension of the Kruskal-Wallis test (a one-way ANOVA design for ranked data), followed by the posthoc Bonferroni adjustment. A value of P < 0.05 per number of comparisons was considered to be significant.
Cloning and Characterization of Eel fshr cDNA
Two populations of double-stranded cDNA were prepared from testicular fragments that had been cultured with (+) or without (–) 1 µg/ml 11-KT for 6 days, and an enriched subtractive (+) or (–) cDNA library was constructed. The nucleotide sequences of the (+) cDNA fragments obtained by cDNA subtraction were determined, and one of the deduced amino acid sequences was similar to FSHR of other species. To clone the full-length cDNA of this clone, a complete cDNA library was screened, and a positive clone was obtained. This clone is 5184 bp in length (DDBJ/EMBL/GenBank accession no. AB360713) and encodes a 660-amino acid residue protein, including a signal peptide composed of 19 amino acids. The extracellular N-terminal domain region consists of 320 amino acids and has seven potential N-linked glycosylation sites (Asn 26, 49, 95, 120, 195, 272, 289) and a total of 13 cysteine residues (Cys 23, 34, 279, 280, 296, 313, 319, 329, 415, 490, 519, 521, 619). The transmembrane domain is represented by 263 amino acids arranged as seven transmembrane-spanning segments typical of a G protein-coupled receptor.
Database searches showed that the deduced amino acid sequence of this clone is similar to FSHR of other species (Fig. 1). Overall, eel fshr shares 58%–65%, 57%, 56%, and 56%–57% similarity, respectively, with the amino acid sequence of FSHR from teleosts, newt, chicken, and mammals. The deduced amino acid sequence of eel fshr was phylogenetically related to FSHR and LH receptor (LHR; Fig. 2). This analysis clearly showed that eel fshr belonged to the FSHR cluster.
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To confirm the presence of eel Fshr, we carried out Western blot analysis on immature eel testes using an anti-eel Fshr antibody (Fig. 3). Eel Fshr was detected as two main bands of 72 and 41 kDa in a purified plasma membrane fraction of immature eel testes before the initiation of spermatogenesis. Using whole testis, however, eel Fshr was detected as only one band of 41 kDa in Western blot analysis (Fig. 4).
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To evaluate how expression changes of eel Fshr during spermatogenesis, Western blot analysis was performed using the anti-eel Fshr antibody (Fig. 4). Testicular eel Fshr expression increased only slightly with the progression of spermatogenesis, and was detected as a band of 41 kDa at all stages of spermatogenesis.
Localization of Eel Fshr in Testis During Spermatogenesis
To determine the distribution of eel Fshr in testis during spermatogenesis, we performed immunohistochemistry using the anti-eel Fshr antibody (Fig. 5). The antibodies stained Sertoli cells surrounding type A or early type B spermatogonia and Leydig cells, which produce steroid hormones during spermatogenesis. However, other Sertoli cells and germ cells were not stained. Preimmune serum used as a negative control did not react to any of the samples.
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Effects of r-eFsh on Spermatogenesis In Vitro
To investigate the action of FSH on spermatogenesis, testicular fragments were cultured with increasing concentrations of r-eFsh (0.0005, 0.005, and 0.05 U/ml) or with 11-KT (10 ng/ml) or hCG (0.005, 0.05, and 0.5 IU/ml) as a positive control for 15 and 36 days. After cultivation, the percentage of cysts of a particular germ cell type was calculated. After 15 days under control conditions, all germ cells were either type A or early type B spermatogonia (Fig. 6). In contrast, after treatment with r-eFsh, late type B spermatogonia were observed, similar to the positive controls. The percentage of cysts of late type B spermatogonia increased after r-eFsh treatment in a dose-dependent manner. After 36 days of treatment with 0.05 U/ml r-eFsh, all stages of germ cells were present (Fig. 7) in the following percentages: type A spermatogonia and early type B spermatogonia, 28.4% ± 6.1%; late type B spermatogonia, 44.1% ± 4.9%; spermatocytes, 25.4% ± 10.0%; spermatids, 1.7% ± 0.2%; and spermatozoa, 0.3% ± 0.3% (Fig. 8).
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Effects of Trilostane (Steroid Hormone Synthesis Inhibitor) on the Action of r-eFsh and hCG
To investigate whether FSH affects spermatogenesis through 11-KT production, eel testes were cultured with trilostane and/or r-eFsh. The testicular fragments were cultured with or without various concentrations of either trilostane (1, 10, and 100 µg/ml), which inhibits 3β-HSD—the enzyme that is indispensable for production of 11-KT—r-eFsh (0.05 U/ml), hCG (0.05 IU/ml), or 11-KT (10 ng/ml) for 15 days (Fig. 9). Before cultivation, all germ cells in the eel testis were type A and early type B spermatogonia. Treatment with r-eFsh or hCG or 11-KT alone stimulated the proliferation of spermatogonia (i.e., increased the percentage of cysts of late type B spermatogonia). However, treatment with r-eFsh or hCG and trilostane significantly decreased the percentage of cysts of late type B spermatogonia in a manner dependent on the dose of trilostane. In contrast, cotreatment with 11-KT and trilostane did not induce significant changes in the percentage of cysts of late type B spermatogonia, regardless of trilostane concentrations.
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In Japanese eel, spermatogenesis is regulated by various hormones and growth factors secreted under the stimulatory action of Gths, which are released from the pituitary. However, under cultivated conditions, Japanese eel have immature testes containing only nonproliferated type A and early type B spermatogonia, because Gth-producing cells in the pituitary are still immature [31]. Although a single injection of hCG can induce complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis, hCG does not induce this process directly, but rather acts through the gonadal biosynthesis of 11-KT, which in turn mediates the initiation of spermatogenesis [6, 25, 32].
There are two types of GTHs in vertebrates, FSH and LH [33, 34]. In higher vertebrates, it is known that LH promotes the production of androgens via stimulation of Leydig cells and that FSH promotes the secretion of various growth factors by Sertoli cells that then stimulate spermatogenesis [35, 36]. In teleosts, it is also established that two types of GTHs, FSH and LH, exist [23, 37–39]. However, the definitive function of each GTH has not been established. In some salmonids, it has been reported that Fsh but not Lh is secreted from the pituitary of immature fish, whereas Lh release is higher during the period of sperm maturation [19, 40]. In addition, it seems that in Coho salmon, Fsh acts at early stages of spermatogenesis, because Fsh is able to stimulate steroid hormone production, similarly to Lh. However, Fsh-stimulated production of steroid hormones decreases toward the period of sperm maturation [23]. In Japanese eel, Fsh may act on early stages of spermatogenesis as well, considering that fshb subunit mRNA is expressed in the pituitary of immature fish, but lhb subunit mRNA is not expressed until much later in the period of sperm release [39].
In Western blot analysis using a purified plasma membrane fraction of immature eel testis, eel Fshr was detected in two forms with two different molecular masses of 72 and 41 kDa. Using proteins extracted from whole testis, however, only a band of 41 kDa was detected. This suggests that the 72-kDa form is the full-length eel Fshr, as also predicted from the deduced amino acid sequence of eel fshr cDNA, whereas the 41-kDa form may represent the extracellular domain of eel Fshr, possibly cleaved from the full-length receptor during the extraction of plasma membrane or testicular proteins. Moreover, to evaluate how expression of eel Fshr changes during spermatogenesis, we performed Western blot analysis on hCG-treated eel testis. Eel Fshr was expressed before the initiation of spermatogenesis and was continuously expressed during all stages of spermatogenesis. It is therefore possible that FSH acts on all stages of spermatogenesis.
To determine the distribution of eel Fshr in testis, we performed immunohistochemistry using an anti-eel Fshr antibody. The antibodies stained Leydig cells, which produce steroid hormones, and Sertoli cells surrounding type A or early type B spermatogonia during spermatogenesis. In some teleosts, Fsh increases at early stages of spermatogenesis [19, 23, 40], and in eel, fshb subunit mRNA is expressed in the pituitary of immature fish [39]. These results suggest that FSH acts on early stages of spermatogenesis via Leydig and/or Sertoli cells.
To understand whether FSH acts on spermatogenesis, we investigated the effects of FSH on in vitro spermatogenesis using r-eFsh produced from a yeast expression system. Adding r-eFsh to culture medium induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis. In Japanese eel, it has been reported that Fsh induces the secretion of 11-KT by immature testis [24]. Therefore, it is possible that the role of FSH is to induce 11-KT secretion, which in turn will stimulate spermatogenesis.
Using trilostane that specifically inhibits 3β-HSD activity, we investigated whether FSH acts on spermatogenesis via the production and secretion of 11-KT in testicular organ culture. Adding r-eFsh and trilostane to the culture medium reduced the percentage of cysts of late type B spermatogonia compared with the treatment with only r-eFsh, and the progress of spermatogenesis was inhibited. These results indicate that FSH stimulates spermatogenesis by triggering the secretion of 11-KT.
In this study, we have shown that Fsh induces spermatogenesis via the release of 11-KT. In males, androgens, including 11-KT, are synthesized by Leydig cells in the testis [41, 42]. In Coho salmon, Fshr is localized to Sertoli cells at all stages of spermatogenesis, whereas Lhr was only found in Leydig cells in spermiating fish [43]. Nevertheless, Fsh promoted the synthesis of androgen in immature and mature testis similarly to Lh [23, 40]. It is therefore possible that Leydig cells express Fshr or that paracrine factors secreted by Sertoli cells upon Fsh stimulation promote Leydig cells androgen production [44]. In the present study, eel Fshr is expressed in Leydig and Sertoli cells surrounding type A and early type B spermatogonia in Japanese eel. These results suggest that FSH directly acts on Leydig cells via FSHR activation and promotes the synthesis of 11-KT.
Although FSHR localizes to Sertoli cells from fish to mammals, including Japanese eel, the clear functions of FSH in Sertoli cells via FSHR activation have not been established. In mouse, the absence of functional Fshb or Fshr genes leads to reduced testis size, but the males are still fertile [11–14]. Moreover, in Japanese eel, all stages of spermatogenesis are induced by 11-KT alone in vitro [6, 27]. These results suggest that FSH also supports testicular development and maintenance of sperm production through the action of Sertoli cells. However, the functions of FSH via Sertoli cells are not clear from this study.
In conclusion, we identified a cDNA encoding a fshr from Japanese eel testis, and eel Fshr was expressed in Leydig and Sertoli cells surrounding type A and early type B spermatogonia. In an in vitro organ culture, although r-eFsh induced complete spermatogenesis, spermatogenesis induced by r-eFsh was inhibited by 3β-HSD inhibitor. These results clearly indicate that FSH stimulates the Leydig cells and induces spermatogenesis via the production and secretion of 11-KT.
ACKNOWLEDGMENTS
We thank Dr. R. W. Schulz (Utrecht University, The Netherlands) and Miss Fritzie T. Celino (Ehime University, Japan) for critical reading of the manuscript.
FOOTNOTES
1Supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry, and Fisheries of Japan, the "Global COE" program by Japan's Society for the Promotion of Science (JSPS), and the Ministry of Education, Culture, Sports, Sciences, and Technology of the Japanese government. 
Correspondence: 2FAX: 81 89 946 9818; e-mail: miutake{at}agr.ehime-u.ac.jp
Received: 23 April 2007.
First decision: 2 June 2007.
Accepted: 28 August 2007.
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