Biology of Reproduction最新文献

Mammalian sexual reproduction critically relies on the generation of haploid gametes following a specialized cell division process known as meiosis. Here, we demonstrate that N-6 Adenine-Specific DNA methyltransferase 1 (N6AMT1) plays a crucial role in the progression of meiosis during spermatogenesis, as follows. N6AMT1 was expressed in germ cells throughout the entire process of spermatogenesis, with a peak in mRNA levels in spermatocytes at the prophase I stage of meiosis. Germ cell-specific deletion of N6amt1 in mice resulted in male subfertility as well as a significant reduction in sperm count. Notably, N6amt1-null spermatocytes exhibited meiotic arrest at prophase I and extensive apoptosis. Chromosome spreading assays revealed that N6amt1 loss impaired meiotic sex chromosome inactivation (MSCI) and delayed DNA double-strand break (DSB) repair. Correspondingly, transcriptomic analysis identified a substantial increase in transcript levels for genes mapping to sex chromosomes in N6amt1-null mutants, consistent with disruptions in MSCI. Moreover, N6AMT1 deficiency led to a significant upregulation in the steady-state mRNA levels of genes involved in the p53 pathway and functionally activated p53 signaling. Through integrated analysis of data from single-cell RNA-seq and bulk RNA-seq experiments, we found that knockout of N6amt1 primarily affected the transcriptomic profiles of normal pachytene spermatocytes. Taken together, our findings demonstrate that N6AMT1 is required for quantitatively normal male fertility in mice and involved in the molecular mechanisms for meiotic progression during spermatogenesis, including MSCI and DSB repair.

CRISP2 is enriched in the male reproductive system of mammals and plays roles in spermatogenesis, sperm motility, and fertilization. Although extensively investigated in rodents and boars, human CRISP2 (hCRISP2) remains poorly studied, particularly concerning its localization in testicular and epididymal tissues and its molecular features. In this study, we used immunofluorescence to determine the localization of hCRISP2 in testis, epididymis, and ejaculated sperm. While no expression was observed in the epididymal epithelium, hCRISP2 was detected at different stages during spermatogenesis. Specifically, hCRISP2 was found in the nucleus of primary spermatocytes and of both round and early elongated spermatids. In elongated spermatids, it was additionally observed in the cytoplasm, the flagellum, and the equatorial segment of the acrosome (EqS). The presence of aggregated material with hCRISP2 immunoreactivity in the apical pole of Sertoli cells suggests that most of the hCRISP2 involved in spermatogenesis is phagocytized by these cells during spermiation. In ejaculated sperm, hCRISP2 was found in the cytoplasmic droplet, flagellum, and EqS, consistent with its described roles in sperm motility and gamete fusion. Native and SDS-PAGE combined with western blot analyses depicted the ability of hCRISP2 to form stable high molecular weight complexes and mass spectrometry revealed that these complexes likely consist exclusively of hCRISP2. Furthermore, we showed that hCRISP2 undergoes only limited post-translational modifications. These findings shed light into the dynamic localization of hCRISP2 throughout spermatogenesis and in ejaculated sperm, as well as its molecular features, enhancing our understanding of its pivotal functional roles and relevance for male fertility.

Oviducts contain various nutrients that provide energy during oocyte development. This study aimed to improve the efficiency of in vitro reproduction using extracellular vesicles (EVs) produced by the oviduct epithelial cells of sika deer (Cervus nippon). Surprisingly, the uptake of deer oviduct epithelial cell extracellular vesicles (DOEC-EVs) by cumulus-oocyte complexes, which were encapsulated by dense cumulus cells (CCs), occurred only in CCs during maturation. Therefore, we hypothesized that DOEC-EVs are transported to oocytes through CCs to exert their effects. We first investigated the effects of DOEC-EVs on the expansion capacity of the cumulus-oocyte complexes, as well as cell cycle progression, proliferation, apoptosis, and lactate and pyruvate levels in CCs, and examined reactive oxygen species levels, mitochondrial function, and key gene expression. The results showed that DOEC-EVs regulated cell cycle progression, promoted proliferation, reduced apoptosis, and improved antioxidant capacity and glycolysis, and through the oocyte first polar body excretion rate, reactive oxygen levels and mitochondrial membrane potential, it was shown that CC promoted in vitro oocyte maturation, improved the antioxidant capacity and mitochondrial function of oocytes, and promoted parthenogenetic embryo development. These results suggest that DOEC-EVs improve the efficiency of oocyte development in deer in vitro by acting on CCs, laying the foundation for further research on in vitro deer reproduction.

The effect of interferon tau (IFNT) on the uterus is critical for maternal recognition of pregnancy in ruminants, while its direct role in luteal function is less well understood. To address this, we performed two experiments. In experiment 1, cattle received intrauterine infusions of either: bovine serum albumin (BSA; vehicle) or vehicle with IFNT from day 14 to 16 of the estrous cycle or vehicle with IFNT from day 14 to 19 or vehicle with IFNT from day 14 to 19 with pregnancy associated glycoprotein (PAG) from day 17 to 19. Corpora lutea (CL) were collected on day 17 or 20 and RNAseq was performed. In experiment 2, cultured luteal steroidogenic cells from cyclic (day 10-12) cattle were treated with IFNT and RNAseq was performed. Treatment with IFNT resulted in luteal changes (in vivo: 130 transcripts; in vitro: 2981 transcripts), while addition of PAG resulted in 13 changed transcripts. Only 31% of the genes that changed in the CL during early pregnancy (Hughes et al., 2020) were regulated by IFNT; these were antiviral and immune regulators. In contrast, 50% of the genes that changed during early pregnancy were not regulated by IFNT and were associated with cellular proliferation and extracellular matrix organization. The remaining 19% of genes were not conclusively identified as either IFNT regulated or non-regulated. This suggests that the temporal changes in the CL during early pregnancy are only partially regulated by IFNT, drawing into question identities of other luteal regulators or the effect of age of CL.

Purpose: To investigate how steroid receptor RNA activator (SRA) regulates follicular development in mice.

Methods: Systemic SRA knockout mice were introduced. SRA expression was reinstated in the anteroventral periventricular nucleus (AVPV) of the hypothalamus using lentiviral vectors. Subsequently, the estrous cycle, serum hormone levels, follicle development, and hypothalamic kisspeptin expression in mice were assessed. Kiss1 promoter activity was tested with a fluorescent reporter system in Neuro-2a cells.

Results: SRA deficiency caused a shift to shorter metestrus and longer diestrus phases, reduced numbers of large antral and preovulatory follicles, increased formation of atretic cyst-like follicles, lowered serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2), and decreased expression of hypothalamic AVPV-kisspeptin in mice. The reinstatement of SRA expression in the AVPV nucleus normalized kisspeptin expression, hormone levels, and follicle development. In Neuro-2a cells, SRA increased Kiss1 transcription upon E2 treatment, a response that was nullified by the estrogen receptor alpha (ERα) inhibitor.

Conclusion: SRA enhances ERα-mediated Kiss1 transcription in the AVPV nucleus to control the kisspeptin-GPR54 system in hypothalamus, essential for regulating ovulation through the hypothalamus-pituitary-ovary axis.

Increasing number of pregnant women are consuming probiotics to promote their own health and that of their unborn fetus. Such supplements are presumed to be safe for pregnant mothers and their unborn offspring. For pregnant mothers, such bioactive compounds might lower the risk of constipation, diarrhea, other gastrointestinal conditions, pre-term birth, and prevent adverse pregnancy outcomes, including gestational diabetes mellitus (GDM) and depression/anxiety. More research is needed to examine potential safety of probiotic consumption during pregnancy and long-term health consequences to offspring. The conceptus can also be indirectly affected by maternal probiotic supplementation through microorganism production of bioactive compounds. The placenta is in direct communication with the underlying uterine tissue. Thus, compounds in the maternal blood can easily transfer across the placenta and impact this hormonally sensitive organ. Select studies suggest that disruptions to the maternal microbiome dramatically affects the placenta. In the current review, we will therefore consider the evidence to date of how maternal probiotic supplementation affects the placenta. Three potential mechanisms we will explore include the possibility that maternal probiotic supplementation might impact the putative placenta microbiome. The second potential mechanism we will consider is that maternal probiotic consumption alters bacterial-derived metabolites, including short-chained fatty acids, polyamines, Vitamin B9, and Vitamin B12. The third potential mechanism to be discussed is that such supplements affect maternal and placental immune responses. Before probiotics are promoted for healthy pregnant women and those with gestational disorders, more studies, including those examining the effects on the placenta, are essential.

We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38.5, and 73.3% respectively (9 h vs. 15 min or 3 h, P < 0.05). Fertilization rates differed significantly (45.9% vs. 85.5%) between freezing extenders. Blastocysts were produced using frozen-thawed semen from each of three stallions and transfer of 9 vitrified-warmed blastocysts to mares yielded 7 embryonic vesicles. Anti-protein tyrosine phosphorylation staining of the entire sperm tail increased over pre-incubation, and sperm both with and without staining in the tail bound to the oocyte cumulus after co-incubation. Using the stain DiSC3(5) and flow cytometric analysis, a population of apparently hyperpolarized sperm was identified at 22 h in fresh sperm that was not seen at any time in frozen-thawed sperm. We conclude that frozen-thawed equine sperm can successfully fertilize oocytes after a shortened pre-incubation time of 9 h, suggesting that the freeze-thawing process induces capacitation-related changes. Our findings on evaluation of pre-incubated sperm indicate that the mechanisms by which frozen-thawed sperm become capable of fertilization may differ from those found in fresh sperm.

The juvenile in vitro embryo transfer (JIVET) technology holds the potential to accelerate livestock breeding. However, its application is limited due to the weak in vitro development of oocytes and embryos from prepubertal lambs. To dissect the regulatory networks of gene expression of sheep in vitro embryos and identify the defects in gene expression in prepubertal lamb embryos during the oocyte-to-embryo transition (OET), full-length RNA sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS) based on trace cells were conducted on in vitro-derived embryos generated from adult sheep and prepubertal lamb oocytes. We found that the maternal mRNA degradation occurred selectively in adult sheep embryos in multiple waves and was most completed until the morula stage. Major embryonic genome activation (EGA) was found to occur at the morula stage. By comparing with the patterns of adult embryos, we observed incomplete maternal mRNA degradation and abnormal EGA in lamb embryos and analyzed their potential molecular mechanisms. Furthermore, we explored dynamic DNA methylation concerning the paternal and maternal genomes during the preimplantation development of sheep embryos, revealing the negative regulatory role of promoter DNA methylation on EGA process. Lamb embryos generally displayed higher DNA methylation levels than adults, potentially repressing the EGA gene expression, especially the genes associated with ribosomal and mitochondrial organization. We also found abnormalities in the methylation status of imprinted genes in lamb embryos. Our findings advance the understanding of sheep in vitro embryo development and offer insights for improving the JIVET technology in livestock.

Endometriosis is a common gynecological disorder, whose pathogenesis remains incompletely understood. Macrophages, a key type of immune cell, are pivotal in the context of endometriosis. This study seeks to explore the interactions between endometriotic cells and macrophages. Quantitative real-time PCR (qRT-PCR) and Western blot experiments were employed to detect phosphatase and tensin homolog (PTEN) expression. Glucose consumption, lactate production, extracellular acidification rate, and oxygen consumption rate levels were used to assess cellular glycolytic capacity. The interaction between conditioned media from ectopic endometrial stromal cells (EESCs) and macrophages was investigated through co-culture experiments. The expression of M2 macrophage marker proteins and inflammatory factors was detected via qRT-PCR, immunofluorescence staining, and enzyme-linked immunosorbent assay. Cellular functions were evaluated using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU), and wound healing assays. We found that PTEN deficiency promoted the glycolytic activity of EESCs. Simultaneously, it significantly promoted the macrophages' polarization toward the M2 phenotype, demonstrated by increased expression of M2 markers (differentiation 206 (CD206), CD163, and (C-C motif) ligand 22 (CCL22)). Further studies revealed that PTEN-deficient EESCs increased the level of CCL2 via promoting glycolytic activity, which was reversed by glycolytic inhibitor. Moreover, lactate and conditioned media from overexpressed CCL2 EESCs facilitated M2 polarization of macrophages, while 2-deoxy-d-glucose reversed the promoting effect. Furthermore, lactate-facilitated macrophages promoted the proliferation and migration abilities of EESCs. PTEN deficiency induces M2 macrophage polarization by promoting glycolytic activity in EESCs, which deepens the knowledge of the pathophysiology of endometriosis and provides novel insights into its treatment.