Chiemi Miura

Involvement of Sex Steroid Hormones in the Early Stages of Spermatogenesis in Japanese Huchen (Hucho perryi )

Abstract In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryiu200a). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17β-estradiol (E2) both between months and among the individuals, E2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17α,20β-DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.

Japanese Eel : A Model for Analysis of Spermatogenesis

Abstract The Japanese eel has two characteristics advantageous for the study of the mechanisms controlling spermatogenesis. One is the possibility of artificial induction of the complete process of spermato-genesis from spermatogonial proliferation to spermiogenesis by exogenous gonadotropin injection, and the other is the possibility of inducing this process in an in vitro testicular organ culture or germ-Sertoli cell coculture system. Using the eel system, we analyzed the control mechanisms of spermatogenesis. In Japanese eel, the whole process of spermatogenesis is regulated by several sex steroid hormones. Spermatogonial stem cell renewal is promoted by estradiol-17β (the natural estrogen in vertebrates). Spermatogonial proliferation can be induced by 11-ketotestosterone, the main androgen in teleost. IGF-I is necessary for the action of 11-ketotestosterone in the initiation of spermatogenesis. The action of 11-ketotestosterone is mediated by other factors, such as activin B, produced by Sertoli cells. Although 11-ketotestosterone also induce meiosis and spermiogenesis, the control mechanisms of these processes are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation, thereby becoming capable of fertilization. Sperm maturation is regulated by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), which is progestogen in teleosts. The 17α,20β-DP acts directly on spermatozoa to activate the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.

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.

Two testicular cDNA clones suppressed by gonadotropin stimulation exhibit ZP2- and ZP3-like structures in Japanese eel

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature eel testes consisting of premitotic spermatogonia. To understand the regulatory mechanisms of spermatogenesis, we have applied a subtractive hybridization method to identify genes in which changes in expression occur after HCG treatment in vivo. The subtraction was carried out 24 hours after HCG injection. Two up‐regulated and six down‐regulated cDNA clones by HCG stimulation were isolated, and named eel spermatogenesis‐related substance (eSRS) 1 to 8. In this paper, down‐regulated cDNA clones of eSRS3 and eSRS4 were sequenced. A homology search showed that eSRS3 and eSRS4 have amino acid sequences similar to those of the ZP‐domains of zona pellucida sperm‐binding protein (ZP)‐2 and 3, respectively. Transcripts of eSRS3 and eSRS4 have been detected only in immature testes and ovaries. Both transcripts disappeared immediately after HCG injection and were not detected in testes throughout the experimental period. To determine whether HCG action on down‐regulation of eSRS3 and eSRS4 transcription is direct or mediated through 11‐ketotestosterone (11‐KT), a spermatogenesis‐inducing steroid in eel, we investigated the effect of HCG and 11‐KT on testicular eSRS3 and eSRS4 mRNA transcription in vitro. Northern blot analysis using poly(A)+ RNA extracted from cultured testis showed that both HCG and 11‐KT suppressed the mRNA transcription of both eSRS3 and eSRS4. We speculate that eSRS3 and eSRS4 may play important roles in the prevention of spermatogenesis in the eel. Mol. Reprod. Dev. 51:235–242, 1998.

cDNA cloning of a stage‐specific gene expressed during HCG‐induced spermatogenesis in the Japanese eel

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin βB subunit, was expressed in the initial phase of spermatogenesis (1–6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3–18 days (two clones), 6–18 days (one clone) and 9–18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full‐length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12–15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG‐induced spermatogenesis in the 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.

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

Abstract 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.

Spermatogenesis in the Japanese Eel

Spermatogenesis, the formation of sperm that is highly adapted for deliveringto its genes to an egg,, is a complex developmental process. It begins with the renewal of spermatogonial stem cells to maintain their own numbers, and the mitotic proliferation of spermatogonia. Then, it proceeds through two meiotic divisions followed by spermiogenesis during which the haploid spermatids developP into spermatozoa. Spermatozoa then undergo maturation to gain fertilizing ability. Even though spermatogenesis is the same in both vertebrates and invertebrates, its control mechanisms are not well understood because there are few appropriate models for analyzing them.

Expression and localization of eel testicular ZP-homologues in female Japanese eels (Anguilla japonica).

Abstract In male Japanese eels, eel spermatogenesis-related substance (eSRS) 3 and 4, having high sequence-similarities to zona pellucida protein (ZP) 2 and ZP3, respectively, are down-regulated by gonadotropin stimulation, with their transcripts disappearing upon the initiation of spermatogenesis. Using Northern blot analysis, we investigated the expression of eSRS3 and 4 mRNA in the developing ovary and the liver of SPH (salmon pituitary homogenate)-injected female eels. Both transcripts were detected in the ovary, but not in the liver. When the eel ovary was subjected to in situ hybridization using eSRS3 and 4 cRNA probes, the cytoplasm of previtellogenic oocytes showed a strong signal in comparison with the weak signal in vitellogenic oocytes. Furthermore, stronger signals were observed in the chromatin-nucleolus and the perinucleolus stages than in the oil-droplet stage. Subsequently, we synthesized peptides that were deduced from eSRS3 and 4 cDNAs and generated specific antibodies against them. Staining of the cytoplasm of oocytes in the previtellogenic stage and of egg envelopes in the vitellogenic stage occurred when these antibodies were used in an immunohistochemical analysis. These expression patterns in the ovary suggest that eSRS3 and 4 are components of the eel egg envelope.

Histological Studies on Early Oogenesis in Barfin Flounder (Verasper moseri)

Abstract There is much information on oogenesis from the resumption of the first meiotic division to oocyte maturation in many vertebrates; however, there have been very few studies on early oogenesis from oogonial proliferation to the initiation of meiosis. In the present study, we investigated the histological changes during early oogenesis in barfin flounder (Verasper moseri). In fish with a total length (TL) of 50mm (TL 50mm fish), active oogonial proliferation was observed. In TL 60mm fish, oocytes with synaptonemal complexes were observed. Before the initiation of active oogonial proliferation, somatic cells which surrounded a few oogonial germ cells, started to proliferate to form the oogonial cysts that accompanied oogonial proliferation. In TL 70mm fish, however, the cyst structure of the oocyte was gradually broken by the invagination of somatic cells, and finally the oocyte became a single cell surrounded by follicle cells. Upon comparison of nuclear size, DNA-synthesizing germ cells could be divided into two types: small nuclear cells and large nuclear cells. Based on histological observation, we propose that the small nuclear cells were in the mitotic prophase of oogonia and the large nuclear cells were in the meiotic prophase of oocytes, and that the nuclear size increases upon the initiation of meiosis.