A rapid activation of immature testis of Japanese eel (Anguilla japonica) by a single injection of human chorionic gonadotropin

The testis of Japanese eel (Anguilla japonica) consists of type A and early type B spermatogonia together with inactive Leydig and Sertoli cells. A single injection of human chorionic gonadotropin induced marked changes in the morphology of the testis and in the serum androgen levels within a period of 72 h. Morphological changes include spermatogonial proliferation, activation of Leydig and Sertoli cells, organization of seminiferous lobules and formation of lobular lumen in the testis. Leydig cells were enlarged, exhibiting characteristics of steroid-producing cells. Sertoli cells become elongated, show signs of high cellular activity and remain in close contact with spermatogonia. The lobular organization was achieved much earlier than the progression of spermatogenesis to late type B spermatogonia. Even 6 h after hCG injection, a significant increase in plasma levels of 11-ketotestosterone was observed, followed by a further time dependent increase. Plasma testosterone levels were also increased after injection, but the increase was much less than that of 11-ketotestosterone.     

Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.     

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.     

Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica

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.     

The structure of the germinal dense bodies (nuages) during differentiation of the male germ line of the teleost,Oryzias latipes

Summary The germinal dense body (GDB) in the teleost, Oryzias latipes, an organelle unique to the cells of germ line, is regarded as a counterpart of nuage material in amphibians and mammals. In the study described herein, GDBs in male germ line cells were examined by electron microscopy. GDBs existed continuously in the cytoplasm of primordial germ cells (PGCs), prespermatogonia, type-A spermatogonia and early type-B spermatogonia. But they became rudimentary in late type-B spermatogonia and early spermatocytes, and no longer occurred in spermatids. Differences in the morphology of GDBs of PGCs and male germ cells were also noted. In PGCs of indifferent gonads, about 50% of GDBs were amorphous bodies of fine electron-dense fibrils, whereas in spermatogonia amorphous bodies decreased in number and GDBs of strand-like structure were more frequent. The change in the morphology of GDBs began when the sex differentiation of gonads became evident, and proceeded gradually in prespermatogonia. No obvious differences in morphology of GDBs were noted between prespermatogonia in the fry at later stages of development and spermatogonia in adult fish.     

The cloning of cyclin B3 and its gene expression during hormonally induced spermatogenesis in the teleost, Anguilla japonica

We cloned cyclin B1, B2, and B3 cDNAs from the eel testis. Northern blot analysis indicated that these cyclin B mRNAs were expressed and increased from day 3 onward after the hormonal induction of spermatogenesis, and that cyclin B3 was most dominantly expressed during spermatogenesis. In situ hybridization showed that cyclin B1 and B2 were present from the spermatogonium stage to the spermatocyte stage. On the other hand, cyclin B3 mRNA was present only in spermatogonia. Although mouse cyclin B3 is expressed specifically in the early meiotic prophase, these results indicate that eel cyclin B3 expression is limited during spermatogenesis to spermatogonia, but is not present in spermatocytes. These facts together suggest that eel cyclin B3 is specifically involved in spermatogonial proliferation (mitosis), but not in meiosis.     

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.     

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.     

Stage-specific apoptosis of male germ cells in the rat: mechanisms of cell death studied by supravital squash preparations

Apoptosis has been proposed as a mechanism by which testis germ cells are removed during normal and various pathological conditions. To establish a new rapid way to detect stage-specific apoptosis in male rat germ cells, their supravital morphology was examined from carefully squashed monolayers of living cells, after several established toxic treatments, using a phase contrast microscope. The results were compared with early detection of apoptosis using annexin V and propidium iodide (PI) stainings. The apoptosis of type-A spermatogonia and round spermatids proceeded in a similar way to somatic cells, while intermediate and type-B spermatogonia, and particularly the dividing spermatocytes, possessed characteristics not entirely typical for apoptosis. Death of elongated spermatids was difficult to assess owing to their compacted chromatin. As the first phases of degeneration seemed different in various germ cell classes, the final stage (karyopycnosis) was similar for most cells. Degenerating cells also showed positive reactions for annexin V and PI. The 'living cell method' provides rapid and accurate possibilities for analysis of stage-specific apoptosis during spermatogenesis. This method is not influenced by artefacts induced by fixation, embedding and sectioning. It may be developed further for routine analyses of the accurate stage-specific effects of various physical and chemical effects on mammalian and human spermatogenesis.     

UCHL-1 protein expression specifically marks spermatogonia in wild bovid testis

Spermatogonia are germ cells that initiate spermatogenesis in mammalian testis and they are the only cells in adult body capable of dividing both mitotically and meiotically. Therefore, isolation and preservation of spermatogonia can provide an alternative method for preservation of genetic pool of endangered animals. To achieve this objective, it is essential to identify markers that can specifically distinguish spermatogonia from other cells in the testis. In the present study, anti-ubiquitin C-terminal hydroxylase-1 (UCHL-1/PGP 9.5) antibody specifically recognized spermatogonia in the testis of wild and domestic bovid. The size of the UCHL-1 protein in various species of bovid testes was identical, and similar to that in mice. Furthermore, UCHL-1 staining could be utilized for the identification of spermatogonia in isolated testicular cells from wild bovids suggesting that UCHL-1 protein expression could be used as a specific marker for spermatogonia in bovid family.     

Human chorionic gonadotropin induces all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica)

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Using a newly developed organ culture system, we obtained evidence that human chorionic gonadotropin (HCG) can induce the entire process of spermatogenesis, in vitro, from spermatogonia to spermatozoa within 24 days. The HCG-induced spermatogenesis in vitro was accompanied by a marked activation of Sertoli cells and Leydig cells, occurring prior to the beginning of spermatogonial proliferation. These results indicate that gonadotropin triggers spermatogenesis in the Japanese eel and further suggest that this effect of gonadotropin is mediated through the actions of testicular somatic cells.     

Steel-Dickie (Sld) mutation affects both maintenance and differentiation of testicular germ cells in mice.

The effects of Steel-Dickie (Sld) mutations on testicular germ cell differentiation were investigated using experimental cryptorchidism and its surgical reversal in mutant, C57BL/6-Sld/+ and wild-type C57BL/6- +/+ mice. In Sld/+ cryptorchid testes the maintenance of undifferentiated type-A spermatogonia was impaired and their numbers decreased. In contrast, the proliferative activity of type-A spermatogonia in the cryptorchid testis of mutant mice appeared normal as judged by their progression through the cell cycle. Surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in +/+ testes. However, the regenerative differentiation of type-A spermatogonia which remained in Sld/+ cryptorchid testes was strongly impaired, particularly at two steps of cellular differentiation, from type-A spermatogonia to intermediate or type-B spermatogonia and at meiotic division. Furthermore, in mutant mice, no significant recovery of testicular weight was observed after surgical reversal compared with +/+ mice.     

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.     

Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis

To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N-glycanase, suggesting the presence of asparagine-linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present. © 1995 Wiley-Liss, Inc.     

Heteroplasmy of the chloroplast genome of Medicago sativa L. cv ‘Regen S’' confirmed by sequence analysis

The heteroplasmy of chloroplast DNA (cpDNA) observed in Medicago sativa L., which involves the presence (type B) or absence (type A) of an Xba I restriction site, was examined using closed fragments covering the variable XbaI site from type-A and type-B cpDNA. The 6.2-kb PstI fragment of DNA from type-A cpDNA (–XbaI) and from type-B cpDNA (+XbaI) was cloned into pUC19 plasmids. EcoRI fragments bearing the variable XbaI site from the type-A and type-B 6.2-kb PstI fragments were subcloned into pUC19. DNA sequences of both types of the 696-bp EcoRI fragments were determined and computer-assisted analysis of the sequence data carried out. Type-A cpDNA was found to differ from type-B cpDNA by 1 base, a G to T conversion, which results in a non-recognition site for XbaI in the type-A cpDNA. The sequence difference was in a non-coding region. Cloning and sequencing of the fragments verified the individual identity of the type-A and type-B cpDNA.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.