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The Tianzhu white yak, a globally rare species, holds immense value as a source for yak materials. While the Fas/FasL pathway is pivotal in granulosa cells apoptosis, its precise molecular workings remain enigmatic. This study endeavours to decipher the role of follicle-stimulating hormone (FSH) in suppressing ovarian granulosa cells (GC) apoptosis in the Tianzhu white yak. Utilizing advanced cell culture techniques, we employed the MTT method, flow cytometry, fluorescence labelling and RT-PCR to investigate the apoptotic effects of FSH on yak GCs. Our results reveal that FSH's inhibitory effect on GC apoptosis follows a normal distribution pattern, peaking at an FSH concentration of 100 ng/mL with an apoptosis inhibition rate of 89.31%. When serum was withdrawn, an FSH concentration of 2 × 10 6  ng/mL reduced apoptosis by 72.84%. Annexin V-FITC staining revealed membrane invaginations, bubble and protrusion formation on the cell surface, and alterations in membrane structure and cell morphology. Flow cytometry analysis further demonstrated that FSH administration prior to early granulosa cell apoptosis had a more profound effect than during gradual apoptosis, both showing a suppressive effect on early follicular granulosa cell apoptosis. A transcription-level analysis conducted 3 h prior to serum withdrawal, with the addition of 100 ng/mL FSH, revealed intricate regulations in the expression of Fas/FasL. Notably, we observed a gradual increase in FasL expression over time, yet the presence of FSH effectively down-regulated FasL expression to baseline levels, without notable changes in Fas expression. Immunocytochemical analysis further confirmed the presence of both Fas and FasL on the cell membrane, nucleus and cytoplasm, with varying intensities depending on the duration of FSH treatment. Our findings suggest that FSH may suppress the apoptotic pathway in follicular primarily by down-regulating FasL expression, indicating that Fas-regulated mitochondrial pathways play a more prominent role compared to death receptor pathways. This study offers a fresh perspective on the mechanism underlying follicular atresia in Tianzhu white yaks and lays a solid theoretical foundation for the expansion of this endangered species' population., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The yak (Bos grunniens) is the most important livestock animal in high‐altitude regions owing to its prominent adaptability to cold conditions, nutritional deficiencies and hypoxia. The reproductive organs exhibit different histological appearances and physiological processes at different reproductive stages. Hypoxia‐inducible factor‐1 alpha (HIF‐1α) is the regulatory subunit of HIF‐1 that crucially regulates the response to hypoxia in mammalian organisms. The goal of our study was to investigate the expression and distribution of HIF‐1α in the primary yak reproductive organs at different reproductive stages. Samples of the ovary, oviduct and uterus of 15 adult female yaks were collected and used in the experiment. The expression and localization of HIF‐1α proteins and mRNA were investigated using quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blot (WB) and immunohistochemistry (IHC). The results indicated that the expression of HIF‐1α protein in the ovary was higher during the luteal phase than during the follicular phase and gestation period (p <.05). In the oviduct, HIF‐1α protein was also more highly expressed during the luteal phase than during the follicular phase and gestation period (p <.01). However, in the uterus, the HIF‐1α protein had stronger expression during the gestation period than during the follicular phase (p <.01) and luteal phase (p <.05). The expression of HIF‐1α mRNA was similar to that of its protein. Immunohistochemical analysis revealed intense immunostaining of HIF‐1α proteins in the follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium and luminal epithelium, foetal villous trophoblast, and epithelia of caruncular crypts. This study showed that the expression of HIF‐1α in the ovary, oviduct and uterus varies according to the stage of the reproductive cycle. This implies that HIF‐1α plays an important role in regulating the stage‐specific physiological function of yak reproductive organs under hypoxic environments. [ABSTRACT FROM AUTHOR]

Apoptosis and autophagy in granulosa cells (GCs) are highly related to follicular development and atresia. It has also been reported that they are related to LncRNA MEG3, miR-23a and apoptosis signal-regulating kinase 1 (ASK-1). However, their relationship to follicular development and the extent to which follicle stimulating hormone (FSH) or luteinizing hormone (LH) can regulate this process remain unknown. Here, we found that ASK1 and JNK were expressed in the GCs of gonadotropin-dependent follicles, and those levels were significantly higher (p < 0.05) in yak Tertiary follicles compared to that of Secondary follicles and Graafian follicles. Then, the effect of LncRNA MEG3 / miR-23a on apoptosis and autophagy via ASK1/JNK (c-Jun N-terminal kinase) in yak GCs was studied. Overexpressing LncRNA MEG3 reduced miR-23a levels and p-967 protein expression, but enhanced ASK1 and JNK mRNA levels as well as t-ASK1, p-845, t-JNK, and p-JNK proteins levels. And Up-regulation of LncRNA MEG3 promoted apoptosis while attenuating autophagy. The targeting relationship between miR-23a and the binding sites of LncRNA MEG3 and ASK1 was also confirmed with the dual luciferase reporter assay. And, the relationship between LncRNA MEG3 and miR-23 a was observed as a negative feedback regulation, and changes in LncRNA MEG3 and miR-23a levels can alter the expression of ASK1/JNK axis in yaks GCs. In addition, FSH (10 μg/mL) or LH (100 μg/mL) ability to reverse the effects of LncRNA MEG3 on miR-23a levels and ASK1/JNK axis-mediated apoptosis and autophagy was verified in yak GCs. This is significantly beneficial for decreasing abnormal follicular atresia for yaks tertiary follicles. [Display omitted] • LncRNA MEG3, miR-23a and ASK-1 were related with apoptosis and autophagy, but their effect on follicules of yak remain unknown. • The level of ASK1 and JNK were higher in yak Tertiary follicles compared to Secondary and Graafian follicles. • LncRNA MEG3 reduced miR-23a levels, but enhanced ASK1-JNK axis levels, and promoted apoptosis while attenuating autophagy. • miR-23a can directly targeted LncRNA MEG3, and the relationship of LncRNA MEG3 and miR-23a was observed as a negative feedback regulation. • FSH (10 μg/mL) and LH (100 μg/mL) ability to reverse the effects of LncRNA MEG3 on miR-23a and ASK1/JNK axis. [ABSTRACT FROM AUTHOR]