CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation

This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1A1-mediated metabolism of xenobiotics by cytochrome P450 on male germ cell differentiation by qRT-PCR and immunocytochemistry. Results showed that 17,742 DEGs were found during differentiation of ESCs into SSCs and enriched in 72 differently significant pathways. Thereinto, the metabolism of xenobiotics by cytochrome P450 was involved in the whole differentiation process of ESCs into SSCs and several candidate DEGs: CYP1A1, CYP3A4, CYP2D6, ALDH3B1, and ALDH1A3 were expressed with the same trend with RNA-Seq. Knockdown of CYP1A1 caused male germ cell differentiation under restrictions. Our findings showed that the metabolism of xenobiotics by cytochrome P450 was significantly different during the process of male germ cell differentiation and was persistently activated when we induced cESCs to differentiate into SSCs with RA in vitro, which illustrated that the metabolism of xenobiotics by cytochrome P450 played a crucial role in the differentiation process of ESCs into SSCs.     

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.     

Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells

Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.     

Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self‐renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT‐PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self‐renewal mechanism of SSCs. Furthermore, the results of tissue‐specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self‐renewal in SSCs and for identifying specific surface markers of SSCs.     

The RNA helicase DHX9 establishes nucleolar heterochromatin,and this activity is required for embryonic stem cell differentiation

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of chromatin conformation and epigenetic patterns. lncRNA expression levels are widely taken as an indicator for functional properties. However, the role of RNA processing in modulating distinct features of the same lncRNA is less understood. The establishment of heterochromatin at rRNA genes depends on the processing of IGS‐rRNA into pRNA, a reaction that is impaired in embryonic stem cells (ESCs) and activated only upon differentiation. The production of mature pRNA is essential since it guides the repressor TIP5 to rRNA genes, and IGS‐rRNA abolishes this process. Through screening for IGS‐rRNA‐binding proteins, we here identify the RNA helicase DHX9 as a regulator of pRNA processing. DHX9 binds to rRNA genes only upon ESC differentiation and its activity guides TIP5 to rRNA genes and establishes heterochromatin. Remarkably, ESCs depleted of DHX9 are unable to differentiate and this phenotype is reverted by the addition of pRNA, whereas providing IGS‐rRNA and pRNA mutants deficient for TIP5 binding are not sufficient. Our results reveal insights into lncRNA biogenesis during development and support a model in which the state of rRNA gene chromatin is part of the regulatory network that controls exit from pluripotency and initiation of differentiation pathways.     

Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice

Self-renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR-486-5p enriched in Sertoli cell and Sertoli cell-derived exosomes. The exosomes mediate the transfer of miR-486-5p from Sertoli cells to SSCs. Exosomes release miR-486-5p, thus up-regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR-486-5p. Overexpression of miR-486-5p in SSCs down-regulates PTEN expression, which up-regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome-mediated transfer of miR-486-5p inhibits differentiation of SSC. Our findings demonstrate that miR-486-5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.     

MicroRNA‐10b regulates the renewal of spermatogonial stem cells through Kruppel‐like factor 4

MicroRNAs (miRs) are functionally important in spermatogenesis, which is the self‐renewal or differentiation of spermatogonial stem cells (SSCs). Here, we report a novel role for miR‐10b in regulating the self‐renewal of mouse SSCs. We showed that miR‐10b was highly expressed in mouse SSCs in vitro and enhanced SSC proliferation. Knockdown of miR‐10b significantly increased the apoptosis of SSCs compared with controls. Kruppel‐like factor 4 was found to be a target gene of miR‐10b in the enhancement of SSC proliferation. These findings further our understanding of the self‐renewal and differentiation of SSCs and provide a basis for the diagnosis, treatment, and prevention of male infertility.     

Downregulated lncRNA RCPCD promotes differentiation of embryonic stem cells into cardiac pacemaker-like cells by suppressing HCN4 promoter methylation

Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.Subject terms: Arrhythmias, Stem-cell research     

LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3

Divergent long noncoding RNAs (lncRNAs) represent a major lncRNA biotype in mouse and human genomes. The biological and molecular functions of the divergent lncRNAs remain largely unknown. Here, we show that lncKdm2b, a divergent lncRNA for Kdm2b gene, is conserved among five mammalian species and highly expressed in embryonic stem cells (ESCs) and early embryos. LncKdm2b knockout impairs ESC self‐renewal and causes early embryonic lethality. LncKdm2b can activate Zbtb3 by promoting the assembly and ATPase activity of Snf2‐related CREBBP activator protein (SRCAP) complex in trans. Zbtb3 potentiates the ESC self‐renewal in a Nanog‐dependent manner. Finally, Zbtb3 deficiency impairs the ESC self‐renewal and early embryonic development. Therefore, our findings reveal that lncRNAs may represent an additional layer of the regulation of ESC self‐renewal and early embryogenesis.     

Expression profile of genes identified in human spermatogonial stem cell‐like cells using suppression subtractive hybridization

Spermatogenesis is the process by which testicular spermatogonial stem cells (SSCs) self‐renew and differentiate into mature sperm in the testis. Maintaining healthy spermatogenesis requires proper proliferation of SSCs. In this study, we sought to identify factors that regulate the proliferation of SSCs. Human SSC (hSSC)‐like cells were isolated from azoospermic patients by a modified culture method and propagated in vitro. After four to five passages, the SSC‐like cells spontaneously ceased proliferating in vitro, so we collected proliferating (P)‐hSSC‐like cells at passage two and senescent (S)‐hSSC‐like cells at passage five. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between the P‐hSSC‐like and S‐hSSC‐like cells. We selected positive clones up‐regulated in P‐hSSC‐like cells using SSH and functionally characterized them by reference to public databases using NCBI BLAST tools. Expression levels of genes corresponding to subtracted clones were analyzed using RT‐PCR. Finally, we confirmed the differential expression of 128 genes in positive clones of P‐hSSC‐like cells compared with S‐hSSC‐like cells and selected 23 known and 39 unknown clones for further study. Known genes were associated with diverse functions; 22% were related to metabolism. Fifteen of the known genes and two of the unknown genes were down‐regulated after senescence of hSSC‐like cells. A comparison with previous reports further suggests that known genes selected, SPP1, may be related to germ cell biogenesis and cellular proliferation. Our findings identify several potential novel candidate biomarkers of proliferating‐ and senescencet‐hSSCs, and they provide potentially important insights into the function and characteristics of human SSCs. J. Cell. Biochem. 110: 752–762, 2010. © 2010 Wiley‐Liss, Inc.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro

Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post‐EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. It also decreased the levels of Leydig cell‐specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA‐Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell‐specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM‐mediated action was reversed by S3I‐201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.