Int. J. Environ. Res. Public Health 2010, 7 2042 follicles produce higher concentrations of E2 than either progesterone or androgen, while the reverse is true for atretic follicles [19,20,22]. It is conceivable that the significant reduction in serum E2 concentrations at 48 hours post PMSG (proestrus) in HU-treated mice is a reflection of fewer healthy follicles progressing towards ovulation compared with their control counterparts. Hence, the reduced ovarian weight observed among HU-treated versus control mice is secondary to fewer corpora lutea, the most prominent tissues in the ovary immediately following ovulation, due to fewer numbers of healthy follicles that ovulated. In this study, fertilization rates of ova recovered from HU-treated and control mice were comparable, suggesting that in vivo exposure of oocytes to HU did not interfere with the fertilization process. Based on data raised in HeLa cells, HU exhibits antiproliferative effect in a dose-dependent manner [23] on proliferating cells and less so on non-proliferating cells. These are expected observations, because HU mainly affects DNA synthesis, thereby interfering with the growth of proliferating cells. Yeo et al. [24] found that HU inhibits the growth of human diploid fibroblasts by inducing increasing p53 and p21 levels in human diploid fibroblast cells. In this study, mice were treated with 30 mg HU/kg once every 24 hours for 28 days. With a half-life of 2–4 hours, it is possible that the daily bioavailable levels of HU reaching the granulosa cells of maturing follicles were not significant enough to totally inhibit proliferation. Consequently, enough granulosa cells were spared to prepare oocytes to respond to hCG for the attainment of the final meiotic maturation stage (MII) and ovulate ova equipped to facilitate normal fertilization. The percentage of embryos generated towards the tail end of HU treatment that attained morulla or blastocyst stage of development was significantly reduced during in vitro culture in the absence of HU compared with their control counterparts. The ability of some in vivo HU-exposed embryos to develop to blastocyst stage indicates resilience to this therapeutic agent by exposed embryos. Embryos being diploid cells are vulnerable to the antiprolifarative action of HU [24]. If this premise is true, none of the embryos expose in vivo to HU would develop to blastocyst stage. Perhaps the duration of exposure of in vivo generated embryos to this therapeutic agent was too short to completely arrest development. Furthermore, the removal of the embryos from in vivo exposure and placement in an in vitro culture medium without further exposure to HU may have reversed any arrest imposed by HU. According to Linke et al. [18] HU-induced depletion of deoxyribonucleotide pools and the consequent cell cycle arrest at late G1/early S-phase is reversible. The other scenario is that the short duration of in vivo exposure of 2-cell embryos to HU may have resulted in uneven arrest of the blastomeres. Because blastomeres at the early cleavage stage are totepotent, one viable blastomere can rescue the entire embryo that can develop to blastocyst stage in the absence of HU during in vitro culture. Experiment 2 and 3 were designed to determine if exposure of 2-cell embryos, not previously exposed to in vivo HU, but to continuous or intermittent HU in vitro affected development. The continuous in vitro culture of embryos in the presence of HU caused a significant percentage of embryo death due to irreversible arrest of blastomere proliferation and subsequent apoptotic degeneration [18,23]. Intermittent cultures of embryos in the presence of HU slightly reduced the percentage of dead embryos compared with those cultured continuously. This is likely due to arrests imposed on the embryos in culture by HU and arrest reversal post wash followed by a 23 hour culture before re-exposure. This regimen of in vitro embryo culture led to only a few more embryos