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Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2. This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine.

Embryo implantation, a critical step during the mammalian reproductive process, requires normal developing blastocysts and a receptive endometrium. Endometriosis, a common pathologically benign gynecological condition, is associated with decreased fertility and reduced endometrial receptivity. The oncoprotein, Gankyrin, has been associated with endometriosis and endometrial cancer. Here, we examined the role of Gankyrin during the process of embryo implantation and found that Gankyrin expression levels were significantly increased during the mid-secretory phase, but unaffected during the proliferative phase in the human endometrium. Using an in vitro cell adhesion assay to examine the cell adhesion rate of BeWo trophoblast spheroids to Gankyrin knockdown or overexpressing human endometrial carcinoma RL95-2 cells, we demonstrated that the adhesion rate was significantly reduced in Gankyrin-knockdown RL95-2 cells, while overexpression of Gankyrin promoted cell adhesion. Furthermore, we found that the downregulation of Gankyrin inhibited STAT3 activation and subsequent matrix metalloproteinase 2 (MMP2) expression, while overexpression led to STAT3 activation and MMP2 expression. In vivo, we found that Gankyrin expression was increased in the endometrium after conception but decreased with the prolongation of gestation time in female mice. siRNA-mediated knockdown of Gankyrin in the uterine horn led to a significant reduction in the number of implanted embryos 9 days post-gestation, which was associated with a decrease in p-STAT3 expression and MMP2 transcription. Taken together, our findings indicate that Gankryin has a potential role in embryo implantation via STAT3 activation.

Menopause is a significant physiological phase that occurs as women's ovaries stop producing ovum and the production of estrogen declines. Human placenta and some amino acids are known to improve menopausal symptoms. In this study, we investigated that porcine placenta extract (PPE) and arginine (Arg), a main amino acid of PPE, would have estrogenic activities in ovariectomized (OVX) mice as a menopause mouse model, human breast cancer cell line (MCF-7) cells, and human osteoblast cell line (MG-63) cells. PPE or Arg significantly inhibited the body weight and increased the vagina weight compared to the OVX mice. PPE or Arg ameliorated the vaginal atrophy in the OVX mice. The levels of 17β-estradiol and the activities of alkaline phosphatase (ALP) were significantly increased by PPE or Arg in the serum of OVX mice. Trabecular bone parameters such as bone mineral density and porosity were also improved by PPE or Arg in the OVX mice. In the MCF-7 and MG-63 cells, PPE or Arg significantly increased the cell proliferation, estrogen receptor β mRNA expression, and estrogen-response elements luciferase activity. Finally, PPE or Arg increased the activations of ALP and extracellular signal-regulated kinase 1/2 in the MG-63 cells. These results indicate that PPE or Arg would have estrogenic and osteoblastic activity. Therefore, PPE or Arg may be useful as new pharmacological tools for treating menopausal symptoms including osteoporosis.

Free Korean abstract: A Korean translation of this abstract is freely available at http://www.reproduction-online.org/content/150/3/173/suppl/DC1.

Spermatogenesis is composed of three distinctive phases, which include self-renewal of spermatogonia via mitosis, spermatocytes undergoing meiosis I/II and post-meiotic development of haploid spermatids via spermiogenesis. Spermatogenesis also involves condensation of chromatin in the spermatid head before transformation of spermatids to spermatozoa. Epigenetic regulation refers to changes of heritably cellular and physiological traits not caused by modifications in the DNA sequences of the chromatin such as mutations. Major advances have been made in the epigenetic regulation of spermatogenesis. In this review, we address the roles and mechanisms of epigenetic regulators, with a focus on the role of microRNAs and DNA methylation during mitosis, meiosis and spermiogenesis. We also highlight issues that deserve attention for further investigation on the epigenetic regulation of spermatogenesis. More importantly, a thorough understanding of the epigenetic regulation in spermatogenesis will provide insightful information into the etiology of some unexplained infertility, offering new approaches for the treatment of male infertility.

Mechanisms by which female stress and particularly glucocorticoids impair oocyte competence are largely unclear. Although one study demonstrated that glucocorticoids triggered apoptosis in ovarian cells and oocytes by activating the FasL/Fas system, other studies suggested that they might induce apoptosis through activating other signaling pathways as well. In this study, both in vivo and in vitro experiments were conducted to test the hypothesis that glucocorticoids might trigger apoptosis in oocytes and ovarian cells through activating the TNF-α system. The results showed that cortisol injection of female mice (1.) impaired oocyte developmental potential and mitochondrial membrane potential with increased oxidative stress; (2.) induced apoptosis in mural granulosa cells (MGCs) with increased oxidative stress in the ovary; and (3.) activated the TNF-α system in both ovaries and oocytes. Culture with corticosterone induced apoptosis and activated the TNF-α system in MGCs. Knockdown or knockout of TNF-α significantly ameliorated the pro-apoptotic effects of glucocorticoids on oocytes and MGCs. However, culture with corticosterone downregulated TNF-α expression significantly in oviductal epithelial cells. Together, the results demonstrated that glucocorticoids impaired oocyte competence and triggered apoptosis in ovarian cells through activating the TNF-α system and that the effect of glucocorticoids on TNF-α expression might vary between cell types.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Mouse embryonic fibroblasts (MEFs) have been previously used as feeder cells to support the growth of human embryonic stem cells (hESCs). In this study, human adult uterine endometrial cells (hUECs), human adult breast parenchymal cells (hBPCs) and embryonic fibroblasts (hEFs) were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells. Cultured hUECs, hBPCs and hEFs were mitotically inactivated and then plated. hESCs (Miz-hES1, NIH registered) initially established on mouse feeder layers were transferred onto each human feeder layer and split every 5 days. The morphology, expression of specific markers and differentiation capacity of hESCs adapted on each human feeder layer were examined. On hUEC, hBPC and hEF feeder layers, hESCs proliferated for more than 90, 50 and 80 passages respectively. Human feeder-based hESCs were positive for stage-specific embryonic antigen (SSEA)-3 and -4, and Apase; they also showed similar differentiation capacity to MEF-based hESCs, as assessed by the formation of teratomas and expression of tissue-specific markers. However, hESCs cultured on hUEC and hEF feeders were slightly thinner and flatter than MEF- or hBPC-based hESCs. Our results suggest that, like MEF feeder layers, human feeder layers can support the proliferation of hESCs without differentiation. Human feeder cells have the advantage of supporting more passages than when MEFs are used as feeder cells, because hESCs can be uniformly maintained in the undifferentiated stage until they pass through senescence. hESCs established and/or maintained under stable xeno-free culture conditions will be helpful to cell-based therapy.