Estradiol-induced mitogen-activated protein kinase (extracellular signal-regulated kinase 1 and 2) activity in the frog (Rana esculenta) testis

Several lines of evidence support a key role of estradiol-17beta (E(2)) in male fertility. We have used a non-mammalian vertebrate model, the frog Rana esculenta, to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2 )exerts a role in spermatogenesis through the regulation of ERK1/2 activity. ERK1/2 proteins are present in the cytoplasm and nucleus of the primary and secondary spermatogonia (SPG), and in the nucleus of primary spermatocytes. The annual E(2) profile shows a progressive increase during active spermatogenesis with a peak in the month of June. In parallel, ERK1/2 are highly phosphorylated during the period of active spermatogenesis (from April to July) compared with the regressive period (September/October) and winter stasis (from November to March). E(2) treatment induces the proliferation of primary SPG, possibly via the activation of ERK1/2, and this effect is counteracted by the anti-estrogen ICI 182-780.     

Intranuclear appearance of the phosphorylated form of cytoskeleton-associated 350-kDa proteins in U1-ribonucleoprotein regions after growth stimulation of fibroblasts.

Cytoskeleton-associated 350-kDa and 80-kDa polypeptides, which were immunoprecipitated with polyclonal antibody against microtubule-associated protein 1 (MAP-1), were rapidly phosphorylated on mitogenic stimulation of quiescent fibroblasts with serum or growth factors. The enhanced phosphorylation was evident within 5 min and reached a maximum 2 hr after the stimulation. Phosphorylated MAP-1 analogues were first detected in the cytoplasm around the microtubule-organizing center and then in the nucleus by immunofluorescent staining with a monoclonal antibody that recognized the phosphorylated form of MAP-1. The monoclonal antibody reacted with the 350-kDa protein in immunoblot analysis and immunostained intranuclear speckles; both immunoreactions were abolished by treatment with alkaline or acid phosphatase. The nuclear speckles stained by the monoclonal antibody were also stained by anti-U1 small nuclear ribonucleoprotein antibodies on double immunofluorescence, suggesting that the stained regions are sites of maturation of messenger RNA. These results support the idea that part of the cytoskeleton-associated 350-kDa protein is phosphorylated and transferred to the nuclear region of mRNA modification as a common early process after growth stimulation.     

Maintenance of complete but quantitatively reduced spermatogenesis in hypophysectomized monkeys by testosterone alone

In order to investigate whether testosterone can maintain spermatogenesis in the absence of FSH in primates, four cynomolgus monkeys were hypophysectomized and implanted with 20 5-cm-long testosterone-filled silastic capsules within 45 min of pituitary ablation. Thereafter the serum levels of testosterone were elevated about 9-fold over presurgical levels. Testicular volumes declined to 60% of presurgical values. Testicular concentrations of testosterone were 50-180% of presurgical levels. Germ cell numbers were reduced to 30-50% of presurgical values and germ cell ratios suggested that the reduced numbers of all advanced germ cells were due to a decrease in the efficiency of proliferation of B spermatogonia. A fifth monkey was left untreated following hypophysectomy. Its serum testosterone was as low as that of castrated monkeys, and the testicular volume declined to 30% of that before surgery. Primitive spermatogonia were the only germ cells present 13 weeks after surgery. Thus, in primates testosterone alone maintains the complete process of spermatogenesis, however, spermatogonial proliferation is impaired in the absence of FSH.     

Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse

AIM: The aim of this study was to determine the action of bone morphogenetic proteins (BMPs) on testicular cell proliferation during early postnatal life, a definite developmental time at which crucial changes in germ cell and Sertoli cell maturation occur. METHODS: We investigated the effect of BMP2 and BMP7, two factors which belong to the relatively distant decapentaplegic (DPP) and 60 A classes of the large BMP family, upon spermatogonial and Sertoli cell proliferation, and we examined the expression of activin/BMP type II and type I receptors. We used in vitro cultured testis fragments from 7-day-old mice, highly purified populations of somatic and germ cells and total testes from mice of different ages. Cell proliferation was assessed by BrdU labelling and [3H]-thymidine incorporation. Ribonuclease protection assays and Northern blotting were performed to analyse receptor expression. RESULTS AND CONCLUSIONS: We have demonstrated a stimulatory action of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation respectively. ActRIIB is the type II receptor expressed most in spermatogonia, whereas Sertoli cells specifically expressed BMPRIIB, in addition to ActRIIB. By contrast, the presence of ActRIIA was undetectable in either germ or somatic cells. The type I receptors ActRIA, ActRIB and BMPRIA were all found in both cell types, indicating that the observed effect of BMP2 and BMP7 on testicular cell proliferation may be mediated by a number of combinatorial interactions in the receptor complexes. These findings suggest that BMPs are involved in physiological paracrine signalling during the first wave of spermatogenesis.     

Suppression of the intratesticular testosterone is associated with quantitative changes in spermatogonial populations in intact adult rats

To investigate the interactions between testosterone and FSH on quantitative aspects of spermatogenesis, intact adult rats were implanted sc with 0.5- to 50-cm long testosterone-filled silastic capsules (TC) for 8 weeks. Serum testosterone levels were suppressed slightly in rats bearing 1-cm TC implants but were elevated 6- to 30-fold when 5 cm or longer TC implants were used. These changes in serum testosterone were associated with a biphasic, dose-dependent response in testicular testosterone concentrations, reaching a minimum of 10-15% of the control values in rats bearing the 1- or 5-cm TC implants, but rebounding to 80% when 50-cm long TC implants were used. Meanwhile, serum FSH was comparably suppressed by 40-70%. Complete spermatogenesis was observed in all experimental animals with the exception of three rats with 1-cm TC implants in which elongated spermatids were absent or reduced in number. Enumeration of various cell types in the basal compartment of whole mounted seminiferous tubules revealed a 20% increase in type A1 spermatogonia in rats with the 1-cm TC implants. On the other hand, a 15-20% reduction in type B spermatogonia and preleptotene spermatocytes was noted in rats receiving 5-cm or longer TC implants. These results demonstrate that complete spermatogenesis can be maintained in intact animals in the presence of 10-15% of the normal testicular testosterone concentration. Failure to maintain normal testis weight, testicular testosterone concentration, or germ cell number at a 30-fold increase in serum testosterone demonstrates that high levels of serum testosterone may not be beneficial for quantitative spermatogenesis. Furthermore, both quantitative and qualitative differences were observed in different phases of spermatogenesis in the presence of comparable serum FSH and testicular testosterone concentrations. These results suggest that factors other than these two hormones are also involved in the regulation of spermatogenesis.     

Sox3 functions in a cell-autonomous manner to regulate spermatogonial differentiation in mice

The X-linked Sox3 gene encodes a member of the Sry high-mobility group box proteins, which play a role in many developmental processes including neurogenesis and testis development. This study further examined the role of Sox3 in spermatogenesis. Males without Sox3 expression exhibited a similar number of germ cell nuclear antigen-positive germ cells at 1, 5, and 10 d postpartum (dpp) compared to their wild-type littermates, but there was significant germ cell depletion by 20 dpp. However, spermatogenesis later resumed and postmeiotic germ cells were observed by 56 dpp. The VasaCre transgene was used to generate a germ cell-specific deletion of Sox3. The phenotype of the germ cell-specific Sox3 knockout was similar to the ubiquitous knockout, indicating an intrinsic role for Sox3 in germ cells. The residual germ cells in 20 dpp Sox3(-/Y) males were spermatogonia as indicated by their expression of neurogenin3 but not synaptonemal complex protein 3, which is expressed within cells undergoing meiosis. RNA expression analyses corroborated the histological analyses and revealed a gradual transition from relatively increased expression of spermatogonia genes at 20 dpp to near normal expression of genes characteristic of undifferentiated and meiotic germ cells by 84 dpp. Fluorescent-activated cell sorting of undifferentiated (ret tyrosine kinase receptor positive) and differentiated (kit receptor tyrosine kinase-positive) spermatogonia revealed depletion of differentiated spermatogonia in Sox3(-/Y) tubules. These results indicate that Sox3 functions in an intrinsic manner to promote differentiation of spermatogonia in prepubertal mice but it is not required for ongoing spermatogenesis in adults. The Sox3(-/Y) males provide a unique model for studying the mechanism of germ cell differentiation in prepubertal testes.     

Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel

Meiosis is an indispensable process of sexual reproduction. However, detailed information on the regulatory mechanisms that initiate meiosis is not available. Progestins are important steroids regulating final maturation in male and female vertebrates. In male teleosts, it is known that progestin induces spermiation and sperm maturation. However, a role for progestin in early spermatogenesis or meiosis has not yet been described. In this study, we examined the functions of progestin on the initiation of meiosis in male Japanese eel. A natural progestin in teleost fish 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and its receptors were present in the testis at an early stage of spermatogenesis. By using an eel testicular culture system, DHP was shown to induce DNA replication in spermatogonia. Although 11-ketotestosterone, a known initiator of spermatogenesis, also stimulated DNA synthesis in spermatogonia, antibodies against DHP prevented DNA replication when added during the period in which meiosis was initiated. DHP treatment also induced the expression of meiosis-specific markers, such as DmcI and Spo11. Furthermore, Spo11 expression and synaptonemal complexes, specific features of the meiotic prophase, were detected in testicular fragments cultured with DHP in some germ cells that showed morphological characteristics of undifferentiated spermatogonia. We conclude that DHP, a progestin, is an essential factor for the initiation of meiosis.     

Zinc is an essential trace element for spermatogenesis

Zinc (Zn) plays important roles in various biological activities but there is little available information regarding its functions in spermatogenesis. In our current study, we further examined the role of Zn during spermatogenesis in the Japanese eel (Anguilla japonica). Human CG (hCG) was injected into the animals to induce spermatogenesis, after which the concentration of Zn in the testis increased in tandem with the progression of spermatogenesis. Staining of testicular cells with a Zn-specific fluorescent probe revealed that Zn accumulates in germ cells, particularly in the mitochondria of spermatogonia and spermatozoa. Using an in vitro testicular organ culture system for the Japanese eel, production of a Zn deficiency by chelation with N,N,N′,N′-tetrakis (2-pyridylemethyl)ethylenediamine (TPEN) caused apoptosis of the germ cells. However, this cell death was rescued by the addition of Zn to the cultures. Furthermore, an induced deficiency of Zn by TPEN chelation was found to inhibit the germ cell proliferation induced by 11-ketotestosterone (KT), a fish specific androgen, 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17β (E2), an inducer of spermatogonial stem-cell renewal. We also investigated the effects of Zn deficiency on sperm motility and observed that TPEN treatment of eel sperm suppressed the rate and duration of their motility but that co-treatment with Zn blocked the effects of TPEN. Our present results thus suggest that Zn is an essential trace element for the maintenance of germ cells, the progression spermatogenesis, and the regulation of sperm motility.     

Cellular and intracellular distribution of growth hormone in the adult chicken testis

Endocrine actions of growth hormone (GH) have been implicated during the development of adult testicular function in several mammalian species, and recently intracrine, autocrine, and paracrine effects have been proposed for locally expressed GH. Previous reports have shown the distribution of GH mRNA and the molecular heterogeneity of GH protein in both adult chicken testes and vas deferens. This study provides evidence of the presence and distribution of GH and its receptor (GHR) during all stages of spermatogenesis in adult chicken testes. This hormone and its receptor are not restricted to the cytoplasm; they are also found in the nuclei of spermatogonia, spermatocytes, and spermatids. The pattern of GH isoforms was characterized in the different, isolated germ cell subpopulations, and the major molecular variant in all subpopulations was 17 kDa GH, as reported in other chicken extra-pituitary tissues. Another molecular variant, the 29 kDa moiety, was found mainly in the enriched spermatocyte population, suggesting that it acts at specific developmental stages. The co-localization of GH with the proliferative cell nuclear antigen PCNA (a DNA replication marker present in spermatogonial cells) was demonstrated by immunohistochemistry. These results show for the first time that GH and GHR are present in the nuclei of adult chicken germinal cells, and suggest that GH could participate in proliferation and differentiation during the complex process of spermatogenesis.     

Effects of a single injection of a new depot formulation of an LH-releasing hormone agonist on spermatogenesis in adult rats

Adult male Wistar rats were treated with a single injection (500 micrograms s.c.) of a new biodegradable depot formulation of the LH-releasing hormone (LHRH) analogue [D-Ser(But)6]AzGly10-LH-RH (Zoladex; ICI 118,630) to evaluate its potential for inhibiting spermatogenesis. The drug produced a marked (P less than or equal to 0.05) decrease in serum concentrations of FSH, LH and testosterone with a maximum effect 14 days after treatment. Since striking focal histological changes were seen in the testis after only 1 week, at a time when changes in serum gonadotrophins were minimal, there may be a direct effect of the LHRH analogue on spermatogenesis. Degenerative changes in germ cells as well as Sertoli cells could be observed. Flow-cytometric analysis of testicular cell suspensions showed a significant decline in the absolute numbers of haploid cells (spermatids), tetraploid cells (mainly pachytene spermatocytes) and of the numbers of cells in the S-phase of the cell cycle. This suggests that the drug also inhibits proliferation of spermatogonia and/or primary spermatocytes. Testis weight, serum hormone concentrations, and histological and cytological parameters returned to essentially normal values 52 days after the injection. It is concluded that this new method of administration may have practical and pharmacokinetic advantages for the purpose of reversible inhibition of spermatogenesis.     

Testicular development and plasma sex steroid levels in cultured male Senegalese sole Solea senegalensis Kaup

Testicular development and plasma levels of sex steroids (11-ketotestosterone (11-KT), testosterone (T) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)) were investigated for the first time in cultured male Senegalese sole Solea senegalensis. The germ cell dynamics and gonadosomatic index (IG) were monitored. Based on the relative abundance of the different types of germ cells present, the spermatogenetic cycle was divided into five stages: early (I; spermatogonia (SPG)), mid (II; SPG, spermatocytes (SPC) and spermatids (SPD)), and late spermatogenesis (III; SPC, SPD, and spermatozoa (SPZ)), functional maturation (IV; SPD and SPZ), and recovery (V; SPD, SPZ, and SPG). During summer, fish had stage I and V testes and the lowest values in plasma levels of sex steroids and IG. Testicular recrudescence seemed to begin in autumn, as denoted by the first increase in IG and in the levels of 11-KT and T, and the appearance of testes at stage II and III. During winter, the levels of 11-KT and T peaked and soon began to decrease, the IG slightly declined and the proportion of running males (RM) gradually increased. In spring, levels of 11-KT and T continued to decline, the IG slightly increased and the proportion of RM peaked concomitantly with the occurrence of stage IV testes. Plasma levels of 17,20beta-P did not change significantly throughout testicular development. Transformation of SPD into SPZ followed a group-synchronous fashion, a phenomenon which parallels asynchronous oocyte development reported in females. This mechanism would be consistent with the observed small quantity of sperm that can be manually stripped at any one time and other aspects of S. senegalensis reproductive biology.     

The testes of moles (Talpa europaea) retain a considerable microsomal capacity for androgen synthesis during seasonal regression

Seasonal changes in testicular histology and steroidogenesis were investigated in the mole (Talpa europaea). Androgen synthesis was examined by incubating [4-14C]pregnenolone (P) and [4-14C]dehydroepiandrosterone (DHA) with testicular minces in a static incubation system. The metabolites formed were characterized by thin-layer chromatography. Morphological changes were studied by routine histological methods. During sexual quiescence spermatogenesis was arrested. The regressive seminiferous tubules consisted predominantly of Sertoli cells and spermatogonia. On the other hand, histological quantification suggested that season has no significant effects on the number or the nuclear dimensions of Leydig cells in this species. The capacity of the regressive testes (per unit weight) to metabolize P and DHA to testosterone (T) was somewhat greater in regressive (48.5%, 49.4%) than in active (33.2%, 41.6%) testes. The results also suggest that the greater in vitro T production encountered during reproductive quiescence is due possibly to an increase in the activity of 17 beta-hydroxysteroid dehydrogenase (per unit weight). Our data on Leydig cell numbers indicate, however, that the capacity of the individual Leydig cells to produce T is decreased during sexual regression. T. europaea appears to be quite exceptional among seasonally breeding small mammals exhibiting pronounced annual changes in spermatogenesis in that the testes retain a considerable enzymatic capacity to produce testosterone from pregnanes during sexual quiescence. The results suggest that pituitary as well as paracrine regulation of the annual testicular cycle in this species differs from that generally encountered in seasonal breeders.     

Drug transporters, the blood-testis barrier, and spermatogenesis

The blood-testis barrier (BTB), which is created by adjacent Sertoli cells near the basement membrane, serves as a 'gatekeeper' to prohibit harmful substances from reaching developing germ cells, most notably postmeiotic spermatids. The BTB also divides the seminiferous epithelium into the basal and adluminal (apical) compartment so that postmeiotic spermatid development, namely spermiogenesis, can take place in a specialized microenvironment in the apical compartment behind the BTB. The BTB also contributes, at least in part, to the immune privilege status of the testis, so that anti-sperm antibodies are not developed against antigens that are expressed transiently during spermatogenesis. Recent studies have shown that numerous drug transporters are expressed by Sertoli cells. However, many of these same drug transporters are also expressed by spermatogonia, spermatocytes, round spermatids, elongating spermatids, and elongated spermatids, suggesting that the developing germ cells are also able to selectively pump drugs 'in' and/or 'out' via influx or efflux pumps. We review herein the latest developments regarding the role of drug transporters in spermatogenesis. We also propose a model utilized by the testis to protect germ cell development from 'harmful' environmental toxicants and xenobiotics and/or from 'therapeutic' substances (e.g. anticancer drugs). We also discuss how drug transporters that are supposed to protect spermatogenesis can work against the testis in some instances. For example, when drugs (e.g. male contraceptives) that can perturb germ cell adhesion and/or maturation are actively pumped out of the testis or are prevented from entering the apical compartment, such as by efflux pumps.     

Analysis of meiosis in intratesticular germ cells from subjects affected by classic Klinefelter's syndrome.

Azoospermic subjects affected by Klinefelter's syndrome may occasionally show the presence of intratesticular residual foci of spermatogenesis, and the retrieval of mature spermatozoa from the testis may permit fertility and paternity by means of intracytoplasmic sperm injection. Previous studies have demonstrated that these subjects show the presence of an increased incidence of hyperaploid spermatozoa. Here we analyzed, by fluorescence in situ hybridization using specific probes for chromosomes 8, X, and Y, the spermatogenic process and the meiotic progression of 47,XXY germ cells retrieved by fine needle aspiration of the testis in ten azoospermic patients affected by classic Klinefelter's syndrome. All patients had lower testicular volume, higher gonadotropins, and lower testosterone plasma levels compared with control subjects. Cytological analysis of the testicular cells retrieved by fine needle aspiration showed the presence of Sertoli cells only in eight subjects, while germ cells were observed in two patients. In each patient Sertoli cells showed a 47,XXY karyotype, and the same chromosome pattern was observed in spermatogonia and primary spermatocytes of patients presenting a residual spermatogenesis. Secondary spermatocytes, spermatids, and mature spermatozoa showed different sex chromosome patterns, reflecting their origin from 47,XXY spermatogonia. In conclusion, this study demonstrated that, in subjects affected by Klinefelter's syndrome, residual germ cells may be present in the testis and that 47,XXY spermatogonia are able to undergo and complete the spermatogenic process leading to mature spermatozoa. These data further suggest the need to evaluate the sex chromosome status of sperm from patients affected by Klinefelter's syndrome undergoing assisted reproductive techniques.