Granulosa cells (GCs) constitute the largest part of the follicular cell population and constitute the main source of progesterone (P4) and estrogen (E2) [1]. Granulosa cells regulate their own proliferation and differentiation and follicle development by secreting hormones, cytokines, and proteins [2]. The normal proliferation and apoptosis of granulosa cells are key to follicle development [3]. Compared with those of oocytes and theca cells, the proliferation and apoptosis of granulosa cells play a more important role in the development and atresia of follicles because granulosa cells undergo apoptosis before follicular cells and oocytes during follicular atresia [4]. Therefore, analyzing the processes of proliferation, apoptosis, and hormone secretion and the potential mechanisms in granulosa cells is important for understanding follicle development.

Steroid hormone secretion by granulosa cells plays key roles in regulating follicle development and oocyte maturation during follicle development. The formation and atresia of follicles are often accompanied by changes in steroid hormone levels. During transformation to the dominant follicle, the level of E2 derived from granulosa cells increases significantly and the level of P4 derived from granulosa cells decreases significantly; the opposite is true when follicle atresia occurs [1]. During this process, the key genes related to steroid synthesis, StAR, HSD3B1, and CYP19A1, are involved in the regulation of E2 and P4 levels [[5], [6], [7]]. Additionally, mitochondria support various metabolic activities during the steroid hormone synthesis process by providing energy and maintaining cell homeostasis [8]. The activation of the PI3K/AKT signaling pathway can promote human follicular theca cell proliferation and inhibit cell apoptosis; it is also involved in the regulation of steroid hormone synthesis by regulating the expression of steroid hormone synthesis-related genes [9,10].

Growth arrest specific 1 (GAS1), an important regulatory protein of cell growth and differentiation, is involved in biological processes such as embryonic development, cell proliferation, and apoptosis [11,12]. Previous studies have shown that GAS1 is expressed throughout the development process of mammalian follicles and is highly expressed in granulosa cells [13]. GAS1 participates in follicle development by regulating the cell cycle, apoptosis, and cell proliferation, among other processes [14,15]. The loss of GAS1 expression in granulosa cells promotes ovulation, and as a target gene of C/EBPα/β, GAS1 mediates luteinization and ovulation [14]. These findings suggest that GAS1 may play an important role in follicular development and ovarian function. However, whether and how GAS1 affects granulosa cell proliferation, apoptosis, and steroid hormone levels and the underlying mechanisms remain unclear.

In this study, GAS1-specific siRNA was used to inhibit GAS1 expression, the differential gene expression profile mediated by GAS1 was constructed, and key biological processes and pathways that affect granulosa cell functions were identified via transcriptomic sequencing analysis. The effects of GAS1 on proliferation, apoptosis, mitochondrial function, redox homeostasis, and P4 and E2 levels in bovine granulosa cells were investigated, and in combination with a PI3K/AKT pathway inhibitor, the effects of GAS1 on bovine granulosa cell function and the potential mechanism involved were identified. These results are helpful for analyzing the process and mechanism of bovine follicle development at the molecular level and provide a theoretical basis for improving follicle development and female animal fertility.