Neurotensin: A novel mediator of ovulation?

The midcycle luteinizing hormone (LH) surge initiates a cascade of events within the ovarian follicle which culminates in ovulation. Only mural granulosa cells and theca cells express large numbers of LH receptors, and LH-stimulated paracrine mediators communicate the ovulatory signal within the follicle. Recent reports identified the neuropeptide neurotensin (NTS) as a product of granulosa cells. Here, we demonstrate that granulosa cells were the primary site of NTS expression in macaque ovulatory follicles. Granulosa cell NTS mRNA and protein increased after human chorionic gonadotropin (hCG) administration, which substitutes for the LH surge. To identify ovulatory actions of NTS, a NTS-neutralizing antibody was injected into preovulatory macaque follicles. hCG administration immediately followed, and ovaries were removed 48 hours later to evaluate ovulatory events. Follicles injected with control IgG ovulated normally. In contrast, 75% of NTS antibody-injected follicles failed to ovulate, containing oocytes trapped within unruptured, hemorrhagic follicles. Serum progesterone was unchanged. Of the three NTS receptors, SORT1 was highly expressed in follicular granulosa, theca, and endothelial cells; NTSR1 and NTSR2 were expressed at lower levels. Excessive blood cells in NTS antibody-injected follicles indicated vascular anomalies, so the response of monkey ovarian endothelial cells to NTS was evaluated in vitro. NTS stimulated endothelial cell migration and capillary sprout formation, consistent with a role for NTS in vascular remodeling associated with ovulation. In summary, we identified NTS as a possible paracrine mediator of ovulation. Further investigation of the NTS synthesis/response pathway may lead to improved treatments for infertility and novel targets for contraception.

Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates.

Angiogenesis is essential to both ovulation and the formation of the corpus luteum. The thrombospondin (THBS) family of glycoproteins plays diverse roles in regulation of angiogenesis, but the role of these vascular regulators in ovulation and luteinization remain to be elucidated. Using the cynomolgus macaque as a model for human ovulation, we demonstrated that levels of THBS1 mRNA and protein in preovulatory follicle granulosa cells increased after the ovulatory gonadotropin surge, with peak levels just before the expected time of ovulation. THBS1 treatment of monkey ovarian microvascular endothelial cells in vitro stimulated migration, proliferation, and capillary sprout formation, consistent with a pro-angiogenic action of THBS1. Injection of an anti-THBS1 antibody into monkey preovulatory follicles reduced rates of follicle rupture and oocyte release in response to an ovulatory gonadotropin stimulus when compared with control IgG-injected follicles. Interestingly, two of three oocytes from anti-THBS1 antibody injected follicles were germinal vesicle intact, indicating that meiosis failed to resume as anticipated. Follicles injected with anti-THBS1 antibody also showed reduced granulosa cell layer expansion, endothelial cell invasion, and capillary formation when compared to control IgG-injected follicles. Overall, these findings support a critical role for THBS1 in follicular angiogenesis, with implications for both successful ovulation and corpus luteum formation.

Placental Growth Factor Is Required for Ovulation, Luteinization, and Angiogenesis in Primate Ovulatory Follicles.

Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.