Prenatally androgenized female rats develop uterine hyperplasia when adult

Molecular and Cellular Endocrinology 499 (2020) 110610

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Prenatally androgenized female rats develop uterine hyperplasia when adult a,∗

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Silvana Rocío Ferreira , Alicia Alejandra Goyeneche , María Florencia Heber , Giselle Adriana Abruzzesea, Carlos Marcelo Telleriab,1, Alicia Beatriz Mottaa,1 a

Laboratorio de Fisio-Patología Ovárica, Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Medicina, Universidad de Buenos Aires (UBA), Argentina Experimental Pathology Unit, Department of Pathology, Faculty of Medicine, McGill University, 3775 University Street, Montreal, QC H3A 2B4, Canada

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Keywords: Uterine Endometrium Prenatal hyperandrogenism PCOS

Prenatal hyperandrogenization (PH) is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). In this study, we aimed to investigate the impact of prenatal exposure to androgen excess on the uterus when animals reach their adulthood. We found that PH altered the morphology of the uteri that show a hyperplastic morphology with increased total uterine thickness as well as luminal epithelium thickness, with both enhanced and altered distribution of glands as compared with controls. Morphological alterations were associated with an unbalanced homeostasis as assessed by the expression of regulators of cell cycle progression and cell death dynamics. PH also causes disturbances in the cell cycle of the uterine tissue and dysregulates cell death and survival pathways leading to the development of uterine hyperplasia. These findings suggest that PH may have a deleterious effect on the uterus.

1. Introduction Prenatal exposure to androgens during intrauterine development in females impacts on steroid target tissues and lead to reproductive alterations along puberty and adulthood (Cardoso and Padmanabhan, 2018), irregular or absent estrous cycles, abnormal follicular development, and decreased uterine response to hormones that could affect fertility (Padmanabhan et al., 2006; Padmanabhan and Veiga-Lopez, 2014). These features, in combination with other factors such as metabolic disturbances, are common in women diagnosed with Polycystic Ovarian Syndrome (PCOS) (Azziz, 2018; Goodman et al., 2015; Walters et al., 2018). PCOS is a complex endocrine disorder that affects women at their reproductive age. To diagnose PCOS, the last consensus released by the Androgen Excess (AE) and PCOS Society proposed two criteria: 1) clinical and/or biochemical hyperandrogenism; and 2) ovarian dysfunction associated with oligo-anovulation and/or polycystic ovaries (Azziz et al., 2006). This pathology has a multifactorial origin and its etiology remains unknown (Diamanti-Kandarakis et al., 2006; Dumesic et al., 2014, 2007). Nowadays, the most accepted hypothesis proposes that PCOS has an intrauterine origin and is caused by several genetic and environmental factors (de Melo et al., 2015; Fenichel et al., 2017;

Franks et al., 2006; Walters et al., 2018). To recreate PCOS-associated symptoms, prenatal androgen exposure is commonly utilized to induce reproductive and metabolic features of human PCOS in rats (Demissie et al., 2008; Foecking et al., 2008; Zhang et al., 2019), monkeys (Abbott et al., 2017, Abbott et al., 2002) and sheeps (Birch et al., 2003; Foecking et al., 2005; Kelley et al., 2019). Experimental animal research indicates that prenatal androgen excess affects several organs in utero, leading to altered genetic expression in adulthood (Abruzzese et al., 2016; Amalfi et al., 2012; Filippou and Homburg, 2017; Heber et al., 2019). Fetal programming mediated by prenatal hyperandrogenism is related to insulin resistance (Bruns et al., 2004; Recabarren et al., 2006), metabolic syndrome (Cardoso et al., 2015; Demissie et al., 2008), endometrial hyperplasia, and/or cancer (Giudice, 2006; Harris and Terry, 2016). However, how fetal programming induced by prenatal hyperandrogenization affects uterine homeostasis and contributes to the pathogenesis of PCOS is still matter of debate. PCOS women have high rates of pregnancy loss, even if ovulation is restored, or by using assisted reproduction techniques, suggesting a problem in their uterine receptivity (Bahri Khomami et al., 2019; Ramezanali et al., 2016; Tandulwadkar et al., 2014). The uterus is a dynamic organ cyclically exposed to hormones. The

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Corresponding author. Laboratorio de Fisio-Patología Ovárica, Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Medicina, Universidad de Buenos Aires (UBA), Paraguay 2155, Buenos Aires, C1121ABG, Argentina. E-mail address: [email protected] (S.R. Ferreira). 1 Co-principal investigators; these authors jointly supervised this work. https://doi.org/10.1016/j.mce.2019.110610 Received 1 August 2019; Received in revised form 19 September 2019; Accepted 3 October 2019 Available online 04 October 2019 0303-7207/ © 2019 Elsevier B.V. All rights reserved.

Molecular and Cellular Endocrinology 499 (2020) 110610

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Following paraffin embedding and sectioning (5 μm), two sections per sample were stained with hematoxylin and eosin (H&E), whereas subsequent sections were used for immunohistochemistry purposes.

direct influence of hormones on the endometrium regulates its homeostasis. To maintain such homeostasis between cell proliferation, survival, and death within the uterus, all genes and protein-products, which control cell death and proliferation, have to be tightly regulated (Avellaira et al., 2006; Oróstica et al., 2016). Thus, loss of cell cycle control could lead to exacerbated proliferation, which associates to the hyperplasia of the uterus found in PCOS women (Villavicencio et al., 2009). The in utero effect of prenatal hyperandrogenism programs the uterus compromising its functionality. Thus, hormonal disorders, which occur in women with PCOS, are highly associated with infertility, high risk of abortion, and the development of endometrial hyperplasia and/ or endometrial cancer (Chakraborty et al., 2013; Fearnley et al., 2010; Homburg, 2004). In this study, we hypothesized that prenatal androgen excess triggers alterations in the morphology and functionality of the uterus by modulating cell cycle and proliferative/apoptotic processes. We demonstrate that fetal programming, triggered by prenatal hyperandrogenization, causes uterine hyperplasia, and dysregulates uterine cell survival and death in association with loss of fertility.

2.2. Study of the estrous cycle Estrous cycles were monitored daily by vaginal smears beginning at 70 days of age and until decapitation. The control group had normal cycles (4–6 days), whereas those animals prenatally exposed to androgens, i.e. the PH group, displayed prolonged cycles lasting 7 days or more (PH ov), or no cycles at all (PH anov) (Abruzzese et al., 2016; Karim et al., 2003). At 90 days (day of euthanasia) the entire PH anov group remained in diestrus; for that reason, and to allow comparison among groups, all animals were sacrificed on the first diestrus after 90 days of age. 2.3. Morphometric studies Samples stained with hematoxylin and eosin (H&E) were photomicrographed at different magnifications (N = 10/group). The thicknesses of the uteri and luminal and glandular epithelia were accurately measured utilizing tools of the Amscope Software 3.7 (United Scope LLC, Irvine, CA, USA). All measures were expressed in μm.

2. Materials and Methods 2.1. Experimental animal model and ethics statement

2.4. Western blot and quantitative analysis Virgin female rats of the Sprague-Dawley strain were mated with fertile males of the same strain. Three females and one male were housed together under controlled conditions of light (12 h light: 12 h dark) and temperature (23–25 °C). Animals received food (Cooperación SRL, Argentina) and water ad libitum. Day 1 of pregnancy was defined as the day in which spermatozoa were observed in the vaginal lavages. Between days 16 and 19 of pregnancy, rats were hyperandrogenized as previously described (Abruzzese et al., 2016a; Heber et al., 2019). Briefly, pregnant rats (N = 15) received subcutaneous injections of 1 mg of free testosterone (Sigma Chemical Co. St. Louis, MO, USA) dissolved in 100 μl sesame oil (Sigma) on days 16, 17, 18, and 19 of pregnancy, whereas the control group (N = 15) received the same number of injections containing only 100 μl of sesame oil as vehicle. The group of female offspring prenatally treated with testosterone was designated as the prenatally hyperandrogenized group or PH group, whereas the offspring of animals receiving vehicle composed the control or C group. The treatments described did not modify the spontaneous term labor, the female-to-male offspring ratio, or the number of pups per litter. Under the conditions of our animal facility, spontaneous term labor occurs on day 22 of gestation. Pups were culled from litters to equalize group sizes (ten pups/mother). Females pups were separated from males at 21 days of age, and randomly assigned to each assay, which was carried out with the same number of animals from each randomly selected littermate. The age of vaginal opening did not differ between C and PH groups. These animals showed their vaginal opening around day 33 of life (Abruzzese et al., 2019a). PH rats had two phenotypes: irregular ovulatory (PH ov) or anovulatory (PH anov), with a hyperandrogenic state during early puberty and adulthood as evidenced by higher levels of testosterone in the PH groups than in the control group (C = 35.9 ± 13.4 pg/ml; PH ov = 291.0 ± 82.7 pg/ml; and PH anov = 344.5 ± 15.0 pg/ml) (Abruzzese et al., 2016; Heber et al., 2019). All the procedures involving animals were conducted in accordance with the 1996 Animal Care and Use Committee of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). The present study was approved by the Ethics Committee of the Facultad de Medicina, Universidad de Buenos Aires, Argentina. One group of female offspring from each experimental set of animals were weighed, exposed to carbon dioxide, and euthanized by decapitation, whereas another group was used to study fertility. One horn of the uterus was extracted and conserved at −80 °C whereas the other one was fixed in 4% formaldehyde neutral buffered solution for 24 h at 4 °C.

Uterine tissues were lysed and prepared for protein extraction (N = 8/group). The lysis solution contained 50 mM Tris-HCl (pH 7.4), 150 mM NaCl (Sigma), 0.5% NP-40 (Sigma), 1 mM PMSF (Sigma), 1 μg/ ml pepstatin (Sigma), 2 μg/ml aprotinin (Sigma), 2 μg/ml leupeptin (Sigma), 1 mM DTT (Promega, Madison, WI, USA), 1 mM sodium orthovanadate (Sigma), and 50 mM sodium fluoride (Sigma). This buffer was added to 50 mg of uterine tissue for 30 min at 4 °C and subjected to homogenization using a pestle mixer. Uterine lysates were then centrifuged at 14,000 g for 20 min at 4 °C, and the supernatant was considered the whole cell extract, which was assayed for protein content using the bicinchoninic acid method (BCA; Pierce, Rockford, IL, USA). After boiling for 5 min at 95 °C, equal amounts of proteins per group (20 μg) were resolved on 10% or 12% TGX stain-free gels (Bio-Rad Laboratories GmbH, Munchen, Germany) and transferred onto low fluorescence PVDF membranes (Bio-Rad) for 7 min by using the TransBlot Turbo Blotting System (Bio-Rad), following the manufacturer's instructions. The blots were blocked for 1 h in 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST). After blocking, the membranes were probed overnight at 4 °C with the primary antibody in TBST containing 5% (w/v) non-fat dry milk or 5% (w/v) bovine serum albumin (in the case of phosphorylated proteins). The blots were washed 5 × 5 min in TBST and incubated with HRPconjugated secondary antibody for 1 h at room temperature. Blots were washed and developed by chemiluminescence (Clarity Western ECL Substrates, Bio-Rad). Ultraviolet activation of the TGX stain-free gel on a ChemiDoc MP Imaging System (Bio-Rad) was used to control for proper loading. Band densitometry was performed using Image Laboratory Software (Version 6.0, Bio-Rad). When quantified, the intensity of each protein band was normalized to the total protein in individual samples to adjust for unequal loading and transfer (Taylor et al., 2013; Taylor and Posch, 2014; Zhang et al., 2017). The identity of the antibodies used for Western blotting are detailed in Supplementary Table 1. 2.5. Immunohistochemical analysis Paraffin sections (N = 5/group) were deparaffinized and rehydrated through a graded alcohol series followed by antigen retrieval in 10 mM sodium citrate buffer, pH 6.0 for 40 min with steaming (IHC WORLD, Woodstock, MD, USA). Slides were treated with 0.5% Triton X-100 to 2

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previously described (Amalfi et al., 2012). Progesterone was extracted twice with diethyl ether. The progesterone antiserum utilized was highly specific and showed low cross reactivity (Abruzzese et al., 2019a). The serum levels of progesterone obtained were as it follows C = 6.09 ± 4.81 ng/ml; PH ov = 11.49 ± 10.36 ng/ml; and PH anov = 12.86 ± 9.61 ng/ml. The intra- and inter-assay coefficients of variation for the latter assay were, respectively, 10.9% and 12.8%.

allow permeabilization. Thereafter, slides were blocked with 5% normal horse serum (NHS) (Vector Labs, Burlingame, CA, USA) for 20 min at room temperature. The primary antibodies (see list and concentrations on Supplementary Table 1) were diluted in NHS and incubated overnight at 4 °C or for 1 h at room temperature in a humidified chamber. Thereafter, the sections were incubated with 3% hydrogen peroxide to remove endogenous peroxidase activity. After being washed with phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBST), and phosphate-buffered saline (PBS), the samples were incubated for 30 min at room temperature with the secondary antibodies identified in Supplementary Table 1. Target visualization was done with 3,3′-diaminobenzidine tetrahydrochloride (Vector). Negative controls were performed, following the same protocol, except that the tissues were not incubated with primary antibodies. Positive tissue controls were performed in cancer cell lines that are known to express the proteins of interest (data not shown). Slides were viewed on a microscope (Amscope MU1000) and photomicrographed using AmScope Software 3.7 (United Scope LLC). A semiquantitative analysis (N = 5/group) (Lessey et al., 1988) was performed to analyze the immunohistochemistry results. At 40x magnification the slides were evaluated by determination of the distribution of staining within each tissue compartment (luminal epithelium, glandular epithelium, and stroma) and the intensity of staining. The positive staining was assessed in at least 1000 cells per compartment per sample in 10 random fields. The tissue was examined by a blinded observer and confirmed by a second observer. The intensity of staining (i) was assigned the following scores: 0, none; 1, weak; 2, distinct; and 3, strong. The HScore (HS: histology score) was calculated using the following equation: ΣPi (i + 1), where i = 1, 2, or 3, and Pi is the percentage of positively stained cells for each intensity.

2.8. Fertility test To determine fertility, 3-month-old females from all groups were mated with adult males. A mating index was determined by monitoring the presence of vaginal plugs the morning after mating. The fertility index was determined by verifying the birth of live pups 22 days after mating. The numbers of pups were counted. The ability to nurse was analyzed by assessing the survival of the offsprings 96 h postpartum. 2.9. Statistical analysis Statistical analyses were performed to test the number of samples needed for each experiment using Infostat Software (Di Rienzo et al., 2011). The same software was used to test normality of data (ShapiroWilks test) and homoscedasticity (Levene's test). Results are presented as the means ± S.E.M. Statistical analyses were performed using GraphPad Instat® software (GraphPad Software, San Diego, CA, USA). For normally distributed data, differences between groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test, or by two-way ANOVA followed by Bonferroni's multiple comparison test. Fertility test results were analyzed as categorical data with chi-squared test. p-values of less than 0.05 were considered statistically significant.

2.6. In situ detection of apoptosis 3. Results Apoptotic cells were detected using a modified TUNEL assay. Uterine sections (N = 5/group) were dewaxed in xylene and rehydrated through graded ethanol. The methodology was performed as previously detailed (Telleria et al., 2001). In brief, by using the DeadEnd™ Colorimetric System (Promega, Madison, WI) and following the manufacturer's instructions with slight modifications, sections were fixed in 4% paraformaldehyde and after washing in PBS, were subjected to permeabilization treatment with proteinase K (20 μg/ml). After re-fixation and immersion of the slides in equilibration buffer, sections were treated with the TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase (TdT) and biotinylated nucleotides, for 30 min at 37 °C. Negative controls were incubated with a solution lacking TdT. The reaction was terminated after immersing the slides in 2X saline sodium citrate buffer, for 15 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min at room temperature. Horseradish peroxidase-labeled streptavidin was bound to incorporated biotinylated nucleotides and detected by incubation with diaminobenzidine. Finally, the sections were mounted, and analyzed with a light microscope. A positive control was developed by treating the samples with DNAse (Sigma) (Supplementary Fig.1 J). The uterus from a cycling mouse was also used as positive control of apoptosis for the TUNEL technique (Supplementary Fig.1 K).

3.1. Prenatal hyperandrogenization causes morphological changes in the uterus To determine if prenatal hyperandrogenization alters the morphology of the uterus, we analyzed the uterine tissue as a whole, discriminating among epithelium, stroma, and glands. The morphometric analysis revealed that the total uterine thickness increased in the PH ov group when compared with both PH anov and C groups (Fig. 1 A-C & J). The luminal epithelium thickness was increased in the PH ov and PH anov groups when compared to the C group (Fig. 1 D-F & K); such increased epithelial thickness was accompanied by the formation of multiple cell layers with morphologies changing from cuboidal to more rounded. The thickness of the glandular epithelium showed no changes between groups (Fig. 1 L). However, an increased number of glands, and the presence of gland conglomerates, were found in the endometria of both PH groups when compared against C (Fig. 1 G-I, M & N). Particularly, PH females showed irregular shaped glands, whereas the number of glands arranged as conglomerates was higher than in the C group (Fig. 1 N). Of interest, however, the average number of glands per conglomerate did not differ among the groups (Fig. 1 O). Cellular atypia was not visualized in the cells, but cystically dilated glands filled with secretory fluid were found in PH groups (Supplementary Fig. 1 AF).

2.7. Hormone measurements The estradiol to progesterone ratio (E/P) is essential for female hormone balance assessment. As previously reported, serum estradiol levels were quantified by Cobas immunoassay analyzers using an Electro Chemiluminescence ImmunoAssay (ECLIA), following the manufacturer's instructions (Abruzzese et al., 2016). The serum levels of estradiol obtained were as it follows: C = 12.25 ± 2.31 pg/ml; PH ov = 9.69 ± 1.93 pg/ml; and PH anov = 8.05 ± 1.24 pg/ml. On the other hand, progesterone serum levels were measured by RIA as

3.2. Prenatal hyperandrogenization alters the expression of hormones receptors in the uterus To determine how sensitive the uterus is to steroidal action after prenatal androgen exposure, we studied the expression of Androgen Receptors (AR), Progesterone Receptors (PR), and Estrogen Receptors (ER). AR protein expression was studied via immunohistochemistry 3

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Fig. 1. Prenatal hyperandrogenization causes morphological changes in the uterus. Representative photomicrographs of longitudinal sections stained with hematoxylin and eosin of the uterine tissue from control (A, D, G), PH ov (B, E, H), or PH anov (C, F, I) groups. The investigators were blinded to allocation for histological analyses (N = 10/group). Scale bars are indicated in the photomicrographs and represent 200 μm (A-C) and 50 μm (D-I), respectively. Morphometric analysis was achieved through measurement of endometrial thickness (J), luminal epithelium thickness (K), and glandular epithelium thickness (L). Morphometry of glandular tissue was done by measuring different glands average (M), amount of conglomerates in the stroma (N), and glands average per conglomerate (O). Different letters represent statistical difference (p < 0.05). Lu, lumen; Le, luminal epithelial cells; Ge, glandular epithelial cells; Str, stromal cells; Myo, myometrium.

(Fig. 2 F, K); however, the expression of ER beta was increased in both PH ov and PH anov groups when compared against the C group (Fig. 2 G, K). The expression of both isoforms of PR, A and B, was also studied; we found that both isoforms were slightly increased in the PH ov group when compared to the C and PH anov groups (Fig. 2 H-I, K). Finally, estrogen to progesterone ratio (E/P), as a marker of the reproductive hormone environment, showed no differences between the C and the PH groups (Fig. 2 J).

(Fig. 2A–C). Nuclear localization and higher expression of AR was found in both glandular and luminal epithelium and, to a lesser extent, in stroma of the PH anov group, as revealed through the HScore, when compared to other groups (Fig. 2 D). Also, protein expression through western blot showed that AR was increased in the PH anov group when compared to C and PH ov groups (Fig. 2 E, K). In the case of ER, both isoforms alpha and beta were studied because of their opposing actions (Lee et al., 2012; Liu et al., 2002; Madeira et al., 2013). Western blot results showed that ER alpha expression is not different between groups 4

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Fig. 2. Prenatal hyperandrogenization alters the expression of hormones receptors in the uterus. Immunohistochemical detection of AR in paraffin embedded uterine tissue sections of rats in diestrus, from control (A), PH ov (B), or PH anov (C) groups. Scale bars = 50 μm (A-C). As a negative control, the primary antibody was omitted (insert in A). Semiquantitative evaluation of AR protein expression by HScore in the nuclear compartment of glandular epithelial cells from the three groups was studied (D); the values are expressed as HScore (mean ± SEM). Calculation of the HScore is described in Materials and Methods (N = 5/group). Results of Western blotting shown in K are expressed as a fold change (mean ± SEM) for AR (E), ER alpha (F), ER beta (G), PRa (H), and PRb (I). Serum estradiol to progesterone (E/P) ratio (J). Representative images of the western blots are shown in (K). Different letters represent statistical significance among the groups; p < 0.05 (N = 8/group).

group; whereas cytoplasmic localization was increased in both PH groups when compared against the C group (Fig. 3 G, left panel). In the luminal epithelium, however, only cytoplasmic localization of p27 was increased in both PH groups when compared to the C group, whereas nuclear localization was similar across the groups (Fig. 3G, right panel). By western blotting, we observed that the expression of p27 in the uterine tissue was increased in the PH groups (Fig. 3 H, M), and that there was also a significant increase in phospho-CDK2 expression in the PH groups with respect to the C group (Fig. 3 I, M). Moreover, results indicate that p21 levels were decreased in the PH ov group (Fig. 3 J, M)

3.3. Prenatal hyperandrogenization alters the expression of cell cycle regulators in the uterus We studied the expression of cell cycle regulators p27, p21, Cyclin D, Cyclin E and phospho-CDK2 in the uterine tissue by Western blot analysis; p27 was also assessed by immunohistochemistry. Positive p27 staining was observed in both nucleus and cytoplasm of epithelial and stromal cells in all samples (Fig. 3A–F). Semi-quantification through the HScore revealed that nuclear p27 localization in the glandular epithelium of the PH anov group was increased when compared to the C 5

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Fig. 3. Prenatal hyperandrogenization alters the expression of cell cycle regulators in the uterus. Immunohistochemical detection of p27 in paraffin embeeded uterine sections of rats in diestrus, from control (A, D), PH ov (B, E), or PH anov (C, F) groups. Scale bars are indicated in the photomicrographs and represent 50 μm (A-F). As a negative control, the primary antibody was omitted (insert in A) (N = 5/group). Positive nuclear and cytoplasmic staining of p27 were detected in epithelial and stromal cells. Semiquantitative evaluation of p27 protein expression in the nuclear compartment of glandular and luminal epithelial cells from the three groups studied (G). The values are expressed as HScore (mean ± SEM). Results of Western blotting show fold change (mean ± SEM) for p27 (H), phosphoCDK2 (I), p21 (J), Cyclin E (K) and Cyclin D1 (L) in Control, PH ov and PH anov groups. Representative images of the western blots are shown in (M). Different letters represent statistical significance among the groups; p < 0.05 (N = 8/group). In G, in the glandular epithelium *p < 0.001 PH anov vs. PH ov and C; #p < 0.05 C vs. PH ov. In the luminal epithelium, *p < 0.001 C vs. PH ov and PH anov; #p < 0.01 PH ov vs. PH anov.

and no differences in the expression of Cyclin E (Fig. 3 K, M) and Cyclin D1 (Fig. 3 L, M) were found between the experimental groups.

immunohistochemistry. Results showed that the localization of PCNA is nuclear and it is mainly confined to the epithelium of the lumen and glands, with lesser extent to the stroma (Fig. 4A–F). PCNA staining was increased in the luminal epithelium of both PH ov and PH anov groups (Fig. 4 A-C & M) when compared to the C group. Moreover, the expression of phospho-histone H3, a marker of mitosis, showed decreased expression in the luminal epithelium (Fig. 4 G-I & O), whereas the glandular epithelium showed no differences in expression (Fig. 4 J-L &

3.4. Prenatal hyperandrogenization dysregulates cell proliferation in the uterus To study cellular proliferation, we first determined the expression of PCNA (Juríková et al., 2016), in the uterine tissue by 6

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Fig. 4. Prenatal hyperandrogenization dysregulates cell proliferation in the uterus. Immunohistochemical detection of PCNA (A-F) and phospho histone-H3 (PHH3) (G-L) from control (A, D, G, J), PH ov (B, E, H, K), or PH anov (C, F, I, L) groups. Scale bars are indicated in the photomicrographs and represent 50 μm (A-L). Semiquantitative evaluation of protein expression by HScore in glandular and luminal epithelial cells nuclear compartment from the three groups studied is shown for PCNA (M, N) and for pHH3 (O, P). The values are expressed as HScore (mean ± SEM). As a negative control, the primary antibody was omitted (insert in A, D, J). Different letters represent statistical significance among the groups; p < 0.05 (N = 5/group).

P).

2014; Guzeloglu Kayisli et al., 2004; Li et al., 2017). We determined the protein expression of Akt phosphorylated on Ser 473 and Thr 308, denoting activated Akt (Alessi et al., 1996; Guzeloglu Kayisli et al., 2004). Western blot results showed that the PH anov group has an increased expression of phosphorylated Ser 473 without modifications in phosphorylated Thr 308, when compared to the other groups (Fig. 5 A, B & D). Moreover, phosphorylated ERK was found to be also increased

3.5. Prenatal hyperandrogenization alters AKT and ERK phosphorylation in the uterus When activated, Akt acts as a regulator of several processes such as cell cycle, apoptosis, proliferation and cell survival (Fabi and Asselin, 7

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Fig. 5. Prenatal hyperandrogenization alters AKT and ERK phosphorylation in the uterus. Results of Western blotting for relative phosphorylation of AKT on Ser 473 (A) or Thr 308 (B), and on ERK (C) in control, PH ov and PH anov groups. The results are expressed as a fold change (mean ± SEM). Representative images of the western blots are shown in (D). Different letters represent statistical significance among the groups; p < 0.05 (N = 8/group).

assessed apoptosis activity via the TUNEL assay, we did not find dying cells in the uteri of any of the experimental groups (Supplementary Fig. 1 G-I).

in the PH groups (Fig. 5 C, D). 3.6. Prenatal hyperandrogenization does not affect apoptosis dynamics

3.7. Prenatal hyperandrogenization affects fertility

The dynamics of apoptosis was inferred by the measurement of the protein levels of Bax and Bcl-2, as pro- and anti-apoptotic markers, respectively, and by measuring the cell death effectors caspase 3 and PARP, all by Western Blot analysis. We found that the Bax:Bcl-2 ratio showed no differences between the C group and the PH groups (Fig. 6 A, E). However, increased protein levels of total caspase-3 and total PARP were found in the PH groups when compared with the C group (Fig. 6 B, C & E). Moreover, absent or light expression of cleaved forms of caspase-3 and PARP were found in all groups. On the other hand, survivin, an important inhibitor of apoptosis, was increased in both PH groups when compared against the C group. (Fig. 6 D, E). When we

Natural pregnancies were monitored in adult females from all groups for 2 months. Fertility studies demonstrated that the PH anov females were unable to mate or become pregnant, whereas animals of the C group showed vaginal plugs and gave birth to live pups. The PH ov females were able to become pregnant but with a lower efficacy than the C females. No differences in the number of pups delivered per female between PH ov and C females were found. Moreover, the C and the PH ov mothers showed good nursing performance as their pups survived until weaning (Table 1).

Fig. 6. Prenatal hyperandrogenization does not affect apoptosis dynamics. Results of Western blotting for Bax:Bcl-2 ratio (A), Caspase-3 (B), PARP (C), Survivin (D), in control, PH ov and PH anov groups (N = 8/group). The results are expressed as a fold change (mean ± SEM). Representative images of the western blots are shown in (E). Different letters represent statistical significance among the groups. p < 0.05 (N = 8/group). In B, *p < 0.01 PH ov vs. PH anov; **p < 0.001 C vs. PH ov and PH anov; and in C, *p < 0.001 C vs. PH ov and PH anov. 8

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The presence of endometrial hyperplasia is commonly found in women with PCOS (Charalampakis et al., 2016), which is often attributed to a misbalanced estrogen to progesterone ratio (Charalampakis et al., 2016; Prakansamut et al., 2014). We could not find such misbalance in our animal model; however, androgens are also considered causative of uterine changes, although, their function and that of their receptors in the female reproductive tract are less clear. Yet, previous results from other authors demonstrated that the AR likely mediates androgen action and affects the homeostasis of the uterus independently of the expression of ER and PR (Apparao et al., 2002; Cloke and Christian, 2012; Li et al., 2015). Here, we extend those previous findings and found that the expression of AR was increased in the PH anov phenotype, accompanied by hyperandrogenism (Abruzzese et al., 2019b) and glandular hyperplasia. In accordance with our results, other authors reported by using a model of androgen excess during perinatal life an increased staining of AR in the epithelium of the uterus during puberty and adulthood (Guerra et al., 2013). The evaluation of ER showed no differences in ERa levels between groups, but increased ERb levels in both PH groups. These data are in accordance with previous results, which demonstrate that women with PCOS and endometrial hyperplasia present increased ERb/ERa ratio in the uterus (Villavicencio et al., 2006). In order to know whether the cell cycle was programmed by prenatal hyperandrogenization, we studied the expression of the cell cycle inhibitor p27 as it was shown to be regulated by hormones (Shiozawa et al., 1998). The activity of p27 depends on its localization. This localization could be explained by the activation of Akt (phosphorylated in Ser473), which is capable to phosphorylate p27 and relocate it from the nucleus to the cytoplasm (Abbastabar et al., 2018; Besson et al., 2008). The presence of nuclear p27 blocks cell proliferation by inhibiting cyclin E/CDK2 complex (Chen et al., 2018); however, in the cytoplasm, p27 inhibits apoptosis (Philipp-Staheli et al., 2001). In that context, we found that p27 was increased in both PH groups; yet, a differential pattern of expression was found between the glandular and the luminal epithelia. In the glandular epithelium, cytoplasmic p27 was increased in both PH groups, whereas nuclear p27 was increased only in the anovulatory group. With respect to the luminal epithelium, an increased expression of p27 in the cytoplasm was found in both PH groups, which could prevent apoptosis from taking place. Thus, higher levels of phosphorylated Akt (Ser 473) could explain, at least in part, the cytoplasmic localization of p27 and its contribution to the survival of cells in the PH anov group. Furthermore, the increased levels of survivin, may prevent the uterine tissue from undergoing apoptosis in both PH groups, thus favoring cell survival. Studies of women with PCOS, and of PCOS accompanied with uterine hyperplasia, revealed that CDK2 activity in the endometrium is not altered (Villavicencio et al., 2009). In that sense, the elevated levels of presumably active phospho-CDK2 that we found in the PH groups, could be associated with the lower levels of the CDK2 inhibitor p21 found in the PH ovulatory group, in which the total thickness of the uterus was more exacerbated. It is known that p21 inhibits DNA synthesis through suppression of CDK activity or upon binding to PCNA (Lu et al., 1998; Waga et al., 1994). Moreover, it was suggested that apoptosis and p21, acting in a cooperative but independently manner, may play a negative role in the regulation of cell proliferation in the endometrium (Toki et al., 1998). Apoptosis and proliferation processes within the uterus are balanced along the estrous cycle. However, it was suggested that if the estrous cycle does not progress correctly and signaling pathways are dysregulated due to an altered expression of reproductive steroid hormones, the development of an unbalanced state might lead to the impairment of the uterine function (Plaza-Parrochia et al., 2017). In our study, cell death effectors as cleaved caspase-3 and PARP were found to be negligible in all groups, revealing that apoptosis is not likely taking place to counterbalance the hyperplastic phenotype found in both PH groups. Moreover, during the secretory phase of the estrous cycle, p27 increases, and cell proliferator markers decrease in

Table 1 Study of the fertility of prenatally androgenized females.

C PH ov PH anov

Mating indexb

Fertility indexc

Pups/female/ litterd

Ability to nurse

10/12 11/22a 0/9

9/12 8/22a 0/9

9 ± 0.97 7.75 ± 0.97 –

100% 100% –

a

Indicate significant differences p < 0.05 vs. C. Mating index was determined as the number of females with vaginal plug over the total of females. c Fertility index was determined as the number of females that delivered live pups over the total of females. d Number of pups per female per litter, through natural mating. b

4. Discussion The uterus is a major female organ that undergoes continuous synchronized changes during the estrous cycle. An impaired influence of hormones could alter the normal functioning of the uterus leading to the development of different pathologies. In particular, the influence of androgens in the uterus is not fully understood. To study the effect of fetal programming in the uterus of animals prenatally exposed to androgens, we reproduced a rodent model of prenatal hyperandrogenization, which has been previously described (Abruzzese et al., 2016; Amalfi et al., 2012; Heber et al., 2013). With this animal model we obtained two different phenotypes, irregular ovulatory and anovulatory, both accompanied with hyperandrogenism, ovarian cysts, and metabolic misbalances (Abruzzese et al., 2019b). Using this model, we addressed the question of whether tissue homeostasis is altered in the uterus of prenatally hyperandrogenized adult rats. We show that prenatal androgen excess reprograms several signaling pathways in the uterus, which were associated with alterations in uterine morphology. These findings suggest that although androgen excess leads to alterations in the uterine tissue, such alterations are independent of the presence of irregular ovulatories or anovulatories estrous cycles. These results would indicate that despite the magnitude of the ovulatory dysfunction, the uterus of prenatally hyperandrogenized animals showed generalized alterations, including histopathologic changes and abnormal morphology, evidenced by increased glandular density (glandular hyperplasia), multiple luminal epithelial cell layers accompanied with changes in cell phenotype, and altered distribution of glands. Other authors have reported, by using a perinatal model of androgen excess with a lower dose of testosterone propionate but with prolonged exposure when compared to our model, a decreased height of the glandular epithelium during puberty. However, this change was reversed at 75 days of life. Moreover, during adulthood no differences were found in the glandular and luminal epithelial height, number of glands and endometrium height, which revealed that uterine physiology was not impaired under the conditions of the study (Guerra et al., 2014). These results could be explained due to the fact that hormonal serum levels were found to be within the normal range, implying that reproductive alterations are dependent on the hormonal environment. All the uterine morphologic alterations found, accompanied with the ovarian and metabolic misbalances previously reported (Abruzzese et al., 2016; Heber et al., 2019), may impact on the normal functioning of the uterine tissue, risking the ability to maintain a pregnancy. Moreover, as other authors reported, animals treated with dihydrotestosterone, a non-aromatizable androgen, or with synthetic androgens, showed similar alterations in the uterine histomorphology (Nantermet et al., 2005; Simitsidellis et al., 2016), such as those showed by us injecting testosterone, suggesting that alterations found in the uterus are independent from aromatization of androgens to estrogens. Taken together, our findings indicate that prenatal androgen excess is able to induce endometrial hyperplasia. 9

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the uterus, favoring nesting ()(Abbastabar et al., 2018). Furthermore, we found that the cell proliferation marker PCNA was increased in the luminal epithelium of the PH groups, and, together with the increased phospho-ERK, may suggest activation of the mitogenic pathway in the face of non-compensatory apoptosis. The presence of luminal epithelial cell proliferation (as assessed by PCNA expression), in conjunction with increased cell survival (reflected by activated Akt and increased survivin), apparently not counterbalanced by increased cell death in the lumen of the prenatally hyperandrogenized adult females, could be associated with low receptivity, implantation failure, and/or cancer development as described in women with PCOS (Gao et al., 2014). In summary, our results demonstrate that fetal programming, through a hyperandrogenic environment, affects the maintenance of uterine homeostasis leading to a hyperplastic state, in association with anovulation and mating failure, likely increasing the risk of developing a more severe pathology.

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Funding This research was supported by Agencia Nacional de Ciencia y Técnica (ABM Grant number 557/2012; 689/2013; 632/2016), and grant number 35635 from the Canada Foundation for Innovation (CMT). Silvana Ferreira was the recipient of a scholarship from the Emerging Leaders in the Americas Program (ELAP), provided with the support of the Government of Canada. Alicia Goyeneche was supported by a Hartland Molson Fellowship from the Research Institute, McGill University Health Centre. Author contributions Silvana Rocío Ferreira: Formal analysis, Investigation, Methodology. Alicia Alejandra Goyeneche, María Florencia Heber and Giselle Adriana Abruzzese: Investigation, Methodology. Carlos Marcelo Telleria and Alicia Beatriz Motta: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing -original draft, Writing - review & editing. Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Acknowledgments We want to thank Enzo Cuba and Marcela Marquez for their technical support in the animal care. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mce.2019.110610. References Abbastabar, M., Kheyrollah, M., Azizian, K., Bagherlou, N., Tehrani, S.S., Maniati, M., Karimian, A., 2018. Multiple functions of p27 in cell cycle, apoptosis, epigenetic modification and transcriptional regulation for the control of cell growth: a doubleedged sword protein. DNA Repair 69, 63–72. https://doi.org/10.1016/j.dnarep. 2018.07.008. Abbott, D.H., Dumesic, D.A., Franks, S., 2002. Developmental origin of polycystic ovary syndrome - a hypothesis. J. Endocrinol. 174, 1–5. https://doi.org/10.1677/joe.0. 1740001. Abbott, D.H., Rayome, B.H., Dumesic, D.A., Lewis, K.C., Edwards, A.K., Wallen, K., Wilson, M.E., Appt, S.E., Levine, J.E., 2017. Clustering of PCOS-like traits in naturally hyperandrogenic female rhesus monkeys. Hum. Reprod. (Oxf.) 32, 923–936. https:// doi.org/10.1093/humrep/dex036. Abruzzese, G.A., Heber, M.F., Campo Verde Arbocco, F., Ferreira, S.R., Motta, A.B.,

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