Biology of Reproduction最新文献

The movement of ions, such as calcium and zinc, is a well-known regulator of oocyte maturation and fertilization. The specific contribution of potassium ions is less well understood. We tested whether acute disruption of K+ homeostasis perturbs oocyte maturation. A preliminary screen identified PA-6 (IK1/KIR2.x inhibitor) as a robust inhibitor of oocyte maturation. We compared effects of PA-6 to Valinomycin (K+ ionophore) and tetraethylammonium chloride (TEAC; broad voltage-gated K+ channel blocker). PA-6 and Valinomycin completely prevented germinal vesicle breakdown (GVBD) and polar body extrusion. The nuclear envelope remained intact but with the chromatin stretching across the nucleus (PA-6) or remained semi condensed in the nucleus (Valinomycin). TEAC permitted GVBD but 66% were arrested at MI, and both MI and MII spindles were abnormal. Thallium-based flux assays showed higher K+ transport in GV than MII oocytes with PA-6 increasing transport in GV only. Valinomycin reduced transport in MII, but not GV oocytes. TEAC enhanced transport in both stages with delayed onset at GV. PA-6 did not elevate cAMP, while Valinomycin and TEAC modestly increased cAMP. Only PA-6 increased mitochondrial membrane potential, while only Valinomycin increased plasma membrane potential. Both PA-6 and Valinomycin blunted depolarization-induced Zn2+ accumulation. Surprisingly, KCNJ2/4/12 transcripts were detected in cumulus oocyte complexes but not in denuded oocytes. However, KCNJX immunoreactivity was present in oocytes, indicating uncertainty about the PA-6 targets in oocytes. Collectively, these data identify K+ flux as a central regulator of oocyte maturation that integrates plasma membrane excitability, organelle function, chromatin remodeling, spindle integrity, and zinc dynamics.

To investigate how copper deficiency during pregnancy affects placental structure, metabolism, and trophoblast function, contributing to fetal growth restriction (FGR). Pregnant C57BL/6N mice were treated with ammonium tetrathiomolybdate to induce copper deficiency, with two different dosages (30 and 60 mg·kg-1·d-1) administered daily from gestational day 1 to day 14. On day 15, assessments were made on fetal growth, placental development, and spatial metabolomics. In parallel, trophoblast cells (HTR8/SVneo) were subjected to copper chelation or SLC31A1 knockdown to model copper deficiency in vitro. Cell invasiveness and proliferation were evaluated using appropriate assays, along with the measurement of molecular markers to assess the impact of copper deficiency. Copper deficiency significantly reduced maternal serum copper levels, leading to FGR, as evidenced by shorter crown-rump lengths, lower fetal weights, and altered fetal-to-placental weight ratios. Structural abnormalities in the placental junctional zone, including reduced size and altered morphology, were observed. Metabolomic analysis revealed disrupted lipid metabolism, with alterations in glycerophospholipids and fatty acids, and lipid droplet accumulation. Copper deficiency impaired trophoblast migration and invasion, linked to decreased MMP2 and MMP9 expression in vivo and in vitro. In vitro studies also showed altered lipid metabolism in SLC31A1-knockdown trophoblast cells. Copper deficiency disrupts placental structure and lipid metabolism, impairs trophoblast function, and contributes to FGR, highlighting the critical role of copper in fetal development and maternal health.

Estrogen stimulates corticotropin-releasing hormone (CRH) expression in endometrial tissue, and CRH signaling contributes to inflammation and pain, suggesting a mechanistic role in endometriosis pathophysiology. We previously identified elevated CRH receptor-1 (CRHR1) levels in endometriotic lesions in a rat model. Here, we tested the hypothesis that antagonizing CRHR1 with pexacerfont, an orally bioavailable small-molecule antagonist, would reduce endometriosis-associated pain and lesion activity. Beginning 25 days after disease induction, rats received pexacerfont (10 mg/kg), vehicle, or elagolix (14 mg/kg) in 7-day treatment cycles separated by 7-day drug-free intervals up to day 60 after endometriosis onset. Pexacerfont significantly reduced mechanical and inflammatory pain (34% and 38% decreases, respectively) and decreased nerve growth factor, vascular endothelial growth factor, Ki67, and IL-6 and TNF-α mRNA expression within lesions. Unlike elagolix, pexacerfont did not alter peritoneal leptin-to-weight or serum FSH-to-LH ratios, indicating minimal disruption of gonadal endocrine signaling. Notably, pexacerfont reduced macroscopic adhesion scores by half, a therapeutic effect not observed with either vehicle or elagolix. CRHR1 antagonism, therefore, suppressed neuro-inflammatory and proliferative pathways while limiting lesion-associated angiogenesis and postoperative adhesion development. These findings position CRHR1 as a mechanistically distinct, non-hormonal therapeutic target for endometriosis and highlight pexacerfont's potential to reduce both pain and adhesions.

Post-ovulatory supplementation of progesterone (P4) to cows has been associated with enhanced conceptus elongation. The aim of the present study was to investigate the impact of P4 supplementation on peri-implantation conceptus signaling including the secretion of interferon tau (IFNT), the maternal response to IFNT (mRNA abundance of interferon-stimulated genes [ISG; ISG15 and MX2]), and circulating pregnancy-associated glycoproteins (PAGs). Two cohorts (A and B) of primiparous Angus beef cows were synchronized to estrus, artificially inseminated (d0), and assigned randomly to receive intravaginal P4 device (CIDR, 1.38 mg) from d3 to d12 or remain as untreated controls. Blood and cervical cytobrush were collected from d3 to d24 and a uterine cytobrush was collected on d24 from Cohort A to assess circulating concentrations of P4, PAG, mRNA abundance of ISG, and concentrations of IFNT, respectively. Blood and cytobrush (cervical and uterine) were collected from Cohort B on d18 to assess circulating concentrations of P4, mRNA abundance of ISG, and concentrations of IFNT, respectively. The mRNA abundance of ISG was assessed in luteal biopsies on d18 and d22. There was no effect of P4 supplementation on concentrations of IFNT in the uterus on d18. There was no effect of supplemental P4 on mRNA abundance or timing of ISG in endometrial, cervical, or luteal tissue. The earlier detection of PAG in circulation and lower uterine fluid concentrations of IFNT on d24 in P4-supplemented cows suggests advanced trophoblast differentiation and enhanced conceptus development beyond previously reported advances in conceptus elongation alone.

Several zona pellucida 3 (Zp3)-Cre driver mouse lines are used to enable conditional loss-of-function studies in oocytes. The Knowles Zp3-Cre line, currently maintained live at the Jackson Laboratory, is the most widely published. We recently found that the transgene expressed in the Knowles line contains a truncated metallothionein 1 (Mt1) sequence that is expressed at high levels in oocytes from transgenic mice. This finding led us to search for an alternative Zp3-Cre line that did not express an exogenous Mt1 transcript. We tested a second transgenic Zp3-Cre line and then created our own transgenic Zp3-Cre line, neither of which was oocyte-specific as documented by crossing to the tdTomato reporter line. Similar testing confirmed the exquisite oocyte specificity of the Knowles Zp3-Cre line. An alternative to Zp3-Cre is to use the growth differentiation factor 9 (Gdf9)-iCre line, which is reported to be oocyte-specific and expressed beginning at the primordial follicle stage. This line similarly showed high somatic tissue Cre expression. Reasoning that a knock-in approach would ensure oocyte specificity, we used a CRISPR/Cas9 approach to insert Cre into the endogenous Zp3-Cre locus. However, crosses of this knock-in line with tdTomato females revealed high somatic tissue expression. Both the Zp3-Cre knock-in and Gdf9-iCre alleles, when paternally inherited, induced Cre expression by the blastocyst stage, explaining the broad tissue distribution. We conclude that the Knowles Zp3-Cre transgenic line remains the best model for generating oocyte-specific expression, though investigators should be aware of the spurious Mt1 expression from the transgene.

Davies and colleagues provide a new perspective on maternal recognition of pregnancy in ruminants, challenging long-held assumptions and stimulating debate that will ultimately advance our understanding of pregnancy recognition.

In recent years, the study of bovine sperm epigenetics has garnered increasing attention alongside research on biomarkers associated with dairy cattle fertility. Male gametes not only transmit the paternal haploid genome to the offspring through fertilization, but also convey epigenetic components, such as DNA methylation, small noncoding RNA, histone variants, and histone modifications to offspring. This epigenetic information may transmit an acquired phenotype leading to intergenerational inheritance. The ongoing worldwide decline in dairy herd fertility affecting both males and females causes significant economic losses for dairy farmers. Previous scientific efforts to address this issue primarily targeted genetic aspects, identifying numerous fertility-related quantitative trait locus and single-nucleotide polymorphisms. However, since fertility is influenced by genetic, epigenetic, and environmental factors, this review highlights the importance of identifying sperm epigenetic markers as additional tools for evaluating and predicting cattle fertility. Identifying bovine sperm epigenetic markers is thus urgently needed to enhance fertility assessment and mitigate global dairy herd decline and economic losses.

We hypothesized that in dairy cattle maternal energy restriction applied during two gestational windows (up to day 80 or 120 of gestation) impairs ovarian and cardiovascular development in juvenile female offspring. We also investigated the role of maternal leptin and testosterone in developmental programming in calves. Holstein-Friesian heifers were randomly assigned to one of three experimental groups; starting 10 days before artificial insemination, they were individually fed at (i) 0.6 of their maintenance energy requirements (M) up to day 80 (Nutrient Restricted, NR80) or (ii) day 120 of gestation (NR120); (iii) 1.8 M until day 120 of pregnancy (Control). Plasma leptin concentrations increased transiently in nutritionally restricted heifers pregnant with a single female calf, but maternal testosterone concentrations were not influenced by diet. Calves had similar body growth, but daughters of NR80 and NR120 had impaired ovarian development, as assessed by reduced gonadal weight, fewer surface antral and primary follicles, and recovered cumulus-oocyte complexes, as well as lower circulating anti-Müllerian hormone concentrations. Cardiovascular morphology and function in the offspring were not influenced by maternal diet, as determined by peripheral arterial blood pressure, echocardiography, post-mortem heart weight, and aortic circumference. Regardless of its duration (until day 80 or 120 of gestation), nutritional restriction resulted in a similar alteration of ovarian development in juvenile progeny, but cardiovascular development was unaltered. Evidence suggests that the window of development that encompasses the peri-ovulatory period to the first 2.6 months of gestation is critical in ovarian programming and that maternal leptin may be involved.

Sturgeons (order Acipenseriformes) represent one of the most ancient lineages of Actinopterygii. Presently, most sturgeon species are classified as critically endangered. In this study, a novel ovarian cell line which contained germline cells derived from the sterlet (Acipenser ruthenus) was established, designated as the ARO cell line. Germline cells in the cell line were identified using marker gene analysis, alkaline phosphatase activity assay and immunofluorescence staining. Transmission electron microscope observation showed that previtellogenic oocytes existed in the ARO cell line. Flow cytometry analysis indicated the cell line maintained meiotic competence to generate DNA reduced ploidy (diploid) oocytes. Under three-dimensional culture condition, germinal vesicle stage, germinal vesicle breakdown stage and meiosis I oocytes were found. Furthermore, the cell line exhibited sustained proliferation and self-organization into reconstituted ovaries (rOvaries). Transcriptome analysis of rOvaries revealed that rOvaries had formed cell junctions and normal intercellular signaling, and possessed normal endocrine function and oocyte-producing capacity. In summary, the established ARO cell line exhibited stable passaging over multiple generations in vitro, was capable of sustainably generating oocytes, and was expected to be applicable to the establishment of ovarian organoids.