Effect of rapamycin treatment on oocyte in vitro maturation and embryonic development after parthenogenesis in yaks

Yaks (Bos grunniens) are the main livestock in the Qinghai – Tibetan Plateau [1]. They are the main sources of income for pastoralists as they provide milk, meat, hides and so on, and also regarded as an important asset in relation to the status of individuals, dowry, and roles during religious ceremonies [2]. Due to harsh natural environmental conditions on the Plateau, such as coldness, hypoxia, and limited feed, growth and reproduction potentials of yaks are adversely affected. Generally, the natural reproduction efficiency of yaks is low [1], and this has great detrimental influence on the economy and income of herdsmen in pastoral areas of the Qinghai – Tibetan Plateau [3].

Autophagy is ubiquitous in the development and aging process of organisms, which is mainly manifested as the removal of redundant or damaged organelles by eukaryotic cells. Under physiological conditions, cells can realize material reuse and self-renewal through autophagy, albeit low level, to ensure the normal process of basic life activities such as metabolism. Under metabolic stress of energy or oxygen deficiency, autophagy is at a high level. Many researches have shown that autophagy is a protective mechanism in many diseases, but it can also lead to cell injury, thus autophagy is akin to “double-edged sword”. So, it is imperative to understand the regulatory mechanism of autophagy and the important role it plays in maintaining normal cell homeostasis, body function, and injury reduction [4,5]. Rapamycin target protein (mTOR) is a highly conserved Ser/Thr kinase in mammalian cells, belonging to the phosphatidylinositol kinase-related kinase (PIKK) family. The mTOR signaling pathway can aggregate and integrate stimulus signals from nutrition, growth factors, energy, and environmental stresses [6,7]. Rapamycin is a macrolide antibiotic and immunosuppressant. It binds to the cytoplasmic receptor FKBP12 to form a complex which can bind to the FRB domain of the carboxyl terminal of mTOR protein to form a ternary complex. This gives the kinase catalytic domain of mTOR the ability to approach and phosphorylate the downstream target proteins, thus inhibiting the biological function of mTOR [[8], [9], [10]]. The autophagy pathway can be activated by AMPK signaling, but is normally inhibited by the mammalian target of rapamycin (mTOR) pathway. The critical role of autophagy in oocyte maturation and preimplantation development has been reported. Previous studies have reported that autophagy mediates follicle growth, atresia and differentiation [11]. Vilser et al. [12] found that decreased autophagic repair in granulosa cells overages them, leading to follicular atresia. Beclin1 is not only a key gene regulating autophagy, but also a tumor suppressor gene. In ovaries lacking or with knockout Beclin1 gene the function of granulosa cells to secrete progesterone was defective [13]. Recent studies reported that autophagy of oocytes was involved in a large number of follicular atresia [14] and autophagy was a dynamic process during in vitro maturation of porcine [15]. There was also reported that rapamycin treatment increased the expression of Beclin1 and LC3-II mRNA, and emphasized that rapamycin induced autophagy could promote oocyte maturation [16]. Song et al [17] observed that rapamycin induced autophagy increased transcription level of Bcl-xL, increase blastocyst formation rate and cell survival rate, and improve the maturation of nuclear and cytoplasm of oocytes in vitro through the study of autophagy induced by rapamycin in immature porcine oocytes. Abnormal autophagy has been reported in many in vitro cultured oocytes and embryos [18,19]. Strategies to control autophagy have been effectively employed to treat these diseases, and improve in vitro embryonic development processes. Autophagy, as an important self-protection mechanism in cells, plays a key role in female mammalians developmental processes. However, the roles of rapamycin have not been fully studied in yak oocyte maturation and embryonic development.

Therefore, the objective of this experiment is to study the effect of rapamycin treatment on in vitro maturation of yak oocytes and parthenogenetic embryonic development. We also seek to evaluate the effects of rapamycin on mitochondrial membrane potential, ROS and early apoptosis of oocytes in order to understand the regulatory mechanism of rapamycin on yak oocytes and provide a scientific basis for improving yak oocyte quality and follicle development.

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