DNA Methylation Mediates the Transcription of STAT4 to Regulate KISS1 During Follicular Development

Maturation of follicles is the primary condition for the initiation of puberty, and excessive apoptosis of granulosa cells (GCs) will hinder the normal development of follicles in pigs. Signal Transducer and Activator of Transcription 4 (<i>STAT4</i>) plays an important role in cell proliferation and apoptosis. However, the mechanism of DNA methylation regulating <i>STAT4</i> transcription and affecting follicle development in pigs remains unclear. To resolve this problem, we constructed a <i>STAT4</i> overexpression vector and interference fragment to explore the effects of <i>STAT4</i> on GC function and investigate the effects of changes in methylation status of the <i>STAT4</i> promoter region on cell function and kisspeptin-1 (<i>KISS1</i>) expression, as well as the <i>STAT4</i> effects on the development of the follicles of pigs and mice in vitro. We found that the expression of <i>STAT4</i> decreased, while DNA methylation of the <i>STAT4</i> promoter region increased with the growth of the follicles. After overexpression of <i>STAT4</i>, the apoptosis of GCs was increased but the proliferation, cell cycle and estrogen secretion of GCs were inhibited. When GCs were treated with DNA methyltransferase inhibitor (5-Aza-CdR), the methylation of the <i>STAT4</i> promoter region decreased, resulting in a significant increase in the expression of <i>STAT4</i>. Consequently, the expression of <i>KISS1</i> was inhibited. At the same time, the expressions of genes related to cell proliferation, cell cycle and estrogen secretion signaling pathways decreased, while the expressions of genes related to the apoptosis signaling pathway increased. After infection with the <i>STAT4</i> lentiviral vector (LV-<i>STAT4</i>) in follicles of mice, the expression of <i>STAT4</i> in ovaries of mice significantly increased, and the expression of <i>KISS1</i> was significantly decreased. The capillaries on the surface of follicles were constricted, the age of puberty onset in mice was delayed while the levels of GnRH, LH, FSH and E2 in serum were decreased. In conclusion, we found that reduced methylation status of the <i>STAT4</i> promoter region promoted the transcription of <i>STAT4</i> and then inhibited the expression of <i>KISS1</i>, as well as promoted the apoptosis of GCs and ultimately inhibited the normal development of follicles in mammals.