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In animal assisted reproductive technology, the production of high-quality oocytes is crucial. The yak, having lived in the Qinghai-Tibet Plateau for an extended period, has reproductive cells that are regulated by hypoxia-inducible factor 1α (HIF-1α). This study aimed to investigate the impact of HIF-1α on yak oocyte maturation and early embryonic development in vitro through the regulation of autophagy. The in vitro maturation process of yak oocytes involved the addition of the HIF-1α inducer DFOM and the inhibitor LW6 to examine their effects on yak oocyte maturation, early embryonic development, cell autophagy, cytochrome P450s (CYP450s) enzyme expression, and cumulus diffusion factors. The findings revealed that DFOM significantly upregulated the expression of HIF-1α, resulting in increased the cumulus diffusion area, elevated first polar body expulsion rate of oocytes, enhanced mitochondrial and actin levels, decreased ROS production, and reduced early apoptosis levels of oocytes. Moreover, DFOM promoted the expression of autophagy-related proteins, CYP450s enzymes, and cumulus diffusion factors, thereby enhancing oocyte maturation and early embryonic development. Conversely, LW6 exhibited opposite effects. The inhibition of autophagy levels with 3-MA during DFOM treatment yielded similar outcomes. Furthermore, reducing autophagy led to increased apoptosis levels at all stages of early embryonic development, as well as a significant decrease in total cell number and ICM/TE ratio of blastocysts. Studies have shown that during the in vitro maturation of yak oocytes, HIF-1α can affect the cumulus expansion area of oocytes by regulating autophagy, the first polar body excretion rate, mitochondrial level, actin level, ROS and early apoptosis level, the CYP450s enzyme, and the expression of cumulus expansion factors, thereby improving the in vitro maturation and early embryonic development of yak oocytes. These findings offer valuable insights into the reproductive regulation mechanism of yaks in hypoxic environments and suggest potential strategies for the advancement of yak assisted reproductive technology.

The yak is a unique creature that thrives in low-oxygen environments, showcasing its adaptability to high-altitude settings with limited oxygen availability due to its unique respiratory system. However, the impact of hypoxia on alveolar type II (AT2) epithelial cell proliferation in yaks remains unexplored. In this study, we investigated the effects of different altitudes on 6-month-old yaks and found an increase in alveolar septa thickness and AT2 cell count in a high-altitude environment characterized by hypoxia. This was accompanied by elevated levels of hypoxia-inducible factor-1α (HIF-1α) and epidermal growth factor receptor (EGFR) expression. Additionally, we observed a significant rise in Ki67-positive cells and apoptotic lung epithelial cells among yaks inhabiting higher altitudes. Our in vitro experiments demonstrated that exposure to hypoxia activated HIF-1α, EGF, and EGFR expression leading to increased proliferation rates among yak AT2 cells. Under normal oxygen conditions, activation of HIF-1α enhanced EGF/EGFR expressions which subsequently stimulated AT2 cell proliferation. Furthermore, activation of EGFR expression under normoxic conditions further promoted AT2 cell proliferation while simultaneously suppressing apoptosis. Conversely, inhibition of EGFR expression under hypoxic conditions had contrasting effects. In summary, hypoxia triggers the proliferation of yak AT2 cells via activation facilitated by the HIF-1α/EGF/EGFR signaling cascade.

The aim of this study was to investigate protein regulation at different time points during the in vitro maturation of yak oocytes. Yak oocytes at GV, MI, and MII stages were collected during in vitro maturation, and differential proteomics sequencing was performed using iTRAQ technology. GO functional classification indicated that the differential proteins were closely associated with biological processes such as “metabolic processes”, and molecular events such as “binding” molecular-function-related categories were active. KOG analysis showed that energy-metabolism-related activities were vigorous during oocyte development from the GV phase to MI phase, and genetic material preparation activities were more active when oocytes developed from the MI stage to MII stage. KEGG pathway analysis showed that the PPAR metabolic pathway, Hippo signaling pathway, and ECM–receptor interaction and metabolic pathway were enriched from the GV to the MI stages. The PI3K-Akt, TGF-β, and phagosome pathways were enriched from the MI stage to the MII stage. These results indicate that transient dynamic changes occurred in the proteome during the maturation of yak oocytes, and the physiological functions mediated by these were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII was helpful in further analyzing the molecular regulatory mechanism of yak oocyte maturation.

Fas-associated factor 1 (FAF1), a member of the Fas family, is involved in biological processes such as apoptosis, inflammation, cell proliferation and proteostasis. This study aimed to explore the biological role of FAF1 in testicular tissue at different ages (juveniles (1 and 2 years old), adults (3, 4, 6, and 7 years old) and old-aged animals (11 years old)) and ovaries during different reproductive cycle phases (follicular, luteal, and pregnancy phases). FAF1 mRNA, relative protein expression and protein expression localization were determined in testes and ovaries using real-time quantification, WB and immunohistochemistry (IHC), respectively. Real-time quantification of testis tissues showed that the relative expression of FAF1 mRNA in testis tissues at 3, 4 and 7 years of age was significantly higher than of those in other ages, and in ovarian tissues was significantly higher in luteal phase ovaries than those in follicular and pregnancy phase ovaries; follicular phase ovaries were the lowest. WB of testis tissues showed that the relative protein expression of FAF1 protein was significantly higher at 11 and 7 years of age; in ovarian tissue, the relative protein expression of FAF1 protein was significantly higher in follicular phase ovaries than in luteal and pregnancy phase ovaries, and lowest in luteal phase ovaries. The relative protein expression of FAF1 at 3, 4 and 7 years of age was the lowest. IHC showed that FAF1 was mainly expressed in spermatozoa, spermatocytes, spermatogonia and supporting cells; in ovarian tissue, FAF1 was expressed in ovarian germ epithelial cells, granulosa cells, cumulus cells and luteal cells. The IHC results showed that FAF1 mRNA and protein were significantly differentially expressed in testes of different ages and ovarian tissues of different reproductive cycle phases, revealing the significance of FAF1 in the regulation of male and female B. grunniens reproductive physiology. Furthermore, our results provide a basis for the further exploration of FAF1 in the reproductive physiology of B. grunniens.

Exosomes in the follicular fluid can carry and transfer regulatory molecules to recipient cells, thus influencing their biological functions. However, the specific effects of yak follicular fluid exosomes on 2-hydroxyestrodiol (2-OHE2) secretion remain unknown. Here, we investigated whether yak follicular fluid exosomes can increase 2-OHE2 secretion through the activation of autophagy in cumulus cells (YCCs). In vitro cultured YCCs were treated with yak follicular fluid exosomes for 6, 12, and 24 h. The effects of yak follicular fluid exosomes on autophagy and 2-OHE2 secretion were evaluated through real-time quantitative fluorescence PCR (RT-qPCR), Western blotting (WB), transfected with RFP-GFP-LC3, immunohistochemistry, and ELISA. To further investigate whether 2-OHE2 secretion was related to autophagy, YCCs were administered with yak follicular fluid exosomes, 3-methyladenine (3-MA), and rapamycin (RAPA). The results revealed that treatment with yak follicular fluid exosomes activated autophagy in YCCs and increased 2-OHE2 secretion. Conversely, the inhibition of autophagy with 3-MA blocked these effects, suggesting that autophagy has an important role in 2-OHE2 secretion in YCCs. Treatment of YCCs with rapamycin showed similar results with yak follicular fluid exosomes as there was an increase in 2-OHE2 secretion due to the activation of autophagy in the treated cumulus cells. Our results demonstrate that autophagy is enhanced by yak follicular fluid exosomes, and this is associated with an increase in 2-OHE2 secretion in YCCs.

Hsp70 and Hsp90 play an important role in testis development and spermatogenesis regulation, but the exact connection between Hsp70 and Hsp90 and metabolic stress in cattle is unclear. Here, we focused on the male cattle–yak and yak, investigated the expression and localization of Hsp70 and Hsp90 in their tissues, and explored the influence of these factors on development and metabolism. In our study, a total of 54 cattle (24 cattle–yaks and 30 yaks; aged 1 day to 10 years) were examined. The Hsp90 mRNA of the cattle–yak was first cloned and compared with that of the yak, and variation in the amino acid sequence was found, which led to differences in protein spatial structure. Using real-time quantitative PCR (RT-qPCR) and Western blot (WB) techniques, we investigated whether the expression of Hsp70 and Hsp90 mRNA and protein are different in the cattle–yak and yak. We found a disparity in Hsp70 and Hsp90 mRNA and protein expression in different non-reproductive organs and in testicular tissues at different stages of development, while high expression was observed in the testes of both juveniles and adults. Moreover, it was intriguing to observe that Hsp70 expression was significantly high in the yak, whereas Hsp90 was high in the cattle–yak (p < 0.01). We also examined the location of Hsp70 and Hsp90 in the testis by immunohistochemical (IHC) and immunofluorescence (IF) techniques, and the results showed that Hsp70 and Hsp90 were positive in the epithelial cells, spermatogenic cells, and mesenchymal cells. In summary, our study proved that Hsp70 and Hsp90 expressions were different in different tissues (kidney, heart, cerebellum, liver, lung, spleen, and testis), and Hsp90 expression was high in the testis of the cattle–yak, suggesting that dysplasia of the cattle–yak may correlate with an over-metabolism of Hsp90.

Due to its rich nutritional value, yak milk is an important food source in the alpine pastoral areas. However, yaks have a low milk yield. The Hippo pathway participates in cell proliferation and organ development. We aimed to determine the regulatory mechanism of this pathway in yak mammary cells. A greater understanding of how the expression of its essential genes influence the reproductive cycle could lead to improvements in lactation performance. The expression levels of the key genes MST1, LATS1, YAP1, and TEAD1 were detected by quantitative real-time PCR, Western blotting, and immunohistochemistry during the growth, lactation, and dry periods (GP, LP and DP, respectively). The MST1 and LATS1 mRNA and protein expression level was highest during GP and lowest during LP. The YAP1 and TEAD1 mRNA and protein expression level decreased from GP to LP and DP. MST1 and LATS1 were expressed in the cytoplasm whereas YAP1 and TEAD1 were expressed in the nucleus and cytoplasm, respectively. The differential expression of MST1, LATS1, YAP1, and TEAD1 expression in the yak mammary gland during different developmental stages strongly suggests that they play an important role in the regulation of developmental functions under different physiological conditions.

Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes, exerts a direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤2 years juvenile (y-c, n = 3; yak, n = 3) and ≥3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher, but not statistically significant, in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

The main reproductive organs undergo different histological appearances and physiological processes under different reproductive statuses. The variation of these organs depends on a delicate regulation of cell proliferation, differentiation, and apoptosis. Extracellular signal-regulated kinases1/2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) super family. They have important roles in regulating various biological processes of different cells, tissues, and organ types. Activated ERK1/2 generally promotes cell survival, but under certain conditions, ERK1/2 also have the function of inducing apoptosis. It is widely believed that ERK1/2 play a significant role in regulating the reproductive processes of mammals. The goal of our research is to investigate the expression and distribution of ERK1/2 in the yak’s main reproductive organs during different stages. In the present study, samples of the ovary, oviduct, and uterus of 15 adult female yak were collected and used in the experiment. The ERK1/2 proteins, localization, and quantitative expression of their mRNA were investigated using immunohistochemistry (IHC), western blot (WB) and relative quantitative real-time polymerase chain reaction (RT-PCR). The results indicated that ERK1/2 proteins and their mRNA were highly expressed in the ovary of the luteal phase and gestation period, in the oviduct of the luteal phase, and in the uterus of the luteal phase and gestation period. Immunohistochemical analysis revealed a strong distribution of ERK1/2 proteins in follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium, and luminal epithelium. These results demonstrated that the expression of ERK1 and ERK2 proteins and their mRNA in the yak’s ovary, oviduct, and uterus varies with the stage of the reproductive cycle. The variation character of ERK1 and ERK 2 expression in the yak’s main reproductive organs during different stages implies that they play an important role in regulating the reproductive function under different physiological statuses.