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.     

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.     

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.     

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.     

Pulsatile gonadotropin-releasing hormone treatment of men with idiopathic hypogonadotropic hypogonadism

OBJECTIVES/METHODS: To induce testicular growth and spermatogenesis, 11 patients with idiopathic hypogonadotropic hypogonadism were treated with long-term subcutaneous pulsatile gonadotropin-releasing hormone (GnRH) administration. Three patients had a history of undescended testes. Patients who did not respond to therapy with a sufficient increase in serum testosterone or spermatogenesis were offered additional injections with hCG or, after discontinuation of GnRH, either combined therapy with hCG and hMG or recombinant FSH. RESULTS: During treatment testicular volume and serum levels of FSH, LH and testosterone increased. Semen analysis revealed the presence of spermatogenesis in 9 of the 11 patients (8 on GnRH alone and in 1 when hCG/hMG was subsequently instituted), and 7 pregnancies have resulted thus far. CONCLUSION: Pulsatile GnRH therapy is a well-tolerated and effective therapy for the induction of spermatogenesis in some men with idiopathic hypogonadotropic hypogonadism. It appears that a significant fraction of them should be treated for a minimum of 1-2 years to maximize testicular growth and achieve spermatogenesis. Cryptorchidism was a negative prognostic factor.     

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.     

Androgen secretion and characteristics of testicular HCG binding in cryptorchid rats

Response of the cryptorchid testis to gonadotrophic stimulation was assessed by comparison of the androgen production capability in vivo and in vitro with that of the normal scrotal testis. Serum androgen concentrations in cryptorchid rats were similar to those in normal rats, and the incremental increase 60 min after 50 i.u. hCG (i.v.) was about 7-fold for both groups. Basal and hCG-stimulated androgen production in vitro was higher for abdominal testes (557 and 3286 ng/pair) than for scrotal tests (157 and 504 ng/pair). Specific binding of hCG by testicular homogenates was slightly higher (P < 0.05) for cryptorchid testes when expressed per unit weight, but Scatchard analysis indicated that although hCG binding affinities did not differ (Ka = 2 x 10(10) M-1), hCG binding capacity of cryptorchid testes was only 75 ng, compared to 219 ng for scrotal testes. These data indicate that a discrepancy exists between androgen production in vivo and in vitro by cryptorchid testes and that normal serum androgen concentrations are maintained in the presence of decreased numbers of testicular LH/hCG receptors.     

hCG-induced maturation of the seminiferous epithelium in hypogonadotropic men

The effect of long-term hCG administration on sperm output was evaluated in a study in 3 hypogonadal patients with a selective deficiency of gonadotrophins (LH and FSH). The diagnosis of complete hypogonadotropic hypogonadism was based on clinical and hormonal findings as well as testicular histology. Pubertal maturation took place gradually during hCG therapy. 2 out 3 patients, who were azoospermic before treatment, had spermatozoa in their ejaculate after 12 and 24 months of therapy respectively. These effects on spermatogenesis were reversed after hCG withdrawal for 4 months and the patients again became azoospermic. This azoospermia was not reversed by testosterone (T) replacement therapy, or by addition of HMG to T. In vitro, the crude hCG preparation stimulated cAMP accumulation in rat Sertoli cell cultures indicating that this hCG preparation possesses an 'FSH-like' action. The present findings indicate that hCG therapy alone can induce and maintain spermatogenesis in some patients with complete hypogonadotropic hypogonadism.     

Estradiol-17beta triggers luteinizing hormone release in the protandrous black porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotropin-releasing hormone control.

We investigated the mechanism of estradiol-17beta (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17beta also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17beta potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2) induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2in the sex change observed in the protandrous black porgy.     

Control of spermatogenesis resumption in post-diapausing larvae of the codling moth

The lepidopteran primary spermatocytes produce first eupyrene (nucleated) and later apyrene (anucleated) spermatozoa. The shift to apyrene commitment of the spermatocytes is related to an apyrene-spermatogenesis-inducing factor (ASIF) becoming active towards pupation. During diapause, the primary spermatocytes lyse and spermatogenesis ceases. The renewal of the dichotomous spermatogenesis in the testes of post-diapausing, last-instar larvae of the codling moth was studied in vivo and in vitro. In vivo, the post-diapausing larvae resume the two types of spermatogenesis. Since ASIF activity is related to pupation, the earliest apyrene spermatids appear one day before pupation, as in non-diapausing larvae. In vitro, renewal of spermatogenesis occurs if 20-hydroxy-ecdysone is added to the medium, but only eupyrene spermatids occur since the testes are explanted before ASIF activity has started. These spermatids are unreduced and develop directly from primary spermatocytes which do not undergo meiotic divisions. Moreover, only flagella develop in these spermatids and the nuclei remain spherical. Post-diapause resumption of spermatogenesis is thus a complex process in which meiosis-blocking and meiosis-deblocking factors, ecdysteroids, and the ASIF play regulative roles.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility

The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.     

Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA distributions in maldescended testes: hyperdiploid aneuploidy without evidence of germ cell neoplasia.

Fine-needle aspiration biopsies and surgical biopsies were obtained from maldescended testes of 149 consecutive men. The aspirates were subjected to quantitative DNA flow cytometry and the surgical biopsy to histological evaluation. From more than 80% of the gonads, sufficient material was obtained for both examinations. A significant hyperdiploid cell population with a mean DNA index of 1.23 (range 1.17-1.31) was found in six gonads. Hyperdiploid aneuploidy was found in gonads without, as well as with, complete spermatogenesis. In none of the six cases did the surgical biopsy show evidence of early testicular neoplasia by morphology or by immunohistochemical methods with antibodies against carcinoma in situ. This indicates that aneuploidies in maldescended testes do not necessarily indicate malignancy. It may be speculated that hyperdiploid aneuploidy is related to the development of preneoplastic lesions.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.