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

The mammalian ovary is comprised of follicles at various stages of growth and is responsible for gamete and steroid hormone production. Conservation of ovarian function is vital for female reproductive and general health, and disruption can lead to infertility and/or ovarian senescence. Stressors affect the ovary by inflicting damage to the primordial oocyte and/or inducing follicular activation thereby depleting the follicular reserve and disrupting biological pathways or defense mechanisms necessary for maintaining proper ovarian function. Cyclophosphamide (CPA) is a chemotherapy drug that biotransforms into an ovotoxic metabolite phosphoramide mustard (PM) via hepatic biotransformation. Our previous findings demonstrated that PM (60 µM) exposure depleted primordial follicles as well as all follicle types in cultured postnatal day (PND) 4 rat ovaries. With the advancement of science and medicine, there has been a decline in mortality rates of females who receive chemotherapy. The viability and health of the ovary post-chemotherapy treatment is of major concern for female cancer survivors due to the harsh effects of anti-neoplastic agents. An ovarian defensive mechanism would challenge the adverse effects of PM-induced ovotoxicity. We hypothesized that the ovarian defense response to PM-induced damage involves both the ABCB1 and ABCC1 proteins to excrete PM from the ovary and that their regulation involved the PI3K pathway. ABCB1 and ABCC1 are involved phase III chemical biotransformation, having enzymatic activity that requires ATP derived energy for the transportation of endogenous and exogenous compounds. Briefly, PND4 rat ovaries were cultured in PM (60 µM), LY294002 (20 µM), or vehicle control (1% DMSO) followed by protein isolation and western blotting. Adult female mice (15 wks) were intraperitoneally (i.p) dosed once with sesame oil (SO) or PM (95%; 25 mg/kg), euthanized 3 days post dosing followed by removal of ovaries and fixation in paraformaldehyde (4%). Ovaries were sectioned and mounted on slides followed by immunohistochemistry (IHC) performance to localize ABCC1 and ABCB1 proteins. PM exposure decreased ABCB1 protein abundance in PND4 cultured rat ovaries, however, no impact was observed in ABCC1 in either PND4 or cultured ovarian rat granulosa cell models. Localization of ABCB1 and ABCC1 in PND4 ovaries was observed in the oocyte cytoplasm post PM exposure. ABCB1 was localized in granulosa cells, interstitial tissue, and the oocyte of adult mice ovaries, and there was striking ABCB1 oocyte peri-cytoplasmic and peri-nuclear immunofluorescence staining. ABCC1 was localized in the nucleus of oocyte in PND4 ovaries, but confined to theca cells, oocyte cytoplasm, and interstitial tissue of adult mouse ovaries. Additionally, pre-ovulatory follicles expressed ABCC1 in the oocyte peri-cytoplasmic membrane in control animals, but this staining pattern was absent in PM-exposed mice. In regards to PI3K, inhibition resulted in down regulation of both ABCB1 and ABCC1, however, PI3K activation via kit ligand treatment had no effect on ABCB1 or ABCC1. Heat stress has a negative impact on agriculture livestock production. Our previous studies suggested that HS alters ovarian steroidogenic signaling impacting the productivity of reproduction in swine (Nteeba et al., 2015). This thesis investigated whether SULT1E1 and ABCC1 were involved in metabolism and excretion of 17β-estradiol synthesis during HS. It was hypothesized that HS alters SULT1E1 and ABCC1 impacting 17β-estradiol. Briefly, pre-pubertal gilts were exposed to thermoneutral (TN; 20°C) or heat stress (HS; 35°C) conditions for 5, 7, or 35 d with an additional group of thermoneutral pair-fed (PFTN) gilts included to eliminate bias of feed intake followed by protein isolation, RNA isolation, western blotting, and qRT-PCR. Additionally, post-pubertal gilts were synchronized in their follicular phase with Matrix feeding and exposed to TN (20.3°C) or HS (26-32°C) conditions for 5 d followed by protein isolation, RNA isolation, western blotting, and qRT-PCR. We found that chronic HS (35 d) of pre-pubertal gilts increased both ABCC1 and SULT1E1 protein abundance without having an effect at the transcriptional level. HS during the follicular phase in post-pubertal estrous synchronized gilts resulted in reduction of ABCC1 mRNA levels and increased levels of SULT1E1 protein, but no impact on ABCC1 protein was observed. The data generated from these studies suggest that PI3K plays a critical role in ovarian phase III biotransformation enzymes providing evidence that the ovary has the ability to conduct phase III drug metabolism and that SULT1E1 and ABCC1 are active in the ovary and may alter 17β-estradiol metabolism during HS