Epigenetic regulation in male germ cells

In recent years, it has become increasingly clear that epigenetic regulation of gene expression is critical during spermatogenesis. In this review, the epigenetic regulation and the consequences of its aberrant regulation during mitosis, meiosis and spermiogenesis are described. The current knowledge on epigenetic modifications that occur during male meiosis is discussed, with special attention on events that define meiotic sex chromosome inactivation. Finally, the recent studies focused on transgenerational and paternal effects in mice and humans are discussed. In many cases, these epigenetic effects resulted in impaired fertility and potentially long-ranging affects underlining the importance of research in this area.     

Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis

The study of mammalian meiosis is complicated by the timing of meiotic events in females and by the intermingling of meiotic sub-stages with somatic cells in the gonad of both sexes. In addition, studies of mouse mutants for different meiotic regulators have revealed significant differences in the stringency of meiotic events in males versus females. This sexual dimorphism implies that the processes of recombination and homologous chromosome pairing, while being controlled by similar genetic pathways, are subject to different levels of checkpoint control in males and females. This review is focused on the emerging picture of sexual dimorphism exhibited by mammalian germ cells using evidence from the broad range of meiotic mutants now available in the mouse. Many of these mouse mutants display distinct differences in meiotic progression and/or dysfunction in males versus females, and their continued study will allow us to understand the molecular basis for the sex-specific differences observed during prophase I progression.     

Voltage-operated calcium channels in male germ cells

The acrosome reaction is a key event in fertilization. Current models for induction of the acrosome reaction incorporate a necessary influx of Ca(2+), which is mediated by agonist-induced gating of ion channels in the sperm plasma membrane. The difficulty of applying electrophysiological techniques to spermatozoa has severely hampered studies on the expression of functional ion channels in these cells. However, during the last few years, a combination of molecular and physiological techniques (applied to immature spermatogenic cells) has elucidated both the expression of Ca(2+) channels in male germ cells and their role in induction of the acrosome reaction. It now appears that a range of voltage-operated Ca(2+) channels, similar to those that occur in somatic cells, is expressed in spermatozoa. Male rodent germ cells express a low-voltage activated (T-type) channel that is regulated by membrane potential and provides the primary Ca(2+) influx mechanism in zona pellucida-stimulated spermatozoa. In human spermatozoa, similar channels are apparently expressed, but their function in induction of the acrosome reaction has yet to be established. A range of other, high voltage-activated channels also appear to be present in rodent and human spermatozoa, but their roles are not yet known. In this review, the structure and characteristics of voltage-operated Ca(2+) channels are outlined and the evidence for their expression and function in male germ cells is assembled and discussed.     

Chromatoid body and small RNAs in male germ cells

The chromatoid body (CB) is a germ granule in the cytoplasm of postmeiotic haploid round spermatids that is loaded with RNA and RNA-binding proteins. Following the discovery of small non-coding RNA-mediated gene regulation and the identification of PIWI-interacting RNAs (piRNAs) that have crucial roles in germ line development, the function of the CB has slowly begun to be revealed. Male germ cells utilise small RNAs to control the complex and specialised process of sperm production. Several microRNAs have been identified during spermatogenesis. In addition, a high number of piRNAs are present both in embryonic and postnatal male germ cells, with their expression being impressively induced in late meiotic cells and haploid round spermatids. At postmeiotic stage of germ cell differentiation, the CB accumulates piRNAs and proteins of piRNA machinery, as well as several other proteins involved in distinct RNA regulation pathways. All existing evidence suggests a role for the CB in mRNA regulation and small RNA-mediated gene control, but the mechanisms remain uncharacterised. In this review, we summarise the current knowledge of the CB and its association with small RNA pathways.     

Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated gamma-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.     

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1 x Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D(28k) (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.     

Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.     

17beta-estradiol induces Akt-1 through estrogen receptor-beta in the frog (Rana esculenta) male germ cells

Several lines of evidence support the key role of estrogens in male fertility. Here, we investigate the regulation of the serine/threonine kinase Akt-1 in the frog (Rana esculenta) testis during the annual sexual cycle and, whether 17beta-estradiol (E2) exerts a role in the Akt-1 activity. Akt-1 has been shown to be the mediator of growth factor-dependent cell proliferation, survival, and metabolism in a variety of cell types. First, we demonstrate by immunohistochemistry, the presence of estrogen receptor-beta (ERbeta), and Akt-1 in the spermatogonia (SPG), spermatocytes (SPC), and spermatids (SPT). Western-blot analysis revealed that ERbeta isoform (molecular weight 55 kDa) was highly expressed in May (reproductive period) with respect to January and November (winter stasis); in parallel, Akt-1 (molecular weight 60 kDa) is highly phosphorylated (Ser-473) during the period of active spermatogenesis (May) compared with the winter stasis (January and November). In addition, in vitro experiments demonstrate that E2 treatment induces the activation of Akt-1, and this effect is counteracted by the anti-estrogen ICI 182-780. In conclusion, our data show that E2 induces Akt-1 phosphorylation (Ser-473) possibly via ERbeta in frog (R. esculenta) male germ cells.     

高通量基因表达谱的应用

基因表达谱可以描绘特定情况下组织、细胞中所表达的全套基因及其丰度,它从mRNA水平上反映出组织或细胞特异性表型和表达模式.目前基于高通量的基因表达谱主要有基因芯片和数字基因表达谱(DGE).本文主要介绍了基因芯片和DGE的原理、特点,并对其在作物科学、动物科学和乳品科学中的应用做了简要列举.     

Adult-only exposure of male rats to a diet of high phytoestrogen content increases apoptosis of meiotic and post-meiotic germ cells

Apoptosis plays a critical role in regulating sperm production. Removal of androgens and gonadotropins, or estrogen administration induces germ cell apoptosis. It is hypothesized that dietary phytoestrogens increase apoptosis of developing germ cells, decreasing sperm production. This study aimed to test this in rats fed a high phytoestrogen diet only during adulthood. Male Wistar rats used in this study were offspring of females maintained on a low phytoestrogen diet prior to conception through to weaning. After weaning, juveniles were fed the same low phytoestrogen diet into adulthood. A cohort of males were transferred to a high phytoestrogen diet for 24 days and subsequently testes were collected from all animals. In the high phytoestrogen fed group, homogenization-resistant sperm counts were significantly decreased, as were epididymal sperm counts. Morphometric analysis determined round and elongated spermatid volumes to be significantly decreased, but seminiferous tubule lumen diameters to be significantly increased. TUNEL analysis determined that apoptosis of spermatocytes and round spermatids was significantly greater in the high phytoestrogen fed rats. Neither plasma gonadotropin concentrations nor testicular testosterone were altered. In conclusion, exposure of the adult male rat to a high phytoestrogen diet disrupts spermatogenesis, increasing germ cell apoptosis. This effect is independent of the hypothalamo-pituitary-testicular axis and is likely due to disruption of estrogen's actions in the testis.     

牡蛎提取物对雄性大鼠性功能的影响

目的 探究牡蛎提取物对正常雄性大鼠和半去势大鼠的性功能的影响。方法 将40只雄性SD (Sprague Dawley)大鼠随机分为4组, 各组分别灌胃受试物或纯净水, 连续灌胃30 d后进行交配试验, 并测定大鼠阴茎勃起时间。将50只雄性大鼠随机分为5组, 除正常对照组外, 其余大鼠摘除右侧睾丸, 各组分别灌胃受试物或纯净水, 连续灌胃30 d, 然后称重雄性大鼠的包皮腺、精囊腺和前列腺,测定勃起潜伏期、血清皮质醇、促黄体生成素和睾酮水平。结果 与正常对照组相比, 牡蛎提取物低、中、高剂量组均能提高大鼠的射精次数(P<0.05), 高剂量牡蛎提取物能同时提高大鼠的捕捉次数和射精率(P<0.05)。与半去势大鼠模型对照组相比, 牡蛎提取物中、高剂量组的大鼠包皮腺系数有明显增高(P<0.05), 促黄体生成素含量显著降低(P<0.01), 高剂量组大鼠的血清皮质醇含量升高(P<0.05)。结论 牡蛎提取物具有一定的雄激素样作用, 能改善大鼠性功能。     

Short communication: lack of stage-specific embryonic antigen-1 expression by bovine embryos and primordial germ cells

The objective of this study was to determine whether stage-specific embryonic antigen-1, a cellular marker commonly used to identify murine undifferentiated embryonic cells, is also a useful marker for bovine pluripotent cells. Expression of stage-specific embryonic antigen-1 was examined by indirect immunohistochemistry on bovine preimplantation embryos and on primordial germ cells contained in the genital ridge. Expression of stage-specific embryonic antigen-1 was not observed in any of the cleavage-stage bovine embryos examined, including one-cell, two-cell, four-cell, eight-cell, morula, and blastocyst stages, nor in tissue sections of bovine genital ridges collected from embryos on d 34, 37, and 40 of gestation. As expected, expression of stage-specific embryonic antigen-1 was detected on murine preimplantation embryos and on murine teratocarcinoma cells. Results of this study indicate that, unlike in the mouse, stage-specific embryonic antigen-1 is not a useful cellular marker for pluripotent bovine embryonic cells or bovine primordial germ cells.     

Purification of mouse primordial germ cells by Nycodenz

Primordial germ cells are important cells for the study of germ cell lineage. It has proved difficult to obtain highly purified primordial germ cells for preparation of a specific antibody. In the present study, a new method for purifying mouse primordial germ cells was developed using a Nycodenz gradient. Furthermore, the polyclonal anti-mouse primordial germ cells IgG derived from mouse primordial germ cells was prepared. As this IgG reacted only with primordial germ cells obtained at day 12.5 after mating, this antibody appeared to recognize the stage-specific antigen of primordial germ cells. One reason that a continuous primordial germ cell marker has not been obtained is because the purity of the primordial germ cells used has been too low to prepare the antibody. This new method represents a significant improvement in the purification of primordial germ cells; it is simpler than previous methods, and produced mouse primordial germ cells with a purity of more than 95%. In addition, the separation reagent Nycodenz is non-toxic and achieved separation of primordial germ cells without attachment of antibodies against the primordial germ cell membrane surface. This new purification method and stage-specific antibody will be useful for the analysis of the mechanisms of primordial germ cell migration.     

Different expression and activity of the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

Two catalytic isoforms of the Na,K-ATPase, alpha1 and alpha4, are present in testis. While alpha1 is ubiquitously expressed in tissues, alpha4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase alpha isoforms in male germ cell biology, we have studied the expression and activity of alpha1 and alpha4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that alpha1 and alpha4 undergo differential regulation during development. Whereas alpha1 exhibits only modest changes, alpha4 increases with gamete differentiation. The most drastic changes for alpha4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of alpha4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the alpha1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase alpha1 and alpha4 isoforms are functional in diploid, meiotic and haploid male germ cells, alpha4 being significantly upregulated during spermatogenesis. These results support the importance of alpha4 in male gamete differentiation and function.