Int. J. Environ. Res. Public Health 2010, 7 2041 Figure 8. Effect of Intermittent in vitro Exposure of Embryos to Bioavailable HU (18 µg/mL) on Their Ability to Develop to Blastocyst Stage; n = 60 per Group. Results are expressed in percentages (HU = treated embryos; Control = vehicle treated embryos. Asterisks indicate a significant difference from controls (P < 0.05). 4. Discussion In Experiment 1, we wanted to determine if HU treatment of adult female mice compromised the ability of the in vivo exposed ovary to function maximally. In this experiment, we observed that ovarian weight, the key predictor of normal folliculogenesis (E2 production) and ovulation rate were significantly reduced in HU-treated adult mice compared with their control counterparts, indicating hypogonadism among HU-treated mice. We have shown that HU is an endocrine disruptive chemical [12] and could have very specific effects on the different tissues within the ovary. Because of the intense interdependency of the different tissues within the ovary, perturbation of the function of one or all the tissues in the ovary by HU can compromise normal folliculogenesis, ovulatory process, fertilization of ovulated ova and subsequent embryo development. During each ovarian cycle, follicular development beyond the early antral stages is absolutely dependent upon FSH [16] for both proliferation and differentiation of granulosa cells and the synthesis of E2 via aromatization of androgen from the thecal tissue [17]. The resulting secondary follicles are them recruited for possible selection as dominant follicles that are triggered to ovulate under the stimulatory influence of E2-regulated LH surge. It is likely that the reduction in ovulation rate observed in this study among HU-treated versus control mice resulted from the inhibition of granulosa cell ribonucleotide reductase, resulting in the depletion of deoxyribonucleotide pools and subsequently arresting granulosa cell proliferation at late G1/early S-phase [18]. Furthermore, HU-induced reduction in DNA synthesis [1] can induce granulosa cell death among HU-treated versus controls. Such actions of HU on granulosa cells can negatively impact FSH-induced proliferation and differentiation of these cells and consequently lead to reduced numbers of follicles recruited for possible selection as dominant follicles; hence the reduced ovulation rate among HU-treated compared with control mice. Numerous studies have measured steroid hormones in follicular fluid as a method for distinguishing between healthy and atretic follicles [19-22]. The inference drawn from these studies is that healthy