Neurotensin expression, regulation, and function during the ovulatory period in the mouse ovary

Abstract

The LH surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day old PMSG-primed mice before (0 h) and after administration of human chorionic gonadotropin (hCG; an LH-analog) across the periovulatory period (4, 8, 12, and 24 h). In response to hCG, Nts expression rapidly increased 250-fold at 4 h, remained elevated until 8 h and decreased until 24 h. Expression of Nts receptors for Ntsr1 remained unchanged across the periovulatory period, Ntsr2 was undetectable whereas Sort1 expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Nts regulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Nts expression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Nts induction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Nts silencing approach (si-Nts) followed by RNA sequencing. RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a and Smtnl2 as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Nts and that Nts expression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.

Neurotensin modulates ovarian vascular permeability via adherens junctions

“…4 Neurotensin mRNA is highly expressed by granulosa cells of the ovulatory follicle in response to the LH surge, and all three known NTS receptors are expressed by follicle cells. [3][4][5] In the present study, we highlight increased red blood cell accumulation in the luteinizing follicle after blockade of NTS receptors with SR142948; similar red blood cell accumulation was observed after NTS neutralization. 4 These observations led to the hypothesis that NTS acts at follicular endothelial cells to regulate the permeability of the developing vasculature.…”

“…Neurotensin was recently identified as a LH‐stimulated paracrine mediator of ovulation 4 . Neurotensin mRNA is highly expressed by granulosa cells of the ovulatory follicle in response to the LH surge, and all three known NTS receptors are expressed by follicle cells 3–5 . In the present study, we highlight increased red blood cell accumulation in the luteinizing follicle after blockade of NTS receptors with SR142948; similar red blood cell accumulation was observed after NTS neutralization 4 .…”

“…88,89 NTS, however, is rapidly induced by the LH surge and expressed at high levels until ovulation. [3][4][5] NTS may serve as a critical counterbalance to the increased permeability during aggressive ovulatory angiogenesis and mediate appropriate regulation of permeability during the transition from the stable preovulatory follicle to mature corpus luteum.…”

“…1 Recent studies have identified neurotensin (NTS) as a paracrine mediator of ovulation. [3][4][5] This 13-amino acid peptide is produced primarily by the granulosa cells in response to the LH surge and is transiently but highly upregulated around the time of ovulation in the human, monkey, rat, and mouse ovary. [3][4][5] NTS exerts its effect directly on ovarian endothelial cells.…”

“…[3][4][5] This 13-amino acid peptide is produced primarily by the granulosa cells in response to the LH surge and is transiently but highly upregulated around the time of ovulation in the human, monkey, rat, and mouse ovary. [3][4][5] NTS exerts its effect directly on ovarian endothelial cells. The pro-angiogenic activities of migration and endothelial sprout formation are increased in cultured primary monkey ovarian microvascular endothelial cells (mOMECs) treated with NTS.…”