Generation of male germ cells from induced pluripotent stem cells （iPS cells）： an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem （iPS） cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitrodifferentiation and in vivotransplantation. Embryoid bodies （EBs） were obtained from iPS cells using leukaemia inhibitor factor （LIF）-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in StraSand Vasa mRNA in the EBs derived from iPS cells, iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells （SSCs）, as evidenced by their expression of VASA, as well as CDH1 and GFRal, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Generation of male germ cells in vitro from the stem cells

Infertility has become a serious disease since it affects 10%–15%of couples worldwide,and male infertility contributes to about 50%of the cases.Notably,a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China.As such,it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility.Stem cells,including embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs),spermatogonial stem cells(SSCs),and mesenchymal stem cells(MSCs),possess the abilities of both self-renewal and differentiation into germ cells.Significantly,much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions,e.g.,the coculturing with Sertoli cells,three-dimensional culture system,the addition of growth factors and cytokines,and/or the overexpression of germ cell-related genes.In this review,we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us,and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.

Damaged male germ cells induce epididymitis in mice

Epididymitis can be caused by infectious and noninfectious etiological factors.While microbial infections are responsible for infectious epididymitis,the etiological factors contributing to noninfectious epididymitis remain to be defined.The present study demonstrated that damaged male germ cells(DMGCs)induce epididymitis in mice.Intraperitoneal injection of the alkylating agent busulfan damaged murine male germ cells.Epididymitis was observed in mice 4 weeks after the injection of busulfan and was characterized by massive macrophage infiltration.Epididymitis was coincident with an accumulation of DMGCs in the epididymis.In contrast,busulfan injection into mice lacking male germ cells did not induce epididymitis.DMGCs induced innate immune responses in epididymal epithelial cells(EECs),thereby upregulating the pro-inflammatory cytokines such as tumor necrosis factor-a(7/VF-a),interleukin-6(/L-6),and interleukin-ip(IL-ip) as well as the chemokines such as monocyte chemotactic protein-1(MCP-1),monocyte chemotactic protein-5(MCP-5),and chemokine ligand-10(CXCL10).These results suggest that male germ cell damage may induce noninfectious epididymitis through the induction of innate immune responses in EECs.These findings provide novel insights into the mechanisms underlying noninfectious epididymitis,which might aid in the diagnosis and treatment of the disease.

No Evidence of Effect on Male Mice Germ Cells After Acute Treatment With Thiram

Thiram is a dithiocarbamate compound widely used for industrial processes and agriculture. Animal studies reveal that this compound may afftct the male reproductive system. Aim of this study was to test, using sensitive testicular parameters, whether thiram directly affects germinal cells. For this purpose, B6C3F1 mice were intraperitoneally injected with thiram in oil (single dose:75 mg/kg; repeated five daily doses: 25 mg/kg).Although both treatments were toxic, none of the parameters examined, i.e., testis weighi, spermatid head number,specific enzyme levels at different times after treatment (14, 28, 35, 56 days) showed significant variations from the controls, On the contrary, in the positive controls (treated with chlorambucil), a marked reduction of sperm head number as well as a deerease of lactate dehydrogenasex and sorbitol dehydrogenase activity letels were evidenced at day 28, with a tendency to recover at day 35. Under these conditions thiram did not cause cytotoxicity on differentiating spermatogonia and on late spermatocyte stages of mice gonads

Transplantation of Goat Bone Marrow Mesenchymal Stem Cells (gMSCs) Help Restore Spermatogenesis in Endogenous Germ Cells-Depleted Mouse Models

Mesenchymal stem cells （MSCs） derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs （gMSCs） and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these ceils were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid （RA）, as analysed by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.

Single-cell RNA sequencing of mitoticarrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

Background:Germ cell mitotic arrest is conserved in many vertebrates,including birds,although the time of entry or exit into quiescence phase differs.Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch.However,mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage.Results:We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens.Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages(E12,E16,and hatch),we found that metabolic and signaling pathways were regulated,and that the epigenome was reprogrammed during mitotic arrest.In particular,we found that histone H3K9 and H3K14 acetylation(by HDAC2)and DNA demethylation(by DNMT3B and HELLS)led to a transcriptionally permissive chromatin state.Furthermore,we found that global DNA demethylation occurred gradually after the onset of mitotic arrest,indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome.DNA hypomethylation persisted after hatching,and methylation was slowly re-established 3 weeks later.Conclusions:We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching.This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described.Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.