Ovulatory Signals Stimulate Active Theca Cell Migration
Poster #: 194
Session/Time: A
Author:
Megan A. G. Sage, PhD
Mentor:
Diane M Duffy, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Theca cells are an integral component of the ovarian follicle. Theca cells synthesize all androgens produced by the follicle. Androgens are then converted into estrogens by granulosa cells. After the ovulatory luteinizing hormone (LH) surge, oocyte release occurs concomitantly with the rapid transformation of the follicle remnant into the corpus luteum. During this process, theca cells integrate into the granulosa cell layer. Whether theca cell movement during ovulation is the result of passive relocation or if it is an active, intentional process of migration has not been investigated. We hypothesize that theca cells actively migrate during ovulation in response to hCG and/or hCG-stimulated local mediators of ovulation. Granulosa cells of the ovulatory follicle produce neurotensin (NTS) in response to the LH surge or the LH receptor ligand hCG, and local action of NTS is essential for ovulation to occur. Theca cells express receptors for LH/hCG as well as NTS, so either stimulus may alter theca cell migration.
METHODS:
To determine the role of hCG and hCG-stimulated NTS in theca cell migration in vivo, a vehicle control or a NTS receptor antagonist was injected into the adult female cynomolgus macaque ovulatory follicle (n=3-4/treatment), and hCG was administered systemically. After 48 hours, the ovaries were removed and fixed for immunofluorescent detection of theca cells. Primary theca cells isolated from monkey ovaries (n=3-4 lines/experiment) were cultured in vitro and allowed to migrate through a porous membrane towards hCG or NTS. Membranes were fixed and stained, and the number of migrated cells was quantified. Theca cells were also seeded onto beads and allowed to migrate through 3D-matrix in the presence of hCG or NTS. Migratory distance and number of migratory projections were determined. Theca cell-coated beads were fluorescently stained to assess cell morphology and migratory structures.
RESULTS:
In vivo, hCG stimulated theca cell invasion from the surrounding stroma into the ovulatory follicle. Follicle injection of a NTS receptor antagonist reduced the hCG-stimulated increase in theca cell invasion by 66%, demonstrating that both hCG and hCG-stimulated NTS can promote theca cell migration in vivo. In vitro, hCG and NTS increased theca cell migration 66% above basal migration and 71% above basal migration, respectively. NTS-stimulated theca cell migration was blocked in the presence of a NTS receptor antagonist in vitro. In 3D-matrix, NTS increased both theca cell migration distance by 77% and the number of theca cell projections by 74% over basal levels. In addition, NTS-treated theca cells formed more migratory structures such as filopodia.
CONCLUSION:
Theca cells actively migrate in response to hCG and NTS during ovulation. These findings indicate that LH/hCG-stimulated theca cell migration in vivo may be dependent upon locally-produced NTS. Future investigation will query the mechanism by which theca cells migrate and the role of extracellular matrix in ovulatory migration. These studies will contribute to our overall understanding of theca cell function, and may shed light on the development of future fertility-improving treatments or contraceptive strategies. Supported by a generous product donation from Organon and NIH HD097675 to DMD and TEC.
Theca cells are an integral component of the ovarian follicle. Theca cells synthesize all androgens produced by the follicle. Androgens are then converted into estrogens by granulosa cells. After the ovulatory luteinizing hormone (LH) surge, oocyte release occurs concomitantly with the rapid transformation of the follicle remnant into the corpus luteum. During this process, theca cells integrate into the granulosa cell layer. Whether theca cell movement during ovulation is the result of passive relocation or if it is an active, intentional process of migration has not been investigated. We hypothesize that theca cells actively migrate during ovulation in response to hCG and/or hCG-stimulated local mediators of ovulation. Granulosa cells of the ovulatory follicle produce neurotensin (NTS) in response to the LH surge or the LH receptor ligand hCG, and local action of NTS is essential for ovulation to occur. Theca cells express receptors for LH/hCG as well as NTS, so either stimulus may alter theca cell migration.
METHODS:
To determine the role of hCG and hCG-stimulated NTS in theca cell migration in vivo, a vehicle control or a NTS receptor antagonist was injected into the adult female cynomolgus macaque ovulatory follicle (n=3-4/treatment), and hCG was administered systemically. After 48 hours, the ovaries were removed and fixed for immunofluorescent detection of theca cells. Primary theca cells isolated from monkey ovaries (n=3-4 lines/experiment) were cultured in vitro and allowed to migrate through a porous membrane towards hCG or NTS. Membranes were fixed and stained, and the number of migrated cells was quantified. Theca cells were also seeded onto beads and allowed to migrate through 3D-matrix in the presence of hCG or NTS. Migratory distance and number of migratory projections were determined. Theca cell-coated beads were fluorescently stained to assess cell morphology and migratory structures.
RESULTS:
In vivo, hCG stimulated theca cell invasion from the surrounding stroma into the ovulatory follicle. Follicle injection of a NTS receptor antagonist reduced the hCG-stimulated increase in theca cell invasion by 66%, demonstrating that both hCG and hCG-stimulated NTS can promote theca cell migration in vivo. In vitro, hCG and NTS increased theca cell migration 66% above basal migration and 71% above basal migration, respectively. NTS-stimulated theca cell migration was blocked in the presence of a NTS receptor antagonist in vitro. In 3D-matrix, NTS increased both theca cell migration distance by 77% and the number of theca cell projections by 74% over basal levels. In addition, NTS-treated theca cells formed more migratory structures such as filopodia.
CONCLUSION:
Theca cells actively migrate in response to hCG and NTS during ovulation. These findings indicate that LH/hCG-stimulated theca cell migration in vivo may be dependent upon locally-produced NTS. Future investigation will query the mechanism by which theca cells migrate and the role of extracellular matrix in ovulatory migration. These studies will contribute to our overall understanding of theca cell function, and may shed light on the development of future fertility-improving treatments or contraceptive strategies. Supported by a generous product donation from Organon and NIH HD097675 to DMD and TEC.