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. 2023 Apr 12;16:74. doi: 10.1186/s13048-023-01141-1

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© The Author(s) 2023

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Androgen-induced exosomal mir-379-5p release as a determinant of rat granulosa cell mir-379-5p content and proliferation. A & B Irrespective of DHT treatment, GW4869 at 20 nM effectively inhibited exosome release, as evident by increased granulosa cellular content and reduced conditioned medium level of CD63 protein (exosome marker; Supplementary Fig. 3). The inhibition of exosome release increased granulosa cell mir-379-5p content (normalized to U6; DHT/control ratio) and down-regulate cell proliferation (MCM2) induced by DHT; C DHT increased granulosa cell cortactin protein content in granulosa cells from preantral, but not antral follicle; D cortactin overexpression in combination with DHT significantly increased exosome release in antral follicle granulosa cells and reduced granulosa cell mir-379-5p content (normalized to U6). Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats;1 mg/d, subcutaneous injection, for 3 consecutive days) or antral follicles (Equine chorionic gonadotropin–injected 21-day old rats; 10 IU, intraperitoneal injection, animals sacrificed 48 h post-injection). Exosome release was inhibited with GW4869 pre-treatment (0, 10 and 20 mM, 24 h) before DHT (0 to 1 µM, 24 h and 36 h). Exosomes were isolated from granulosa cell conditioned medium by differential centrifugations. To induce exosome release in antral follicle stage, granulosa cells were infected with adenoviral sense full-length cortactin cDNA or adenoviral control vector at 50 MOI (24 h), and then cultured with DHT (0 to 1 µM, 24 h and 36 h). Protein extraction and Western blotting were performed as described previously [15]. Signal intensities generated on the film were measured densitometrically using Image J and normalized over that of GAPDH or β-Actin. U6 RNA was used to normalize miR-379-5p content. Results are expressed as means ± SEM (n = 3 replicates, each from 2 rats). Data were analyzed by (A & B) two-way ANOVA and tukey post hoc; (C & D) three-way ANOVA and tukey post hoc. *P < 0.05; **P < 0.01, ***P < 0.001