Spermatogenesis-preventing substance in Japanese eel

Under fresh-water cultivation conditions, spermatogenesis in the Japanese eel is arrested at an immature stage before initiation of spermatogonial proliferation. A single injection of human chorionic gonadotropin can, however, induce complete spermatogenesis, which suggests that spermatogenesis-preventing substances may be present in eel testis. To determine whether such substances exist, we have applied a subtractive hybridisation method to identify genes whose expression is suppressed after human chorionic gonadotropin treatment in vivo. We found one previously unidentified cDNA clone that was downregulated by human chorionic gonadotropin, and named it 'eel spermatogenesis related substances 21' (eSRS21). A homology search showed that eSRS21 shares amino acid sequence similarity with mammalian and chicken Müllerian-inhibiting substance. eSRS21 was expressed in Sertoli cells of immature testes, but disappeared after human chorionic gonadotropin injection. Expression of eSRS21 mRNA was also suppressed in vitro by 11-ketotestosterone, a spermatogenesis-inducing steroid in eel. To examine the function of eSRS21 in spermatogenesis, recombinant eSRS21 produced by a CHO cell expression system was added to a testicular organ culture system. Spermtogonial proliferation induced by 11-ketotestosterone in vitro was suppressed by recombinant eSRS21. Furthermore, addition of a specific anti-eSRS21 antibody induced spermatogonial proliferation in a germ cell/somatic cell co-culture system. We conclude that eSRS21 prevents the initiation of spermatogenesis and, therefore, suppression of eSRS21 expression is necessary to initiate spermatogenesis. In other words, eSRS21 is a spermatogenesis-preventing substance.     

Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica)

In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitrol 1-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis

The developmental transition from a residential, immature ‘yellow’ eel to a migratory, maturing adult ‘silver’ eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed ‘chisel-shaped’ snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

Analysis of male sterile mutations in the mouse using haploid stage expressed cDNA probes

A differential hybridization screening procedure has identified cDNAs which correspond to RNAs which are expressed in mouse testis and at lower levels in liver and spleen. The sensitivity of this procedure is such that approximately 0.5% of 1.4 X 10(4) cDNA clones are revealed as "testis specific". We have focused on ten cDNA clones which have been used to identify RNAs expressed in the haploid phase of spermatogenesis. Using Northern blots to analyse RNA isolated from the testes of mutant mice (Tfm/Y and Sxr/+) blocked at specific stages in spermatogenesis or RNA from sexually immature mice, 8 clones have been identified which correspond to RNAs expressed uniquely or at much higher levels in meiotic or post meiotic cells.     

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

A novel stage-specific antigen is expressed only in early stages of spermatogonia in Japanese eel,Anguilla japonica testis

We produced an antibody that recognized only early stages of spermatogonia in Japanese eel testis. This antibody (anti-spermatogonia-specific antigen-1, anti-SGSA-1) recognized a band of about 38 kDa in Western blot analysis of extracts from eel testis. This antigen was observed by immunohistochemistry only in type-A and early type-B spermatogonia and could not be seen in the late type-B spermatogonia, which appeared after the initiation of spermatogenesis by a single injection of human chorionic gonadotropin. Immunoreactive SGSA-1 was absent in spermatocytes, spermatids, spermatozoa, Sertoli cells, and interstitial Leydig cells. Similarly, this antigen was also detected only in type-A/primary spermatogonia in the testes of two species of teleosts, medaka (Oryzias latipes) and tilapia (Oreochromis niloticus), as well as a toad (Xenopus laevis). These results imply that the disappearance of SGSA-1 in late type-B/secondary spermatogonia is a critical step in the progression of spermatogenesis, and indicate that anti-SGSA-1 is a useful marker for analysis of the molecular mechanism controlling the differentiation of spermatogonia in lower vertebrates. Mol. Reprod. Dev. 51:355–361, 1998. © 1998 Wiley-Liss, Inc.     

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis.

The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.     

Annual changes in testicular development and plasma sex steroids in the captive male dojo loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

Testicular development in the captive male dojo loach Misgurnus anguillicaudatus was examined monthly in relation to the levels of plasma sex steroids [testosterone (T), 11-ketotestostrone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (DHP)]. On the basis of testicular histology, the annual gonadal cycle was found to be divisible into 3 periods: the recovery and proliferation period, which mainly consists of early spermatogenic testis from August to November (reproductive phase I); the preparation period for the next spawning period, which mainly consists of late spermatogenic testis from December to April (reproductive phase II); and the mature period, characterized by a high proportion of mature testis from May to July (reproductive phase III). Individual variability in testicular development was high, and continuous spermatogenesis was observed throughout the year. High levels of plasma T, 11-KT, and DHP were observed during reproductive phase III. 11-KT began to increase in February, while T was present at low levels in reproductive phase II. These results suggest that the physiologically active season of testis development for breeding in the dojo loach is from May to July, although spermatogenesis occurs throughout the year.     

PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica)

Spermatogenesis can be initiated by a single injection of human chorionic gonadotropin (hCG) into the cultivated Japanese eel, which produces only spermatogonia in the testis. To isolate the genes responsible for regulating spermatogenesis, we performed a differential mRNA display using poly (A)+ RNA extracted from the testes at different time points after hCG injection. Among several cDNA clones, the expression of which was initiated before the onset of meiosis, one clone has high homology with the proliferating cell nuclear antigen (PCNA). In this study, we investigated the protein expression of eel PCNA and found for the first time in any species that two forms (32-kDa and 36-kDa) of PCNA are present in the testis. Although the 36-kDa form existed in both the testis and spleen, the 32-kDa form was specifically expressed in the testis. In contrast to the appearance of 36-kDa PCNA 1 day after the hCG treatment, the 32-kDa PCNA appeared only 9 days after the hCG treatment, at which time active spermatogonial proliferation occurred in the testis. Both the 32- and 36-kDa forms were recognized by antibodies raised against different epitopes of PCNA, and their N-terminal amino acid sequences were identical. The 36-kDa form, but not the 32-kDa form, was recognized by antibodies against phosphoamino acids. These results suggest that the two PCNA proteins are the same molecule with different chemical modifications, including phosphorylation. We discuss the roles of these two forms of PCNA in the spermatogenesis of the Japanese eel.     

Differential expression of bcl-2 and bcl-x during chicken spermatogenesis

We report the isolation and characterization of a chicken testis bcl-x_L cDNA coding for a long bcl-x protein with a hydrophobic tail, and the expression of bcl-2 and bcl-x during chicken spermatogenesis. Bcl-2 is highly expressed in embryonic and immature testes enriched in spermatogonia and barely detectable in mature testes, where most of the cells are meiotic and postmeiotic. Bcl-x is expressed in both mature and immature testes, but in a lesser amount in mature testes. Differential expression of bcl-2 and bcl-x during spermatogenesis is consistent with the reported different susceptibility to apoptosis of spermatogonia, and meiotic and postmeiotic cells. Mol. Reprod. Dev. 47:26–29, 1997. © 1997 Wiley-Liss, Inc.