Presentations

Abstract

Introduction: Neurotensin (NTS), a 13-amino acid peptide, is produced within the ovarian follicle and is essential for ovulation to occur. Granulosa cells produce NTS in response to the ovulatory surge of luteinizing hormone (LH). Theca cells of the ovarian follicles are responsible for producing androgens, which granulosa cells convert to estrogens. Both theca and granulosa cells express NTS receptors. Theca steroidogenesis-the production of steroid hormones such as progesterone (P4), androstenedione (A4), and testosterone (T)-is known to be stimulated by LH or human chorionic gonadotropin (hCG) via the LH/CG receptor (LHCGR). LHCGR activation impacts steroidogenesis by modulating cholesterol utilization and steroidogenic enzyme expression and activity. We hypothesize that NTS stimulates theca cell androgen production by altering cholesterol and/or steroidogenic enzyme expression.

Methods: Ovaries were removed from adult, female cynomolgus macaques (Macaca fascicularis) after a modified ovarian stimulation protocol, and the theca layers were isolated from antral (2-4 mm) follicles. Theca tissue was minced to uniform size and cultured in serum-free media with or without NTS or hCG. Media was collected after 24 or 48 hours and analyzed for A4, T, and P4 concentrations via ELISA. LDL uptake after 4 hours with or without NTS or hCG was determined via LDL ELISA of lipoproteins extracted from theca tissue homogenate. Total and esterified cholesterol after 24 or 48 hours treatment with or without NTS or hCG was determined via colorimetric detection of theca tissue homogenate in a modified cholesterol quantification assay using resorufin. Theca tissue RNA was isolated after 4 hours with or without NTS or hCG and analyzed for expression of steroidogenic protein mRNA after reverse transcription and qPCR, and mRNAs of interest were normalized to expression of GAPDH. For culture of proliferating theca cells, theca cells were enzymatically dispersed from surrounding matrix and enriched by culture under conditions ideal for theca cells. Lipid droplet contents of cultured theca cells were quantified and visualized after 24 hours with or without NTS or hCG using Oil Red O staining. For lipid quantification, Oil Red O stain was extracted from theca cells and relative stain intensity was quantified via absorbance at 492 nm. Theca lipid droplets were visualized via microscopy of theca cells stained with Oil Red O and counterstained with hematoxylin.

Results: NTS increased A4 and T concentrations in theca tissue media after 24 hours in a dose-dependent manner. However, NTS treatment did not significantly alter A4 or T after 48 hours compared to basal. In contrast, NTS did not alter P4 media accumulation after 24 or 48 hours compared to basal. As expected, hCG increased A4, T, and P4 media accumulation at both 24 and 48 hours compared to basal. Neither NTS nor hCG significantly altered theca tissue LDL uptake, total cholesterol content, or esterified cholesterol content compared to basal. The quantity and appearance of isolated theca cell lipid droplet contents were also unaffected by NTS or hCG compared to basal. Theca tissue expression of CYP17A1 and STAR mRNA were unaffected by NTS or hCG compared to basal.

Conclusion: These results indicate that NTS stimulates production of androgens but not progesterone in theca cells. While LHCGR stimulation via hCG increased production of all theca steroids, neither NTS nor hCG were found to significantly affect LDL uptake, cholesterol, lipid droplets, or CYP17A1 or STAR mRNA at the selected time points. While cholesterol and steroidogenic protein mRNA are prominent areas of steroidogenic regulation, there are other processes where NTS and hCG could be acting to alter theca steroid production. LHCGR has also been found to regulate theca steroidogenesis by augmenting enzyme activity levels via protein kinase A (PKA). Accordingly, NTS and hCG may regulate enzyme activity without alteration in enzyme mRNA levels. In addition, neither NTS nor hCG appear to increase steroidogenesis primarily through regulation of cholesterol utilization/availability. Androgen production is a key function of theca cells that is necessary for ovulation and corpus luteum formation. These data implicate NTS as a novel regulator of theca androgen production, and the alignment of follicular NTS production with ovulation suggests a key role for this regulation in ovarian function. Future studies will explore possible regulation of other steroidogenic enzymes (e.g., CYP11A1, HSD3B, HSD17B) and investigate enzyme activity as a potential focal point of steroidogenic regulation.