Initiation Of Meiosis Research Articles

P-705 Homozygous missense variant in MEIOSIN causes premature ovarian insufficiency

Abstract Study question Are the gene variants involved in meiosis initiation responsible for premature ovarian insufficiency (POI)? Summary answer MEIOSIN variant participates in the pathogenesis of human POI by impairing meiosis due to insufficient transcriptional activation of essential meiotic genes. What is known already Meiosis is the key event for the establishment of the ovarian reserve, and several gene defects impairing meiotic homologous recombination have been found to contribute to the pathogenesis of POI. However, the genes involved in meiosis initiation have not been associated with POI. Study design, size, duration Retrospective genetic study. An in-house whole exome sequencing (WES) database of 1,030 idiopathic POI patients was screened for variations of meiosis initiation genes. Participants/materials, setting, methods Gene variations involved in meiosis initiation were screened in the in-house WES database of 1,030 POI cases. The pathogenicity of the variation was verified by in vitro experiments, including protein structure prediction and dual-luciferase reporter assay. The effect of the variant on ovarian function and meiosis was demonstrated through histological analyses in a point mutation mouse model. Main results and the role of chance A homozygous variant of MEIOSIN that initiates meiosis via the retinoic acid dependent pathway was identified in a patient with idiopathic POI. The dual-luciferase reporter assay revealed that the variant adversely affected the transcriptional function of MEIOSIN in upregulating meiotic genes. Further knock-in mice with the homologous mutation confirmed that the variation impacted the meiotic prophase I program and accelerated oocyte depletion. Limitations, reasons for caution Further studies are needed to explore the role of other meiosis initiation genes in the pathogenesis of POI. Wider implications of the findings The present study firstly identified pathogenic MEIOSIN variants in patients with POI. Although the causative variation in meiotic initiation genes is rare in POI, our study expanded the spectrum of causative genes and elucidated the different mechanisms in human infertility. Trial registration number Not available

Gonadal morphogenesis in the South American toad Rhinella Arenarum (Anura, Bufonidae) unveils an extremely delayed rate of sex differentiation.

Among anurans, Bufonids are recognized for their retarded sex differentiation. However, few studies have addressed gonadal morphogenesis in this family. Here, we analyzed the early gonadogenesis in laboratory-reared Rhinella arenarum. Few germ cells were identified in the genital ridge at Gosner stage 26. At metamorphosis, somatic cells and germ cells were observed in the outer region of the undifferentiated gonad, whereas the central region was occupied by stromal tissue. A cortico-medullary organization was first recognized on Day 7 postmetamorphosis. The cortex was composed of germ cells and encompassing epithelial cells, whereas the medulla contained cells presumptively derived from the coelomic epithelium. Medullary somatic cells formed metameric knots along the length of the undifferentiated gonad. Consequently, a series of 12-14 gonomeres became recognizable externally. The first sign of ovarian differentiation was observed on Day 15 postmetamorphosis, when a cavity was formed within each gonomere. In contrast, testes were recognized by a uniform distribution of germ cells and intermingled somatic cells, as the division into cortex and medulla was lost. By Day 50 postmetamorphosis, the gonadal metameric organization was still apparent both in the ovaries and testes. Follicles containing diplotene oocytes were observed within the ovary. In the testis, an incipient lobular architecture was recognized without initiation of meiosis within the seminiferous cords. These observations reveal an extremely delayed gonadal development in R. arenarum, not reported previously for other anuran species. In addition, the late differentiation of the gonads contrasted with the early appearance of follicles in the Bidder's organ. Lastly, we observed that delayed metamorphs exhibited an undifferentiated gonad, demonstrating that gonadogenesis in this species is more dependent on somatic development than on age.

Genetic Regulation of Mitosis-Meiosis Fate Decision in Plants: Is Callose an Oversighted Polysaccharide in These Processes?

Timely progression of the meiotic cell cycle and synchronized establishment of male meiosis in anthers are key to ascertaining plant fertility. With the discovery of novel regulators of the plant cell cycle, the mechanisms underlying meiosis initiation and progression appear to be more complex than previously thought, requiring the conjunctive action of cyclins, cyclin-dependent kinases, transcription factors, protein-protein interactions, and several signaling components. Broadly, cell cycle regulators can be classified into two categories in plants based on the nature of their mutational effects: (1) those that completely arrest cell cycle progression; and (2) those that affect the timing (delay or accelerate) or synchrony of cell cycle progression but somehow complete the division process. Especially the latter effects reflect evasion or obstruction of major steps in the meiosis but have sometimes been overlooked due to their subtle phenotypes. In addition to meiotic regulators, very few signaling compounds have been discovered in plants to date. In this review, we discuss the current state of knowledge about genetic mechanisms to enter the meiotic processes, referred to as the mitosis-meiosis fate decision, as well as the importance of callose (β-1,3 glucan), which has been unsung for a long time in male meiosis in plants.

Intrafollicular Retinoic Acid Signaling Is Important for Luteinizing Hormone-Induced Oocyte Meiotic Resumption.

It has been clear that retinoic acid (RA), the most active vitamin A (VA) derivative, plays a central role in governing oocyte meiosis initiation. However, it has not been functionally determined if RA participates in luteinizing hormone (LH)-induced resumption from long-lasting oocyte meiotic arrest, which is essential for haploid oocyte formation. In the present study, using well-established in vivo and in vitro models, we identified that intrafollicular RA signaling is important for normal oocyte meiotic resumption. A mechanistic study indicated that mural granulosa cells (MGCs) are the indispensable follicular compartment for RA-prompted meiotic resumption. Moreover, retinoic acid receptor (RAR) is essential for mediating RA signaling to regulate meiotic resumption. Furthermore, we found zinc finger protein 36 (ZFP36) is the transcriptional target of RAR. Both RA signaling and epidermal growth factor (EGF) signaling were activated in MGCs in response to LH surge, and two intrafollicular signalings cooperate to induce rapid Zfp36 upregulation and Nppc mRNA decrease, which is critical to LH-induced meiotic resumption. These findings extend our understanding of the role of RA in oocyte meiosis: RA not only governs meiotic initiation but also regulates LH-induced meiotic resumption. We also emphasize the importance of LH-induced metabolic changes in MGCs in this process.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Conserved H3K27me3-associated chromatin remodelling allows STRA8 but not MEIOSIN expression in mammalian germ cells.

Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other. In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

The adverse effects of 17β-estradiol immersion during gonadal differentiation on ovarian development of female Takifugu rubripes

Estrogen exposure during early-life stages has been found to delay ovarian development in female fish, even after a long-term depuration period. However, the mechanisms underlying the disordered ovarian development remain unclear. In this study, the larvae of tiger puffer Takifugu rubripes were exposed to 0 (control) and 10 μg/L 17β-estradiol (E2) from 20 to 90 days post-hatch (dph) and maintained in clear seawater until 180 dph. Genetic females collected at 90 and 180 dph were identified by analyzing a sex-associated SNP. Then, the ovarian structure, gonadosomatic index (GSI), the maximum follicle area and the mRNA levels of genes involving in cell cycle (ckd2, ckd4, cdk6, ccna2, ccnd2, cdkn1b and cdkn2c), meiosis initiation (sycp3, rec8, spo11, and dmc1), follicle formatiaon (bmp2, hnrnpk, hmp15, gdf9, nobox and figla) and apoptosis (bax and bcl-2) were analyzed between control and E2-exposed females. The results show that, no structure difference in ovaries was observed between control and E2-treated females at 90 dph, but the primary growth follicles in E2-treated females were observed to be fewer in number than control at 180 dph. Both ovarian weight and GSI of E2-treated females were significant lower than the control at 90 and 180 dph, while there was no significant different in the maximum follicle area between control and E2-treated females at neither 90 or 180 dph. Additionally, the E2 exposure suppressed the expression of sycp3, rec8, spo11, dmc1, bmp2, hnrnpk and bcl-2 at 90 dph, but the mRNA levels of those genes in E2-treated females showed no significant different with the control at 180 dph. The reduced mRNA levels of sycp3, rec8, spo11 and dmc1 might result in disrupted meiosis, and suppression the expression of bmp2 and hnrnpk affected follicle formation. Then, abnormal meiosis initiation and follicle formation might further promote apoptosis as indicated by the decrease in mRNA levels of bcl-2, which ultimately contributed to less number of follicles and low GSI value in E2-treated females.

Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50–150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.Graphical

Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary.

Normalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed. Six candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively. Our findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

BDE-209 induced spermatogenesis disorder by inhibiting SETD8/H4K20me1 related histone methylation in mice

Past studies have observed that decabromodiphenyl ether (BDE-209) induces reproductive and developmental toxicity, but the specific mechanism remains unclear. Based on our previous work, male mice were orally given BDE-209 at 75 mg/kg/d via continuous exposure for one spermatozoon development period (50 days) and then stopping exposure for another 50 days. The mouse spermatocyte line GC-2spd was used to examine the toxic effects of BDE-209 on histone methylation and spermatogenesis. The findings indicated that BDE-209 damaged testis and epididymis structure, induced spermatogenic cell apoptosis, and decreased sperm quantity and quality after the 50-day exposure. Furthermore, BDE-209 lowered the levels of SETD8/H4K20me1 and activated the upstream signaling of DNA damage response (Mre11/Rad50/NBS1), thereby causing spermatogenic cell cycle arrest and apoptosis. Downregulation of meiotic promoter Stra8 was associated with a decrease in SETD8 after BDE-209 exposure. After stopping the exposure for 50 days, reproductive system damage and meiosis and cell cycle inhibition due to histone methylation did not improve. In vitro experiments revealed that Setd8 overexpression upregulated the histone methylation and Stra8 expression but did not promote the cell cycle in GC-2 cells. Therefore, BDE-209 exposure impaired spermatogenesis by affecting SETD8/H4K20me1-linked histone methylation and inhibiting meiosis initiation and cell cycle progression, thereby resulting in long-term male reproductive toxicity.

Foetal exposure to the bisphenols BADGE and BPAF impairs meiosis through DNA oxidation in mouse ovaries

Many endocrine disruptors have been proven to impair the meiotic process which is required for the production of healthy gametes. Bisphenol A is emblematic of such disruptors, as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing use of BPA alternatives raises concerns for public health. Here, we investigated the effects of foetal exposure to two BPA alternatives, bisphenol A Diglycidyl Ether (BADGE) and bisphenol AF (BPAF), on oogenesis in mice. These compounds delay meiosis initiation, increase the number of MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by changes in gene expression in foetal premeiotic germ cells and aberrant mRNA splicing of meiotic genes. We observed an increase in DNA oxidation after exposure to BPA alternatives. Specific induction of oxidative DNA damage during foetal germ cell differentiation causes similar defects during oogenesis, as observed in 8-oxoguanine DNA Glycosylase (OGG1)-deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damage. The supplementation of BPA alternatives with N-acetylcysteine (NAC) counteracts the effects of bisphenols on meiosis. Together, our results propose oxidative DNA lesion as an event that negatively impacts female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for numerous environmental pro-oxidant pollutants, and its discovery, could lead to reconsider the adverse effect of bisphenol mixtures that are simultaneously present in our environment.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

Recycling amino acids ensures meiosis and seed development.

Nitrogen (N) nutrition and meiosis demand large amounts of energy and widely affect crop yield. Recently, Yang and colleagues connected both processes by demonstrating that meiosis initiation depends on the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) system, whereas meiotic defects of the etfβ mutant can be rescued using N supplementation.

Rice GLUCAN SYNTHASE-LIKE5 promotes anther callose deposition to maintain meiosis initiation and progression.

Callose is a plant cell wall polysaccharide whose deposition is spatiotemporally regulated in various developmental processes and environmental stress responses. The appearance of callose in premeiotic anthers is a prominent histological hallmark for the onset of meiosis in flowering plants; however, the biological role of callose in meiosis remains unknown. Here, we show that rice (Oryza sativa) GLUCAN SYNTHASE LIKE5 (OsGSL5), a callose synthase, localizes on the plasma membrane of pollen mother cells (PMCs) and is responsible for biogenesis of callose in anther locules through premeiotic and meiotic stages. In Osgsl5 mutant anthers mostly lacking callose deposition, aberrant PMCs accompanied by aggregated, unpaired, or multivalent chromosomes were frequently observed and, furthermore, a considerable number of mutant PMCs had untimely progress into meiosis compared to that of wild-type PMCs. Immunostaining of meiosis-specific protein HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS2 in premeiotic PMCs revealed precocious meiosis entry in Osgsl5 anthers. These findings provide insights into the function of callose in controlling the timing of male meiosis initiation and progression, in addition to roles in microsporogenesis, in flowering plants.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits.

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

The function of Foxo1 in spermatogonia development is independent of PI3K/PTEN signaling

Spermatogenesis is a highly coordinated process that initiates shortly after birth and continues throughout the lifespan of male animals. Foxo1 is a transcription factor and is involved in many biological processes. It has been reported that the inactivation of Foxo1 in gonocytes during the embryonic stage causes the defects of spermatogenesis. In the present study, we found that the inactivation of Foxo1 in spermatogonia after birth also caused germ cell loss and male infertility. We found that the initiation of meiosis was not affected; however, the germ cell development was arrested after meiosis and lack of mature spermatozoa in the cauda epididymis. We also found that the proliferation of Foxo1-deficient spermatogonia stem cells was significantly reduced under in vitro conditions. Further study revealed that inactivation of Pten in postnatal spermatogonia using Stra8-Cre did not affect germ cell development and the subcellular location of FOXO1 in Pten-deficient spermatogonia. This study demonstrated that Foxo1 was involved in the development of spermatogonia after birth and the function of Foxo1 was probably not regulated by PI3K/PTEN signaling.

Human-specific gene CT47 blocks PRMT5 degradation to lead to meiosis arrest

Exploring the functions of human-specific genes (HSGs) is challenging due to the lack of a tractable genetic model system. Testosterone is essential for maintaining human spermatogenesis and fertility, but the underlying mechanism is unclear. Here, we identified Cancer/Testis Antigen gene family 47 (CT47) as an essential regulator of human-specific spermatogenesis by stabilizing arginine methyltransferase 5 (PRMT5). A humanized mouse model revealed that CT47 functions to arrest spermatogenesis by interacting with and regulating CT47/PRMT5 accumulation in the nucleus during the leptotene/zygotene-to-pachytene transition of meiosis. We demonstrate that testosterone induces nuclear depletion of CT47/PRMT5 and rescues leptotene-arrested spermatocyte progression in humanized testes. Loss of CT47 in human embryonic stem cells (hESCs) by CRISPR/Cas9 led to an increase in haploid cells but blocked the testosterone-induced increase in haploid cells when hESCs were differentiated into haploid spermatogenic cells. Moreover, CT47 levels were decreased in nonobstructive azoospermia. Together, these results established CT47 as a crucial regulator of human spermatogenesis by preventing meiosis initiation before the testosterone surge.

Investigation of Signals and Transcription Factors for The Generation of Female Germ-Like Cells.

Objective Primordial germ cell (PGCs) lines are a source of a highly specialized type of cells, characteristically oocytes, during female germline development in vivo. The oocyte growth begins in the transition from the primary follicle. It is associated with dynamic changes in gene expression, but the gene-regulating signals and transcription factors that control oocyte growth remain unknown. We aim to investigate the differentiation potential of mouse bone marrow mesenchymal stem cells (mMSCs) into female germ-like cells by testing several signals and transcription factors. Materials and Methods In this experimental study, mMSCs were extracted from mice femur bone using the flushing technique. The cluster-differentiation (CD) of superficial mesenchymal markers was determined with flow cytometric analysis. We applied a set of transcription factors including retinoic acid (RA), titanium nanotubes (TNTs), and fibrin such as TNT-coated fibrin (F+TNT) formation and (RA+F+TNT) induction, and investigated the changes in gene, MVH/ DDX4, expression and functional screening using an in vitro mouse oocyte development condition. Germ cell markers expression, (MVH / DDX4), was analyzed with Immunocytochemistry staining, quantitative transcription-polymerase chain reaction (RT-qPCR) analysis, and Western blots. Results The expression of CD was confirmed by flow cytometry. The phase determination of the TNTs and F+TNT were confirmed using x-ray diffraction (XRD) and scanning electron microscope (SEM), respectively. Remarkably, applying these transcription factors quickly induced pluripotent stem cells into oocyte-like cells that were sufficient to generate female germ- like cells, growth, and maturation from mMSCs differentiation. These transcription factors formed oocyte-like cells specification of stem cells, epigenetic reprogramming, or meiosis and indicate that oocyte meiosis initiation and oocyte growth are not separable from the previous epigenetic reprogramming in stem cells in vitro. ConclusionResults suggested several transcription factors may apply for arranging oocyte-like cell growth and supplies an alternative source of in vitro maturation (IVM).

Germline FOXJ2 overexpression causes male infertility via aberrant autophagy activation by LAMP2A upregulation

Spermatogenesis is a complex biological process that produces haploid spermatozoa and requires precise regulation by many tissue-specific factors. In this study, we explored the role and mechanism of Fork head box J2 (FOXJ2, which is highly expressed in spermatocytes) in the regulation of spermatogenesis using a germline-specific conditional Foxj2 knock-in mouse model (Stra8-Cre; Foxj2tg/tg mouse). Foxj2 overexpression in mouse testes led to spermatogenesis failure, which started at the initiation of meiosis, and resulted in male infertility. Lysosomes and autophagy-related genes were upregulated in Stra8-cre; Foxj2tg/tg mouse testes and the number of autolysosomes in the spermatocytes in Stra8-cre; Foxj2tg/tg mice was increased. Chromatin immunoprecipitation-PCR and Dual-luciferase reporter assays showed that Lamp2 (encoding lysosome‐associated membrane protein‐2) was a target of FOXJ2. Foxj2 overexpression increased the expression levels of Lamp2a and Hsc70 (70-kDa cytoplasmic heat shock protein) in the Stra8-cre; Foxj2tg/tg mouse testes. Our results suggested that Foxj2 overexpression in the germ cells of mouse testes affects chaperone-mediated autophagy by upregulating LAMP2A, leading to spermatogenesis failure at the initiation of meiosis, thus resulting in male infertility. Our findings provide a new insight into the function of FOXJ2 in spermatogenesis and the significance of autophagy regulation in spermatogenesis.

BPA disrupts meiosis I in oogonia by acting on pathways including cell cycle regulation, meiosis initiation and spindle assembly

The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.