Endocrinology of spermatogenesis in the hypophysectomized ram.

Adult rams were hypophysectomized and treated for 20 days with testosterone (2 X 0.25 g/day), PMSG (2 X 300 i.u./day) or hCG (2 X 250 i.u./day), or for 40 days with testosterone (2 X 0.25 g/day). All treatments maintained a normal concentration of testosterone within the seminiferous tubules. Quantitative histological analysis showed that (1) the differentiation from A0 to A1 spermatogonia was maintained by PMSG or hCG but not completely by testosterone; (2) the transition from intermediate spermatogonia to primary spermatocytes was maintained only by PMSG but not by testosterone or hCG; (3) meiotic prophase and spermiogenesis were maintained by the three hormones but there were qualitative abnormalities in the spermatids. These results suggest that in the ram, the differentiation of renewing stem spermatogonia is under LH control and that the last stages of spermatogonial multiplication, from intermediate to B spermatogonia and to primary spermatocytes, are under the control of the FSH-like activity of PMSG.     

Analysis of the testes of H-Y negative XOSxrb mice suggests that the spermatogenesis gene (Spy) acts during the differentiation of the A spermatogonia

H-Y antigen negative XOSxrb mice, like their H-Y positive XOSxra counterparts, have testes; but, in contrast to XOSxra males, XOSxrb testes almost totally lack meiotic and postmeiotic stages of spermatogenesis. The quantitative analysis of the testes of XOSxrb males and their XY +/- Sxrb sibs, described in the present study, identified two distinct steps in this spermatogenic failure. First, there was a reduction in mitotic activity among T1 prospermatogonia, so that approximately half the normal number of T2 prospermatogonia were produced. Second, there was a dramatic decrease in the number of A3 and A4 spermatogonia and no Intermediate or B spermatogonia. These reductions were also largely due to decreased mitotic activity, there being a shortage of A1 and A2 spermatogonial divisions and no divisions among A3 or A4 spermatogonia. Mitotic activity among the T2 prospermatogonia and the undifferentiated A spermatogonia was normal. This means that the spermatogonial stem cells, which are a subset of the undifferentiated A spermatogonia, are unaffected in XOSxrb mice. Sxrb is now known to have arisen by deletion of DNA from Sxra. It is clear from the present findings that a gene (or genes) present in the deleted DNA plays a major role in the survival and proliferation of the differentiating A spermatogonia.     

Pituitary gonadotropins regulate spermatogonial differentiation and proliferation in the rat‡

The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization of lu teinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells,while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial proliferation as indicated by a decrease in [³H] thymidine incorpor-ation into DNA by purified spermatogonia. Administration of follicle stimulating hormone a/s for 7 days also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone led to a significant reduction in tetraploid and haploid cells. However interestingly,it failed to totally abolish the appearance of these cells. Administration of testosterone (3 mg/day/rat) for 2 days along with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis, (iii) testosterone may be involved in spermatogonia] proliferation providing a mitotic signal and (v) both follicle stimulating hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation. A part of the data was presented at the 16th International Congress of Biochemistry and Molecular Biology, New Delhi, September 1994.     

蝗虫精母细胞减数分裂各时期的识别

蝗虫具有材料易得、染色体大、染色体数相对较少等优点, 被普遍用于观察精子发生中减数分裂各个过程。蝗虫精巢制片简单、快速, 但初学者普遍感到辨别分裂相较为困难。鉴于此, 文章以东亚飞蝗(Locusta migratoria manilensis (Meyen))精母细胞的减数分裂过程为例, 介绍如何根据染色体的数目及染色体构象, 识别减数分裂各个时期。此外, 文章也简要描述了东亚飞蝗精母细胞的有丝分裂及精子形成过程, 供初学者参考。     

Enrichment of the stages of the seminiferous epithelium in vitamin A-replaced-vitamin A-deficient rats

Morphometric study revealed that, at 40 days after the start of vitamin A replacement, A1 spermatogonia and preleptotene spermatocytes appeared in more than 70% of the whole mounts of seminiferous tubules of vitamin A-deficient rats. By 42 days, the appearance of these cell types was reduced by 50%, and A2 and A3 spermatogonia were predominant. By 46 days, A1-A3 spermatogonia appeared in less than 30% of the tubular length while A4, intermediate and B spermatogonia became the major cell types in the basement compartment of seminiferous tubules. The predominance of spermatogonia noted at given times was corroborated by higher frequencies of tubular cross-sections of stages in which that particular type of spermatogonium resides. These results indicate that seminiferous tubules of vitamin A-replaced-vitamin A-deficient rats are 'enriched' for particular stages. Tracing the development of [3H]thymidine-labelled preleptotene spermatocytes revealed normal kinetics of germ cell differentiation in these animals. Furthermore, the spermatogonial proliferations in the vitamin A-replaced-vitamin A-deficient rats were quantitatively normal. We suggest that vitamin A replacement may result in temporal suppression of the differentiation of A2-B spermatogonia, leading to a stimulation or synchronization of certain groups of undifferentiating spermatogonia which undergo active proliferation simultaneously. These synchronized populations of spermatogonia continue to proliferate and differentiate, thus resulting in the stage-enrichments noted at later times.     

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance

p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.     

Influence of stress on gonadotrophin induced testicular recrudescence in the lizard Mabuya Carinata

Administration (ip) of FSH (10 IU/0.1 ml distilled water (dw)/lizard/alternate days/30 days) to adult male lizards, Mabuya carinata, during the early recrudescence phase of the reproductive cycle caused activation of spermatogenic and steroidogenic activity of the testis, as shown by a significant increase in mean number of spermatogonia, primary spermatocytes and spermatids, and serum levels of testosterone, as compared to initial controls. In addition, there were abundant spermatozoa in the lumen of the seminiferous tubules. Interestingly, administration of a similar dosage of FSH to lizards exposed to stressors (handling, chasing, and noise randomly applied, five times a day for 30 days) resulted in a significant increase in mean number of spermatogonia and primary spermatocytes over initial control values, whereas the number of secondary spermatocytes and spermatids and serum levels of testosterone did not significantly differ from those of initial controls, and were significantly lower than FSH treated normal lizards. Further, spermatozoa were infrequently found in the seminiferous tubules of these lizards. Treatment controls (receiving 0.1 ml dw/lizard/alternate days for 30 days) did not show significant variation in mean number of spermatogonia, spermatocytes and spermatids, and serum levels of testosterone from initial controls. Another group of lizards was exposed to stressors and did not receive FSH. These lizards showed a significant decrease in mean number of secondary spermatocytes compared to treatment controls and all other parameters did not significantly differ from those of both control groups. The results reveal that gonadotrophin-induced spermatogonial proliferation occurs under stressful conditions, whereas progress of spermatogenesis beyond primary spermatocyte stage is impaired due to inhibition (under stress) of gonadotrophin induced steroidogenic activity in M. carinata.     

Effect of HCG on the interstitial cells and androgen production in the immature rat testis.

The effect on the interstitial cells in the immature rat testis of administration of HCG for different periods was correlated with testosterone plasma levels. Significant and progressive stimulation of mitosis was observed after 3 days of HCG treatment but stabilization occurred after 5 days. The numbers of precursor fibroblasts had increased by the 5th day and were still increasing by the 10th day of treatment. Numbers of Leydig cells were significantly greater at 5 and 10 days of treatment. Plasma testosterone showed a progressive and continuous increase in all groups. The increase in Leydig cell number is considered to be due to a combination of increased stimulation of mitoses in Leydig cells and differentiation of precursor fibroblasts. Mitosis seems to precede fibroblastic differentiation, but the latter continues when mitotic changes have stabilized. The elevation of plasma testosterone concentrations is probably due firstly to the stimulation of the existing Leydig cells and then to the increase in the number of hormone-secreting cells.     

Congenital defects in the offspring of male mice treated with ethylnitrosourea

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 1.0 X 10(-4), which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.     

Seasonal changes of spermatogonial proliferation in roe deer, demonstrated by flow cytometric analysis of c-kit receptor, in relation to follicle-stimulating hormone, luteinizing hormone, and testosterone.

The roe deer (Capreolus capreolus) is a seasonal breeder. The cyclic changes between totally arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time, c-kit receptor-positive cells in the testis of roe deer. They were immunohistologically identified mainly as spermatogonia. Analysis of the amount of those cells by flow cytometry shows a distinct seasonal pattern, with pronounced differences between cells in the diploid state and in the G2/M phase of mitosis. The specific seasonal pattern of spermatogonial proliferation results in the increased relative abundance of spermatogonia as well as in their increased total number per testis in November and December. This suggests that cell divisions continue on a level sufficient to accumulate spermatogonia during winter. The serum concentrations of LH and FSH showed a peak in spring; testosterone showed a maximum concentration during the rut (July/August). The peak of both gonadotropins seems to precede the period of stimulated spermatogonial proliferation in spring. The testosterone peak coincides with maximal meiotic intensity in August. The results suggest the importance of testosterone for sperm production, and they provide a basis for detailed investigations of regulatory factors of the proliferation of spermatogonia.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

精子发生过程中组蛋白甲基化和乙酰化

精子发生(Spermatogenesis)这一高度复杂的独特分化过程包括精原细胞发育为精母细胞、单倍体精细胞的形成和精子成熟,并以阶段特异性和睾丸特异性基因的表达、有丝分裂和减数分裂以及组蛋白向鱼精蛋白的转变为特征。表观遗传修饰在减数分裂重组、联会复合物的形成、姊妹染色体的结合、减数分裂后精子的变态、基因表达阻遏和异染色质形成过程中发挥着重要作用。其中具有一定组成形式、起抑制作用和/或激活作用的组蛋白甲基化和乙酰化标记,不仅保证了正确的染色体配对和二价染色体的成功分离,并且精确调节减数分裂特异性基因的适时表达。精子发生过程中组蛋白甲基化和/或乙酰化错误会直接影响表观遗传修饰的建立和维持,导致生精细胞异常甚至引发不育。文章旨在对精子发生过程中组蛋白甲基化和乙酰化表观遗传修饰的动态变化及其相关酶的调节机制进行综述,为进一步研究精子发生的表观遗传调控,预防男性不育疾病的发生提供基础资料。     

Maintenance of spermatogenesis by the activated human (Asp567Gly) FSH receptor during testicular regression due to hormonal withdrawal

The first activating mutation of the FSH receptor (FSHR*D567G) was identified in a gonadotropin-deficient hypophysectomized man who exhibited persistent spermatogenesis and fertility with only androgen replacement. We have determined the ability of FSHR* activity to maintain spermatogenesis and/or steroidogenesis during gonadotropin and androgen deprivation in mature transgenic FSHR* mice (Tg(Abpa-FSHR*D567G)1Cmal), hereafter referred to as Tg-FSHR* mice. Testes of untreated adult Tg-FSHR* males were equivalent in weight to nontransgenic controls but exhibited increased total Sertoli cell (24%) and spermatogonia (34%) numbers and nonsignificantly elevated spermatocyte-spermatid numbers (13%-17%). During sustained GNRH1 agonist treatment that markedly reduced (96%-98%) serum LH and testosterone (T) and decreased serum FSH (68%-72%), the testes of GNRH1 agonist-treated Tg-FSHR* mice remained significantly larger than treated nontransgenic controls. After 4 wk of gonadotropin suppression, Sertoli cell numbers were reduced in Tg-FSHR* testes to levels comparable with nontransgenic testes, whereas spermatogonia numbers were maintained at higher levels relative to nontransgenic testes. However, after 8 wk of GNRH1 agonist treatment, the total spermatogonia, spermatocyte, or postmeiotic spermatid numbers were reduced to equivalent levels in Tg-FSHR* and nontransgenic mice. FSHR* effects were further examined in gonadotropin-deficient hypogonadal Gnrh1hpg/Gnrh1hpg (Gnrh1(-/-)) mice during testicular regression following withdrawal of T after maximal T-stimulated spermatogenesis. After 6 wk of T withdrawal, spermatogonia, spermatocyte, and postmeiotic spermatid numbers in Tg-FSHR* Gnrh1(-/-) testes decreased to levels found in untreated Tg-FSHR* Gnrh1(-/-) testes. Basal serum T levels in untreated Tg-FSHR* Gnrh1(-/-) males were 2-fold higher than Gnrh1(-/-) controls, but following T treatment/withdrawal, serum T and epididymal weights declined to basal levels found in nontransgenic Gnrh1(-/-) mice. Therefore, FSHR* was unable to sustain circulating T or androgen-dependent epididymal size or postmeiotic spermatogenic development. We conclude that FSHR* activity enhances Sertoli and spermatogenic development in normal testes but has limited ability to maintain spermatogenesis during gonadotropin deficiency, in which the testicular response provided by the FSHR*D567G mutation resembled typical FSH-mediated but not steroidogenic activity.     

The arrangement of germ cells in the rat seminiferous tubule: an electron-microscope study.

The spermatogonia and early spermatocytes of 13 samples of rat seminiferous epithelium (about 0-05 mm2 each) were mapped from electron micrographs of serial sections. Clones of cells, connected by cytoplasmic bridges (syncytia of 2-100 cells), in various stages of spermatogenic development were identified. Maps of 7 separate areas are illustrated. It is concluded that, contrary to the models of spermatogonial proliferation based on light-microscope observations, regions of seminiferous epithelium which are identical in terms of spermatid and spermatocyte criteria have, in fact, quantitative and qualitative differences in their spermatogonial population. The data are interpreted that for a given epithelial area there is a periodic build-up of spermatogonia which then produce several successive quanta of spermatocytes and when the spermatogonia are depleted the process repeats. That cell numbers less than double following a mitotic cycle has generally been attributed to systematic degeneration. Evidence from electron microscopy indicates, however, that at the mitotic peaks not all the syncytia undergo division but that some remain arrested. Similarly, within a dividing syncytium a few cells do not divide while they advance developmentally with the syncytium as a whole. The observed large size of spermatocyte syncytia further argues against systematic degeneration with its attendant fragmentation of syncytia.     

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.     

Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.     

Loss of connexin 43 in Sertoli cells provokes postnatal spermatogonial arrest,reduced germ cell numbers and impaired spermatogenesis

For the reason that adult Sertoli cell specific connexin 43 knockout (SCCx43KO) mice show arrested spermatogenesis at spermatogonial level or Sertoli cell only tubules and significantly reduced germ cell (GC) numbers, the aims of the present study were (1) to characterize the remaining GC population and (2) to elucidate possible mechanisms of their fading. Apoptosis was analyzed in both, KO and wild type (WT) male littermates during postnatal development and in adulthood using TUNEL. Although GC numbers were significantly reduced in KO at 2 and 8 days postpartum (dpp) when compared to WT, no differences were found concerning apoptotic incidence between genotypes. From 10 dpp, the substantial GC deficiency became more obvious. However, significantly higher apoptotic GC numbers were seen in WT during this period, possibly related to the first wave of spermatogenesis, a known phenomenon in normal pubertal testes associated with increased apoptosis. Characterization of residual spermatogonia in postnatal to adult KO and WT mice was performed by immunohistochemical reaction against VASA (marker of GCs in general), Lin28 and Fox01 (markers for undifferentiated spermatogonia) and Stra8 (marker for differentiating spermatogonia and early spermatocytes). During puberty, the GC component in SCCx43KO mice consisted likely of undifferentiated spermatogonia, few differentiating spermatogonia and very few early spermatocytes, which seemed to be rapidly cleared by apoptosis. In adult KOs, spermatogenesis was arrested at the level of undifferentiated spermatogonia. Overall, our data indicate that Cx43 gap junctions in SCs influence male GC development and differentiation rather than their survival.