FOXO3 and AP1 Expression in Pre-Pubertal Pig Ovaries During Follicle Development.

Pre-pubertal gilts begin follicular development prior to achieving their first normal estrous cycle although the mechanism for ovarian activation is not well understood. Forkhead transcription factor (FOXO3) activity has been associated with oocyte activation, and knockdown of FOXO3 has been demonstrated to induce primordial oocyte activation in pig ovaries xenografted into immuondeficient mice. Activator protein 1 (AP1) is downstream of the FOXO3 pathway and may also contribute to follicular activity. The objective of this study was to determine the expression and activation of FOXO3 and the AP1 subunit, c-jun, relative to follicular activation in pre-pubertal gilts. Ovaries were harvested from pre-pubertal gilts (84 ± 2d of age; 45.9 ± 2 kg BW) that contained either no tertiary follicle activity (naïve) or substantial tertiary follicle activity (advanced; n= 4 per stage of development). One ovary from each pig was frozen in liquid nitrogen and stored at −80oC for RNA and protein analysis. Following normalization to 18S ribosomal RNA, quantitative PCR demonstrated increased (P < .05) mRNA expression of both FOXO3 (7-fold greater) and c-jun (5.5-fold greater) in naïve compared with advanced ovaries. Western blot analysis demonstrated FOXO3 total protein was not different in naïve compared to advanced ovaries. Additionally phosphorylated FOXO3 (Ser 253) was not different between the two stages based on ImageJ quantification. These results indicate a reduction in FOX03 and c-JUN mRNA in pre-pubertal gilt ovaries containing recruited follicles compared to those lacking tertiary follicle development. These results are in agreement with previous studies that demonstrate a decrease in FOXO3 in secondary and growing follicles in the mouse, and suggest a role for down-regulation of FOXO3 and c-JUN during puberty onset in pigs. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-05309 and 2008-35205-18712 from the USDA National Institute of Food and Agriculture