Melatonin Attenuates Oxidative Stress-Induced Apoptosis of Bovine Ovarian Granulosa Cells by Promoting Mitophagy via SIRT1/FoxO1 Signaling Pathway.

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.

Melatonin inhibits the apoptosis of rooster Leydig cells by suppressing oxidative stress via AKT-Nrf2 pathway activation.

Oxidative stress has been described as a key driver of Leydig cell apoptosis. Melatonin has antioxidative and antiapoptotic effects, but the potential effects and mechanism of melatonin on oxidative stress and apoptosis in rooster Leydig cells remain unclear. Our results showed that melatonin biosynthetic enzymes and melatonin receptors were expressed in rooster Leydig cells and their expression were locally inhibited as rooster sexual maturation. We found that melatonin inhibited H2O2-induced apoptosis of rooster Leydig cell by activating the melatonin receptors Mel-1a and Mel-1b. Additionally, melatonin protects mitochondria from damage by reducing the level of oxidative stress in Leydig cells. Melatonin relieved H2O2-induced oxidative stress by significantly reducing intracellular ROS, MDA and 8-OHdG levels and increasing SOD and GSH-Px activities. Simultaneously, melatonin significantly reduced H2O2-induced depolarization of ΔΨm and decreased the release of Cytochrome C and Ca2+. We also observed that melatonin activated the Nrf2 pathway, while Nrf2 silencing abrogated the anti-oxidative and anti-apoptotic effects of melatonin in rooster Leydig cells. Furthermore, melatonin promoted the phosphorylation of AKT, while AKT inhibitor suppressed the Nrf2 pathway activated by melatonin and alleviated the inhibitory effects of melatonin on apoptosis and oxidative stress. In conclusion, melatonin could inhibit apoptosis in rooster Leydig cells by suppressing oxidative stress via activation of the AKT-Nrf2 pathway.

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen.

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.

Quercetin Protects Against Lipopolysaccharide-Induced Intestinal Oxidative Stress in Broiler Chickens through Activation of Nrf2 Pathway.

We investigated the potential ability of quercetin to protect against lipopolysaccharide (LPS)-induced intestinal oxidative stress in broiler chickens and the potential role of the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway. One-day-old broiler chickens (n = 240) were randomized into four groups: saline-challenged broiler chickens fed a basal diet (Con), LPS-challenged broiler chickens on a basal diet (LPS), and LPS-treated broiler chickens on a basal diet containing either 200 or 500 mg/kg of quercetin (Que200+LPS or Que500+LPS). Quercetin (200 mg/kg) significantly alleviated LPS-induced decreased duodenal, jejunal, and illeal villus height and increased the crypt depth in these regions. Quercetin significantly inhibited LPS-induced jejunal oxidative stress, including downregulated reactive oxygen species (ROS), malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, and it upregulated superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. Quercetin relieved LPS-induced jejunal mitochondria damage and upregulated mitochondrial DNA copy number-related gene expression, including cytochrome c oxidase subunit 1 (COX1), ATP synthase F0 subunit 6 (ATP6), and NADH dehydrogenase subunit 1 (ND1). Quercetin attenuated the LPS-induced inhibition of Nrf2 activation, translocation, and downstream gene expression, including heme oxygenase-1 (HO-1), NAD (P) H dehydrogenase quinone 1 (NQO1), and manganese superoxide dismutase (SOD2). Additionally, quercetin attenuated the LPS-inhibition of c-Jun N-terminal kinase (JNK), Extracellular Regulated protein Kinases (ERK), and p38MAPK (p38) phosphorylation in the MAPK pathway. Thus, quercetin attenuated LPS-induced oxidative stress in the intestines of broiler chickens via the MAPK/Nrf2 signaling pathway.

Effect of kisspeptin on the proliferation and apoptosis of bovine granulosa cells.

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT assays showed that 100nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.05). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl (P< 0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.