Ovarian function in mammals is mediated by follicles, which consist of membrane cells, GCs, and oocytes [1]. GCs are the most considerable cell population in developing follicles [2]. They perform critical physiological functions, including rapid proliferation and secretion of steroid hormones and growth factors essential for follicular development and oocyte maturation [3,4]. Estradiol (E2) is secreted by GCs and regulates mammals’ estrous cycle and follicle development [5,6]. E2 is a crucial signal generating the GnRH/LH surge [7]. In addition, E2 is also a marker of follicle quality [8]. E2 synthesis entails a multistep physiological process, which requires the participation of many vital enzymes, including steroidogenic acute regulatory protein (StAR), 3β-hydroxy steroid dehydrogenase (3β-HSD), cytochrome P450 (CYP) family 11 subfamily A member 1 (CYP11A1), cytochrome P450 (CYP) family 17 subfamily A member 1 (CYP17A1) and aromatase (CYP19A1). Mitochondria are involved in E2 synthesis. Some rate-limiting enzymes, such as StAR, 3β-HSD, and CYP11A1, must be in mitochondria to perform routine functions [9]. In addition, the synthesis of E2 requires the consumption of ATP supplied by mitochondria [10]. Therefore, normal mitochondrial function is essential for E2 synthesis.

Genetically encoded molecular clocks endogenously produce circadian rhythm with a periodicity of about 24 h. The components cooperate to regulate mammalian physiology, metabolism, and behavior [11,12]. Cellular circadian clocks rely on a transcriptional-translational feedback loop made of clock genes [13]. Circadian locomotor output cycles kaput (Clock) encodes a transcription factor with a basic-helix-loop-helix PER-ARNT-SIM domain [14]. CLOCK binds to BMALI in the cytoplasm to form a heterodimeric transcription complex. It activates the expression of the repressors Period and Cryptochrome or target genes by binding to E-box elements in nuclear promoters [15]. Circadian rhythms play a critical role in maintaining mammalian fertility [16], and clock genes are expressed in the ovaries of humans [17], mice [18,19], rats [20], goats [21], and pigs [22]. Circadian rhythm disruption is closely related to polycystic ovary syndrome [23]. Irregular circadian rhythm leads to premature ovarian insufficiency in mice [18]. Clock knockdown impairs fertility in mice and leads to decreased release of oocytes and smaller litter size [24]. Moreover, Clock small interfering RNA (siRNA) reduced the number of GCs and inhibited the secretion of E2 in cattle GCs treated with FSH [25]. These results suggest that CLOCK might be associated with the proliferation and steroid hormone secretion of ovarian GCs. However, the underlying mechanisms by which CLOCK regulates the physiological function of ovarian GCs remain unclear, particularly in the pig.

Therefore, in this study, we analyzed the sequence of porcine CLOCK using bioinformatics and investigated its localization and expression in the ovary. Finally, we demonstrated the effect of CLOCK on mitochondrial function and E2 synthesis in porcine ovarian GCs.