Which hormone promotes spermatogenesis

The diluted antiserum strongly reacted with purified antigen, as assessed by Western blot. Antibodies against bacterial proteins in the antiserum were removed by the following method. Plasma membrane fractions of eel testis were obtained by the following method.

Fresh testicular tissue samples were homogenized with 0. The separated proteins were transferred to polyvinylidene difluoride membranes Millipore, Bedford, MA. After washing twice with TBS containing 0. 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. Sections were deparaffinized in xylene and hydrated in a graded ethanol series.

R-eFsh was prepared using the yeast synthesis system according to Kamei et al. 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 ]. 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. 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. AB and encodes a amino acid residue protein, including a signal peptide composed of 19 amino acids.

The extracellular N-terminal domain region consists of amino acids and has seven potential N -linked glycosylation sites Asn 26, 49, 95, , , , and a total of 13 cysteine residues Cys 23, 34, , , , , , , , , , , The transmembrane domain is represented by 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. This analysis clearly showed that eel fshr belonged to the FSHR cluster.

The vertical arrow indicates the predicted signal sequence cleavage sites. Potential N-glycosylation sites are boxed. The positions of the seven transmembrane regions are underlined numbered above with roman numerals. Asterisks indicate cysteine residues. Branch lengths indicate proportionality to the amino acid changes on the branch.

Scale bar shows substitution per site. To confirm the presence of eel Fshr, we carried out Western blot analysis on immature eel testes using an anti-eel Fshr antibody Fig.

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.

Characterisation of eel Fshr using an anti-eel Fshr antibody. Western blot analysis of plasma membrane of immature cultivated eel testis. Numbers on the left represent molecular size markers kDa. Expression of eel Fshr during hCG-induced spermatogenesis determined by Western blot analysis using an anti-eel Fshr antibody. Testicular samples were analyzed at 0, 1, 3, 6, 9, 12, 15, and 18 days after hCG treatment. To evaluate how expression changes of eel Fshr during spermatogenesis, Western blot analysis was performed using the anti-eel Fshr antibody Fig.

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.

To determine the distribution of eel Fshr in testis during spermatogenesis, we performed immunohistochemistry using the anti-eel Fshr antibody Fig. 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. Cellular localization of eel Fshr in testis assessed by immnohistochemistry using a specific anti-eel Fshr antibody. A Testis section stained with hematoxylin and eosin. B Testis immunostained with anti-eel Fshr antibody. C Testis immunostained with preimmune serum. Arrowheads indicate immunoreactivity. To investigate the action of FSH on spermatogenesis, testicular fragments were cultured with increasing concentrations of r-eFsh 0.

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. 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. Effects of r-eFsh in Japanese eel after 15 days in vitro. The percentage of cysts of late type B spermatogonia number of late type B spermatogonia per total number of germ cells. Effects of r-eFsh in Japanese eel after 36 days in vitro. The percentage of germ cell types in Japanese eel after 36 days in vitro. Before cultivation, all germ cells in the eel testis were type A and early 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 KT and trilostane did not induce significant changes in the percentage of cysts of late type B spermatogonia, regardless of trilostane concentrations. Effect of trilostane treatment on r-eFsh and hCG-induced spermatogenesis in Japanese eel in vitro.

The percentage of late type B spermatogonia per total number of germ cells is compared among different treatments. C, controls; TRI, trilostane. 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 KT, which in turn mediates the initiation of spermatogenesis [ 6 , 25 , 32 ].

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 ]. 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 kDa form is the full-length eel Fshr, as also predicted from the deduced amino acid sequence of eel fshr cDNA, whereas the 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 ]. 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 KT by immature testis [ 24 ].

Therefore, it is possible that the role of FSH is to induce KT secretion, which in turn will stimulate spermatogenesis. 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. In this study, we have shown that Fsh induces spermatogenesis via the release of KT.

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In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate.

This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis. Abstract Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro.