Progress 10/01/10 to 09/30/13
Outputs
Target Audience: The target audience was other reproductive biologists interested in the effects of endocrine disputing chemicals (EDCs) on ovarian function who attended the annual meeting of the Society for the Study of Reproduction in Montreal, Canada in July 2013. Changes/Problems: We began the project with Objective 1. Because our initial experiments yielded interesting results, we have focused primarily on Objective 1, proceeding in directions suggested by the results. Therefore, there was little time to focus on Objective 2. However, we have interesting preliminary findings under Objective 2 and hope to secure other funding to continue with it. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? The results have been disseminated thus far by presentation of portions of the results at two annual meetings of the Society for the Study of Reproduction. This disseminated our findings to other reproductive biologists who study the effects of endocrine disruptors and enabled us to get their feedback on our approach and results. Some of the results have also been presented at annual meetings of the Multistate project that the PI participates in (NE1227), again disseminating the findings to other reproductive biologists. In the near future, we plan to disseminate the results further through preparation of one or two manuscripts. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major Activities Completed For Objective 1, experiments were conducted in vitro to determine if phytoestrogens and/or environmental estrogens affect the earliest stages of ovarian follicular development (the formation of follicles and their acquisition of the capacity to initiate follicular growth). In cattle, these stages occur during fetal life and we used methods developed previously by our laboratory for studying follicle formation and early growth in vitro. Since previous experiments in our laboratory provided evidence that these early stages of follicular development are regulated by steroids produced by the fetal bovine ovary, the effects of a phytoestrogen (genistein) and an environmental estrogen (bisphenol A or BPA) on the capacity of fetal ovarian tissue to produce estradiol and progesterone in vitro were tested. Real-time PCR was used to assess the effects of genistein and BPA on fetal ovarian levels of various mRNAs for enzymes and other proteins involved in steroid synthesis and action. To test the hypothesis that genistein and BPA disrupt the production of steroids by preovulatory follicles of adult cattle (Objective 2), preliminary experiments were conducted with cultures of theca and granulosa cells from large, dominant bovine follicles, using methods developed previously by our laboratory for studying the regulation of steroidogenesis in preovulatory follicles of cattle. Specific Objectives Met and Results Achieved For Objective 1, several estrogenic endocrine disrupting chemicals (EDCs) were tested initially for effects on follicle formation and acquisition of the capacity to activate, using an in vitro model in which these events occur in vitro in small pieces of fetal ovarian cortex. Based on those studies, one phytoestrogen (genistein) and one environmental estrogen (BPA) were selected for more extensive study. The most important findings are: 1) Genistein decreased the number of primary follicles in a dose-dependent manner, suggesting that it may inhibit follicle activation, whereas BPA did not affect follicle numbers. 2) When ovarian cortical pieces were cultured with BPA or genistein, both EDCs increased the percentage of multi-oocyte follicles, suggesting that both can disrupt follicle formation. 3) Since steroids produced by fetal ovaries appear to regulate follicle formation and development of the capacity to activate, we tested the effects of BPA and genistein on production of steroids by fetal ovarian tissue. Both EDCs disrupted steroid production in a dose-dependent manner, with BPA decreasing estradiol with no effect on progesterone, whereas genistein increased estradiol and decreased progesterone. Studies to determine how these effects were produced revealed that BPA decreased levels of mRNA for aromatase (CYP19), whereas genistein increased the abundance of mRNA for both CYP11 and CYP19 and for ERalpha. 4) When cortical pieces from fetal ovaries of late gestation (when follicles have developed the capacity to activate) were cultured with BPA, follicle activation was slower than in control cultures, but follicle numbers were normal, whereas genistein decreased the numbers of both primordial and primary follicles. Thus, experiments conducted under Objective 1 have revealed that both genistein and BPA disrupt early follicular development and fetal ovarian steroidogenesis in dose-dependent and specific ways. Most of our efforts were directed at Objective 1, but preliminary experiments were conducted for Objective 2. Those experiments suggest that: 1) BPA exerts dose-dependent inhibitory effects on FSH-stimulated estradiol production by granulosa cells from preovulatory follicles, with no effect on progesterone production by the same cultures. 2) BPA dose-dependently inhibits both control and LH-stimulated production of androstenedione and progesterone by theca interna cells in culture, whereas the highest dose of genistein enhances the stimulatory effect of LH on androstenedione production, with no effects on progesterone. These preliminary results for Objective 2 suggest that both genistein and BPA can disrupt steroidogenesis by bovine preovulatory follicles. Key Outcomes The results described above show that a phytoestrogen (genistein) and an environmental estrogen (BPA) affected follicle formation and activation, fetal steroid production, and the production of steroids by theca and granulosa cells of dominant/preovulatory follicles of cattle. These results in vitro suggest that these important reproductive stages may be affected by exposure to the EDCs in vivo. Genistein is present in some cattle forage and BPA is a key component of plastic and therefore is ubiquitous in the environment. Knowledge of the disruptive effects of these compounds on key ovarian processes may provide useful information for dairy producers and veterinarians and eventually lead to better herd management and nutritional strategies to avoid exposing cattle to EDCs found to have adverse effects and thus, increase reproductive performance.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Fortune, J.E. and M.Y. Yang, 2011. Effects of exposure to phytoestrogens and environmental estrogens on ovarian follicular number and initiation of follicular growth in bovine fetal ovarian cortex in vitro. Published online 21 July, 2011: available from Society for the Study of Reproduction (http://www.ssr.org/11Abstracts.shtml), Abstract #305.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Yang, MY, JE Fortune, 2013. BPA inhibits estradiol production, decreases aromatase mRNA and increases the incidence of multi-oocyte follicles in bovine fetal ovaries in vitro. Biology of Reproduction, Supplement, Proc. of the 46th annual meeting of the Soc. for the Study of Reproduction, Abstract #124.
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Endocrine-disrupting chemicals (EDCs) include phytoestrogens, commonly found in forage and environmental estrogens found in pesticides, plastics, and industrial chemicals. In recent years, concern about potential harmful effects of EDCs on animal and human health and reproduction has increased. The first objective of this project is to determine if phytoestrogens and/or environmental estrogens affect the formation of ovarian follicles and initiation of follicle growth (activation), which occur during early to mid-gestation in cattle. During Year 1 we tested a number of phyto- and environmental estrogens and reported that the phytoestrogen genistein and the environmental estrogen octyphenol (Oct-P) can prevent newly formed (primordial) follicles from gaining the ability to activate. During Year 2, we continued to test these two EDCs and the environmental estrogen bisphenol A (BPA) for effects on early follicular development, using the methods described in last year's report. Accomplishments: 1) Based on the results reported last year, we determined whether the effects of a phytoestrogen (genistein) and two environmental estrogens (BPA and Oct-P) on early follicular development are dose-dependent (0.01, 0.1, 1 uM). Freshly isolated cortical pieces contained mostly primordial follicles, but after 10 days in control medium, most primordial follicles had activated, differentiating into primary follicles, as expected. Genistein and Oct-P decreased the number of primary follicles on day 10, compared with controls, in a dose-dependent manner and Oct-P also decreased the number of primordial follicles; BPA had no effect. These data suggest that genistein and Oct-P may inhibit both the formation of ovarian follicles and their ability to initiate growth in vivo. 2) Since fetal bovine ovaries produce steroid hormones, which appear to regulate the formation of primordial follicles and their capacity to initiate growth, we are examining the effects of estrogenic EDCs on steroid production by bovine fetal ovaries in vitro. Pieces of ovaries obtained between day 80-89 of gestation (i.e. when steroid production is still fairly robust and just before follicle formation begins) were cultured for 6 days with control medium or with graded doses of BPA (0.01, 0.1, 1 uM). BPA dose-dependently inhibited E2 accumulation during culture, but had no effect on P4. These data suggest that BPA disrupts fetal ovarian steroidogenesis. 3) We further tested the hypothesis that BPA exerts this effect by altering the expression of steroidogenic enzymes. Real-time PCR showed that after 6 days of culture BPA did not affect steady-state levels of mRNA for steroidogenic acute regulatory protein, cytochrome P450 11A1, 3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase or cytochrome P450 17A1 in ovarian pieces, but all doses of BPA (0.01 to 1 uM) decreased aromatase mRNA, consistent with the reduced estradiol production. These data suggest that fetal exposure to BPA alters levels of aromatase and thus disrupts estradiol production. Similar studies are underway to test genistein and Oct-P for effects on fetal ovarian steroidogenesis. PARTICIPANTS: 1)J.E. Fortune, Professor Physiology, PI 2)M.Y. Yang, Research Associate, Co-PI TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Primordial follicles comprise the "follicular reserve" which will supply the female with follicles and gametes throughout her reproductive life. Therefore, formation of ovarian follicles and initiation of follicle growth are two critical processes affecting female reproductive lifespan. An increase in our understanding of the effects of exposure to endocrine disrupting chemicals (EDCs; including phytoestrogens and environmental estrogens) on those two critical stages of ovarian follicular development in cattle will be of potential practical importance since it may provide useful information for developing better herd management and nutritional strategies to avoid exposure to harmful EDCs. The results thus far suggest that the estrogenic EDCs genistein and octylphenol reduce the number of growing follicles in fetal bovine ovaries and the environmental estrogen BPA disrupts fetal ovarian production of estradiol. These findings suggest that the exposure of pregnant cattle to estrogenic EDCs may have specific negative effects on fetal ovarian development and hence a potential impact on later fertility of females exposed as fetuses.
Publications
- No publications reported this period
|
Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Endocrine disrupting chemicals (EDCs) include phytoestrogens (biologically active natural substances commonly found in forage) and environmental estrogens found in pesticides, plastics, and industrial chemicals. In recent years, concern about the potential harmful effects of EDCs on animal and human health and reproduction has increased. The first objective of this project is to determine if phytoestrogens and/or environmental estrogens affect the formation of ovarian follicles and initiation of follicle growth in bovine ovaries. These two critical processes occur during early to mid-gestation in cattle. Accomplishments: 1) We tested the hypothesis that exposure to phytoestrogens (equol, coumestrol, genistein, and daidzein) and/or environmental estrogens (bisphenol A [BPA] and octylphenol) during fetal life affects early ovarian follicular development and thus, potentially affects female reproductive lifespan in cattle. Ovaries were obtained from bovine fetuses (n=3) estimated to be day 110-130 of gestation (crown-rump length). Cortical pieces were dissected and then cultured (4 pieces/treatment/fetus) for 10 days with control medium or with 1 uM phytoestrogens (equol, coumestrol, genistein, and daidzein) or environmental estrogens (BPA and octylphenol). Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis. On day 0, freshly isolated cortical pieces contained mostly primordial follicles, whereas after 10 days in culture in control medium, most primordial follicles had activated, differentiating into primary follicles as expected. Neither phytoestrogens nor environmental estrogens (except octylphenol) affected the number of primordial follicles compared with controls. However, both genistein and octylphenol decreased the number of primary follicles. 2) We examined the effects of exposure of bovine fetal ovarian cortex to a lower concentration (0.1 uM) of genistein, BPA, and octylphenol on follicular number and stage after 10 days in vitro. Experimental materials and methods were the same as described above. At 0.1 uM, there were no effects of genistein, BPA and octylphenol on the number of primordial follicles after 10 days in culture. However, genistein and octylphenol, but not BPA, decreased the number of primary follicles, compared with control cultures. We are currently determining whether genistein, BPA and octylphenol have dose-dependent (0.01, 0.1, 1 uM) effects of on the formation of ovarian follicles and initiation of follicle growth in bovine ovaries. The results obtained thus far were disseminated via an abstract (poster) at the annual meeting of the Society for the Study of Reproduction in July 2011 (Portland, OR). PARTICIPANTS: Fortune, J.E. Project Director; Yang, M.Y. Project Co-director TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Primordial follicles comprise the "follicular reserve" which will supply the female with follicles and gametes throughout her reproductive life. Therefore, formation of ovarian follicles and initiation of follicle growth are two critical processes affecting female reproductive lifespan. An increase in our understanding of the effects of exposure to endocrine disrupting chemicals (EDCs; including phytoestrogens and environmental estrogens) on those two critical stages of ovarian follicular development in cattle will be of potential practical importance since it may provide useful information for developing better herd management and nutritional strategies to avoid exposure to harmful EDCs. The observed decreases in the numbers of primary follicles suggest that genistein and octylphenol induce the death of growing follicles in fetal bovine ovaries.
Publications
- Fortune, J.E. and Yang, M.Y. 2011. Effects of exposure to phytoestrogens and environmental estrogens on ovarian follicular number and initiation of follicular growth in bovine fetal ovarian cortex in vitro. Published online 21 July, 2011; available from Society for the Study of Reproduction (http://www.ssr.org/11Abstracts.shtml), Abstract #305.
|
|