Molecular Reproduction and Development最新文献

Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic secretome, and EVs were subsequently harvested. In vitro assays demonstrated that PGE2-preconditioned ET-eMSCs and their EVs inhibited α-smooth muscle actin expression, reduced collagen I deposition, and restored key endometrial markers of receptivity and proliferation. These findings establish the first evidence that PGE2-enhanced ET-eMSC-derived EVs can exert anti-fibrotic effects in an in vitro model of equine endometrial fibrosis. This study provides a robust translational framework for developing targeted regenerative therapies for fibrotic diseases across species, with potential applications in human reproductive medicine.

Phospholipase D2 (PLD2) modulates cytoskeletal dynamics and membrane trafficking processes by converting phosphatidylcholine (PC) into phosphatidic acid (PA) and choline within somatic cells. Nonetheless, the role in oocyte meiosis remains largely unknown. Here, we demonstrate that PLD2 is selectively targeted to the meiotic spindle in mouse oocytes. The knockdown of PLD2 via the specific morpholino oligonucleotides (MOs) or its inhibition with VU 0364739 led to a marked increase in α-tubulin acetylation and induced a meiotic arrest at metaphase I (MI), accompanied by misaligned chromosomes. These defects were effectively rescued by the ectopic expression of Pld2 complementary RNA (cRNA). Furthermore, our findings implicate the Pld2 MO-induced alterations in the AKT-GSK3-Tau signaling cascade in oocytes. The overexpression of a gain-of-function GSK3β mutant (GSK3β^S9A) and a Tau-phosphorylation-enhancing mutant (Tau^P301L) substantially reversed the increased microtubule acetylation and the reduced rate of the first polar body extrusion (PBE) in oocytes lacking PLD2 activity. Additionally, the co-immunoprecipitation revealed a direct physical interaction between PLD2, GSK3β, and Tau in mouse oocytes. Together, PLD2 finely regulates α-tubulin acetylation through the modulation of the AKT-GSK3β-Tau signaling axis, thereby preserving an optimal microtubule dynamic equilibrium and ensuring the fidelity of the spindle apparatus function during oocyte meiosis.

There is a small number of studies describing the uterine biology of Bos indicus cows. We hypothesized that there is a transcriptional signature in endometrial epithelial cells 4 days after estrus (D4) that predicts the ability of the cow to remain pregnant/artificial insemination (AI). Brahman cows were submitted to an estrous synchronization protocol and AI. On D4 cows were submitted to endometrial cytology. Pregnancy/AI was diagnosed on day 30 and endometrial cytology samples were submitted to RNA-seq (n = 32 nonpregnant and n = 32 pregnant). Based on RNA-seq we performed targeted analysis using pathways previously reported in the literature and untargeted analysis using Ingenuity Pathway Analysis. A total of 975 genes were significantly associated (p ≤ 0.1) with pregnancy/AI, 64.9% of them (620/975) showed a negative association. Targeted and untargeted analysis showed a downregulation of Th2 immune response and activation of cholesterol biosynthesis in pregnant cows. Prediction analysis resulted in greater accuracy for the targeted transcripts than the whole transcriptome (0.91 vs. 0.86) but reduced precision (0.64 vs. 0.74). In conclusion, the endometrial receptivity was predominantly marked by an overall reduction in the molecular response. Th2 response and cholesterol biosynthesis are promising pathways to understand uterine biology, and the use of a set of genes rather than a single gene appears to be the future for prediction of pregnancy in bovine.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that play roles in transposon regulation and gene silencing. Follicular atresia, a process involving granulosa cell (GC) apoptosis, is tightly linked to female reproductive efficiency. Previous studies suggested that piR-23 is differentially expressed in porcine healthy (HF) and early atretic (AF) antral follicles, while prostaglandin-endoperoxide synthase 2 (PTGS2), a key enzyme in prostaglandin biosynthesis, may be a potential target of piR-23. This study investigated whether piR-23 regulates GC apoptosis by targeting PTGS2. Porcine GCs were isolated from HF and AF. piR-23 mimics/inhibitors and PTGS2 siRNA were transfected into GCs to assess cell apoptosis via Annexin V-FITC/PI and CCK-8 assays. Dual-luciferase reporter assays validated the targeting of PTGS2 by piR-23, while qRT-PCR and Western blot analyzed PTGS2 expression. piR-23 expression was downregulated in AF GCs. Overexpression of piR-23 significantly reduced GC apoptosis, whereas inhibition of piR-23 promoted apoptosis. PTGS2 expression was upregulated in AF GCs, and its knockdown suppressed GC apoptosis. Dual-luciferase assays showed that piR-23 directly bound to the 3′UTR of PTGS2, reducing its mRNA and protein levels. Cotransfection of piR-23 inhibitor and PTGS2 siRNA reversed the proapoptotic effect of piR-23 inhibition, confirming PTGS2 as a functional target. piR-23 acts as an antiapoptotic regulator in porcine GCs by directly targeting PTGS2. This finding unveils a novel piRNA-mediated regulatory mechanism in porcine follicular atresia.

This study investigated the effect of Bushen Shengjing Decoction (BSSJD) to modulate SCF/c-kit signaling pathway-mediated apoptosis on spermatogenesis in rats with oligoasthenospermia (OAS). The glucoside of Tripterygium wilfordii Hook F was given by gavage administration for OAS modeling in rats. The modeled rats were administered by gavage with BSSJD at different doses. Blood was collected from the abdominal aorta. The effect of BSSJD on OAS rats was determined by measuring serum testosterone (T), estradiol (E2) and luteinizing hormone (LH), epididymal index and testicular index, and sperm concentration and viability. Pathological changes in testicular tissues were observed. Caspase-3, Bax and Bcl-2 mRNA levels, along with SCF and c-kit levels in the testis tissues of rats were measured. BSSJD improved sperm concentration and quality, increased T and E2 levels, and reduced LH levels in rats with OAS. BSSJD alleviated apoptosis by decreasing Bax and caspase-3 and increasing Bcl-2 in testicular tissues of rats with OAS. Isck03 reversed the improvement of spermatogenesis and apoptosis in OAS rats treated with BSSJD. BSSJD improves spermatogenic function in OAS rats, which may act by regulating SCF/c-kit signaling pathway-mediated apoptosis.

This study investigated IZUMO1, IZUMO2, and IZUMO4 expression, localization, and participation in sperm-oocyte interaction, combining standard biochemical (Western immunoblotting and fluorescence immunocytochemistry) and functional (CPA, HZA, SPA) with computational biology approaches (bioinformatics, protein's 3D-structure modelling). Human IZUMO1, IZUMO2, and IZUMO4 transcripts were found to be testis-enriched (HPA), and expressed since puberty (MeDAS). IZUMO2 and IZUMO4 transcripts levels were lower (p < 0.05) in teratozoospermic men (GEO). Human protein forms of ~40 kDa (IZUMO1), ~23 KDa (IZUMO2) and ~25 KDa (IZUMO4) were specifically immunodetected in sperm extracts. IZUMO proteins were immunolocalized in the acrosomal region of acrosome-intact and acrosome-reacted cells. Sperm pre-incubation with anti-IZUMO antibodies resulted in lower CPA rates using anti-IZUMO4 antibodies, lower HZA rates using anti-IZUMO1, anti-IZUMO2, or anti-IZUMO4 antibodies, and lower SPA rates using anti-IZUMO1 or anti-IZUMO4 antibodies (p < 0.05). IZUMO1 and IZUMO4 were immunodetected in mouse sperm, and antibodies also blocked homologous fertilization. Protein modeling using AlphaFold identified potential antigenic regions recognized by the antibodies used in biological assays, which impaired IZUMO1-JUNO and IZUMO4-JUNO protein interactions. This is the first study that reports human IZUMO1, IZUMO2, and IZUMO4 expression/localization and involvement in gamete interaction. Findings presented enhance our understanding of the molecular interactions leading to fertilization and may contribute to male infertility diagnosis and treatment.