Molecular Reproduction and Development最新文献

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.

Enhancing the developmental competence and quality of oocytes cultured in vitro remains challenging, in part due to limited understanding of the structural and molecular regulators of oocyte maturation. Microtubules, as key components of the cytoskeleton, are essential for chromosome segregation and meiotic progression. Tubulin beta 1 class VI (TUBB1) plays crucial roles in cellular stability, yet its involvement in regulating endoplasmic reticulum (ER) stress and mitochondrial function in oocytes via immune checkpoint pathways remains unclear. This study aimed to investigate the role of TUBB1 in regulating ER stress and mitochondrial integrity in oocytes via the transforming growth factor-β1 (TGF-β1)/SMAD family member 3 (Smad3) pathway, and its potential implications for improving assisted reproductive technologies. First, oocytes isolated from ICR female mice were subjected to oxidative stress using hydrogen peroxide (H₂O₂) to investigate TUBB1 expression. Next, lentiviral vectors and a TGF-β1/Smad3 activator (SRI-011381 hydrochloride) were utilized to modulate pathway activity. ER stress, mitochondrial integrity, oxidative stress, and apoptosis were subsequently assessed. H₂O₂ exposure significantly reduced TUBB1 expression, confirming its susceptibility to oxidative stress. TUBB1 silencing exacerbated ER stress (elevated Bax, GRP78, Caspase-12; decreased Bcl-2), increased apoptosis rates, and caused mitochondrial dysfunction evidenced by disrupted mitochondrial morphology; increased Ca²⁺ levels, higher ROS and MDA, and lower SOD activity. Conversely, TUBB1 overexpression or direct activation of the TGF-β1/Smad3 pathway reversed these detrimental effects, significantly reducing apoptosis, normalizing mitochondrial distribution, and alleviating ER and oxidative stress. Furthermore, activation of the TGF-β1/Smad3 pathway in TUBB1-silenced oocytes (rescue group) effectively restored mitochondrial and ER stress parameters to normal levels. Our findings demonstrate that TUBB1 plays a critical role in modulating ER and mitochondrial stress responses in oocytes via the TGF-β1/Smad3 signaling pathway. Targeted regulation of TUBB1 may serve as a promising therapeutic approach to improve oocyte quality in assisted reproductive technologies.

Although gamma-aminobutyric acid (GABA) is known to play a role in the regulation of several physiological functions in vertebrates, the role of GABA in regulating the reproductive axis in viviparous teleosts remains opaque. This study examined the effects of GABA_A and GABA_B receptor agonists, muscimol (MUSC) and baclofen (BAC), on the hypothalamic–pituitary–ovarian (HPO) axis in the black molly Poecilia sphenops. A dose-dependent upregulation of gonadotropin-releasing hormone-I (GnRH-I) mRNA expression in the preoptic hypothalamus was observed in 0.5 and 5 µg MUSC-treated fish compared to experimental controls; however, a significant upregulation of luteinizing hormone-β (LH-β) mRNA expression was observed only at the highest dose. A similar stimulatory effect on GnRH-I mRNA expression was observed in the 5, 10, or 50 µg BAC-treated group compared to experimental controls, but no significant change was observed in LH-β mRNA level. Furthermore, treatment with 0.5 or 5 µg MUSC stimulated the development of all stages of follicles in the ovary except for Stages IV or III, respectively. On the other hand, treatment with 50 µg BAC caused a significant increase in the numbers of Stages I–V follicles. These results were concomitant with a significant increase in some of the embryonic developmental stages and fecundity levels in 0.5 or 5 µg MUSC-treated or 50 µg BAC-treated fish compared to those of experimental controls. Together, these findings suggest that GABA_A and GABA_B receptor agonists produce a stimulatory effect on the HPO axis in a receptor-specific and concentration-dependent manner for the first time in a viviparous teleost.