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. 2021 Oct;99(10):1427-1446.
doi: 10.1007/s00109-021-02104-z. Epub 2021 Jun 28.

Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance

Yuehui Zhang #  1   2 , Min Hu #  2   3   4 , Fan Yang  1 , Yizhuo Zhang  1 , Shuting Ma  1 , Dongqi Zhang  1 , Xu Wang  1 , Amanda Nancy Sferruzzi-Perri  5 , Xiaoke Wu  1 , Mats Brännström  6 , Linus R Shao  7 , Håkan Billig  2
Affiliations

Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance

Yuehui Zhang et al. J Mol Med (Berl). 2021 Oct.

Abstract

In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility. KEY MESSAGES: The proper regulation of uterine androgen receptor (AR) contributes to a normal pregnancy process, whereas the aberrant regulation of uterine AR might be linked to polycystic ovary syndrome (PCOS)-induced pregnancy-related complications. In the current study, we found that during normal rat pregnancy there is a stage-dependent decrease in AR abundance in the gravid uterus and that this is correlated with the differential expression of the endometrial receptivity and decidualization genes Spp1, Prl, Igfbp1, and Hbegf. Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS) or to DHT alone show elevated uterine AR protein abundance and implantation failure related to the aberrant expression of genes involved in endometrial receptivity and decidualization in early to mid-gestation. Treatment with the anti-androgen flutamide, starting from pre-implantation, effectively prevents DHT + INS-induced defects in endometrial receptivity and decidualization gene expression, restores uterine mitochondrial homeostasis, and increases the pregnancy rate and the numbers of viable fetuses. This study adds to our understanding of the mechanisms underlying poor pregnancy outcomes in PCOS patients and the possible therapeutic use of anti-androgens, including flutamide, after spontaneous conception.

Keywords: Androgen receptor; Flutamide; Implantation; Mitochondrial function; Polycystic ovary syndrome; Pregnant uterus.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Exposure to DHT or INS alone or in combination alters body weight, embryo or fetal number, and implantation-related gene expression in rats during pregnancy. Schematic of the experimental design A. Comparison of body weight and embryo or fetal number in pregnant rats treated with DHT and/or INS (B, n = 8/group). The fertility index is the percentage of matings that resulted in pregnancy. In all plots the data are presented as means ± SEM. aP < 0.05, Control (vehicle) group vs. DHT + INS group; bP < 0.01, Control group vs. DHT group; cP < 0.05, DHT + INS group vs. INS group; dP < 0.05, DHT group vs. INS group. *P < 0.05, ***P < 0.001. Time-dependent changes in implantation-related gene expression C. After removing the embryos/fetuses and placenta, uterine tissues from pregnant rats treated with vehicle, DHT + INS, DHT, or INS were used for analyzing genes for uterine receptivity and decidualization by qPCR (n = 4–6/group). In all plots, data are presented as means ± SEM (vs. Control GD 4.5 values). Statistical tests are described in the “Materials and methods” section, and differences between the groups are reported as *P < 0.05, ** P < 0.01, and ***P < 0.001
Fig. 2
Fig. 2
Differential regulation of AR protein in uteri collected from pregnant rats exposed to DHT and/or INS from GD 4.5 to GD 14.5. Time-dependent regulation of AR protein abundance in the pregnant uterus. After removing the embryos/fetuses and placenta, the uterine tissues from pregnant rats treated with control (vehicle), DHT + INS, DHT, or INS were used for analyzing AR and uterine cell marker proteins (cytokeratin, vimentin, and α-smooth muscle actin) by Western blotting. In all plots, the relative mean protein abundance ± SEM (vs. Control GD 4.5 values, n = 5–6/group) was measured by Western blotting with total proteins serving as loading controls. Statistical tests are described in the “Materials and methods” section, and differences between the groups are reported as *P < 0.05, **P < 0.01, and ***P < 0.001. The size representation in kilodaltons (kDa), as determined by a molecular weight ladder, is shown to the right
Fig. 3
Fig. 3
Localization of the AR protein in uteri collected from pregnant rats exposed to DHT and/or INS from GD 4.5 to GD 14.5. Histological appearance of gravid uteri using hematoxylin and eosin (H&E) staining (A1–A4, B1–B4, C1–C4, and D1–D4) and AR localization by immunohistochemistry (A5–A8, B5–B8, C5–C8, and D5–D8) in pregnant rats treated with vehicle A, DHT + INS B, DHT C, or INS D. Images are representative of eight tissue replicates. GD, gestational day; DHT, 5α-dihydrotestosterone; INS, insulin; En, endometrium; Cm, circular myometrium; Lm, longitudinal myometrium; Le, luminal epithelial cells; Ge, glandular epithelial cells; Str, stromal cells; E, embryo; PDZ, primary decidual zone; MT, mesometrial triangle; MD, mesometrial decidua; P, placental disc. Scale bars (100 μm) are indicated in the photomicrographs
Fig. 4
Fig. 4
Chronic treatment with flutamide induces changes in hormones and metabolites in control and DHT + INS-exposed pregnant rats at GD 14.5. Comparison of body weight A, uterine weight B, fertility index C, fetal number D, serum total T E, A4 F, DHT G, DHEA H, SHBG I, P4 J, E2 K, blood glucose concentrations during OGTT L, and area under the curve (AUC) for glucose M in pregnant rats treated with and without DHT and INS [n = 10/group, except for the DHT + INS + flutamide group (with fetuses, n = 7) and the DHT + INS + flutamide (without fetuses) group (n = 3)]. The fertility index is the percentage of matings that resulted in pregnancy. AUC was calculated by the formula [0.5 × (BG0 + BG30)/2 + 0.5 × (BG30 + BG60)/2 + 0.5 × (BG60 + BG90)/2 + 0.5 × (BG90 + BG120)/2], where the BG terms are the blood glucose levels at 0 min, 30 min, 60 min, 90 min, and 120 min. aP < 0.05, control (vehicle) group vs. DHT + INS group; bP < 0.05, control group vs. DHT + INS + flutamide (no fetuses) group; cP < 0.05, DHT + INS group vs. DHT + INS + flutamide (with fetuses) group; dP < 0.05, DHT + INS + flutamide group vs. DHT + INS + flutamide (without fetuses) group. In all plots, data are presented as means ± SEM. Statistical tests are described in the “Materials and methods” section, and differences between the groups are reported as *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 5
Fig. 5
Effects of flutamide on uterine morphology, implantation-related gene expression, and AR, p21WAF1/CIP1, and mitochondrial marker protein expression in control and DHT + INS-exposed pregnant rats at GD 14.5. Histological analysis by H&E staining in vehicle control A, B and DHT + INS-exposed C, D pregnant rats treated with flutamide B, D. The yellow arrowheads indicate infiltrated immune cells in the endometrial gland. Images are representative of 7–10 tissue replicates. Ut, uterus; P, placenta; MT, mesometrial triangle; MD, mesometrial decidua; M, myometrium; En, endometrium; G, gland. Scale bars (100 μm) are indicated in the photomicrographs. After removing the embryos/fetuses and placentas, uterine tissues from control and DHT + INS-treated pregnant rats treated with flutamide were used for analyzing uterine receptivity and decidualization genes by qPCR (F, n = 5–7/group), and the relative abundance of AR, p21WAF1/CIP1, VDAC, and PHB1 proteins was determined by Western blotting (G, 5–6/group). The size representation in kDa, as determined by a molecular weight ladder, is shown to the right. In all plots, data are presented as means ± SEM (vs. control vehicle values). Statistical tests are described in the “Materials and methods” section, and differences between the groups are reported as *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 6
Fig. 6
Effects of flutamide on mitochondrial transcriptional activation, uterine oxidative phosphorylation (OXPHOS) protein expression, and mitochondrial morphology in control and DHT + INS-exposed pregnant rats at GD 14.5. After removing the embryos/fetuses and placentas, uterine tissues from control and DHT + INS-exposed pregnant rats treated with flutamide were used for analyzing mRNA levels of Tfam, Pgc1a, and Nrf1 by qPCR (A, n = 5–7/group), and the relative protein abundance of O2-dependent mitochondrial OXPHOS subunits was determined by Western blotting (B, Complexes I–V, 5–6/group). The size representation in kDa, as determined by a molecular weight ladder, is shown to the right. In all plots, data are presented as means ± SEM (vs. Control vehicle values). Statistical tests are described in the “Materials and methods” section, and differences between the groups are reported as * P < 0.05, ** P < 0.01, and *** P < 0.001. Uterine ultrastructural analysis by TEM in control (C1–C2) and DHT + INS-treated (C3–C4) pregnant rats treated with flutamide (C2 and C4). Images are representative of two tissues per group, and enhanced magnifications are shown in the lower panel of each photomicrograph. DB, decidual basalis; Epi, epithelial cells; Str, stromal cells. Scale bars (2 μm) are indicated in the photomicrographs
Fig. 7
Fig. 7
A schematic representation illustrating our working hypothesis regarding the regulation and activation of gravid uterine AR signaling pathways after combined exposure of hyperandrogenism and INS resistance. The signaling pathway depicted is based on the findings of the current study. There is evidence that cytosolic AR is translocated to and/or exists in the mitochondrion in several cell types and thus might exert effects downstream of the hyperandrogenism, thus leading to mitochondrial dysfunction. Further studies are required to determine whether PCOS-induced uterine cell defects are due to the mitochondrial AR actions during pregnancy
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