The purpose of the present study is to examine the effects of melatonin on apoptosis and oxidative stress in mouse Leydig cells and to elucidate the mechanisms responsible for these effects. Our results indicated that 10 ng/mL of melatonin significantly promoted cell viability, the ratio of EdU-positive (5-Ethynyl-2′-deoxyuridine) cells, and increased the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin D1(CCND1), and cell division control protein 42 (CDC42) (p < 0.05). We also observed that melatonin inhibited apoptosis of mouse Leydig cells, accompanied with increased B-cell lymphoma-2 (BCL-2) and decreased BCL2 associated X (BAX) mRNA and protein expression. Moreover, addition of melatonin significantly decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, while it increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels (p < 0.05). In addition, we also found that melatonin increased the expression of SIRT1 (Silent information regulator 1) (p < 0.05). To explore the role of SIRT1 signaling in melatonin-induced cells, mouse Leydig cells were pretreated with EX527, an inhibitor of SIRT1. The protective effects of melatonin on mouse Leydig cells were reversed by EX527, as shown by decreased cell proliferation and increased cell apoptosis and oxidative stress. In summary, our results demonstrated that melatonin inhibited apoptosis and oxidative stress of mouse Leydig cells through a SIRT1-dependent mechanism.

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 influences physiological processes such as promoting proliferation and regulating cell development and function, and its effects on chicken Sertoli cells are unknown. Therefore, we investigated the effects of melatonin on cell proliferation and its underlying mechanisms in chicken Sertoli cells. Chicken Sertoli cells were exposed to varying melatonin concentrations (1, 10, 100, and 1000 nM), and the melatonin-induced effects on cell proliferation were measured by Cell Counting Kit 8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), real-time qPCR, and western blotting. We found that 1000 nM melatonin significantly (p < 0.05) promoted cell proliferation in chicken Sertoli cells. Furthermore, melatonin significantly (p < 0.05) increased the expression of inhibin alpha subunit (INHA), and the silencing of INHA reversed the melatonin-induced effects on Sertoli cell proliferation. We also found that melatonin activates the extracellular-regulated protein kinase (ERK) signaling pathway. To explore the role of the ERK signaling pathway in melatonin-induced cell proliferation, PD98059 (an inhibitor of EKR1/2) was used to pre-treat chicken Sertoli cells. The melatonin-induced proliferation of chicken Sertoli cells was reversed by PD98059, with decreased cell viability, weakened cell proliferation, and down-regulated expression of the proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1) and INHA. In summary, our results indicate that melatonin promotes the proliferation of chicken Sertoli cells by activating the ERK/inhibin alpha subunit signaling pathway.

The purpose of this study was to determine the effects of apigenin and astragalus polysaccharides on the cryopreservation of bovine semen. Apigenin, astragalus polysaccharides, or their combination were added to a frozen diluent of bovine semen. Afterwards, Computer Assisted Semen Analysis (CASA), membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-Px, MDA, and ROS detection were conducted. The results showed that adding 0.2 mmol/L AP or 0.5 mg/mL APS could improve the quality of frozen sperm. Compared to 0.2 mmol/L AP alone, the combination of 0.2 mmol/L AP and 0.3 mg/mL APS significantly increased the total motility (TM), average path distance (DAP), straight line distance (DSL), average path velocity (VAP), curvilinear velocity (VCL), wobble (WOB), and sperm CAT and SOD levels (p < 0.05), while reducing the ROS and MDA levels (p < 0.05). These results indicated that the addition of 0.2 mmol/L AP or 0.5 mg/mL APS alone has a protective effect on the freezing of bovine semen. Compared to the addition of 0.2 mmol/L AP, a combination of 0.2 mmol/L AP and 0.3 mg/mL APS could further improve the quality of frozen semen.

The objective of this study was to clarify the effect of kisspeptin-10 (kp-10) on the synthesis of progesterone (P4) in bovine granulosa cells (BGCs) and its mechanisms via microRNA 1246 (miR-1246). According to the results, we found that treating with kp-10 for 24 h could increase P4 level, the mRNA expression of the steroidogenesis-related gene steroidogenic acute regulatory protein (StAR), free cholesterol content, and decrease miR-1246 expression in BGCs. Overexpression of miR-1246 significantly inhibited P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs, whereas underexpression of miR-1246 significantly reversed this effect in BGCs. Additionally, overexpression of miR-1246 counteracted the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Conversely, underexpression of miR-1246 enhanced the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Meanwhile, results of dual-luciferase reporter assays indicated that miR-1246 targeted the 3′UTR of StAR in BGCs. These results demonstrated that kp-10 induced P4 synthesis in BGCs by promoting free cholesterol transport via regulating expression of miR-1246/StAR.

It is important to inhibit oxidative stress to maintain sperm motility during cryopreservation. The present study was performed to investigate the effects of supplementing oligomeric proanthocyanidins (OPC) and bamboo leaf flavonoids (BLF) or their combination as an extender for Simmental bull semen freezing. OPC, BLF, or their combination were added to the frozen diluent of bovine semen. Afterwards, computer-assisted semen analysis (CASA), detection of membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-PX, MDA, and ROS were conducted. The results showed that adding 50 mg/L OPC or 4 mg/L BLF could improve the quality of frozen sperm. Compared with 50 mg/L OPC alone, the combination of 50mg/L OPC and 2 mg/L BLF significantly increased the kinematic parameters of sperm, and sperm CAT, GSH-PX and SOD levels (p < 0.05), whereas the MDA of sperm was decreased (p < 0.05). These results indicated that compared to the addition of 50 mg/L OPC alone, a combination of 50 mg/L OPC and 2 mg/L BLF could further improve the quality of frozen semen. The results could provide theoretical data support for the development of a new protective agent and are significant for the cryopreservation of bovine semen in the future.