Molecular control mechanisms of fish spermatogenesis

Fish spermatogenesis, from spermatogonial stem-cell renewal to sperm maturation, is controlled by the sex steroid hormones. Mitotic divisions of spermatogonia can be categorized by spermatogonial stem cell renewal and spermatogonial proliferation. Spermatogonial renewal is regulated by estradiol-17β (E₂; the natural estrogen in vertebrates), and spermatogonial proliferation toward meiosis is promoted by 11-ketotestosterone (11-KT), the main androgen in teleost. The action of E2 and 11-KT is mediated by other factors produced by Sertoli cells; E2 is mediated by spermatogonial stem-cell renewal factor and 11-KT is mediated by spermatogenesis preventing substance and activin B. Although 11-KT also induce meiosis and spermiogenesis, the control mechanisms of these processe are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation. Sperm maturation is regulated by 17α,20β-dihydroxy-4-pregnen-3-one (DHP), which is progestin in teleost. The DHP acts directly on spermatozoa to active the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.     

Sertoli cell proliferation and FSH signalling in African catfish, Clarias gariepinus

Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cyst-forming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.     

Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model

The morphometric study of spermatogenic cysts in sexually mature tilapias, during the evolution of spermatogenesis, showed a dramatic increase in both number of germ cells and cyst volume. However, the opposite trend was observed for germ cell size. Nevertheless, the number of Sertoli cells increased gradually up to leptotene/zygotene cysts, stabilizing thereafter. Based on the number of spermatids supported by each Sertoli cell and compared to mammals, Sertoli cell efficiency in tilapias is remarkably high. Sertoli cell proliferation was frequently observed, mainly in spermatogonial cysts, and probably is the major factor related to the testis growth and the increase in sperm production that normally occurs in adult tilapias. The combined duration of spermatocytes (5 days) and spermiogenic (5–6 days) phases of spermatogenesis in fish kept at 25 °C was 10–11 days. Mainly due to acceleration in meiosis, these two phases lasted a total of 6 days in tilapias kept at 30 °C, in the opposite way, at 20 °C spermatogenesis was arrested at pachytene spermatocytes. To our knowledge, this is the most comprehensive investigation performed up to date on testis morphometry and function in adult tilapias.     

Regulation of gonadotropin subunit genes expression by 11-ketotestosterone during early spermatogenesis in male red seabream, Pagrus major

To examine the roles of gonadal steroids in the regulation of expression of gonadotropin subunit genes, male red seabream were gonadectomized and a sub-group was treated with 11-ketotestosterone (11-KT). Castration of males during the early stage of spermatogenesis elicited a significant increase in FSHβ mRNA levels, which was prevented by 11-KT replacement. By contrast, LHβ mRNA levels were not changed by castration or 11-KT replacement. In addition, administration of 11-KT to sham-operated males suppressed the steady-state FSHβ and LHβ mRNA levels. These results indicate that 11-KT may function as a negative feedback regulator of FSHβ gene expression, and may act through the testis to down-regulate LHβ mRNA levels in male red seabream during this period.     

外源性促性腺激素诱导日本鳗鲡精子发生和成熟的作用机制

用兔抗血清对抗促黄体素生成素受体（LHR）或称绒毛膜促性腺激素受体（CGR）和雄激素受体（AR）进行LHR和AR免疫组织化学定位,以揭示外源性促性腺激素（鲤脑垂体激素和hCG）诱发日本鳗鲡精子发生及其内分泌机制。结果表明，经过注射激素处理后的实验组与注射前的对照组相比较，其精巢发育和精子发生出现十分显著的变化。组织学切片观察显示,激素处理前鳗鲡精巢处于精原细胞增殖期，而两种激素混合注射后第10天，实验组可见精小叶中精原细胞的有丝分裂和初级与次级精母细胞的数量显著的增加。注射后第35天，靠近生殖上皮除有少量精原细胞外，精小叶中有大量初级精母细胞和次级精母细胞和少数精子细胞以及管腔中存在少量精子。在注射后第83天，日本鳗鲡完成了精子发生和精巢发育成熟以及释精。免疫组织化学染色结果进一步揭示，激素处理前，LH受体免疫活性分布在生殖上皮，显示强的免疫阳性反应；激素处理后，LH受体定位在Sertoli细胞和间质细胞以及精原细胞和初级与次级精母细胞的胞膜上，均显示强的免疫阳性反应。激素处理前，雄激素受体定位在生殖上皮和早期生精细胞的胞膜上；激素处理后，AR则定位在这些生精细胞的核或胞质，而精子细胞和精子显示免疫阴性反应。这些结果首次证明了这两种激素诱导鳗鲡精子发生和成熟的作用机制是通过LH受体和雄激素受体的介导。     

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology—transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.     

Colonization,proliferation, and survival of intraperitoneally transplanted yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> spermatogonia in nibe croaker <Emphasis Type="Italic">Nibea mitsukurii</Emphasis> recipient

Recently, we developed an intraspecies spermatogonial transplantation technique in a pelagic egg spawning marine teleost, nibe croaker Nibea mitsukurii. Nibe croaker is an ideal candidate recipient for spermatogonial transplantation since it has a short generation time and small body size. In the present study, yellowtail Seriola quinqueradiata spermatogonia were transplanted into nibe croaker larvae, and the behavior of transplanted spermatogonia in recipient gonads was observed. Three weeks post-transplantation, yellowtail spermatogonia were incorporated into the gonads of 72 out of 88 recipients. An antiproliferating cell nuclear antigen was detected in incorporated yellowtail spermatogonia, suggesting that the xenogenic germ cells were proliferating in recipient gonads. Yellowtail vasa-positive spermatogonia survived for 11 months after transplantation in the gonads of recipient fish. Thus, we showed that the microenvironment in nibe croaker gonads can support the colonization, proliferation, and survival of germ cells derived from a different taxonomic family.     

Cell-cell interactions in the testis of the dogfish: stage-related changes in protein synthesis

Previous studies have shown that the testis of Selachians is a very suited model to study stage-dependent changes in Sertoli cells during spermatogenesis (Dubois and Callard 1989; Sourdaine et al. 1990). In the dogfish testis (here: Scyliorhinus canicula), germ cells, at an identical stage of spermatogenesis, are associated with Sertoli cells to form spermatocysts, which are arranged in zones corresponding to the different stages of spermatogenesis. Using previously described methods for the isolation and culture of spermatocysts from four spermatogenic stages (spermatogonia, spermatocytes, early spermatids and late spermatids; Sourdaine and Jégou 1989; Sourdaine and Garnier 1992) and electrophoresis techniques (1D and 2D-SDS-PAGE) we have investigated the [³⁵S] methionine incorporation into proteins in the dogfish testis. Our results indicate that protein synthesis reaches a maximum in spermatocysts with spermatocytes. Marked stage-related changes of protein synthesis and secretion were also observed on the autoradiograms of 1D and 2D-SDS-PAGE. Further investigations of the paracrine control of germ cells on Sertoli cell protein synthesis requires the identification of specific Sertoli cell proteins in the dogfish.     

Testicular responsiveness to gonadotropic hormonein vitro and Leydig and Sertoli cell ultrastructure during pubertal development of male African catfish (Clarias gariepinus)

The gonadotropin (GTH)-stimulated testicular androgen secretionin vitro and the ultrastructure of Leydig and Sertoli cells was studied during the pubertal development in male African catfish. Testicular weight increased from less than 1 mg in the ninth week of age to nearly 600 mg in the 28th week. Immature testes (stage I: spermatogonia) were highly sensitive to GTH and secreted very high amounts of androgens per mg of tissue. The secretion per mg tissue decreased gradually in stages II (spermatogonia and spermatocytes) and III (spermatogonia, spermatocytes, and spermatids), but precipitously in stage IV (all germ cell stages, including spermatozoa). However, due to the testicular weight gain, the total androgen output per pair of testes increased slightly in stage III and strongly in stage IV. The sensitivity to GTH decreased with the appearance of haploid germ cells in stage III. Leydig cells but not Sertoli cells showed the ultrastructural characteristics of steroid producing cells. Leydig cell morphology did not change in stages I–III, while in stage IV, more smooth endoplasmic reticulum was present. The ultrastructural characteristics of Sertoli cells did not change prominently. Thus, spermatogonial multiplication and spermatocyte formation takes place when the testicular steroidogenic system is highly active and responsive to GTH; whereas the differentiation of haploid germ cells is accompanied by a reduced responsiveness to GTH and by the secretion of several-fold lower androgen amounts per mg of tissue.     

Role of the sertoli cell in spermatogenesis: TheSqualus testis model

In the dogfish sharkSqualus acanthias different germ cell stages are topographically segregated within the testis. Using this species we have developed methods for the isolation and culture of Sertoli cells from premeiotic, meiotic and post-meiotic stages of spermatogenesis and present preliminary evidence for stage-dependent variations in cell morphology and behavior, thymidine incorporation, protein synthesis and steroidogenesis. The goal of future studies is to determine how maturational changes are regulated in Sertoli cells and, in turn, to elucidate Sertoli cell-germ cell interactions.     

Histopathology of gonadal neoplasms in cyprinid fish from the lower Great Lakes of North America

Abstract. The hybrids of carp, Cyprinus carpio L., and goldfish, Carassius auratus (L.), collected from the lower Great Lakes between 1978 and 1981 exhibited epizootics of gonadal neoplasms which were rare in the parental species. The gonadal histology of male and female carp captured for this study was typical of a successfully reproducing population but the hybrids appeared to be sterile. In male hybrids meiosis was often completed, but spermatids usually degenerated in early spermiogenesis. In female hybrids, vitellogenesis was frequently completed but there was no evidence of final maturation or ovulation. Proliferations of undifferentiated germ cells and stromal cells were evident in the tumour nodules of both the male and female hybrids and were considered to represent neoplastic transformations. Proliferations of Sertoli cells were common in males and in the male portion of intersex gonads, which were present in some hybrids. The Sertoli cells of these individuals were well differentiated, formed tubules and accumulated large stores of lipid. Extensive degeneration of germinal cells was often apparent in areas of Sertoli cell proliferation as were numerous, large, melano-macrophage centres. Gonadal stromal cell tumours did not form tubules or accumulate lipid. The spindle cells of these neoplasms could be found in homogeneous sheets or intermixed to varying degrees with the early stages of germ cells and/or connective tissue elements.     

Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish. αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.     

Seasonality of reproduction in male spotted murrel <Emphasis Type="Italic">Channa punctatus</Emphasis>: correlation of environmental variables and plasma sex steroids with histological changes in testis

The present study was undertaken to develop a comprehensive understanding of how environmental cues and sex steroids relate with cyclic changes in spermatogenesis in freshwater spotted snakehead Channa punctatus that is nutritious and economically important. The seasonal histological changes in testis and annual profile of gonadosomatic index (GSI) of C. punctatus delineated the testicular cycle into four phases: regressed (December–March), preparatory (April–June), spawning (July and August) and postspawning (September–November). Among environmental variables, correlation and regression analyses exhibited an important relationship between photoperiod and testicular weight while role of rainfall was seen confined to spawning. The seasonal profile of plasma sex steroids when correlated with cyclic changes in spermatogenesis in spotted snakehead, testosterone (T) seems to be involved in controlling the major events of spermatogenesis from renewal of stem cells to spawning of spermatozoa. Another important androgen prevalent in teleosts, 11-ketotestosterone (11-KT), was high during preparatory phase, suggesting that 11-KT in addition to T plays an important role in progression of spermatogenesis and spermiation in C. punctatus. However, 11-KT was not seen to be associated with milt production and release of spermatozoa during spawning. Plasma profile of estradiol-17β (E₂) during different reproductive phases revealed the involvement of E₂ in repopulation of stem cells during postspawning phase and in maintaining quiescence of testis during regressed phase.     

Immunohistochemical localization of rainbow trout androgen receptors in the testis

SUMMARY: We examined the distribution of two rainbow trout androgen receptors (rtAR: rtAR-α and rtAR-β) in the testis immunohistochemically using a specific antibody to clarify the target cells of androgen in spermatogenesis. Positive rtAR immunoreactivity in paraffin-embedded sections was revealed using microwave treatment, and was detected in the nuclei of Sertoli cells, Leydig cells, and other interstitial cells. The presence of rtAR in Leydig cells suggested that fish androgens regulate Leydig cell activity in an autocrine fashion similar to mammalian androgens. In addition, we found that not all Leydig cells exhibited rtAR immunoreactivity in the mature testis by double staining using anti-3β-hydroxysteroid dehydrogenase (3β-HSD) antibody. Furthermore, rtAR immunoreactivity was also detected in the nuclei of spermatogonia, spermatocytes, and spermatids. The intensity of rtAR immunoreactivity in the nuclei of spermatogonia seemed to be weaker than those of spermatocytes and spermatids. These results suggested that androgens act directly on both germ cells and somatic cells in the regulation of spermatogenesis in the rainbow trout.     

Differentiation and development of Leydig cell, and induction of spermatogenesis during testicular differentiation in the Japanese eel, Anguilla japonica

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight^-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.     

Analysis of germ cell proliferation, apoptosis, and androgenesis in the Lake Van fish (Chalcalburnus tarichi) during testicular development

In the present study, the testis histology, gonadosomatic index (GSI), germ cell proliferation and apoptosis, and the plasma 11-ketotestosterone (11-KT) and testosterone (T) levels of male Chalcalburnus tarichi were analyzed. According to the histological examinations of the specimens that were caught between February 2009 and January 2010, three testicular stages were determined. Those stages were as follows: (1) recrudescence or prespawning (July–April), (2) spawning (May–June), and (3) postspawning (July). It was observed that the GSI increased gradually, starting from the recrudescence stage, and it reached peak values at the spawning stage, while the lowest values were in the postspawning. Germ cell proliferation in the testis was detected using a proliferating cell nuclear antigen (PCNA), and germ cell apoptosis was detected by transferase dUTP nick end labeling staining. The germ cell PCNA and apoptosis index values were calculated. It was indicated that germ cell proliferation was observed in all of the testicular stages. The highest germ cell PCNA index (PI) levels were detected in July, August, and September, which then dropped in October and stabilized between February and April. The lowest PI values were detected in the spawning stage (May–June). Germ cell apoptosis was observed in all of the months, and the highest apoptotic index values were detected in August, September, October, May, and June. Plasma 11-KT and T levels were at their highest levels in May and June, and it was detected as stabile in the other months. There was a correlation between GSI, PI, and plasma androgen levels. In conclusion, the present data illustrate testicular development stages for C. tarichi and show changes in the level of GSI and sex steroid biosynthesis through spermatogenesis.     

Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red sea bream Pagrus major

ABSTRACT:   In order to investigate the influence of estrogen and androgen on reproductive activities of male teleosts, male red sea bream were implanted with silicone capsules containing estradiol-17β (E2), testosterone (T) or 11-ketotestosterone (11-KT) in immature and early spermatogenic stages. One month after implantation of either E2 or T, the gonadosomatic index decreased in accordance with testicular regression in both stages. Implantation of E2 decreased circulating 11-KT levels but did not affect gonadotropin (GTH) subunits, follicle stimulating hormone-β (FSHβ), luteinizing hormone-β (LHβ), α glycoprotein subunit (αGSU) gene expression, and serum LH levels in both stages. Alternatively, T decreased serum 11-KT and LH levels, and FSHβ and LHβ mRNA levels in the early spermatogenic stage but not in the immature stage. These results suggest E2 may directly inhibit testicular development through the suppression of 11-KT production. Meanwhile, T may decrease serum 11-KT levels through the suppression of FSH and LH secretion, resulting to inhibition of testicular development in the early spermatogenic stage. Treatment with 11-KT did not affect the testis in either stage, whereas 11-KT increased LHβ and αGSU mRNA levels in immature, and decreased FSHβ mRNA levels in the early spermatogenic stage. These results suggest that 11-KT may have different effects on GTH subunit gene expression in each reproductive stage.     

Androgen secretion activity of recombinant follicle-stimulating hormone of Japanese eel, Anguilla japonica in immature and maturing eel testes

The androgen secretion activities of recombinant Japanese eel follicle-stimulating hormone (rjeFSH) were investigated in immature and maturing eel testes. The rjeFSH stimulated testosterone (T) and 11-ketotestosterone (11-KT) secretion in immature testis but not in maturing testis. This result suggests that eel FSH plays an important role through the sex steroid secretion in immature testis rather than in maturing testis.