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. 2020 Jan 23;10(1):1021.
doi: 10.1038/s41598-020-57672-w.

Ovarian mitochondrial dynamics and cell fate regulation in an androgen-induced rat model of polycystic ovarian syndrome

Reza Salehi  1 , Hannah L Mazier  1   2 , Anne-Laure Nivet  1   2 , Arkadiy A Reunov  3 , Patricia Lima  1 , Qi Wang  1 , Arianna Fiocco  2   4 , Ciro Isidoro  5 , Benjamin K Tsang  6   7
Affiliations

Ovarian mitochondrial dynamics and cell fate regulation in an androgen-induced rat model of polycystic ovarian syndrome

Reza Salehi et al. Sci Rep. .

Abstract

In this study, we investigated in an androgenized rat model the involvement of autophagy and mitochondrial dynamics in granulosa cells in the pathogenesis of polycystic ovarian syndrome (PCOS) and its modulation by exogenous gonadotropin (eCG). We found 5α-dihydrotestosterone (DHT) treatment reduces ovarian length and weight with predominantly late antral and/or preovulatory stage follicles and no corpora lutea. DHT increased the population of large lysosomes (>50 micron) and macroautophagy, an event associated with granulosa cell apoptosis. Increased granulosa cell Dynamin Related Protein 1 (Drp1) content in the DHT group was accompanied by increased circular and constricted, but reduced rod-shaped, mitochondria. eCG eliminated all atypical follicles and increased the number of late antral and preovulatory follicles with less granulosa cell apoptosis. eCG-treated rats had a higher proportion of connected mitochondria, and in combination with DHT had a lower proportion of circular and constricted mitochondria than rats treated with DHT alone, suggesting that eCG induces mitochondrial fusion and attenuates fission in granulosa cells. In summary, we observed that DHT-induced up-regulation of Drp1 is associated with excessive mitochondrial fission, macroautophagy and apoptosis in granulosa cells at the antral stage of development in an androgenized rat model for PCOS, a response partially attenuated by exogenous gonadotropin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
One-month DHT model demonstrates ovarian changes as in PCOS, but not the systemic changes. Rats were surgically implanted with sham control capsule or DHT (83 μg/d) time-release capsules for 1-, 2- or 3- month. (A) DHT treatment significantly increases body weight in 2 and 3 months, but not in 1 month (n = 8 rats per experimental group at each time point); (B) insulin sensitivity index significantly reduces in 2-month model, but not in 1- and 3-month models (n = 8 rats per experimental group at each time point); (C) DHT treatment dysregulates the estrus cycle in 1-, 2- and 3-month DHT treated rat model (n = 3 rats per experimental group at each time point); (D) Ovarian width significantly reduces in 1-, 2- and 3-month DHT treated rat model (n=8 rats per experimental group at each time point); (E) TUNEL (brown) and Methyl green coloration of ovarian paraffin sections from 1-, 2-, 3-month control and DHT rats. Data are presented as mean ± SEM, and analyzed by two-way ANOVA and Bonferroni post hoc test. **P < 0.01; ***P < 0.001 vs. CTL.
Figure 2
Figure 2
DHT reduced ovarian length and weight, responses reverted by gonadotropin injection. Rats were surgically implanted with sham control capsule or DHT (83 μg/d) time-release capsules for 1-month and injected with eCG (20 IU i.p./rat) 48 h prior to sacrifice. Fresh ovaries from each rat were measured for length (A) and weight (B). Data are presented as mean ± SEM of eight independent replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test. ***P < 0.001 vs. CTL. ##P < 0.01 vs. DHT alone.(C) representative sections of whole ovaries in each treatment group were visualized by H&E staining and variations in ovarian structural features were observed. PAF, preantral follicle; EAF, early antral follicle; POvF, preovulatory follicle; CL, corpus luteum; AtyF, atypical follicle.
Figure 3
Figure 3
DHT induced granulosa cell apoptosis, an effect attenuated by gonadotropin. (A) Representative images of TUNEL positive (+) preantral (PAF), early antral (EAF), late antral (LAF) and preovulatory follicles (POvF) from a 1-month DHT-induced rat ovary. (B) After 1-month DHT implantation (83 μg/d) and 48 h after eCG injection (20 IU i.p./rat), granulosa cell apoptosis was determined in PAF, EAF, LAF and POvF of whole ovarian sections by fluorescent TUNEL assay, and was expressed as a ratio of mean number of TUNEL+ follicles over total ovarian follicles per rat* (*Rat = 1 ovary per rat/3 representative slides from whole ovary/3 sections per slide). Data are presented as mean ± SEM of three independent replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test. *P < 0.05; **P < 0.01 vs. CTL. #P < 0.01 vs. DHT. ++P < 0.01 vs. DHT.
Figure 4
Figure 4
DHT treatment increased the population of large lysosomes (>50 micron). (A,i-iv) Representative examples of autophagic structures (autolysosome <0.5 micron, autolysosome >0.5 micron, autophagosome and mitophagic structures) using transmission electron microscopy (TEM) images. (B) After 1-month DHT implantation (83 μg/d) and 48 h following eCG (20 IU i.p./rat), TEM images of whole ovaries from each treatment group were captured and autophagic structures was assessed in granulosa cells. A minimum of 68 cells was assessed per treatment group. Data are presented as mean ± SEM of three independent replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test. ***P < 0.001 vs. CTL.
Figure 5
Figure 5
DHT treatment induces the accumulation of LC3 in Granulosa cells. After 1-month DHT implantation (83 μg/d) and eCG injection (20 IU/rat) 48 h prior to sacrifice, granulosa cells were isolated from rat ovaries and Western blots were performed to determine the protein content of LC3 (isoforms I and II) and of p62. Quantification of autophagy markers versus loading control as well as LC3II:LC3I ratio is represented in the histograms. Data are presented as mean ± SEM of six independent replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test.
Figure 6
Figure 6
DHT-induced circular and constricted mitochondrial appearance were reduced by gonadotropin. (A, i–iii) Representative examples of mitochondrial phenotypes (rod-shaped, circular, constricted or connected appearance) in transmission electron microscopy (TEM) images. (B) After 1-month DHT implantation (83 μg/d) and 48 h following eCG (20 IU i.p./rat), TEM images of whole ovaries from each treatment group were captured and mitochondrial morphology was assessed. A minimum of 275 mitochondria was assessed per treatment group. Data are presented as mean ± SEM of three replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CTL; #P < 0.05, ###P < 0.001 vs. DHT; +P < 0.05, +++P < 0.001 vs. eCG. Scale bar = 500 nm.
Figure 7
Figure 7
Granulosa cells in one-month DHT-treated rats exhibited increased total Drp1 content. After 1-month DHT implantation (83 μg/d) and 48 h after eCG (20 IU i.p./rat), granulosa cells were isolated and protein contents of phospho-Drp1 Ser616, phospho-Drp1 Ser637 and total Drp1 were assessed by Western blot. Data are presented as mean ± SEM of six independent replicate experiments, and analyzed by two-way ANOVA and Bonferroni post hoc test.
Figure 8
Figure 8
Hypothetical model of showing androgen excess-induced changes in granulosa cell mitochondrial dynamics in PCOS and the influence of gonadotropic stimulation. (A) In PCOS, androgen excess results in the up-regulation of Drp1, leading to increased mitochondrial fission. This dysregulation in mitochondrial fission results in increased autophagy and apoptosis, leading to early antral follicular growth arrest; (B) Gonadotropin stimulation attenuates mitochondrial fission, apoptosis and follicular growth arrest in PCOS.
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