(352) Morphology of Corpus Luteum in RhoAd/d (RhoAf/fPgrCre/+) Mice During Estrous Cycle

Vaishnavi Harsha Chennareddy¹; Jackson Kyle Sundgren¹; Jonathan Matthew Hancock^1,2; Taylor Elijah Martin^1,2; Xiaoqin Ye^1,2

1. Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA, USA

2. Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, USA

 

Abstract Text:

The corpus luteum (CL) is a transient endocrine gland normally developed from an ovulated follicle, in which the remaining granulosa and thecal cells differentiate into luteal cells accompanied by intense angiogenesis. It has a life cycle of development, maintenance, and regression, and it is the main site for progesterone (P4) synthesis to support early pregnancy. In the absence of pregnancy, the CL will undergo luteolysis to begin the next estrous cycle. Our lab has been studying the in vivo functions of RhoA (Ras homolog family member A, a small GTPase) in the uterus using RhoA^d/d (RhoA^f/fPgr^Cre/+) mice, which have conditional deletion of RhoA in the progesterone receptor (PR)-expressing cells. RhoA^d/d mice have normal ovulation but impaired CL development and reduced P4 levels during early pregnancy. It was then demonstrated that RhoA and PR were colocalized in the luteal cells of control mice, causing RhoA deletion in the luteal cells of RhoA^d/d mice.

 
I am a CURO (Center for Undergraduate Research Opportunities) student at the University of Georgia and CURO scholarship recipient working with the PhD students in the lab. My current project is to investigate the CLs in the RhoA^d/d ovaries during the estrous cycle. The ovaries are being collected from the set of RhoA^f/f (control) and RhoA^d/d mice (6-8 weeks old) undergoing ovariectomy for studying local functions of RhoA in the uterus (indicated in an abstract by Sundgren JK et al). Within 30 minutes prior to ovariectomy, we collect vaginal flushing from each mouse for vaginal cytology to determine the estrous stage. During ovariectomy, one ovary from each mouse is fixed in Hollande’s fixative (modified Bouin’s) and the other ovary is flash-frozen. So far, I have processed four fixed ovaries, with one each from RhoA^f/f and RhoA^d/d mice at proestrus and metestrus, respectively, for paraffin sectioning. I have collected serial paraffin sections and stained the 5^th and 15^th sections from each ovary with hematoxylin and eosin (H & E). H & E staining indicates that there are visible CLs in the RhoA^f/f ovaries at proestrus and metestrus, and the RhoA^d/d ovary at proestrus, but no identifiable CL in the RhoA^d/d ovary at metestrus. In the RhoA^f/f control ovary at proestrus, the CLs have clear luteal cords (endothelial cells surrounding a group of luteal cells) and the luteal cells have dense cytoplasm; while in the RhoA^f/f control ovary at metestrus, the luteal cords become less clear and the cytoplasm of luteal cells have less density, most likely due to large lipid droplets dissolved during the staining process. In the RhoA^d/d ovary at proestrus, the CLs have the appearance more like the RhoA^f/f control CLs at metestrus, indicating defective luteal cell differentiation and/or premature luteolysis. I am processing more ovaries for histology. I will employ immunohistochemistry and immunofluorescence to detect proteins in P4 steroidogenesis (e.g., StAR), vasculature (e.g., Col IV), and cytoskeleton (e.g., beta-actin) in the RhoA^f/f and RhoA^d/d CLs. These studies will reveal the in vivo roles of RhoA in regulating CL morphology and functions during the estrous cycle.