Oxidative-stress-induced apoptosis of granulosa cells is considered to be a main driver of follicular atresia. Increasing evidence suggests a protective effect of melatonin against oxidative damage but the mechanism remains unclear. The aim of this study is to investigate the effects of melatonin on mitophagy and apoptosis of bovine ovarian granulosa cells under oxidative stress, and to clarify the mechanism. Our results indicate that melatonin inhibited H₂O₂-induced apoptosis and mitochondrial injury of bovine ovarian granulosa cells, as revealed by decreased apoptosis rate, reactive oxygen species (ROS) levels, Ca²⁺ concentration, and cytochrome C release and increased mitochondrial membrane potential (ΔΨm). Simultaneously, melatonin promoted mitophagy of bovine ovarian granulosa cells through increasing the expression of PTEN-induced putative kinase 1 (PINK1), PARKIN, BECLIN1, and LC3II/LC3I; decreasing the expression of sequestosome 1 (SQSMT1); and promoting mitophagosome and lysosome fusion. After treatment with a mitophagy inhibitor CsA, we found that melatonin alleviated apoptosis and mitochondrial injury through promoting mitophagy in bovine ovarian granulosa cells. Furthermore, melatonin promoted the expression of silent information regulator 1 (SIRT1) and decreased the expression level of forkhead transcription factors class O (type1) (FoxO1). By treatment with an SIRT1 inhibitor EX527 or FoxO1 overexpression, the promotion of melatonin on mitophagy as well as the inhibition on mitochondrial injury and apoptosis were reversed in bovine ovarian granulosa cells. In conclusion, our results suggest that melatonin could promote mitophagy to attenuate oxidative-stress-induced apoptosis and mitochondrial injury of bovine ovarian granulosa cells via the SIRT1/FoxO1 signaling pathway.

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O₂⁻) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m⁶A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m⁶A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m⁶A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m⁶A modification.

Melatonin has been proved to be involved in testosterone synthesis, but whether melatonin participates in testosterone synthesis by regulating miRNA in Leydig cells is still unclear. The purpose of this study is to clarify the mechanism of melatonin on Leydig cells testosterone synthesis from the perspective of miRNA. Our results showed that melatonin could significantly inhibit testosterone synthesis in rooster Leydig cells. miR-7481-3p and CXCL14 were selected as the target of melatonin based on RNA-seq and miRNA sequencing. The results of dual-luciferase reporter assays showed that miR-7481-3p targeted the 3′-UTR of CXCL14. The overexpression of miR-7481-3p significantly inhibited the expression of CXCL14 and restored the inhibitory role of melatonin testosterone synthesis and the expression of StAR, CYP11A1, and 3β-HSD in rooster Leydig cells. Similarly, interference with CXCL14 could reverse the inhibitory effect of melatonin on the level of testosterone synthesis and the expression of StAR, CYP11A1, and 3β-HSD in rooster Leydig cells. The RNA-seq results showed that melatonin could activate the PI3K/AKT signal pathway. Interference with CXCL14 significantly inhibited the phosphorylation level of PI3K and AKT, and the inhibited PI3K/AKT signal pathway could reverse the inhibitory effect of CXCL14 on testosterone synthesis and the expression of StAR, CYP11A1 and 3β-HSD in rooster Leydig cells. Our results indicated that melatonin inhibits testosterone synthesis by targeting miR-7481-3p/CXCL14 and inhibiting the PI3K/AKT pathway.

Mercury, a prevalent heavy metal, negatively impacts oocyte maturation. However, the exact mechanism by which methylmercury chloride (MMC) affects this process remains elusive. The present study found that MMC administration triggered meiotic failure in oocytes by disrupting cumulus cell expansion, leading to compromised spindle apparatus and altered chromosomal architecture, which are crucial for oocyte development. This disruption is characterized by abnormal microtubule organization and defective chromosome alignment. Additionally, MMC exposure caused oxidative stress-induced apoptosis due to mitochondrial dysfunction, as indicated by decreased mitochondrial membrane potential, mitochondrial content, mitochondrial DNA copy number, and adenosine triphosphate levels. Proteomic analysis identified 97 differentially expressed proteins, including P62, an autophagy marker. Our results confirmed that MMC induced autophagy, particularly through the hyperactivation of the mitochondrial autophagy to remove damaged and normal mitochondria. The mitochondrial reactive oxygen species (ROS) scavenger Mito-TEMPO alleviated oxidative stress and mitochondrial autophagy levels, suggesting that mitochondrial ROS initiates this autophagic response. Notably, MMC activates mitochondrial autophagy via the monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signal pathway due to mitochondrial dysfunction. In vivo studies in mice revealed that MMC exposure decreased reproductive performance, attributed to excessive mitochondrial autophagy leading to reduced oocyte quality. Overall, these findings demonstrate that MMC exposure impairs oocyte maturation via the hyperactivation of mitochondrial autophagy induced by mitochondrial dysfunction.

Microbiota in the reproductive tract of cattle play a vital role in maintaining normal reproduction. However, the information on microbiota in different parts of reproductive tracts with different genetic background is few. The aim of the present study was to describe and compare the microbiota in vagina, cervix and uterus of Yanbian cattle and Yanhuang cattle. The results showed that microbial diversity increases from the vagina to the uterus. The top three bacterial phyla in bovine reproductive tract were Proteobacteria, Firmicutes and Bacteroidetes, accounting for more than 85%. From the vagina to the uterus, the relative abundance of Proteobacteria gradually decreased, while that of Firmicutes gradually increased. Phylum-level Firmicutes and genus-level UCG_010 were significantly enriched in the uterus of Yanbian cattle and Yanhuang cattle. Comparing the same parts of the two breeds, it was found that there was no significant difference in alpha diversity, but significant differences in beta diversity. In addition, microbiota with significant differences in the relative abundance of the reproductive tract were found. These findings lay a foundation for a comprehensive understanding of the structure of the genital tract microbiota of cows and its regulatory mechanisms.

Dimethyl sulfoxide (DMSO) is widely utilized in the vitrification of oocytes, but DMSO exhibits concentration-dependent toxicity, which can compromise oocyte developmental potential by disrupting key cellular processes. This study reports the first successful use of cold shock protein B (CspB protein) as a substitute for DMSO in vitrification solutions for oocyte vitrification. Combining dynamics simulations and experimental validation, we demonstrated CspB’s ability to inhibit ice crystallization and recrystallization by stabilizing its position at the ice–water interface and reducing ice formation rates. Recombinant CspB was successfully expressed and shown to bind to the oolemma. In vitrification solutions, CspB (1–2 mg/mL) effectively reduced ice crystal size and enabled a significant reduction or complete replacement of DMSO. This strategy markedly improved the post-thaw survival rates of both mouse and bovine metaphase II (MII) oocytes. Furthermore, oocytes vitrified with an optimized formulation (15% ethylene glycol + 2 mg/mL CspB) exhibited developmental competence (cleavage and blastocyst rates), oxidative stress markers (ROS, GSH), mitochondrial function (membrane potential and content), and apoptosis levels (Caspase-3/9) comparable to those treated with a standard DMSO-containing system. Transcriptomic analysis revealed that CspB’s cryoprotection involves the modulation of the mTOR signaling pathway. This role was functionally confirmed, as activation of mTOR abolished CspB’s beneficial effects, reinstating oxidative damage, mitochondrial dysfunction, and apoptosis. Thus, the CspB protein replaces DMSO with direct ice crystal formation suppression and mTOR-mediated oxidative stress regulation. This study offers a protein-based alternative to conventional permeable cryoprotectants. This approach holds promise for improving reproductive biotechnologies across species.