(319) Unfolding Ovarian Neurotensin Action During Preovulatory Period

Ketan Shrestha¹, Michelle A. Wynn¹, Hilaree N. Frazier², Misung Jo¹, Diane M. Duffy³, Thomas E. Curry Jr¹.

¹Department of Obstetrics & Gynecology, University of Kentucky, Lexington, KY;

 ²Department of Pharmacology and Nutritional Science, University of Kentucky, Lexington, KY;

³Department of Biomedical and Translational Sciences, Eastern Virginia Medical School, Old Dominion University, Norfolk, VA.

Abstract Text:

Neurotensin (NTS) is a small neuropeptide that rapidly increases in the ovaries of multiple species in response to the luteinizing hormone (LH) surge during ovulation. Blocking NTS activity results in luteinized unruptured follicles in the non-human primate ovary, suggesting that NTS functions as a paracrine mediator in the ovulatory follicle. However, the precise role of NTS in the ovulatory process is still not fully understood. Thus, to better understand the role of NTS in the ovulatory process, this study investigated the molecular mechanisms of NTS action in the process of ovulation, focusing on its downstream gene regulation and metabolic effect on mouse granulosa cells. To accomplish this, the present study used the broad spectrum NTS receptor (NTSR) antagonist (SR142948) to block NTS action and then determine ovarian outcomes. To validate the ability of the NTSR antagonist to block NTS action, experiments were performed to examine NTS regulated genes in the absence or presence of the NTSR antagonist. Treating mouse granulosa cells with human chorionic gonadotropin (hCG; LH analog) increased the expression of Nts as well as genes regulated by NTS: Elongation factor for RNA polymerase II (Ell2), and Radical s-adenosyl methionine domain containing 2 (Rsad2).  However, the addition of the NTSR antagonist significantly reduced their expression, reinforcing the role of NTS in the regulation of Ell2 and Rsad2 and demonstrating the ability of the NTSR antagonist to block ovarian NTS action. To explore NTS action in metabolic processes, glucose uptake and glucose utilization in mouse granulosa cells were assessed using fluorescently labeled glucose (2-NBDG). Cells treated with either forskolin (FKS; 10 µM) + phorbol 12-myristate 13-acetate (PMA; 20 nM) or NTS (25 µM) exhibited reduced glucose uptake. Moreover, the addition of the NTSR antagonist to cells treated with FSK+PMA or NTS had no effect on glucose uptake. In parallel to glucose uptake, we also examined the rate of glucose being utilized by mouse granulosa cells under these conditions. Similar to that of glucose uptake, we observed reduced glucose utilization in cells treated with either FKS+PMA or NTS alone. NTSR antagonist counteracted the inhibitory effect of FKS+PMA on glucose utilization. However, no such reversal was observed in the NTS+NTSR antagonist group, suggesting that NTS-mediated metabolic regulation in granulosa cells is distinct from that induced by FKS+PMA. Further investigating NTS action using mice lacking Nts (Nts null mice), we observed morphological differences in cumulus-oocyte complexes (COCs) from Nts null mice. The COCs from Nts null mice exhibited uneven COC expansion. Thus, we carried out morphological examination in vitro of the expanding COCs isolated from wild-type and Nts null mice. Epidermal growth factor (EGF), a known COC expansion ligand, successfully increased COCs expansion in both wild-type and Nts null mice compared to untreated COCs (control group). However, COCs from Nts null mice exhibited reduced expansion capacity compared to wild-type COCs, suggesting that NTS contributes to optimal COC expansion. These findings provide new insights into the role of NTS in the ovulatory process, including its influence on gene expression, metabolic regulation in granulosa cells, and COCs expansion.