The effect of gonadotropin-releasing hormone analogues on the preservation of ovarian function against cyclophosphamide-induced damage in adult mice

Objective: To assess the effect of gonadotropin-releasing hormone analogue (GnRHa) on the preservation of ovarian function against cyclophosphamide-induced gonadal toxicity. Materials and Methods: In a controlled, experimental study, 64 female mice were divided into four groups: control (C), triptorelin acetate (T), cyclophosphamide (CY), and triptorelin plus cyclophosphamide (T+CY) groups. Mice in the group (T) were subcutaneously injected with GnRHa (triptorelin acetate) in a dose of 0.5 mg/kg daily for 21 days. In contrast, mice in the (CY) group and (T+CY) group were injected intraperitoneally with 75 mg/kg of CY on day 15. After 21 days, half of the mice in each group were sacrificed, and their ovaries were removed. The rest of the mice in each group were left without any intervention for an additional 21 days, and the same procedures were repeated to assess the ovarian follicles. Results: There was significant depletion of ovarian follicles in the CY group compared to the control group (p<0.05). There were significant decreases in the number of secondary and antral follicles at late stage as compared to early stage in the CY group (p<0.05). There was also a significant increase in the number of primordial and primary follicles in the T+CY group as compared with the CY group early post-treatment, while the increase was significant in all follicles after 42 days (p<0.05). Conclusion: Cyclophosphamide destroys primordial and primary follicles at an early stage while damage in secondary and antral follicles was prominent after 42 days. Triptorelin acetate reduces the toxic effect of CY; it has early and late protective effects and preserves ovarian function in mice

Novel Strategies in Cancer Prevention and Fertility Preservation with Tamoxifen

Women at high risk for breast cancer are often also at high risk for ovarian cancer, reflecting similar risk factors and suggesting intertwined disease pathways and common prevention targets. A novel strategy to overcome obstacles in preventing ovarian neoplasia (low incidence, lack of specific disease markers, and difficulties in tissue sampling), the deadliest gynecologic cancer, may be to develop a prevention strategy that targets breast and ovarian cancer simultaneously. Tamoxifen, a selective estrogen receptor modulator, reduces hormone responsive breast cancer risk by 50% but its effects on risk of ovarian cancer, also hormonal responsive, are unclear. The goals of this work were to 1) develop and characterize a preclinical model of concurrent breast and ovarian cancer and 2) use this dual cancer model to examine the efficacy of tamoxifen to prevent both breast and ovarian cancer. Mammary carcinogens [7,12-dimethylbenz[α]anthracene (DMBA), N-methyl-N-nitrosourea and estradiol (Ey2)] were tested separately in combination with local ovarian DMBA administration to determine the best combined treatment to induce mammary and ovarian cancer concurrently and effectively in the rat. Results showed that systemic Ey2 and ovarian DMBA promoted the highest incidence of dysplasia in the mammary gland and ovary and elevated levels of mammary Ki-67 and cyclooxygenase 2 (COX-2) mimicking the human disease. Next, the ability of tamoxifen to prevent mammary and ovarian cancer simultaneously was evaluated. Tamoxifen which inhibited mammary carcinogenesis and normalized levels of Ki-67 and COX-2, had no effect on (neither accelerated nor inhibited) ovarian cancer progression. In addition, carcinogen treatment increased levels of stem cell markers, Oct-4 and aldehyde dehydrogenase-1, in the mammary gland; interestingly, this expansion was not reversed by tamoxifen. Intriguingly, while examining ovaries from this study, we serendipitously discovered an apparent protective effect of tamoxifen against DMBA-induced follicular destruction and this effect was further investigated. Chemotherapy and environmental toxicants (e.g. DMBA) deplete ovarian follicles and often lead to accelerated ovarian aging and premature ovarian failure; however, there is no established treatment that can protect the ovary from these toxic insults. In vivo, rats were treated with tamoxifen and DMBA or cyclophosphamide (the most ovotoxic chemotherapy) and total numbers of follicles in the ovary were determined. In vitro, ovarian organ culture and oocyte culture were carried out to examine local effects of tamoxifen on DMBA-induced follicle loss and doxorubicin-induced oocyte fragmentation, respectively. We demonstrated for the first time that tamoxifen protects ovarian follicles against not only DMBA- but also chemotherapy (cyclophosphamide and doxorubicin)-induced ovarian damage. Clinically, tamoxifen has already been tested for safe use as an adjuvant therapy for several cancers; therefore, if translated into clinical use, these results may have immediate impact on options for fertility preservation and quality of life in young female cancer patients undergoing chemotherapy. The long term goals of this work are to 1) use the dual cancer model to screen for promising agents that decrease risks for both breast and ovarian cancer and 2) examine the mechanism by which tamoxifen inhibits toxicant-induced ovarian follicle loss

Ovarian metabolism of xenobiotics

At birth, the mammalian ovary contains a finite number of primordial follicles, which once depleted, cannot be replaced. Xenobiotic exposures can destroy primordial follicles resulting in premature ovarian failure and, consequently, early entry into menopause. A number of chemical classes can induce premature ovarian failure, including environmental, chemotherapeutic and industrial exposures. While our knowledge on the mechanistic events that occur in the ovary with chemical exposures is increasing, our understanding of the ovary\u27s capacity to metabolize such compounds is less established. This review will focus on three chemicals for which information on ovarian metabolism is known: trichloroethylene, 7,12-dimethylbenz[a]anthracene and 4- vinylcyclohexene. The current state of understanding of ovarian bioactivation and detoxification processes for each will be described

Mechanisms of Ovarian Atresia Induced by Xenobiotic Exposures

The focus of this chapter is the mechanisms of apoptosis that occur during “normal” (physiologically-induced) or “abnormal” (chemical- or pathology-induced) attrition of ovarian oocytes. One of the primary functions of the ovary is development and maturation of oocytes, which occur within a follicular structure. During fetal development of the ovary, primordial germ cells (oogonia) are formed and become oocytes when they stop dividing, and are arrested at the diplotene stage (prophase) of the first meiotic division. At this stage the oocyte is surrounded by a single layer of flattened somatic cells (pre-granulosa cells) and a basement membrane to form primordial follicles (Hirshfield, 1991), the most immature follicular stage of development. As a result, the lifetime supply of oocytes is set at the time of birth, and is irreplaceable. Association of the granulosa cells with the oocyte is critical for maintenance of oocyte viability and follicle development (Buccione et al., 1990). Development and progression of a recruited follicle also requires the appropriate expression of numerous factors, including critical members of the transforming growth factor β superfamily, such as growth differentiation factor 9 (GDF9) and bone morphogenic protein (BMP15) (Paulini and Melo 2011). Characterization of GDF9 function has a demonstrated a required role in promoting somatic cells of the follicle to undergo mitosis and initiates paracrine signaling between the oocyte and the follicular cells surrounding it (Carabatsos et al. 1998; Hreinsson et al. 2002; Nilsson and Skinner 2002). The impaired fertility in GDF9 mice appears to primarily be a result of loss of function in somatic cells since oocytes of GDF knockout mice remain capable of undergoing in vitro maturation and progressing to metaphase II of meiosis (Carabatsos et al. 1998)

Impact of coadministration of apigenin and bone marrow stromal cells on damaged ovaries due to chemotherapy in rat: An experimental study

Background: Apigenin is a plant-derived flavonoid with antioxidative and antiapoptotic effects. Bone marrow stromal cells (BMSCs) are a type of mesenchymal stem cells (MSCs) that may recover damaged ovaries. It seems that apigenin may promote the differentiation of MSCs. Objective: The aim of this study was to investigate the effect of coadministration of apigenin and BMSCs on the function, structure, and apoptosis of the damaged ovaries after creating a chemotherapy model with cyclophosphamide in rat. Materials and Methods: For chemotherapy induction and ovary destruction, cyclophosphamide was injected intraperitoneally to 40 female Wistar rats (weighing 180–200 gr, 10 wk old) for 14 days. Then, the rats were randomly divided into four groups (n = 10/each): control, apigenin, BMSCs and coadministration of apigenin and BMSCs. Injection of apigenin was performed intraperitoneally and BMSC transplantation was performed locally in the ovaries. The level of anti-mullerian hormone serum by ELISA kit, the number of oocytes by superovulation, the number of ovarian follicles in different stages by H&amp;E staining, and the expression of ovarian Bcl-2 and Bax proteins by western blot were assessed after four wk. Results: The results of serum anti-mullerian hormone level, number of oocytes and follicles, and Bcl-2/Bax expression ratio showed that coadministration of apigenin and BMSCs significantly recovered the ovarian function, structure, and apoptosis compared to the control, BMSC, and apigenin groups (p &lt; 0.001). Conclusion: The results suggest that the effect of coadministration of apigenin and BMSCs is maybe more effective than the effect of their administrations individually on the recovery of damaged ovaries following the chemotherapy with cyclophosphamide in rats. Key words: Apigenin, Bone marrow stromal cells, Chemotherapy, Ovary, Regeneration

All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

The irreplaceable mammalian primordial follicle represents the basic unit of female fertility, serving as the primary source of all developing oocytes in the ovary. These primordial follicles remain quiescent, often for decades, until recruited into the growing pool throughout a woman's adult reproductive years. Once recruited, <1% will reach ovulation, with the remainder undergoing an apoptotic process known as atresia (Hirshfield, 1991). Menopause, or ovarian senescence, occurs when the pool of primordial follicles becomes exhausted

Investigation of TAp63 gene expression and follicle count using melatonin in cisplatin-induced ovarian toxicity

Background: Premature ovarian failure is among the most important side effects of chemotherapy during reproductive period. Preserving ovarian function is gradually gaining importance during oncologic treatment. The present study aims to investigate the potential of melatonin to protect from cisplatin-induced ovarian toxicity in rats.Methods: Twenty-nine female rats were divided to three groups: Saline control group (group 1), cisplatin group (group 2), and cisplatin and melatonin group (group 3). While the rats in groups 2 and 3 were administered 5 mg/kg single dose of cisplatin via intra-peritoneal (IP) route, the rats in group 3 were started on melatonin (20 mg/kg IP) before cisplatin administration and continued during 3 consecutive days. Ovaries were removed one week after cisplatin administration in all groups. Blood samples were obtained before the rats were decapitated. Histological evaluation, follicle count, and classification were performed. TAp63 mRNA expression was evaluated using mRNA extraction and real-time polymerase chain reaction (PCR) method. Serum estradiol (E2) and anti-Mullerian hormone (AMH) values were measured with enzyme immune-assay technology.Results: While primordial follicles were seen to decrease in group 2 as compared to group 1 (p=0.023), primordial follicle count was observed to be preserved significantly in melatonin group as compared to group 2 (p=0.047). Moreover, cisplatin-induced histo-pathological morphology was preserved in favor of normal histology in melatonin group. A significant difference was not observed between groups with regard to mean serum AMH and E2 values (p=0.102 and p=0.411, respectively). While TAp63 gene expression significantly increased in group 2 as compared to control group (p=0.001), we did not detect a statistically significant difference in cisplatin and melatonin group, although gene expression decreased (p=0.34).Conclusions: We conclude that concurrent administration of melatonin and cisplatin may protect from ovarian damage

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