Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells.

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM.

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells. HuR inhibitor MS-444 arrested embryonic development at 2-cell stage. Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444. MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA. STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.

Comprehensive Transcriptomic Analysis of Mouse Gonadal Development Involving Sexual Differentiation, Meiosis and Gametogenesis.

BACKGROUND: Mammalian gonadal development is crucial for fertility. Sexual differentiation, meiosis and gametogenesis are critical events in the process of gonadal development. Abnormalities in any of these events may cause infertility. However, owing to the complexity of these developmental events, the underlying molecular mechanisms are not fully understood and require further research. RESULTS: In this study, we employed RNA sequencing to examine transcriptome profiles of murine female and male gonads at crucial stages of these developmental events. By bioinformatics analysis, we identified a group of candidate genes that may participate in sexual differentiation, including Erbb3, Erbb4, and Prkg2. One hundred and two and 134 candidate genes that may be important for female and male gonadal development, respectively, were screened by analyzing the global gene expression patterns of developing female and male gonads. Weighted gene co-expression network analysis was performed on developing female gonads, and we identified a gene co-expression module related to meiosis. By alternative splicing analysis, we found that cassette-type exon and alternative start sites were the main forms of alternative splicing in developing gonads. A considerable portion of differentially expressed and alternatively spliced genes were involved in meiosis. CONCLUSION: Taken together, our findings have enriched the gonadal transcriptome database and provided novel candidate genes and avenues to research the molecular mechanisms of sexual differentiation, meiosis, and gametogenesis. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s12575-019-0108-y.

Ablation of the MiR-17-92 MicroRNA Cluster in Germ Cells Causes Subfertility in Female Mice.

BACKGROUND/AIMS: Oogenesis is a highly complex process that is intricately regulated by interactions of multiple genes and signaling molecules. However, the underlying molecular mechanisms are poorly understood. There is emerging evidence that microRNAs contribute to oogenesis. Here, we aimed to investigate the role of miR-17-92 cluster in regulating oogenesis. METHODS: The miR-17-92 cluster was genetically ablated in germ cells of female mice by applying the Cre-loxp system for conditional gene knockout. Mating experiment, superovulation and histological analysis were used to assess the fertility of the model female mice. TUNEL assay was used to identify apoptotic cells in ovaries. The expression level of apoptosis- and follicular atresia- related genes was evaluated by qRT-PCR. Western blotting was performed to detect protein expression. Bioinformatics software and dual luciferase reporter assay were applied to predict and verify the target of miR-17-92 cluster. RESULTS: Deletion of miR-17-92 cluster in germ cells of female mice caused increased oocyte degradation and follicular atresia, perturbed oogenesis, and ultimately led to subfertility. Genes involved in follicular atresia and the mitochondrial apoptotic pathway were obviously up-regulated. Furthermore, we verified that miR-19a regulated oogenesis at the post-transcriptional level by targeting Bmf in the ovaries of miR-17-92 cluster conditional knockout female mice. CONCLUSION: The miR-17-92 cluster is an important regulator of oogenesis. These findings will assist in better understanding the etiology of disorders in oogenesis and in developing new therapeutic targets for female infertility.

Effects of bisphenol A on ovarian follicular development and female germline stem cells.

Bisphenol A (BPA), one of the most frequently detected emerging pollutants in the environment, has been implicated in adverse effects in male and female reproduction at extremely low concentrations. This study aimed to investigate the effects and potential mechanism of BPA on mouse ovarian follicular development and female germline stem cells (FGSCs). Female CD-1 adult mice were administered gradient concentrations of BPA (12.5, 25, and 50 mg/kg/day) by intraperitoneal injection. We found that the number of atretic ovarian follicles was significantly increased at high BPA concentrations. Additionally, the numbers of primordial follicles, primary follicles, and corpus luteum (CL) were significantly reduced at high BPA concentrations. Interestingly, the number of FGSCs was remarkably reduced in BPA-treated ovaries. Furthermore, the increased apoptotic rate of FGSCs in vitro was triggered by BPA accompanied by increased BPA concentrations. To investigate the mechanism of BPA in ovarian follicular development, 193 differentially expressed proteins were identified in BPA-treated ovaries by the isobaric tags for relative and absolute quantification-coupled 2D liquid chromatography-mass spectrometry technique. A total of 106 proteins were downregulated and 85 proteins were upregulated. Among these proteins, the apoptosis-related protein SAFB-like transcriptional modulator (SLTM) was remarkably upregulated, and this result was consistent with western blotting. Taken together, our results suggest that an ovarian follicular development, especially, the development of primordial follicles, primary follicles, and the CL, is inhibited by high BPA concentrations, and the ovarian follicle atresia is initiated by BPA through upregulated expression of SLTM. Furthermore, BPA induces apoptosis of cultured FGSCs. The effect of BPA on ovarian follicular development and FGSCs, especially the effect on FGSCs, suggests a novel mechanism of how BPA causes female 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.

Reproductive chemical database: a curated database of chemicals that modulate protein targets regulating important reproductive biological processes.

Recent studies have shifted the spotlight from adult disease to gametogenesis and embryo developmental events, and these are greatly affected by various environmental chemicals, such as drugs, metabolites, pollutants, and others. Growing research has highlighted the critical importance of identifying and understanding the roles of chemicals in reproductive biology. However, the functions and mechanisms of chemicals in reproductive processes remain incomplete. We developed a comprehensive database called the Reproductive Chemical Database (RCDB) ( https://yu.life.sjtu.edu.cn/ChenLab/RCDB ) to facilitate research on chemicals in reproductive biology. This resource is founded on rigorous manual literature extraction and precise protein target prediction methodologies. This database focuses on the delineation of chemicals associated with phenotypes, diseases, or endpoints intricately associated with four important reproductive processes: female and male gamete generation, fertilization, and embryo development in human and mouse. The RCDB encompasses 93 sub-GO processes, and it revealed 1447 intricate chemical-biological process interactions. To date, the RCDB has meticulously cataloged and annotated 830 distinct chemicals, while also predicting 614 target proteins from a selection of 3800 potential candidates. Additionally, the RCDB offers an online predictive tool that empowers researchers to ascertain whether specific chemicals play discernible functional roles in these reproductive processes. The RCDB is an exhaustive, cross-platform, manually curated database, which provides a user-friendly interface to search, browse, and use reproductive processes modulators and their comprehensive related information. The RCDB will help researchers to understand the whole reproductive process and related diseases and it has the potential to promote reproduction research in the pharmacological and pathophysiological areas.

Metformin promotes female germline stem cell proliferation by upregulating Gata-binding protein 2 with histone ß-hydroxybutyrylation.

BACKGROUND: Metformin as a first-line clinical anti-diabetic agent prolongs the lifespan of model animals and promotes cell proliferation. However, the molecular mechanisms underlying the proliferative phenotype, especially in epigenetics, have rarely been reported. The aim of this study was to investigate the physiological effects of metformin on female germline stem cells (FGSCs) in vivo and in vitro, uncover ß-hydroxybutyrylation epigenetic modification roles of metformin and identify the mechanism of histone H2B Lys5 ß-hydroxybutyrylation (H2BK5bhb) in Gata-binding protein 2 (Gata2)-mediated proliferation promotion of FGSCs. METHODS: The physiological effects of metformin were evaluated by intraperitoneal injection and histomorphology. The phenotype and mechanism studies were explored by cell counting, cell viability, cell proliferation assay and protein modification omics, transcriptomics, chromatin immunoprecipitation sequencing in FGSCs in vitro. RESULTS: We found that metformin treatment increased the number of FGSCs, promoted follicular development in mouse ovaries and enhanced the proliferative activity of FGSCs in vitro. Quantitative omics analysis of protein modifications revealed that H2BK5bhb was increased after metformin treatment of FGSCs. In combination with H2BK5bhb chromatin immunoprecipitation and transcriptome sequencing, we found that Gata2 might be a target gene for metformin to regulate FGSC development. Subsequent experiments showed that Gata2 promoted FGSC proliferation. CONCLUSION: Our results provide novel mechanistic understanding of metformin in FGSCs by combining histone epigenetics and phenotypic analyses, which highlight the role of the metformin-H2BK5bhb-Gata2 pathway in cell fate determination and regulation.

YTHDF1 phase separation triggers the fate transition of spermatogonial stem cells by activating the IκB-NF-κB-CCND1 axis.

N6-methyladenosine (m6A) modification controls cell fate determination. Here, we show that liquid-liquid phase separation (LLPS) of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a pivotal m6A "reader" protein, promotes the transdifferentiation of spermatogonial stem cells (SSCs) into neural stem cell-like cells by activating the IκB-nuclear factor κB (NF-κB)-CCND1 axis. The inhibition of IκBα/ß mRNA translation mediated by YTHDF1 LLPS is the key to the activation of the IκB-NF-κB-CCND1 axis. Disrupting either YTHDF1 LLPS or NF-κB activation inhibits transdifferentiation efficiency. Moreover, overexpression of the YTH domain of YTHDF1 inhibits the activation of the IκB-NF-κB-CCND1 axis by promoting IκBα/ß mRNA translation. Overexpression of the tau-YTH fusion protein reactivates IκB-NF-κB-CCND1 axis by inhibiting the translation of IκBα/ß mRNAs, and tau LLPS is observed, which can restore transdifferentiation efficiency. Our findings demonstrate that the protein-RNA LLPS plays essential roles in cell fate transition and provide insights into translational medicine and the therapy of neurological diseases.

Metformin Promotes Proliferation of Mouse Female Germline Stem Cells by Histone Acetylation Modification of Traf2.

Female germline stem cells (FGSCs) are adult stem cells that can both self-renew and differentiate into mature oocytes. Although small-molecule compounds are capable of regulating the development of FGSCs, the effects and mechanisms of action of metformin, a commonly used drug for diabetes, on FGSCs are largely unknown. Here, we found that metformin promoted the viability and proliferation of FGSCs through H3K27ac modification. To elucidate the mechanism by which metformin promoted FGSCs proliferation, Chromatin Immunoprecipitation Sequencing of histone 3 lysine 27 acetylation (H3K27ac) in FGSCs was performed with or without metformin-treatment. The results indicate that metformin modulates FGSCs via the mitogen-activated protein kinase (MAPK) signaling pathway, and tumor necrosis factor receptor associated factor 2 (Traf2) was identified as an important target gene for H3K27ac modification during FGSCs proliferation. Subsequent experiments showed metformin promoted FGSCs proliferation by H3K27ac modification of Traf2 to regulate MAPK signaling. Our findings deepen understanding of how H3K27ac modifications regulate FGSCs development and provide a theoretical basis for the prevention and treatment of premature ovarian failure, polycystic ovary syndrome, infertility, and related diseases.

Stella Regulates the Development of Female Germline Stem Cells by Modulating Chromatin Structure and DNA Methylation.

Female germline stem cells (FGSCs) have the ability to self-renew and differentiate into oocytes. Stella, encoded by a maternal effect gene, plays an important role in oogenesis and early embryonic development. However, its function in FGSCs remains unclear. In this study, we showed that CRISPR/Cas9-mediated knockout of Stella promoted FGSC proliferation and reduced the level of genome-wide DNA methylation of FGSCs. Conversely, Stella overexpression led to the opposite results, and enhanced FGSC differentiation. We also performed an integrative analysis of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), high-throughput genome-wide chromosome conformation capture (Hi-C), and use of our published epigenetic data. Results indicated that the binding sites of STELLA and active histones H3K4me3 and H3K27ac were enriched near the TAD boundaries. Hi-C analysis showed that Stella overexpression attenuated the interaction within TADs, and interestingly enhanced the TAD boundary strength in STELLA-associated regions. Taking these findings together, our study not only reveals the role of Stella in regulating DNA methylation and chromatin structure, but also provides a better understanding of FGSC development.

Three-dimensional genome structure shapes the recombination landscape of chromatin features during female germline stem cell development.

BACKGROUND: During meiosis of mammalian cells, chromatin undergoes drastic reorganization. However, the dynamics of the three-dimensional (3D) chromatin structure during the development of female germline stem cells (FGSCs) are poorly understood. METHODS: The high-throughput chromosome conformation capture technique was used to probe the 3D structure of chromatin in mouse germ cells at each stage of FGSC development. RESULTS: The global 3D genome was dramatically reorganized during FGSC development. In topologically associating domains, the chromatin structure was weakened in germinal vesicle stage oocytes and still present in meiosis I stage oocytes but had vanished in meiosis II oocytes. This switch between topologically associating domains was related to the biological process of FGSC development. Moreover, we constructed a landscape of chromosome X organization, which showed that the X chromosome occupied a smaller proportion of the active (A) compartment than the autosome during FGSC development. By comparing the high-order chromatin structure between female and male germline development, we found that 3D genome organization was remodelled by two different potential mechanisms during gamete development, in which interchromosomal interactions, compartments, and topologically associating domain were decreased during FGSC development but reorganized and recovered during spermatogenesis. Finally, we identified conserved chromatin structures between FGSC development and early embryonic development. CONCLUSIONS: These results provide a valuable resource to characterize chromatin organization and for further studies of FGSC development.

Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three-dimensional chromatin structure.

OBJECTIVES: This study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development. MATERIALS AND METHODS: FGSCs were induced to differentiate into meiosis in differentiation medium. RNA sequencing was performed to analysis the difference of transcription level. High-through chromosome conformation capture sequencing (Hi-C) was performed to analysis changes of three-dimensional chromatin structure. Chromosome conformation capture further confirmed a spatial chromatin loop. ChIP-qPCR and dual luciferase reporter were used to test the interaction between Stimulated by retinoic acid gene 8 (STRA8) protein and Trip13 promoter. RESULTS: Compared with FGSCs, the average diameter of STRA8-positive germ cells increased from 13 µm to 16.8 µm. Furthermore, there were 4788 differentially expressed genes between the two cell stages; Meiosis and chromatin structure-associated terms were significantly enriched. Additionally, Hi-C results showed that FGSCs underwent A/B compartment switching (switch rate was 29.81%), the number of topologically associating domains (TADs) increasing, the average size of TADs decreasing, and chromatin loop changes at genome region of Trip13 from undifferentiated stage to meiosis-initiation stage. Furthermore, we validated that Trip13 promoter contacted distal enhancer to form spatial chromatin loop and STRA8 could bind Trip13 promoter to promote gene expression. CONCLUSION: FGSCs underwent chromatin structure remodelling from undifferentiated stage to meiosis-initiation stage, which facilitated STRA8 binding to Trip13 promoter and promoting its expression.

Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation.

BACKGROUND: During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). RESULTS: We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and "bivalent" (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (µChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. CONCLUSION: This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

Spermidine induces cytoprotective autophagy of female germline stem cells in vitro and ameliorates aging caused by oxidative stress through upregulated sequestosome-1/p62 expression.

BACKGROUND: Autophagy is required for oogenesis and plays a critical role in response to aging caused by oxidative stress. However, there have been no reports on regulation of cytoprotective autophagy in female germline stem cells (FGSCs) in response to aging caused by oxidative stress. RESULTS: We found that Spermidine (SPD) significantly increased protein expression of autophagy markers microtubule-associated protein 1 light chain 3 beta-II (MAP1LC3B-II/LC3B-II) and sequestosome-1/p62 (SQSTM1/p62), and evoked autophagic flux in FGSCs. Moreover, SPD increased the number and viability of FGSCs in vitro. Further, we found that SPD significantly reduced basal or hydrogen peroxide (H2O2)-induced up-regulated protein expression of the aging markers, cyclin dependent kinase inhibitor 2A (p16/CDKN2A) and tumor protein 53 (p53). After knockdown of p62 in FGSCs, p16 protein levels were significant higher compared with controls. However, protein p16 levels were not significantly changed in p62 knockdown FGSCs with SPD treatment compared with without SPD. Moreover, SPD significantly changed the expression of autophagy-related genes and pathways in FGSCs, as shown by bioinformatics analysis of RNA sequencing data. Additionally, SPD significantly inhibited AKT/mTOR phosphorylation. CONCLUSIONS: SPD induces cytoprotective autophagy in FGSCs in vitro and ameliorates cellular senescence of FGSCs induced by H2O2. Furthermore, SPD can ameliorate cellular senescence of FGSCs through p62. SPD might induce autophagy in FGSCs via the PI3K/Akt pathway. Our findings could be helpful for delaying aging of female germ cells due to oxidative stress and preserving female fertility.

Identifying cortical specific long noncoding RNAs modified by m6A RNA methylation in mouse brains.

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation. Increasing evidence shows that cortical development is regulated by both mRNAs and long noncoding RNAs (lncRNAs), which also are modified by N6-methyladenosine (m6A). Patterns of m6A-methylation in lncRNAs in the developing cortex have not been uncovered. Here we reveal differentially expressed lncRNAs and report stage-specific m6A-methylation patterns in lncRNAs expressed in mouse embryonic (E) and postnatal (P) cortices using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs show temporal differential expression, and display genic distribution in the genome. Interestingly, we detect temporal-specific m6A-methylation with consensus m6A motif GGACU in the last exon in most lncRNAs. And m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs that have no significantly differential expression in E- and P-stages. Furthermore, the transcript abundance has a positive correlation between the m6A genic lncRNAs and their nearest m6A methylated mRNAs. Our work reveals a fundamental expression reference of lncRNAs and their nearest mRNAs, and highlights an importance of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in the developing cortex.

Comparison of RNA m6A and DNA methylation profiles between mouse female germline stem cells and STO cells.

N6-methyladenosine (m6A) methylation modification is the most prevalent and abundant internal modification of eukaryotic mRNAs. Increasing evidence has shown that mRNA m6A plays important roles in the development of stem cells. However, to the best of our knowledge, no reports about the roles of mRNA m6A in mouse female germline stem cells (mFGSCs) have been published. In this study, we compared the genome-wide profiles of mRNA m6A methylation and DNA methylation between FGSCs and sandosinbred mice (SIM) embryo-derived thioguanine and ouabain-resistant (STO) cells. qRT-PCR revealed that the expression levels of mRNA m6A-related genes (Mettl3, Alkbh5, Ythdf1, Ythdf2, Ythdc1, and Ythdc2) in FGSCs were significantly higher than those in STO cells. m6A RNA immunoprecipitation sequencing (MeRIP-seq) data further showed that the unique m6A-methylated mRNAs in FGSCs and STO cells were related to cell population proliferation and somatic development, respectively. Additionally, knockdown of Ythdf1 inhibited FGSC self-renewal. Comparison of methylated DNA immunoprecipitation sequencing (MeDIP-seq) results between FGSCs and STO cells identified that DNA methylation contributed to FGSC proliferation by suppressing the somatic program. These results suggested that m6A regulated FGSC self-renewal possibly through m6A binding protein YTHDF1, and DNA methylation repressed somatic programs in FGSCs to maintain FGSC characteristics.

C28 induced autophagy of female germline stem cells in vitro with changes of H3K27 acetylation and transcriptomics.

There are a few studies indicating that small molecular compounds affect the proliferation, differentiation, apoptosis, and autophagy of female germline stem cells (FGSCs). However, whether small molecular compound 28 (C28) affect development of FGSCs remains unknown. In this study, we found that C28 reduced the viability and proliferation of FGSCs, respectively. Additionally, western blotting showed that the expression of autophagy marker light chain 3 beta II (LC3B-II) was significantly increased and expression of sequestosome-1 (SQSTM1) was significantly reduced in C28-treated groups. Immunofluorescence showed that, in C28-treated groups, the number of LC3B-II-positive puncta was increased significantly. These results indicated that C28 induced autophagy of FGSCs in vitro. Furthermore, data from Chromatin Immunoprecipitation Sequencing for H3K27ac showed that autophagy-related biological processes such as regulation of mitochondrial membrane potential, Golgi vesicle transport, and cellular response to reactive oxygen species were different after C28-treated. In addition, RNA-Seq showed that the expression of genes (Trib3, DDIT3, and ATF4) related to endoplasmic reticulum (ER) stress was enhanced by C28. These results suggest that the changes of H3K27ac and ER stress might be associated with C28-induced FGSC autophagy.

Offspring production of ovarian organoids derived from spermatogonial stem cells by defined factors with chromatin reorganization.

Introduction: Fate determination of germline stem cells remains poorly understood at the chromatin structure level. Objectives: Our research hopes to develop successful offspring production of ovarian organoids derived from spermatogonial stem cells (SSCs) by defined factors. Methods: The offspring production from oocytes transdifferentiated from mouse SSCs with tracking of transplanted SSCs in vivo, single cell whole exome sequencing, and in 3D cell culture reconstitution of the process of oogenesis derived from SSCs. The defined factors were screened with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into induced germline stem cells (iGSCs) using high throughput chromosome conformation. Results: We demonstrate successful production of offspring from oocytes transdifferentiated from mouse spermatogonial stem cells (SSCs). Furthermore, we demonstrate direct induction of germline stem cells (iGSCs) differentiated into functional oocytes by transduction of H19, Stella, and Zfp57 and inactivation of Plzf in SSCs after screening with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into iGSCs, which was highly similar to female germline stem cells. We observed that although topologically associating domains were stable during SSC conversion, chromatin interactions changed in a striking manner, altering 35% of inactive and active chromosomal compartments throughout the genome. Conclusion: We demonstrate successful offspring production of ovarian organoids derived from SSCs by defined factors with chromatin reorganization. These findings have important implications in various areas including mammalian gametogenesis, genetic and epigenetic reprogramming, biotechnology, and medicine.

Molecular cloning and expression of a new gene, GON-SJTU1 in the rat testis.

BACKGROUND: Spermatogenesis is a complex process involving cell development, differentiation and apoptosis. This process is governed by a series of genes whose expressions are highly regulated. Male infertility can be attributed to multiple genetic defects or alterations that are related to spermatogenesis. The discovery, cloning and further functional study of genes related to spermatogenesis is of great importance to the elucidation of the molecular mechanism of spermatogenesis. It is also physiologically and pathologically significant to the therapy of male infertility. METHODS: GON-SJTU1 was identified and cloned from rat testis by cDNA library screening and 3'-and 5'-RACE. The products of GON-SJTU1 were assessed by Northern and Western blotting. The expression of GON-SJTU1 was also examined by In situ hybridization and immunohistochemistry. RESULTS: Here we identified and cloned a new gene, GON-SJTU1, with the biological process of spermatogenesis. GON-SJTU1 is highly expressed in the testis from day 1 to 15 and then decreased, suggesting that GON-SJTU1 might be a time-related gene and involved in the early stage of spermatogenesis. And the expression of GON-SJTU1 in the testis occurred in some male germ cells, particularly in gonocytes and spermatogonial stem cells. CONCLUSION: GON-SJTU1 may play a role in the biological process of spermatogenesis.