OVARIAN FRAGMENT CULTURE WITH 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To evaluate the culture of rhesus monkey ovarian fragments with 2,3,7,8-tetrachlorodibenzo-p-dioxin and assess effects on follicle number and size. There has been limited investigation of the effects of the toxicant dioxin (TCDD) on the reproductive function of non-human primates. We have demonstrated the presence of functional TCDD- or aromatic hydrocarbon (AH)-receptor capable of binding DNA in rhesus monkey ovarian tissue, localized that binding to follicles, and reported TCDD-related altered production of sex steroids by ovarian fragments in culture. We now describe the effects of TCDD exposure on ovarian follicle size and number in the latter model system. Three fresh monkey ovaries (Macaca mulatta; WNPRC; Madison, WI) were cleaned of excess tissue, rinsed (PBS), weighed and quartered into fragments. Fragments were placed in separate Falcon organ culture dishes and incubated (37¿C, 5% CO2, 98% humidified air) in culture medium (phenol red-free DMEM/F12, FBS, 50 ug/ml gentamycin) containing either dioxane (CTL), or picomolar (pM), nanomolar (nM), or micromolar (¿M) concentrations of TCDD for up to 96 hours. All fragments were weighed, stored frozen (-80 ¿C), cryosectioned (10 ¿M) and stained with hematoxylin and eosin. Quantification of follicles by size was performed using light microscopy. Although variability was noted between animals, analyses show that fragments exposed to TCDD tended to have a decreased total number of follicles when compared to control fragments (%CTL; ¿M:78%; nM:57%; pM:65%), especially in the mid-sized range (greatest cross sectional area of 104  106 ¿M2 ;%CTL; ¿M:68%; nM:44%; pM:59%). We hope to use this approach as a model for studying environmental contaminants on monkey fertility. Supported, in part, by ASP to LKC, and NIH P51 RR000167, RR15459-01, RR020141-01 to WNPRC. Funding ended before this reporting period but there are publications reported here and additional publications pending.