Presentations

Abstract

Introduction: The luteinizing hormone (LH) surge is the kick start for ovulation. Theca cells are critical for ovarian steroidogenesis, both in the ovulatory follicle and the corpus luteum that forms after ovulation. Neurotensin (NTS), a tridecapeptide, is highly expressed in the ovarian follicle in response to the ovulatory LH surge. NTS has been shown to promote ovulation and luteinization in vivo. Receptors for NTS, including NTSR1 and SORT1, have also been identified in many ovarian cells including theca cells. This study tested the hypothesis that NTS initiates theca cell proliferation and migration into the ovarian follicle during ovulation.

Methods: To study the effects of NTS in vivo, vehicle or a NTS receptor antagonist was injected into preovulatory Macaca fascicularis follicles. Administration of human chronic gonadotropin (hCG) immediately followed, a substitute for the LH surge, and ovaries were removed 48 hours later, with ovulation anticipated at 40 hours. Additional ovaries were collected without the administration of hCG to serve as a pre-hCG control. All ovaries were then processed for histology and examined via immunofluorescent detection of the theca cell protein CYP17. Images of the ovulatory follicle were used to quantify CYP17 positive cells and assess migration of CYP17+ cells into the luteinizing follicle. Replicating populations of primary theca cells were isolated from M. fascicularis ovaries treated in vitro with NTS (0.05-50 µM) or NTS with the addition of a NTS receptor antagonist. Post exposure, theca cells were collected and processed for detection of NTS and NTS receptor RNA (by qPCR) and protein (by western blot and immunohistochemistry). Immunohistochemistry was also used to quantify proliferating cells via Ki67 immunodetection, and a transwell migration assay was used to quantify theca cell migration.

Results: In follicles of pre-hCG ovaries, CYP17+ theca cells were sparce and located in a punctuate pattern in the ovarian stroma surrounding the basement membrane, consistent with the expected appearance of a large, pre-ovulatory follicle. 48 hours after vehicle intrafollicular injection and systemic hCG administration, theca cells of the ovulated, luteinizing follicle were abundant and present both in the stroma and in the luteinized granulosa cell layer. Intrafollicular injection of a general NTS receptor antagonist, NTSR1 antagonist, or SORT1 antagonist reduced or eliminated the movement of theca cells past the basement membrane and into the granulosa cell layer. NTS receptor antagonist injection also reduced the number of theca cells when compared to the vehicle injected follicles. To determine if NTS acts directly at theca cells via the NTSR1 and SORT1 receptors to promote migration and proliferation; isolated theca cells were studied in vitro. Cultured theca cells expressed NTS, NTSR1, and SORT1 mRNA and protein. NTS treatment stimulated theca cell proliferation in vitro, and NTS-stimulated proliferation was reduced to basal levels by inclusion of NTS receptor antagonists. Theca cell migration also increased in response to NTS, and receptor antagonists reduced migration to basal levels. Since NTS receptor antagonists limited theca cell migration in vivo, we considered that components of the ovarian extracellular matrix may also influence theca cell migration. Collagen III and laminin each reduced theca migration in the presence of NTS, but NTS stimulated migration on fibronectin.

Conclusion: This study is the first to demonstrate that NTS acts directly at theca cells to promote changes associated with ovulation and luteinization. Theca cells of the M. fascicularis ovulatory follicle express NTS and the NTS receptors NTSR1 and SORT1. Our in vivo and in vitro findings suggest that NTS acts directly at theca cells to promote migration and proliferation via interactions with NTSR1 and SORT1 receptors. The lack of NTS-stimulated migration on collagen III and laminin matrixes in vitro suggests that NTS stimulates theca cell migration in a matrix dependent manner in vivo. Prior studies demonstrated that follicular NTS is required for follicle rupture and oocyte release. The current studies further elucidate the role of NTS in the ovary and expands our knowledge of theca cells and their potential roles during ovulation.