Involvement of gonadotropins in the induction of hypertrophy-hyperplasia in the interstitial tissues of ovaries in neonatally diethylstilbestrol-treated mice

https://doi.org/10.1016/j.reprotox.2011.10.013Get rights and content

Abstract

Neonatally diethylstilbestrol (DES) treatment causes hypertrophy-hyperplasia in the interstitial tissue of mouse ovaries. To understand the induction mechanism of the hypertrophy, mRNA expression involved in steroidogenesis in the ovary of neonatally DES-treated mice was examined. The expression of StAR and Cyp11a1 was significantly reduced while Cyp19 and Sf-1 were stimulated in the ovary of neonatally DES-treated 3-month-old mice. Expression of those genes was not different between DES- and oil-treated mice after the gonadotropins treatment. Lhb in the pituitary of 3-month-old neonatally DES-treated mice was significantly decreased. Finally, ovaries from DES-treated mice transplanted to neonatally oil-treated hosts had developing follicles at several stages and corpora lutea, whereas grafted ovaries from neonatally oil-treated mice in 3-month-old neonatally DES-treated hosts showed lipid accumulation in the interstitial tissue. Thus, hypertrophy and accumulation of lipid droplets in interstitial cells of neonatally DES-treated mice is caused by impaired steroidogenesis due to the alterations of gonadotropins levels.

Highlights

► Neonatally diethylstilbestrol (DES) treatment causes hypertrophy-hyperplasia in the ovary. ► mRNA expression involved in steroidogenesis in neonatally DES-treated mice was altered. ► Lhb in the pituitary of neonatally DES-treated mice was decreased. ► After the gonadotropins treatment, gene expression was similar to that of oil-treated mice. ► Hypertrophy was caused by impaired steroidogenesis due to the changes of gonadotropins levels.

Introduction

Diethylstilbestrol (DES), a synthetic estrogen, had been prescribed for preventing abortion, however, women who were exposed to DES in utero developed vaginal clear-cell adenocarcinoma [1]. In mice, perinatal exposure to natural or synthetic estrogens, including DES, results in reproductive abnormalities such as absence of corpora lutea (CL), hypertrophy-hyperplasia of interstitial tissues, induction of polyovular follicles in the ovary and persistent vaginal cornification [2], [3], [4], [5], [6].

Hypertrophy-hyperplasia of interstitial tissues is already found in the ovary of 3-month-old prenatally DES-exposed mice, and a dramatic increase in lipid droplets in the interstitial tissue as determined by Oil Red O stain [3]. In a study of estrogen receptor α knockout (αERKO) mice, it was shown that the induction of hypertrophy-hyperplasia of interstitial tissues was mediated by ERα [7]. The absence of CL in the ovary of neonatally DES-treated mice is caused by lack of a luteinizing hormone (LH) surge [8], [9], [10]. In addition, plasma levels of testosterone (T) in neonatally DES-treated mice are lower than those in the controls, and ovariectomy, adrenalectomy or a combination of both surgeries shows no effect [11]. These results suggest that alterations in the hypothalamic–pituitary–gonadal (HPG) axis may affect steroidogenesis in the interstitial and theca cells of neonatally DES-treated mouse ovaries.

Several studies have shown that disruption of genes involved in steroidogenesis results in alterations of steroid levels and progressive increases in lipid deposits with age [12], [13], [14]. In theca and interstitial cells, cholesterol obtained from plasma lipoproteins is transferred by steroidogenic acute regulating (StAR) protein from the outer mitochondrial membrane to the inner membrane where cytochrome P450 side-chain cleaving enzyme (CYP11A1) is located [15], [16]. CYP11A1 converts cholesterol to pregnenolone [15], [16]. Ovaries from 8-week-old Star knockout (KO) mice retain a few scattered follicles and are largely composed of vacuolated stromal cells that stain with Oil Red O [12], [13]. In addition, ovaries of Cyp11a1 KO mice treated with daily injections of corticosteroids are similar to those of wild-type (WT) mice during the neonatal period, however, lipid accumulation is increased in interstitial cells around follicles at 13 days of age [14]. These results suggest that the failure of steroidogenesis leads to lipid accumulation in interstitial cells with age. Pregnenolone is converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD) and/or dehydroepiandrosterone by C17-hydroxylase (CYP17A1) and then converted to androstendione by CYP17A1 and/or 3β-HSD, respectively [15], [16]. Androstenedione synthesized in theca cells diffuses to granulosa cells and it is converted to T by 17β-hydroxysteroid dehydrogenase (17β-HSD) or estrone by aromatase (CYP19) and then 17β-estradiol (E2) by 17β-HSD [15], [16]. Steroidogenesis is tightly regulated by the HPG axis. In theca and interstitial cells, androstenedione is produced for supplying androgen for granulosa cells to synthesize E2 in response to LH. Follicle stimulating hormone (FSH) is required for preantral to later-stage follicle development [17]. FSH also regulates the expression of CYP19 [18], [19] in developing follicles where FSH receptors are located. The LH surge down-regulates this FSH-induced increase in Cyp19 expression [20] and triggers ovulatory changes in the preovulatory follicle, including luteinization of the granulosa and theca cells and production of progesterone following the expression of Star and Cyp11a1 [21], [22].

The orphan nuclear receptors SF-1 and LRH-1 regulate critical genes in the reproductive axis and steroidogenesis [23], [24]. In the ovary, SF-1 is localized in theca, interstitial and granulosa cells [23], [25], [26] and activates promoters of Star, Cyp11a1, Cyp11b1 and Cyp21 [27], [28], through the consensus T/CAAGGTCA sequence. LH reduces SF-1 expression in preovulatory granulosa cells [29]. Granulosa-specific KO mice of Sf-1 are infertile and show hemorrhagic cysts (HC) and the absence of CL in the ovary [30] similar to the phenotype of αERKO or Cyp19 KO mice, suggesting that Sf-1KO mice have the defect in the synthesis of estrogen in the ovary. LRH-1 is mainly localized in granulosa cells and CLs, and regulates Cyp19 [23]. Mice lacking Lrh-1 in granulosa cells are sterile due to anovulation [31]. Lack of Lrh-1 also results in an increase of intrafollicular E2 levels with elevated Cyp19, and decreases of Star and Cyp11a1 in granulosa cells. These results suggest that SF-1 and LRH-1 have critical roles in the ovary. Since both SF-1 and LRH-1 are expressed in granulosa cells and have similar actions [24], [32], distinct differences between the actions of SF-1 and LRH-1 within the ovary are still unclear [33].

In the present study, we aimed to identify the timing of lipid droplets accumulation in interstitial cells and examined the mRNA expression involved in steroidogenesis in the ovary of neonatally DES-treated mice. We hypothesized that the altered HPG axis caused the lipid accumulation in the ovary of neonatally DES-treated mice. Therefore, ovaries from neonatally DES-treated mice were grafted under the renal capsule of ovariectomized neonatally oil-treated mice and vice versa was also examined. In addition, the serum levels of E2 in neonatally oil- and DES-treated mice were measured to assess steroidogenesis in the ovary and the expression of gonadotropin-related genes in the anterior pituitary was examined.

Section snippets

Animals and treatments

C57BL/6J mice (CLEA Japan, Tokyo, Japan) were maintained on a 12-h light/12-h dark cycle (lights off at 2000 h) with controlled temperature (25 °C) and mice were given a commercial diet (MF, Oriental Yeast Co., Tokyo, Japan) and fresh tap water ad libitum. All animals were maintained in accordance with the NIH guide for the care and use of laboratory animals. All experiments were approved by the institutional animal care committee of the Yokohama City University. Female pups were given daily

Lipid accumulation in the ovary and involvement of ERα

Ovaries from 1-month-old neonatally oil- or DES-treated mice did not stain with Oil Red O (data not shown). CL in the ovaries of oil-treated mice were weakly stained with Oil Red O at 1.5 and 3 months of age (Fig. 2A and B). The interstitial and theca cells in the ovaries of neonatally DES-treated 1.5- and 3-month-old mice (Fig. 2A and B) stained with Oil Red O. In addition, the interstitial tissues of 3-month-old neonatally DES-treated mice showed medullary tubule-like structures. No CL was

Discussion

Ovulation and steroidogenesis have begun by 1.5 months of age in neonatally oil-treated (Neo Oil) mice, since CL were observed in their ovaries. In contrast, CL were not found but lipid droplets were observed in the ovary of 1.5- and 3-month-old neonatally DES-treated (Neo-DES) mice, suggesting a lack of LH surge caused by neonatal DES treatment as previously reported [8].

The expression of Star and Cyp11a1 was decreased in the ovary of 1.5- and 3-month-old Neo DES mice due to the alterations of

Conflict of interest statement

There is no conflict of interest.

Acknowledgements

We thank Dr. Yayoi Ikeda, Yokohama City University School of Medicine, for supplying the rabbit polyclonal antibody against bovine SF-1, and Dr. Raphael Guzman, Department of Molecular Cell Biology and Cancer Research Laboratory of University of California at Berkeley, for his critical reading of this manuscript.

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    Grant support: This work was partially supported by a Grant-in-Aid for Scientific Research (B) (T.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants for Strategic Research Projects at Yokohama City University (Nos. K19020, K2109, G2201 and W18005 to T.S.), a Health Sciences Research Grant from the Ministry of Health, Labor and Welfare, Japan (T.I.), and a Grant for Support of the Collaborative Study at NIBB (T.S).

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