Endogenous acetaldehyde toxicity during antral follicular development in the mouse ovary

https://doi.org/10.1016/j.reprotox.2012.01.001Get rights and content

Abstract

The biosynthesis of androgens requires multiple steps and during the conversion of pregnenolone to 17α-hydroxypregnenolone and dehydroepiandrosterone (DHEA) by CYP17a1. Acetaldehyde is potentially formed as a by-product in theca cells during antral follicular development. In this study, acetaldehyde level was significantly increased after eCG stimulation and reached a maximum level at 36-h post-eCG. By 48 h, the level of acetaldehyde decreased in association with the induction of aldehyde dehydrogenase (ALDH) type 1 family members. When immature mice were co-injected with the ALDH inhibitor, cyanamide, and eCG, the expression of genes involved in the differentiations of granulosa cells was suppressed and the number of ovulated oocytes was reduced. The in vitro studies showed that ALDH inhibitors prevented FSH-induced granulosa cell differentiation. These results indicate that acetaldehyde is generated as a by-product during steroidogenesis and can exert toxic effects to impair the differentiation of granulosa cells, reduce ovulation and decrease oocyte quality.

Highlights

► We analyzed the kinetic changes of acetaldehyde level in ovary during follicular development. ► The level was transiently increased as a byproduct in a steroidogenic pathway. ► However, the level was decreased in an ALDH dependent manner. ► The reduction of acetaldehyde level was required for granulosa cell differentiation during follicular development.

Introduction

The pituitary gonadotropins, FSH (follicular stimulating hormone) and LH (luteinizing hormone) are essential for the follicular development to preovulatory stage [1]. In growing follicles LH activates its cognate receptor (LHCGR) present in the theca cell layer external to the basement membrane and stimulates androgen biosynthesis from cholesterol [2], [3]. The androgens are then converted to estrogens by the aromatase (CYP19a1) enzyme that is induced in granulosa cells of antral follicles [1], [4], [5]. Estrogen, mainly estradiol 17β (E2) acts on ESR2 (estrogen receptor beta; ERβ) that is expressed in granulosa cells, and enhances FSH-mediated granulosa cell proliferation and differentiation (LHCGR induction) and follicle growth to the preovulatory stage [4], [6], [7]. Elevated serum E2 activates neuronal estrogen receptor alpha (ERS1) inducing GnRH synthesis/release, leading to the LH surge and the initiation of ovulation and luteinization [8], [9], [10]. Esr2 [11] and Esr1 mutant mice [12] are subfertile or infertile due to impaired ovarian and hypothalamic functions.

The biosynthesis of androgens (C17) from cholesterol (C21) in theca cells depends on multiple steps and involves not only the generation of functional steroids but also specific catabolic by-products. For example, the generation of pregnenolone leads to the release of the by-product isocaproaldehyde [13]. The conversion of pregnenolone to 17α-hydroxypregnenolone and dehydroepiandrosterone (DHEA) or progesterone to 17α-hydroxyprogesterone and androstendione by CYP17a1 (Cytochrome P450 17α-hydroxylase/C17–20 lyase) [14], [15] yields acetaldehyde [16]. Both ioscaproaldehyde and acetaldehyde can be toxic to cells and therefore are metabolized rapidly by specific reductase and dehydrogenase enzymes.

The toxic activity of isocaproaldehyde in steroidogenic cells was first reported by Lefrancois-Martinez et al. [13] who showed that an aldo-keto-reductase like protein (Akr1b7) is expressed in adrenal cells and catalyzes the breakdown of endogenous isocaproaldehyde. The transfection of Akr1b7 antisense cDNA to murine adrenocortical Y1 cells, reduced the number of living cells, and the toxicity was decreased by the treatment with aminoglutethimide, an inhibitor of CYP11A1, the cholesterol side chain cleavage enzyme. Therefore, in adrenal cells, ACTH increases the expression and activity of both CYP11A1 and AKR1B7, which produce and immediately catabolize the toxic isocaproaldehyde, respectively [17], [18], [19]. However, Akr1b7 knockout mice are fertile [20] suggesting that other enzyme(s) play a redundant role in metabolizing isocaproaldehyde [20]. Alternatively, the toxic activity may not be sufficient to alter granulosa cell functions or induce granulosa cell death during follicular development.

Although acetaldehyde is a potent cellular toxin that can act to suppress DNA repair, cell proliferation, adhesion and differentiation [21], [22], most research is limited to understanding its actions in alcohol toxicity in the liver. Specifically, alcohol is metabolized to acetaldehyde by alcohol dehydrogenate (ADH) in liver [23], [24], [25]. The endogenous acetaldehyde is neutralized by aldehyde dehydrogenase (ALDH) to acetyl-CoA, and then to carbon dioxide and water [26]. Mutations of the Aldh gene in human is associated with decreased dehydrogenase activity, increased of acetaldehyde levels and cell toxicity [27]. Thus, this metabolic pathway is essential for the maintenance of viable cells and normal liver functions. However there is no report about endogenous acetaldehyde toxicity and the expression of ALDH family members during antral follicular development. In this study, we analyzed the levels of acetaldehyde present in the mouse ovary during antral follicular development when steroidogenesis is increased. Moreover, we detected the induction of specific ALDH family members in the ovary, and investigated the role of ALDH activity in follicular development by using a broad ALDH inhibitor, cyanamide [28] or specific ALDH type I inhibitor, disulfiram [29].

Section snippets

Materials

Equine and human chorionic gonadotropins (eCG and hCG) were purchased from Asuka Seiyaku (Tokyo, Japan). Ovine follicle stimulating hormone (FSH) and luteinizing hormone (LH) were a gift from the National Hormone and Pituitary Program (Rockville, MD). DMEM:F12 medium, penicillin-streptomycin were from Invitrogen (Carlsbad, CA, USA); fetal bovine serum (FBS) from Life Technologies Inc. (Grand Island, NY); oligonucleotide poly-(dT) from Invitrogen, and AMV reverse transcriptase, Taq polymerase

Acetaldehyde levels in the ovary increase during hormonal stimulation of antral follicular development

Acetaldehyde is potentially synthesized as a by-product of CYP17A1 (Fig. 1A). To determine the levels of acetaldehyde in ovary during follicular development, whole ovaries were collected from immature (day 15, 20, or 23) and d23 mice prior to and at 0, 12, 24, 36 or 48 h after eCG. The ovaries were lysed to analyze the acetaldehyde concentrations using f-kit acetaldehyde (Roche Diagnostics). Levels of acetaldehyde were less than 0.1 mg/g at day 15 and 23 or at 12 h after eCG stimulation. However,

Discussion

The biosynthesis of steroids not only generates critical hormones required for reproductive success but also specific catabolic by-products, such as isocaproaldehyde (derived during the conversion of cholesterol to pregnenolone by CYP11A1) and acetaldehyde (CYP17A1 dependent conversions) that are potentially cytotoxic to gonadal cells. In this study, we document that acetaldehyde is produced in the mouse ovary in response to gonadotropin (eCG or hCG) stimulation, most likely by theca cell

Disclosure

The authors have nothing to disclose.

Acknowledgements

FSH and LH ware kindly provided by Dr. A.F. Parlow, the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease, USA. The authors are grateful to Dr. Z. Liu, Baylor College of Medicine and Mr. N. Noma, Hiroshima University, for technical assistance, and Dr. Y. Kuwabara, Nihon Medical University for giving technical suggestions to culture mouse ovary.

Grant: This work was supported in part, by Grant-in-Aid for Scientific Research (Nos. 21688019,

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