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8 November 2022 Neurotensin expression, regulation, and function during the ovulatory period in the mouse ovary
Ketan Shrestha, Linah Al-Alem, Priscilla Garcia, Michelle A. A. Wynn, Patrick R. Hannon, Misung Jo, Jenny Drnevich, Diane M. Duffy, Thomas E. Curry Jr
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Abstract

The luteinizing hormone (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 pregnant mare serum gonadotropin 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). 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.

Summary Sentence

Nts expression increased 250-fold 4 h after hCG in mouse ovaries and is regulated through classical hCG signaling pathways. Using siRNA followed by RNAseq, Ell2, Rsad2, Vps37a, and Smtnl2 were identified as NTS downstream targets in granulosa cells.

Graphical Abstract

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© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Ketan Shrestha, Linah Al-Alem, Priscilla Garcia, Michelle A. A. Wynn, Patrick R. Hannon, Misung Jo, Jenny Drnevich, Diane M. Duffy, and Thomas E. Curry Jr "Neurotensin expression, regulation, and function during the ovulatory period in the mouse ovary," Biology of Reproduction 108(1), 107-120, (8 November 2022). https://doi.org/10.1093/biolre/ioac191
Received: 7 March 2022; Accepted: 11 October 2022; Published: 8 November 2022
KEYWORDS
granulosa cells
neurotensin
ovulation
RNASeq
silencing
siRNA
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