CELL-SPECIFIC EXPRESSION AND ACTIVATION OF LKB1 IN THE RAT OVARY

Abstract

The tumor suppressor LKB1 regulates cell metabolism and proliferation, and is an upstream kinase for adenosine monophosphate-activated kinase (AMPK). LKB1 is itself activated via phosphorylation by other kinases, including protein kinase A. Mutation of LKB1 is associated with the development of tumors in the gastrointestinal and reproductive tract, including ovarian carcinomas. Interestingly, AMPK, a target of LKB1, is a proposed regulator of ovarian hormone production and oocyte maturation. These observations indicate important roles of LKB1 in regulating ovarian functions. However, while one report describes the association of LKB1 with the meiotic spindle of isolated, maturing mouse oocytes, little information is available regarding the cell-specific expression and activation of LKB1 in the rat ovary. This study examined the levels of total LKB1 and activated LKB1 (phosphorylated at Ser 431; pLKB1) in immature rat ovaries following stimulation with equine chorionic gonadotropin (eCG), followed 52 hours later by an ovulatory dose of human chorionic gonadotropin (hCG). Liver (positive control for LKB1 expression) and ovaries were harvested from rats at different time points (n = 4 rats/time point). From each rat, one ovary was fixed and processed for immunohistochemical analysis, while the other ovary was homogenized and protein extracts prepared. Immunoblot analysis of total LKB1 levels in liver and whole ovary homogenates revealed a predominant immunoreactive signal of 52 kDa, corresponding to the reported size of LKB1, and an additional signal of about 82 kDa. Both signals were shown to be specific for LKB1 by preabsorption of antibody with antigenic peptide. Digital image analysis was performed to assess potential changes in LKB1 expression during hormone treatments. However, no significant changes in total LKB1 levels were detected by immunoblot analysis during follicle development, ovulation, and luteinization. Immunohistochemical analysis revealed strong cytoplasmic LKB1 immunoreactivity in oocytes and theca cells, with lower immunoreactivity in granulosa and luteal cells. Faint pLKB1 immunoreactivity was detected in the cytoplasm of granulosa, theca, and luteal cells. There were no apparent effects of hormone treatment on LKB1 or pLKB1 levels in granulosa cells of developing follicles. Interestingly, strong pLKB1 immunoreactivity was found associated with the germinal vesicle (GV), but not cytoplasm, of primary oocytes in developing follicles. Eight hours after administration of hCG, oocytes in preovulatory follicles lacked GV and exhibited only faint pLKB1 immunoreactivity. Together, these findings demonstrate differences in LKB1 protein levels in different ovarian cell types, and reveal that activated LKB1 is primarily associated with the GV of primary oocytes. The latter observation is consistent with a role of LKB1 in maintaining meiotic arrest, presumably by activation of AMPK. Further studies are required to determine the functional roles and mechanisms of action of LKB1 in the ovary. (poster)