LncRNA Gm2044 promotes 17β‐estradiol synthesis in mpGCs by acting as miR‐138‐5p sponge

Long noncoding RNAs (lncRNAs) have been demonstrated to play vital roles in mammalian reproduction. Our previous research revealed that lncRNA Gm2044 is highly expressed in mouse spermatocytes and regulates male germ cell function. The gene annotation database BioGPS shows that Gm2044 is not only highly expressed in testicular tissue but also in ovarian tissue, which suggests that Gm2044 may be involved in female reproductive development. In this study, we confirmed that lncRNA Gm2044 promotes 17β‐estradiol synthesis in mouse pre‐antral follicular granulosa cells (mpGCs). Furthermore, bioinformatics methods, western blot, and the luciferase assay proved that Gm2044 functions as a miR‐138‐5p sponge to inhibit the direct target of miR‐138‐5p, Nr5a1, which enhances 17β‐estradiol synthesis through cyp19a1 activation. Taken together, our results provide an insight into the mechanistic roles of lncRNA Gm2044 for 17β‐estradiol synthesis by acting as competing‐endogenous RNAs to modulate the function of mpGCs. Studying the potential lncRNAs, which regulate estradiol release, will be beneficial for the diagnosis and treatment of steroid hormone‐related disease.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation

Spermatogonial stem cells are required for the initiation of spermatogenesis and the continuous production of sperm. In addition, they can acquire pluripotency and differentiate into derivatives of the three embryonic germ layers when cultured in the appropriate conditions. Therefore, understanding the signaling pathways that lead to self-renewal or differentiation of these cells is of paramount importance for the treatment of infertility, the development of male contraceptives, the treatment of testicular cancers, and ultimately for tissue regeneration. In this report, we studied some of the signaling pathways triggered by glial cell line-derived neurotrophic factor (GDNF), a component of the spermatogonial stem cell niche produced by the somatic Sertoli cells. As model systems, we used primary cultures of mouse spermatogonial stem cells, a mouse spermatogonial stem cell line and freshly isolated testicular tubules. We report here that GDNF promotes spermatogonial stem cell proliferation through activation of members of the Src kinase family, and that these kinases exert their action through a PI3K/Akt-dependent pathway to up-regulate N-myc expression. Thus, to proliferate, spermatogonial stem cells activate mechanisms that are similar to the processes observed in brain stem cells and lung progenitors.     

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh–Venus GS cell line, to our knowledge, is the first one expressing a germ cell‐specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation. genesis 51:498–505. © 2013 Wiley Periodicals, Inc.     

Silencing lncRNA DUXAP8 inhibits lung adenocarcinoma progression by targeting miR-26b-5p

Lung adenocarcinoma (LUAD), a common type of lung cancer, has become a popularly aggressive cancer. Long noncoding RNAs (lncRNAs) play a critical role in the pathogenesis of human cancers, while the function of double homeobox A pseudogene 8 (DUXAP8) in LUAD remains to be fully inquired. Therefore, our study was conducted to elucidate the DUXAP8 expression in LUAD and its mechanism on the biological features of LUAD cells. Loss-of-function experiments were performed to assess the function of DUXAP8 proliferation and apoptosis of H1975 and A549 cells. Functionally, silencing DUXAP8 inhibited proliferation and induced apoptosis of LUAD cells. Mechanistically, further correlation assay indicated a negative association between miR-26b-5p and DUXAP8 expression. Subsequently, we testified that DUXAP8 exerted its role in the progression and development of LUAD through targeting miR-26b-5p. In summary, our results elucidated that that DUXAP8 promoted tumor progression in LUAD by targeting miR-26b-5p, which provide a novel therapeutic target for diagnosis and therapy of LUAD.     

DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice

The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and DROSOPHILA: Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.     

Epigenetic modifications and self-renewal regulation of mouse germline stem cells

Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.     

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.     

LncRNA MAGI2-AS3 inhibits hepatocellular carcinoma cell proliferation and migration by targeting the miR-374b-5p/SMG1 signaling pathway

Long noncoding RNAs (lncRNAs) have been proven to play critical roles in cancer progression. Recently, lncRNA MAGI2-AS3 has been revealed to be a tumor suppressor and inhibit cell growth by targeting the Fas/FasL signalling pathway in breast cancer. However, the role and underlying mechanism of MAGI2-AS3 in hepatocellular carcinoma (HCC) remain largely unknown. In the current study, we found that MAGI2-AS3 expression is downregulated in HCC tissues and closely associated with some clinical characteristics (tumor size, lymph node metastasis, and TNM stage) and poor overall survival. Overexpression of MAGI2-AS3 inhibits HCC cell proliferation and migration in vitro, while impedes tumor growth in vivo accordantly. In addition, our data suggest that MAGI2-AS3 could function as an endogenous sponge of miR-374b-5p by directly binding to it and suppressing its expression. Furthermore, miR-374b-5p upregulation could restore the inhibitory effect of MAGI2-AS3 on HCC cells processes. Moreover, suppressor with morphogenetic effect on genitalia family member 1 (SMG1) is positively regulated by MAGI2-AS3 via absorbing miR-374b-5p in HCC cells. More important, SMG1 knockdown reverses the suppressive function of MAGI2-AS3 in HCC cell processes. Taken together, we reveal a functional MAGI2-AS3/miR-374b-5p/SMG1 axis that suppresses HCC progression, potently suggesting a new road for HCC treatment.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Notch-1, c-kit and GFRalpha-1 are developmentally regulated markers for premeiotic germ cells

Culture, transfection and immortalization of mouse germ line stem cells, germ cell transplantation and grafting of testicular tissue are milestones of recent biotechnological breakthroughs. Alone and in combination they offer new pathways to explore the cellular mechanisms responsible for pluripotency and the requirements of cells to enter the germ line. Efficient markers, isolation and culture systems as well as transfection approaches are developed to elucidate the molecular and cellular mechanisms leading to the development of male germ line cells. Here, we describe the localization pattern of c-kit, Notch-1 and GFRalpha-1 in postnatal, immature and adult testes. All three proteins are potentially useful markers for spermatogonial characterization and enrichment. First attempts and various future perspectives to use spermatogonial stem cells as pathway for the introduction of transgenes are discussed.     

Functional analysis of stem cells in the adult rat testis

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.     

Stra 8基因的激活与精原干细胞的特异性分化研究

视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8基因的表达特性,我们从小鼠的基因组中克隆了Stra8基因的启动子序列(1.4kb)。将Stra8基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8的转录,还有生殖细胞特异表达基因CyclinA8和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。     

Long-term culture of male germline stem cells from hamster testes

Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.     

精原干细胞与睾丸生殖衰老的研究进展

雄性睾丸内精子的生成及其质量随年龄增长逐渐降低。精原干细胞是精子生成的起点,其数量和质量决定了精子的生成,而精原干细胞niche是调节精原干细胞自我更新与分化的重要因素。在衰老过程中,干细胞微环境退化,精原干细胞自我更新和分化失衡,被认为是衰老导致睾丸生殖功能衰退的的主要因素。本文将综述衰老引起的精原干细胞与niche变化及其对生殖的影响相关研究进展。