Testosterone suppresses spermatogenesis in juvenile spermatogonial depletion (jsd ) mice

Male juvenile spermatogonial depletion (jsd/jsd) mice are sterile because of a failure of spermatogonial differentiation. We have previously reported the recovery of spermatogonial differentiation by suppressing the levels of gonadotropins and testosterone with Nal-Glu, a GnRH antagonist. To determine whether suppression of testosterone or the gonadotropins was responsible for spermatogenic recovery, we examined the effect of supplementation of LH or FSH along with Nal-Glu treatment. Systemic administration of flutamide, an androgen receptor antagonist, was also examined. LH supplementation elevated both serum and intratesticular testosterone levels and suppressed the recovery of spermatogonial differentiation in a dose-dependent manner. Supplementation with FSH did not affect either testosterone levels or spermatogonial differentiation. Furthermore, the mice treated with flutamide showed some recovery of spermatogonial differentiation. The overall findings revealed that testosterone action mediated by androgen receptors suppressed the spermatogonial differentiation in jsd/jsd mice and suggested that spermatogonial differentiation in the jsd mutant is highly sensitive to testosterone suppression.     

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta‐hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down‐regulation of the steroid hormones synthesis pathway. Furthermore, qRT‐PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway.     

Genetic factors contributing to defective spermatogonial differentiation in juvenile spermatogonial depletion (Utp14bjsd) mice

Male mice that are homozygous for the juvenile spermatogonial depletion (jsd) mutation in the Utp14b gene undergo several waves of spermatogenesis. However, spermatogonial differentiation ceases and in adults, spermatogonia are the only germ cells that remain. To understand further the blockage in spermatogonial differentiation in Utp14b(jsd) mutant mice, we correlated the rate and severity of spermatogonial depletion and the restoration of spermatogenesis following the suppression of testosterone or elevation of testicular temperature with the genetic background. Testes from Utp14b(jsd) mutant mice on B6, C3H, and mixed C3H-B6-129 (HB129) genetic backgrounds all showed steady decreases in the numbers of normal spermatogonia between 8 wk and 20 wk of age. The percentages of tubules with differentiating germ cells were higher and the spermatogonia were more advanced in C3H- background than in B6- or HB129-background Utp14b(jsd) mice. Genetic crosses showed that the source of the Y chromosome was a major factor in determining the severity of spermatogonial depletion in Utp14b(jsd) mutant mice. When Utp14b(jsd) mutants were subjected to total androgen ablation or unilateral cryptorchidization, spermatogenic development recovered markedly in the C3H and HB129 background but showed less recovery in the B6-background mice. The differences noted between the strains in terms of the severity of spermatogonial depletion were not dependent upon testosterone level or scrotal temperature but correlated with the magnitudes of the effects of elevated temperature on normal and Utp14b(jsd) mutant spermatogenic cells. Thus, the abilities of germ cells in certain strains to survive elevated temperatures may be related to their abilities to maintain some degree of differentiation potential after the Utp14b(jsd) gene is mutated.     

Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection.

Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.     

Expression of Mirlet7 family microRNAs in response to retinoic acid-induced spermatogonial differentiation in mice

Spermatogonial differentiation is orchestrated by the precise control of gene expression involving retinoic acid signaling. MicroRNAs have emerged as important regulators of spermatogenesis, and here we show that the Mirlet7 family miRNAs are expressed in mouse spermatogonia and spermatocytes. Retinoic acid significantly leads to the induction of Mirlet7 miRNAs through suppression of Lin28. We further confirmed both in vitro and in vivo that expressions of Mycn, Ccnd1, and Col1a2, which are targets of Mirlet7, were downregulated during spermatogonial differentiation. These results suggest that Mirlet7 family miRNAs play a role in retinoic acid-induced spermatogonial differentiation.     

Expression of the ubiquitin proteasome system in neonatal rat gonocytes and spermatogonia: role in gonocyte differentiation

The ubiquitin proteasome system (UPS) consists of a cascade of enzymatic reactions leading to the ubiquitination of proteins, with consequent degradation or altered functions of the proteins. Alterations in UPS genes have been associated with male infertility, suggesting the role of UPS in spermatogenesis. In the present study, we questioned whether UPS is involved in extensive remodeling and functional changes occurring during the differentiation of neonatal testicular gonocytes to spermatogonia, a step critical for the establishment of the spermatogonial stem cell population. We found that addition of the proteasome inhibitor lactacystin to isolated gonocytes inhibited their retinoic acid-induced differentiation in a dose-dependent manner, blocking the induction of the spermatogonial gene markers Stra8 and Dazl. We then compared the UPS gene expression profiles of Postnatal Day (PND) 3 gonocytes and PND8 spermatogonia, using gene expression arrays and quantitative real-time PCR analyses. We identified 205 UPS genes, including 91 genes expressed at relatively high levels. From those, 28 genes were differentially expressed between gonocytes and spermatogonia. While ubiquitin-activating enzymes and ligases showed higher expression in gonocytes, most ubiquitin conjugating and deubiquitinating enzymes were expressed at higher levels in spermatogonia. Concomitant with the induction of spermatogonial gene markers, retinoic acid altered the expression of many UPS genes, suggesting that the UPS is remodeled during gonocyte differentiation. In conclusion, these studies identified novel ubiquitin-related genes in gonocytes and spermatogonia and revealed that proteasome function is involved in gonocyte differentiation. Considering the multiple roles of the UPS, it will be important to determine which UPS genes direct substrates to the proteasome and which are involved in proteasome-independent functions in gonocytes and to identify their target proteins.     

犏牛精子发生阻滞的比较转录组研究

犏牛是牦牛与普通牛的杂交后代,其雄性不育是牦牛杂交改良中的一大难题.运用高通量测序技术对健康成年犏牛和牦牛的睾丸组织进行转录组测序和比较研究,探讨了犏牛激素调节、精子发生及细胞凋亡等相关基因的表达情况.结果表明,犏牛、牦牛睾丸组织中分别有17784和18529个基因表达,在犏牛中表达显著上调和下调的基因分别有5000和4089个.犏牛睾丸组织中睾酮合成相关基因和抑制素基因表达均显著上调,认为前者的表达上调可能促进睾丸内睾酮分泌和后者的表达,而后者的表达上调可能分别抑制和几乎不影响脑垂体前叶合成、分泌促卵泡刺激素和黄体生成素.比较睾丸组织中细胞标记基因在两种牛间的表达差异,发现犏牛精原干细胞、支持细胞、间质细胞、肌样细胞(睾丸纤维化)和已分化精原细胞的标记基因分别呈显著表达上调和下调,而减数分裂后期或精子形成期呈极显著下调.精原干细胞自我更新与分化异常可能导致犏牛精子发生障碍,认为其与视黄酸信号通路障碍密切相关.Syce3,Fkbp6和Dmrt7等在犏牛睾丸组织中极显著表达下调与同源染色体间、尤其是性染色体间的联会复合体数量减少有关.Spo11和Dmc1分别参与双链断裂和同源修复过程,其在犏牛睾丸中表达下调分别可能使联会复合体减少和同源修复失常.参与高度浓缩细胞核的相关基因,尤其是Tnp2,Hmgb4和H1fnt等几乎不表达,其调控表达基因Crem,GRTH/DDX25等极显著表达下调,该现象与犏牛生精细胞最终只能分化至圆形精子细胞阶段的结果相符.促凋亡相关基因,如p53,TNF-α,Trail,Bmp8b,Bax,Caspase-3,Caspase-6和Caspase-7表达均显著上调,而抑凋亡基因,如survivin,Bcl-2等显著下调,这可能是导致犏牛睾丸组织中有更多的生精细胞发生凋亡的原因之一.通过对生殖相关基因的表达分析研究,为揭示犏牛雄性不育机理以及牦牛杂交改良研究提供了理论基础.     

Key factors in the regulation of fetal and postnatal Leydig cell development

The primary function of testicular Leydig cells is the production of androgens to promote sexual differentiation in the fetus, secondary sexual maturation at puberty, and spermatogenesis in the adult. The fetal and postnatal (adult) populations of Leydig cells differ morphologically and have distinct profiles of gene expression. As postnatal Leydig cells differentiate, they transition through three discrete maturational stages characterized by decreasing proliferative rate and increasing testosterone biosynthetic capacity. In this review, we discuss the development of both fetal and postnatal Leydig cells and review the regulation of this process by some of the key hormones and growth factors.     

Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells

The objective of this study was to further understand the genetic mechanisms of vitamin A deficiency (VAD) induced arrest of spermatogonial stem-cell differentiation.Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. These observations led us to the hypothesis that VAD affects not only germ cells but also somatic cells.To investigate the effects of VAD on spermatogenesis in mice we used adult Balb/C mice fed with Control or VAD diet for an extended period of time (6–28 weeks). We first observed the chronology, then the extent of the effects of VAD on the testes. Using microarray analysis of isolated pure populations of spermatogonia, Leydig and Sertoli cells from control and VAD 18- and 25-week mice, we examined the effects of VAD on gene expression and identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis.Our results provide a more precise definition of the chronology and magnitude of the consequences of VAD on mouse testes than the previously available literature and highlight direct and indirect (via somatic cells) effects of VAD on germ cell differentiation.     

Developmental regulation of expression of the lactate dehydrogenase (LDH) multigene family during mouse spermatogenesis

Expression of the Lactate Dehydrogenase (LDH) genes during various stages of spermatogenesis was studied by using a combination of Northern blot analyses and in situ hybridization techniques. These studies have indicated that developmentally programmed expression of all three functional LDH genes occurs during differentiation of germ cells. The LDH/C (ldh-3) gene was expressed exclusively during meiosis and spermiogenesis, beginning in leptotene/zygotene spermatocytes and continuing through to the elongated spermatids. LDH/C (ldh-3) gene expression was accompanied by transient expression of the LDH/A (ldh-1) gene in pachytene spermatocytes and round spermatids. The LDH/B (ldh-2) gene was expressed mainly in Sertoli and spermatogonial cells. By using somatic cell hybrids, the LDH/C (ldh-3) gene has been mapped to mouse chromosome 7, establishing that it is syntenic with the LDH/A (ldh-1) gene locus. Experimental observations made in this study provide new insight into the order and sequence of events involved in the regulation of gene expression of the LDH gene family during spermatogenesis.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

Characterization of spermatogonial stem cells lacking intercellular bridges and genetic replacement of a mutation in spermatogonial stem cells

Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14(+/-) spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.     

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.     

Patterns of importin-alpha expression during Drosophila spermatogenesis

Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, -alpha2, and -alpha3. We found that each Imp-alpha is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.     

Hormone pretreatment enhances recovery of spermatogenesis in rats after neutron irradiation.

Previous studies showed that a 6-week pretreatment of rats with testosterone plus estradiol enhanced the recovery of spermatogenesis 9 weeks after gamma irradiation, resulting in a dose-modifying factor (DMF) of about 2. To test whether the effect of the hormone treatment was mediated through changes in oxygen tension, thiol levels or DNA repair, we irradiated the testes of rats with neutrons, which depend less on these factors than does low-LET radiation for their cytotoxic action. Control rats and rats treated with testosterone plus estradiol were irradiated with 0.7-2.7 Gy of cyclotron-generated high-energy neutrons. The recovery of spermatogenesis was assessed 9 weeks after irradiation by testis weights, sperm counts and the tubule repopulation indices. Greater recovery of spermatogenesis was observed for all end points, with a DMF of about 2 for rats treated with testosterone plus estradiol compared to the irradiated, cholesterol-treated rats. The equal protection factors for neutrons and gamma rays indicate that oxygen, thiols and repair of DNA damage are unlikely to be involved in the protective effect of the hormone treatment.