Regulation of proliferation, apoptosis, hormone secretion and gene expression by acetyl-L-carnitine in yak (Bos grunniens) granulosa cells

As the basis of female reproduction, the main functions of the ovaries are follicle development and ovulation as well as hormone secretion. The growth and development of follicles is an important component of fertility in female animals. Follicles contain oocytes and granulosa cells (GCs), and the essence of follicular growth and development is the proliferation and differentiation of GCs [1]. GCs are linked to oocytes through oocyte-derived factors, and GCs are important because they provide steroid hormones and gonadotropin receptors for ovarian and follicle growth and development [2] and they provide nutrition to oocytes through gap junctions, which in turn affects the reproductive capacity of females [3]. GC apoptosis is involved in the induction of follicular atresia [[4], [5], [6]].

The organism produces reactive oxygen species (ROS) during aerobic processes, and a large accumulation of ROS causes oxidative stress, leading to inflammation development, senescence and even apoptosis of cells [7]. ROS are one of the factors that trigger follicular atresia [8]. Lipid droplets, as important organelles of cells, are involved in the lipid metabolism of cells [9]. The metabolism of lipids has an important role in animal reproduction. Oocytes are rich in lipid droplets, which play a crucial role in subsequent maturation as well as in the development of the embryo [10]. Triglycerides gradually decrease with cell maturation, and lipid metabolism serves as an important source of energy for oocyte development during the maturation of porcine oocytes in vitro [11].

Acetyl-l-carnitine (ALC) is the acetyl ester of the trimethylated amino acid l-carnitine (LC), and as a natural derivative of LC, it can be easily converted to LC when needed [12]. ALC plays an important role in the oxidation of fatty acid metabolic pathways as an effective substrate for mitochondrial energy metabolism [13] and is also a strong antioxidant with a wide range of pharmacological properties, such as antioxidant, antiapoptotic [14], anti-inflammatory [15] and lipid metabolism-promoting properties [16]. Xu et al. [17] reported that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases steroid hormone production, thereby improving the quality of mature oocytes and improving embryonic development in vitro in buffalo. Supplementation with ALC during in vitro maturation (IVM) improves buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes [18]. Treatment of oocytes from prepubertal lambs with ALC during IVM increased the blastocyst rate and altered the number, volume, and distribution of mitochondria, vesicles, and lipid droplets [12].

Cell proliferation is regulated by cell cycle-related genes and apoptosis-related genes, and CCND, CCNE and CCNB are all cycle-critical proteins [19]. PCNA acts as a marker of cell proliferation and positively regulates the level of cell proliferation [20]. Compared with healthy follicles, GCs in atretic follicles had reduced expression of NRF-1, increased BAX expression and an increased ratio of BAX to BCL-2 expression [21], indicating that changes in the mitochondria-associated gene expression patterns in GCs may lead to follicular atresia during follicle development. Antioxidant enzymes such as manganese superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) ameliorate apoptosis due to ROS accumulation [22]. The biosynthesis of P4 and E2 is a complex process regulated by multiple proteins and key enzymes (StAR, CYP19A1 and HSD3B1), and the biosynthesis of P4 and E2 is positively and linearly related to the expression of these genes [23].

Yak (Bos grunniens) is an irreplaceable livestock species that lives mainly on the Tibetan Plateau at altitudes ranging from 2500 to 5000 m. Yaks provide meat and milk where few other farm animals will survive because of extreme climatic conditions such as low oxygen content in air and cold temperature [24,25]. However, reproduction in yaks is low as a result of seasonal breeding, delayed puberty and a lower frequency of estrus [26]. The in vitro oocyte maturation rate and embryo development to morula and blastocyst rates are also low [27]. Currently, the effects of ALC on oocyte competence have been studied in sheep and buffalo [12,17]. GCs are important somatic cells in follicles that are involved in follicular development, but the role of ALC in GCs is not clear. Therefore, in this experiment, different concentrations of ALC were added to the culture medium to investigate its effects on the proliferation, ROS, lipids, hormone secretion and related genes of yak GCs and to provide a theoretical basis for the use of ALC to improve oocyte IVM in yaks.

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