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MORE Programs Poster Presentation

December 1, 2006

Title: Expression and Localization of LKB1 in the Rat Ovary

Authors: Mehnaaz B. Haniff and Dr. Philip S. LaPolt

ABSTRACT:

The presence and function of the serine/threonine kinase LKB1 has been observed in a wide variety of physiological processes; implicating roles of LKB1 in the regulation of cell viability, metabolism, and tumorigenesis. Of particular interest to this study, LKB1 acts as an upstream kinase for the AMP-activated protein kinase (AMPK), which is thought to be a regulator of ovarian hormone production and oocyte maturation. However, little information is available regarding the regulated, cell-specific expression of LKB1 in the ovary. The objective of this study is to investigate the regulated expression and cell-specific localization of LKB1 and phosphorylated LKB1 (p-LKB1) in the rat ovary using western immunoblotting and immunohistochemistical techniques. Based on studies indicating that AMPK activity contributes to ovarian hormone production, it is hypothesized that both LKB1 and p-LKB1 levels will be higher in granulosa cells of maturing and luteinizing follicles than in immature follicles. It is also hypothesized that total LKB1 and p-LKB1 will also be expressed in the oocyte, and that p-LKB1 will be found at high levels during oocyte maturation.

To examine the expression of LKB1 during different phases of ovarian function, follicular development was induced in immature Sprague Dawley rats via administration of equine chorionic gonadotropin (eCG), followed 52 h later by human chorionic gonadotropin (hCG) to induce ovulation and luteinization. Ovaries and liver (positive control for LKB1 expression) were harvested from rats at different time points, and protein extracts prepared. Protein extracts from each time point were run on SDS-PAGE gels and electrophoretically transferred to PVDF (polyvinylidene fluoride) nylon membranes. Membranes were then used in immunoblot analyses using a primary antibody for LKB1 or p-LKB1 protein and a secondary antibody for detection of primary antibody-bound-LKB1 or p-LKB1. In addition, blots were analyzed using an antibody for glyceraldehyde phosphate dehydrogenase (GAPDH) to determine equal loading of lanes. Preliminary immunoblot analyses revealed a predominant immunoreactive signal of about 52 kDa in liver and whole ovarian tissue, corresponding to the expected size of LKB1. Recent data suggest that LKB1 levels appear to be lowest in untreated immature ovaries, and increase after administration of eCG and hCG. The second ovary from each stage was embedded in paraffin, sectioned in 8 ┬Ám sections, and subjected to immunohistochemistry for cellular localization analysis of LKB1 and p-LKB1. Immunohistochemistry analysis with LKB1 revealed increased signal intensity in oocytes of pre-ovulatory follicles in comparison to primary oocytes of the immature ovary. Also, signal intensity was stronger in theca cells than granulosa cells of preovulatory follicles. LKB1 is also present in corpora lutea cells, implicating a role of LKB1 in luteal hormone production. These findings suggest that LKB1 expression is regulated in a cell-specific manner during hormone-induced follicular and oocyte maturation, ovulation, and luteinization.


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