Biology of Reproduction最新文献

Dcaf17, also known as DDB1- and CUL4-associated factor 17, is a member of the DCAF family and acts as the receptor for the CRL4 ubiquitin E3 ligase complex. Several previous studies have reported that mutations in Dcaf17 cause Woodhouse-Sakati Syndrome (WSS), which results in oligoasthenoteratozoospermia (OAT) and male infertility. As a model to explore the role of Dcaf17 in the male reproductive system, we created Dcaf17-deficient male golden hamsters using CRISPR-Cas9 technology, the results of which demonstrate that deletion of Dcaf17 led to abnormal spermatogenesis and infertility. To uncover the underlying molecular mechanisms involved, we conducted single-cell RNA sequencing (scRNA-seq) analysis to evaluate the effect of Dcaf17 deficiency on transcriptional levels in spermatogenic cells during various stages of spermatogenesis. These data emphasize the significant regulatory role played by Dcaf17 in early spermatogenic cells, with many biological processes being affected, including spermatogenesis, and protein degradation. Dysregulation of genes associated with these functions ultimately leads to abnormalities. In summary, our findings highlight the critical function of Dcaf17 in spermatogenesis and male fertility and clarify the specific stage at which Dcaf17 exerts its effects, while simultaneously providing a novel animal model for the study of Dcaf17.

Trophoblast stem cells (TSCs), derived from the trophectoderm of the blastocyst, are used as an in vitro model to reveal the mechanisms underlying placentation in mammals. In humans, suitable culture conditions for TSC derivation have recently been established. The established human TSCs (hTSCs) differentiate efficiently toward two trophoblast subtypes: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). However, the efficiency of differentiation is lower in macaque TSCs than in hTSCs. Here, we demonstrate that the activation of Wnt signaling downregulated the expression of inhibitory G protein and induced trophoblastic lineage switching to the STB progenitor state. The treatment of macaque TSCs with a GSK-3 inhibitor, CHIR99021, upregulated STB progenitor markers and enhanced proliferation. Under the Wnt signaling-activated conditions, macaque TSCs effectively differentiated to STBs upon dbcAMP and forskolin treatment. RNA-seq analyses revealed the downregulation of inhibitory G protein, which may make macaque TSCs responsive to forskolin. Interestingly, this lineage switching appeared to be reversible as the macaque TSCs lost responsiveness to forskolin upon the removal of CHIR99021. The ability to regulate the direction of macaque TSC differentiation would be advantageous in elucidating the mechanisms underlying placentation in non-human primates.

This study aimed to understand the physiological mechanisms regulating parturition and to identify potential biomarkers to predict onset of birth. Additionally, we compared hormone profiles between cows with shorter and longer gestation lengths. Twenty-eight days before due date until 3d postpartum, cows (n = 18) were blood sampled daily. Circulating concentrations were measured for progesterone (P4) and estradiol (E2) by RIA, testosterone, prostaglandin F2α metabolite (PGFM), cortisol, pregnancy-specific protein B (PSPB) by ELISA and lactate concentrations by colorimetric assay. At end of gestation, P4 decreased from d-14 to d-4 (from 3.6 to 1.4 ng/mL), most likely from rapid loss of placental P4 production (64% of decline in 24 h). A second rapid decrease in P4 to undetectable concentrations was observed from d-2 to parturition (from 1.4 to 0.1 ng/ml; most likely luteal origin) corresponding to increase in PGFM from d-2 to parturition (249.7 to 2868.4 pg/mL). Estradiol and PSPB increased ~8-fold from ~13d before parturition with acute rise in E2 but not PSPB (45% vs 13% in first 24 h). Testosterone decreased slightly during the same period. Cortisol and lactate increased only at calving. Comparison of cows with shorter vs longer gestation, when data were normalized to parturition day, a difference was detected in circulating E2 and PGFM patterns, but not P4 and PSPB. Thus, the first significant hormonal changes associated with parturition begin at d-14 with E2 and PSPB as two clear biomarkers of impending parturition. Cows with shorter and longer gestation had hormonal differences indicative of identifiable earlier placental maturation.

The accurate diagnosis of non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) is crucial for selecting appropriate clinical treatments. This study aimed to investigate the pivotal role of miRNAs in circulating plasma extracellular vesicles (EVs) in distinguishing between NOA and OA, as well as uncovering the signaling pathways involved in azoospermia pathogenesis. In this study, differential expression of EV miR-513c-5p and miR-202-5p was observed between NOA and OA patients, while the selenocompound metabolism pathway could be affected in azoospermia through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The predictive power of these microRNAs was evaluated using ROC-AUC analysis, demonstrating promising sensitivity, specificity, and area under the curve values. A binomial regression equation incorporating circulating plasma levels of EVs miR-202-5p and miR-513c-5p along with follicle-stimulating hormone was calculated to provide a clinically applicable method for diagnosing NOA and OA. This study presents a potentially non-invasive testing approach for distinguishing between NOA and OA, offering a possibly valuable tool for clinical practice.

Mammalian preimplantation development culminates in the formation of a blastocyst which undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive ICM cells, a lack of Fgf4 expression and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.

Sperm maturation depends on exposure to specific microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles (EVs) could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the EVs from different regions of the epididymis can serve as a form of paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to EVs collected from upstream (i.e. caput) segments. The impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that EVs from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to EV supplementation). Of note, expression of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were regulated by the presence of EVs. Together, our findings comprise the first report of paracrine control of segmental gene regulation by epididymal EVs in any species. These results contribute to a better understanding of epididymis biology and could lead to techniques to enhance or suppress male fertility.

Environmental pollution is an inevitable ecological issue accompanying the process of socialization, with increasing attention to its impacts on individual organisms and ecological chains. The reproductive system, responsible for transmitting genetic material in animals, is one of the most sensitive systems to environmental toxins. Research reveals that Sertoli cells are the primary target cells for the action of environmental toxins. Different environmental toxins mostly affect the blood-testis barrier and lead to male reproductive disorders by disrupting Sertoli cells. Therefore, this article provides an in-depth exploration of the toxic mechanisms of various types of environmental toxins on the male testes. It reveals the dynamic processes of tight junctions in the blood-testis barrier affected by environmental toxins and their specific roles in the reconstruction process.

Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes.

Prior studies showed that mice deficient in the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in synthesis of the thiol antioxidant glutathione (GSH), have decreased ovarian GSH concentrations, chronic ovarian oxidative stress, poor oocyte quality resulting in early preimplantation embryonic mortality and decreased litter size, and accelerated age-related decline in ovarian follicle numbers. Global deficiency of the catalytic subunit of this enzyme, Gclc, is embryonic lethal. We tested the hypothesis that granulosa cell- or oocyte-specific deletion of Gclc recapitulates the female reproductive phenotype of global Gclm deficiency. We deleted Gclc in granulosa cells or oocytes of growing follicles using Gclc floxed transgenic mice paired with Amhr2-Cre or Zp3-Cre alleles respectively. We discovered that granulosa cell-specific deletion of Gclc in Amhr2Cre;Gclc(f/-) mice recapitulates the decreased litter size observed in Gclm-/- mice, but does not recapitulate the accelerated age-related decline in ovarian follicles observed in Gclm-/- mice. In addition to having lower GSH concentrations in granulosa cells, Amhr2Cre;Gclc(f/-) mice also had decreased GSH concentrations in oocytes. By contrast, oocyte-specific deletion of Gclc in Zp3Cre;Gclc(f/-) mice did not affect litter size or accelerate the age-related decline in follicle numbers, and these mice did not have decreased oocyte GSH concentrations, consistent with transport of GSH between cells via gap junctions. The results suggest that GSH deficiency at earlier stages of follicle development may be required to generate the accelerated follicle depletion phenotype observed in global Gclm null mice.