Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries

Abstract

Abstract4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD-and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12d in vehicle control, VCD (30 μM), or DMBA (1 μM), ± PI3 kinase inhibitor LY294002 (20 μM) or its inactive analog LY303511 (20 μM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P < 0.05) small primary and larger follicles beginning on d4. VCD caused primordial and small primary follicle loss (P < 0.05) beginning on d6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P > 0.05) at any time, but did cause loss (P < 0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P < 0.05) on d6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P < 0.05) than with DMBA alone. These results support that 1) PI3 kinase mediates primordial to small primary follicle recruitment, 2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and 3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

Mechanisms of Ovarian Atresia Induced by Xenobiotic Exposures

“…VCD at a concentration of 30 µM induces significant loss of primordial and small primary follicles following 6 days of exposure in cultured PND 4 rat ovaries (Figure 3; Devine et al, 2002;Keating et al, 2009). A key gene identified to be altered by VCD in a microarray study was the PI3K pathway member, c-Kit.…”

“…Data represent mean healthy follicle number ± SE; n = 5 ovaries per treatment; * Different from control, P < 0.05. Adapted from Keating et al, 2009 with journal permission.…”

Phase III biotransformation enzyme involvement in the ovarian response to ovotoxic environmental exposures

“…PI3K activation is regulated by the binding of kit ligand produced by granulosa cells to the oocyte receptor c-Kit [79][80][81][82]. We have previously demonstrated that PI3K inhibition altered ovotoxicity caused by VCD and DMBA [62,83]. Inhibition of PI3K completely prevented VCD-induced depletion of primordial follicles but accelerated that induced by DMBA, to such an extent that essentially no primordial follicle oocytes could be identified [62].…”

“…Activin produced by GC amplifies FSH secretion by boosting formation of GnRH receptors [62,63]. However, activin is inhibited by inhibin and follistatin.…”

“…We have also discovered that both the xenobiotic biotransformation enzymes transcriptional regulators, arylhydrocarbon receptor (AHR) and nuclear factor erythroid-related factor 2 (NRF2), as well as phase I (microsomal epoxide hydrolase (EPHX1)) and phase II (Glutathione S-transferase isoforms pi (GSTP) and mu (GSTM)) enzymes are downstream of the phosphatidylinositol-3 kinase (PI3K) pathway [55,57]. Indeed, inhibition of PI3K prevented ovotoxicity induced by 4-vinylcyclohexene diepoxide (VCD) but accelerated that of dimethylbenz[a]anthracene (DMBA) [62], potentially due to increased EPHX1 [57]. Although the basal function of ovarian ABCB1 and ABCC1 have not been well characterized, considering their function in non-ovarian tissues, their necessity in phase III chemical metabolism for intracellular protection of the ovary seems logical.…”