This study is a comprehensive analysis of the factors contributing to infertility in Gen-treated female mice; a schematic summarizing the experiments performed is shown in . The data presented herein demonstrate that eggs from neonatally Gen-treated females are morphologically indistinguishable from those of controls and are capable of being fertilized in vitro, developing to the blastocyst stage, and developing to term after transfer to control recipients. We conclude that the complete infertility of neonatally Gen-treated and superovulated females cannot be explained on the basis of poor egg quality. Although other investigations have demonstrated that environmentally relevant chemicals such as bisphenol A can cause disruptions in oocyte meiosis [18
], this does not seem to be the case with neonatal exposure to Gen. Several estrogenic compounds induce multioocyte follicles in the mouse, with a proposed mechanism being disruption of the gonadotropin-estrogen-inhibin/activin system that regulates folliculogenesis [23
]. Disruption of endocrine and paracrine mechanisms of folliculogenesis could explain suboptimal egg quality. In one multioocyte model, eggs from multioocyte follicles with prenatal exposure to high-dose DES have reduced fertilization competence compared with eggs from single-oocyte follicles [19
]. However, in the DES-exposed mice, the multioocyte follicles have many more oocytes per follicle (≤28) than are observed in our neonatal Gen model (≤6), likely due to the much higher dose and estrogenic activity of the DES administered. Our results indicate that the existence of the multioocyte phenotype does not necessarily indicate that all eggs from such ovaries are of lower quality. Of note, our experiments were performed when the Gen-treated females were 6–9 wk of age. Because oocyte quality can decrease with age [24
] and higher-quality oocytes may be recruited to develop earlier in reproductive life, it is possible that Gen treatment affects oocyte quality but that this would not be observed in young females.
FIG. 4. Schematic diagram of the experimental design. All female mice were superovulated, and egg-cumulus cell masses were collected 14 h after hCG administration for in vitro fertilization (IVF) and immunohistochemistry (IHC) experiments. For embryo development, (more ...)
Because the infertility phenotype could not be explained on the basis of poor egg quality, we examined fertilization in vivo and progressive development of preimplantation embryos to determine their developmental competence within the reproductive tract. Although almost all of the eggs were eventually fertilized, there was a delay in fertilization of Gen-treated eggs compared with controls. This delay could be explained by differences in the timing of mating, progression of sperm through the reproductive tract, or timing of capacitation or by alterations in the cumulus cell matrix or zona pellucida properties caused by the oviductal environment that delay successful sperm-egg interactions. Further studies to determine the precise timing of fertilization in vivo and the factors that delay this event may provide insights into effects of the female reproductive tract on sperm function in vivo.
Once placed in culture, pronuclear-stage embryos flushed from Gen-treated mice progressed efficiently to the two-cell stage; however, they did not progress as well as controls thereafter, with a small but significant reduction in development to the blastocyst stage (). This difference was not observed when eggs from Gen-treated mice were fertilized in vitro, suggesting that even a very short exposure to the oviductal environment during the time of fertilization was enough to adversely affect early embryo development. However, blastocysts generated from these pronuclear-stage embryos and then transferred into control recipients generated pups equally as well as controls, and these pups were apparently healthy, with all live pups surviving to weaning. These findings suggest that, although there is a very small difference in development from pronuclear stage to blastocyst stage, the embryos that do survive are fully competent to develop.
Detailed examination of preimplantation embryo development in vivo demonstrated a loss of about 50% of the embryos between the two-cell and four-cell stages. This reduction in embryo number was attributed to exposure to the oviductal environment because it did not occur if the eggs were fertilized and cultured in vitro. It is clear that the environment in which preimplantation embryos develop has a strong effect on embryo development and survival. Numerous growth factors that act in both autocrine and paracrine fashions promote preimplantation embryo development [26
]. In addition, other components (including ions, steroids, prostaglandins, energy substrates, and amino acids) of culture media or oviductal fluids strongly influence preimplantation development and can even influence postnatal development and adult health [27
It is notable that the failure of development in vivo occurred during the two-cell to four-cell transition. This is reminiscent of the “two-cell block” phenomenon in which embryos arrest at the two-cell stage when cultured in suboptimal media in vitro but can complete development if transferred back to the oviduct [29
]. The two-cell block can be caused by environmental toxic effects, deficiency, or imbalance [30
]; any or all of these could explain the poor development in vivo observed in our study. Deficiencies in the oviducts of the Gen-treated mouse could include lack of required energy substrates, osmolytes, or amino acids that are provided in vitro by the KSOM/AA culture medium. Tumor necrosis factor, a proinflammatory cytokine normally produced by the oviduct, causes decreased proliferation and increased apoptosis in preimplantation embryos unless these effects are countered by the presence of certain growth factors [31
]. An imbalance in this or a similar system could cause poor development in vivo.
The two-cell to four-cell stage transition is also the timing of the maternal to zygotic transition in the mouse, as well as the time when a failure of zygotic genome activation becomes manifest by an arrest in development. Several events critical for continued embryogenesis occur during this time frame, including degradation of maternal mRNAs, chromatin remodeling, development of a transcriptionally repressive state, and expression of zygotic mRNAs [33
]. Whether specific pathways involved in zygotic genome activation or chromatin remodeling are disrupted because of factors within (or lacking from) the oviductal environment of Gen-treated females will be addressed in future experiments.
The reduction in embryo number after the two-cell stage could account for the fewer implantation sites observed previously in neonatally Gen-treated mice [9
]. However, the presence of morphologically normal-appearing blastocyst-stage embryos, albeit a reduced number, in the uterus of Gen-treated mice suggests that an additional mechanism besides loss of cleavage-stage embryos contributes to the infertility of Gen-treated females. Indeed, we found that the uterus of pseudopregnant Gen-treated females did not support normal implantation of control blastocysts. One explanation for this finding could be that the Gen-treated mice had suboptimal estrogen or progesterone levels that would cause decreased uterine receptivity. However, it was shown previously that there is no difference between control and Gen-treated mice in serum levels of estrogen, progesterone, or testosterone on Days 6, 8, and 10 of pregnancy [9
]. Instead, the poor uterine receptivity is likely due to diminished endometrial responsiveness to normal levels of circulating steroid hormones. This would not be surprising because the uterus of Gen-treated mice does not respond fully to estrogen stimulation at puberty [34
], and implantation is dependent on the presence of functional estrogen receptor α that mediates estrogen responses in the uterus [35
]. Taken together, these data suggest that the uterus of Gen-treated females has compromised responsiveness to hormonal cues, leading to abnormal uterine receptivity and failed implantation or fetal resorption.
It is also possible that factors from the oviductal fluid influence the ability of the endometrium to support implantation. In women undergoing in vitro fertilization procedures, fluid from hydrosalpinges (fluid-filled Fallopian tubes) has a negative effect on pregnancy establishment after embryo transfer to the uterine cavity and may be associated with early miscarriage [36
]. It is unknown if this is because of detrimental effects on uterine receptivity or direct effects on the transferred embryos. The negative effects are abrogated if the hydrosalpinx is removed and are not observed if there is no tubal patency between the tube and uterus, suggesting that luminal transit of the fluid into the endometrial cavity is required [36
In summary, eggs from Gen-treated females are developmentally competent, despite the multioocyte follicle phenotype observed in their ovaries. However, cumulative defects in the reproductive tract result in infertility. First, fertilization seems to be delayed in Gen-treated mice, and although the mechanism is unknown, this could lead to altered developmental timing. Second, the oviductal environment contributes to infertility seen in Gen-treated mice because more than half of the embryos were lost during this time in early embryo development. Third, even if the embryos survive the oviductal environment, the reproductive tract is not capable of sustaining pregnancy, as demonstrated by embryo transfer findings. Taken together, these data show that neonatal Gen exposure adversely affects the function of the female reproductive tract such that the cumulative effect is complete infertility.