4-vinylcyclohexene diepoxide: a model chemical for ovotoxicity

Abstract

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) has been shown to cause selective destruction of ovarian small pre-antral (primordial and primary) follicles in rats and mice by accelerating the natural, apoptotic process of atresia. Chemicals that destroy primordial follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). Initial studies using in vivo exposure of rats determined that VCD specifically targets primordial and primary (small pre-antral) follicles and that repeated dosing is required. Through a method of isolation of ovarian small follicles, biochemical and molecular studies determined that intracellular pro-apoptotic pathways are activated following VCD dosing in rats. Subsequently an in vitro system using cultured whole neonatal rat ovaries was developed to provide more mechanistic information. That approach was used to demonstrate that the cell survival c-kit/kit ligand signaling pathway is the direct target for VCD-induced ovotoxicity. Specifically, VCD directly interacts with the oocyte-associated c-kit receptor to inhibit its autophosphorylation, and thereby impair oocyte viability. The cellular and molecular approach developed to determine these findings is described in this article.

Figure 1

The occupational chemical, 4-vinylcyclohexene (VCH) is a source of exposure in the manufacture of rubber tires, flame retardants, insecticides, plasticizers, and antioxidants. VCH represents the parent form of the compound, and can be metabolized by cytochrome p450 enzymes to the diepoxide (VCD) which is the ovotoxic form of the chemical. Formation of the bioactive form (VCD) can occur in the liver, although the ovary itself expresses the enzymes capable of this conversion.

Figure 2

The mammalian ovary is a heterogeneous mixture of dynamic structures. The basic unit is the ovarian follicle which contains an oocyte surrounded by somatic cells. The most immature form of the follicle is termed primordial (surrounded by a single layer of squamous granulosa cells). Upon activation, the follicle goes through several stages of development: primordial to primary (layer of cuboidal granulosa cells); primary to secondary (granulosa cell layer proliferates, theca interna cells form); and secondary to pre-ovulatory (antrum develops). The pre-ovulatory follicle is responsible for production of 17β-estradiol. Following ovulation, remaining cells differentiate to become a corpus luteum which produces progesterone required for implantation and maintenance of pregnancy. 4-vinylcyclohexene diepoxide (VCD) specifically targets primordial and primary follicles in producing its ovotoxic effects.

Figure 3

The effect of exogenous growth factors on VCD-induced ovotoxicity. A variety of growth factors have been shown to be important for development and survival in small pre-antral follicles. Postnatal day 4 rat ovaries were exposed to growth factors ± VCD (30 μM) for 8 days. Growth factors tested were: kit ligand (KITLG), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), platelet-derived growth factor isoform B (PDGFB), growth factor 9 (GDF9), and bone morphogenic factor 4 (BMP4). Black bars=oocyte targeted. Grey bars=granulosa cell targeted. Primordial follicles were counted and represented as a % of control (*P < 0.05, different from control).

Figure 4

The KIT/KITLG pathway in primordial and small primary follicles participates in oocyte viability and survival. Kit ligand (KITLG) produced by the granulosa cells binds to KIT (tyrosine kinase receptor) on the oocyte. Binding of KITLG to KIT leads to activation of protein kinase activity, KIT autophosphorylation, followed by a series of cellular responses via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Active PI3K signals to 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which phosphorylates and activates the protein kinase AKT. pAKT then translocates to the nucleus and activates a variety of pathways important for inhibition of apoptosis and promotion of cell survival.

Figure 5

Time course of the effect of VCD on the KIT signaling pathway. mRNA and protein samples were examined from neonatal rat ovaries ± VCD (30 μM) on days 2, 4, 6, and 8 of culture. Relative to controls, two days of VCD exposure decreased post-translational events (phosphorylation of KIT and AKT). Relative to controls, four days of VCD exposure decreased in mRNA encoding KIT, along with a decrease in AKT mRNA (post-transcriptional effects). Relative to controls, six days of VCD exposure increased in mRNA encoding KITLG and caused small follicle loss.