Effect of follicle-stimulating hormone and luteinizing hormone on apoptosis, autophagy, and the release and reception of some steroid hormones in yak granulosa cells through miR-23a/ASK1 axis.

Follicle-stimulating hormone (FSH), luteinizing hormone (LH), miR-23a, apoptosis signal-regulating kinase 1(ASK1)/c-Jun N-terminal kinase (JNK), autophagy and apoptosis play crucial roles in follicular development. However, their role in yak granulosa cells (GCs) remains unknown. Therefore, we examined the effect of miR-23a, ASK1, FSH, and LH on apoptosis, autophagy, and the release and reception of some steroid hormones in these cells. Our results showed that miR-23a overexpression significantly increased the abundance of Beclin1, the LC3II/I ratio, and the number of Ad-mRFP-GFP-LC3-labeled autophagosomes, and decreased p62 abundance. Additionally, Bax abundance and the number of terminal deoxynucleotidyl transferase deoxynucleotide triphosphate nick end labeling-positive cells were reduced, while Bcl2 expression was increased. Overexpression of miR-23a also significantly increased the abundance of estradiol receptor α (ER-α) and ß (ER-ß) and the concentrations of estradiol (E2), progesterone (P4) in yak GCs. Here, treating yak GCs with miR-23a decreased ASK1 expression, which regulates ASK1/JNK-mediated apoptosis, autophagy, E2 and P4 levels, and ER-α/ß abundance. In contrast, treatment of yak GCs with FSH (10 µg/mL) and LH (100 µg/mL) increased miR-23a abundance, regulating the subsequent effect on ASK1/JNK-mediated apoptosis, autophagy, ER-α/ß abundance, and E2 and P4 concentrations. In conclusion, miR-23a enhances autophagy in yak GCs, attenuates apoptosis, and increases ER-α/ß abundance and E2 and P4 concentrations by downregulating ASK1. Additionally, FSH and LH can regulate these effects of miR-23a by altering its expression. These results provide important insights that can inform the development of strategies to reduce abnormal follicular atresia and improve the reproductive rate of yaks.

Expression of Rad51 and the histo-morphological evaluation of testis of the sterile male cattle-yak.

Meiotic recombination is key to the repair of DNA double-strand break damage, provide a link between homologs for proper chromosome segregation as well as ensure genetic diversity in organisms. Defects in recombination often lead to sterility. The ubiquitously expressed Rad51 and the meiosis-specific DMC1 are two closely related recombinases that catalyze the key strand invasion and exchange step of meiotic recombination. This study cloned and sequenced the coding region of cattle-yak Rad51 and determined its mRNA and protein expression levels, evaluated its molecular and evolutionary relationship as well as evaluated the histo-morphological structure of testes in the yellow cattle, yak and the sterile cattle-yak hybrid. The Rad51 gene was amplified using PCR, cloned and sequenced using testicular cDNA from yak and cattle-yak. Real-time PCR was used to examine the expression levels of Rad51/DMC1 mRNA in the cattle, yak and cattle-yak testis while western blotting, immunofluorescence and immunohistochemistry were used to assess the protein expression and localization of Rad51/DMC1 protein in the testicular tissue sections. The results revealed that the mRNA and protein expression of Rad51 and DMC1 are extremely low in the male cattle-yak testis with a corresponding higher incidence of germ cell apoptosis. There was also thinning of the germinal epithelium possibly due to the depletion of the germ cells leading to the widening of the lumen area of the cattle-yak seminiferous tubule. Our findings provide support for the hypothesis that the low expression of Rad51 and DMC1 may contribute to the male hybrid sterility in the cattle-yak.

Quantitative measurement of in vitro phagocytosis of apoptotic granulosa cells by monocytes in mice / 生理学报

To establish a method for quantitative measurement of phagocytosis, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were observed by microscope after Wright's staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were progressing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio of monocytes which attached to apoptotic granulosa cells to those which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in the state of engulfment, and this time point was named as 'half phagocytic period'. Regression analysis showed that the equation of linear regression was y = -0.247x +1.644 (y represents Attachment/Engulfment ratio, x represents co-culture time), R(2)=0.912, F=31.095, P=0.011 (<0.05), T= -5.576, P=0.011 (<0.05). In conclusion, the present mode of phagocytosis in vitro can be used as a method to quantitatively assay some effective factors such as medicines which could enhance or restrain phagocytosis.