In the present study, we were interested in using an array-based approach to identify BPA-induced changes in gene expression at the time when germ cells are entering meiosis in the fetal ovary. However, before embarking on this analysis, we tested the utility of the approach—specifically, whether we could reliably detect temporal changes in expression levels of genes known to be involved in different aspects of early meiosis. The results of that analysis were compelling: Virtually all meiosis-specific genes, but relatively few others, that we tested exhibited significant increases in expression over the time period associated with the earliest events of meiosis. Furthermore, the earliest temporal change was observed for Stra8, a gene thought to be a key player regarding entry into the meiotic pathway.
These results are consistent with those of previous array-based analyses of the meiotic transcriptome in humans and mice from our own (see, e.g., [29
]) and other groups (see, e.g., [30
]). Taken together, these studies demonstrate that array-based analyses can be used to monitor temporal changes in gene expression at successive stages of the meiotic prophase. Furthermore, they provide an important new approach for the identification of novel meiotic genes. For example, in our analysis, no fewer than 208 of the 265 genes exhibiting significant increases in expression between 12 and 14.5 dpc (Supplemental Table S1) are genes for which little or no information regarding function is available.
Importantly, our initial analysis of known meiotic genes also provided confidence that the array-based approach could identify BPA-induced changes in gene expression. Thus, in subsequent analyses, we compared gene expression profiles in the fetal ovaries in fetuses from pregnant females exposed to low levels of BPA and from placebo controls over the 4-day gestational interval that encompasses meiotic entry. Our results demonstrate that exposure to low levels of BPA—levels that are below the current U.S. Food and Drug Administration “safe” dose for daily exposure and that are thought to be analogous to current human exposure levels [32
]—induces gene expression changes in the developing fetal ovary within 24 h of the onset of exposure, and with time, increasing numbers of genes exhibit significant BPA-associated alterations in expression. Over the entire 3.5-day exposure window, no fewer than 7192 genes were significantly up- or down-regulated following BPA exposure.
It is notable that the BPA-induced changes in gene expression were subtle, with almost all differences observed being less than 1.6-fold. This is consistent with our previous cytological analyses in which we detected significant, but subtle, BPA-associated effects on meiotic prophase [12
]. Specifically, at the 20-ng/g level, we observed a 3-fold increase in the incidence of subtle synaptic defects, a 10% increase in recombination rates, and a slight increase in length of the SC, the meiosis-specific scaffold on which recombination occurs. The nature of the expression changes is also consistent with our cytological observations, because expression of one quarter of the genes on our list of known and putative meiotic genes was significantly altered by BPA (), and all 16 of the meiosis-specific genes used in our analysis of the control data were subtly up-regulated in ovaries from BPA-exposed females. Because our most recent cytological studies suggest that the level of meiotic disturbance is directly correlated with the dose of BPA (Lawson and Hunt, unpublished data), it will be important in future expression studies to increase the BPA dose and ask whether changes of greater magnitude are also induced in this setting.
Frequently, reverse transcription PCR analysis of small sets of genes is used to validate the findings of expression array studies. In this case, our major finding—that the majority of early meiotic genes are up-regulated by BPA—is itself a validation of our previous cytological studies. As discussed above, those studies indicated increases in SC length and recombination levels following BPA exposure. Given those changes, the up-regulation of SC (i.e., Sycp1, Sycp2, Sycp3, Syce1, Syce2, and Tex12) and recombination machinery genes (i.e., Spo11, Dmc1, Mei1, Msh4, and Msh5) observed in the present analysis makes perfect biological sense: Increased availability of the necessary reagents would allow formation of a longer SC and more recombination.
In our cytological studies, we also found that the Esr2
-knockout female mouse phenocopied most of these BPA-induced meiotic defects [12
]. However, an additional abnormality—end-to-end associations between nonhomologous chromosomes—was observed in pachytene cells, but not in the Esr2
knockout mouse, following BPA exposure. Thus, we postulated that BPA was acting by two distinct mechanisms in the fetal ovary and that the end-to-end association defect resulted from a disruption of the cytoskeleton that impaired normal chromosome movement at the onset of meiosis. Intriguingly, our gene expression studies revealed four genes up-regulated by BPA at 14.5 dpc that are potentially involved in cytoskeletal regulation. These include Dock3
, a guanine nucleotide exchange factor that may be involved in cytoskeletal reorganization; the coiled-coil domain-containing proteins (Ccdc79
), which have roles in actin cytoskeletal processes; and Emerin, a nuclear membrane protein. Thus, our expression results also strongly support the conclusion from our meiotic studies that the effect of BPA on the fetal ovary is complex and likely involves several distinct mechanisms. However, simply confirming that BPA alters the expression of these specific players will not allow us to understand the potential effect of BPA on the oocyte cytoskeleton. This requires a detailed analysis of the way that chromosomes move on the nuclear envelope and undergo synapsis in the presence and absence of BPA, and these studies are currently ongoing in our laboratory.
The consistency of the findings between our meiotic and expression studies is notable, but our expression data do not provide a direct means of unraveling the role that estrogen signaling through ESR2 plays in the transition from germ cell to meiocyte in the fetal ovary. In retrospect, this is perhaps not surprising. By comparison with the male, the period of germ cell differentiation preceding meiotic entry remains poorly characterized in the female. Female germ cell differentiation is rapid, with the entire period of germ cell proliferation, meiotic commitment, and meiotic entry spanning a period of only several days. Thus, the time points used in our analysis (12, 12.5, 13.5, and 14.5 dpc) represent a developmental window that includes a multitude of important events. At the earliest stages of BPA exposure (11–12.5 dpc), germ cells are populating the genital ridges and undergoing mitotic proliferation (for review, see [34
]). By 13.5 dpc, the first wave of oocytes is entering meiotic prophase [35
]. The event commonly perceived as the “onset” of meiosis—the initiation of synapsis between homologous chromosomes—is preceded by a host of complex events in female germ cells, including X-reactivation, major chromatin remodeling events, premeiotic DNA replication, establishment of meiosis-specific cohesion complexes, and prealignment of homologous chromosome in preparation for synapsis and SC formation. Thus, it is not particularly surprising that a host of different genes are up- or down-regulated by BPA at different time points during this relatively short time frame.
Characterizing the molecular signals that control the germ cell-to-meiocyte transition in the female is also hampered by the fact that germ cell differentiation occurs during fetal development and the germ cell population is heterogeneous, with meiotic onset in the posterior gonad lagging a full 24–48 h behind that in the anterior region. Furthermore, unlike the male, where physical separation methods make it possible to obtain purified populations of germ cells and meiocytes, the size of the developing fetal ovary makes it difficult to isolate germ cells in the female. Thus, major changes in expression that are limited to germ cells may be masked by contamination from somatic cells, and vice versa. This will have the effect of dampening any temporal changes in expression levels involving one of the two cell types but, additionally, will make it less likely that we can detect germ cell or somatic cell-specific effects that carry over from one time point to the next. In addition, in our previous analyses of mouse oogenesis [12
], we detected significant BPA-associated effects on meiosis. However, the methodology is highly sensitive, and at the dosage level used in both the previous and present studies (20 ng/g), the effects of BPA, while highly significant, were relatively subtle. Specifically, at the 20-ng/g level, we observed a 3-fold increase in the incidence of subtle synaptic defects and a 10% increase in recombination rates. Importantly, in contrast to the meiotic phenotypes induced by the knockout of individual meiotic genes (for review, see [36
]), in which a block at a specific meiotic stage or a clear and consistent meiotic defect is induced, low-level BPA exposure induces subtle meiotic defects in a significant proportion of cells. Thus, expression level differences of the magnitude observed in the present study are consistent with the subtle, but significant, defects observed in our previous meiotic analyses. Clearly, in future expression studies, it will be important to increase the BPA dose and ask whether changes of greater magnitude are induced. Thus, although the findings reported here demonstrate that BPA induces changes in gene expression in the fetal ovary and provide data that can be mined by us and others, a careful molecular dissection of the germ cell-to-meiocyte transition is badly needed.
Potential Long-Term Consequences of BPA Exposure to the Fetal Ovary
In addition to providing candidate genes that will help unravel the effects of BPA on the fetal ovary, our results raise the possibility that BPA may affect female fertility by yet another mechanism. Specifically, our GO analysis revealed a striking down-regulation of mitotic/cell-cycle genes, raising the possibility that BPA exposure immediately before meiotic entry might act to shorten the reproductive life span of the female. That is, meiotic entry is preceded by successive rounds of mitotic proliferation that expand the germ cell population. Thus, if BPA exposure causes premature meiotic entry, this could act to reduce the total pool of fetal oocytes. In turn, this would have important reproductive ramifications, because females exposed to BPA in utero would be at an increased risk not only of producing aneuploid eggs and embryos [12
] but also of premature reproductive senescence. Studies to test this hypothesis are currently underway.
Although to our knowledge this is the first report of an effect on primordial germ cells in the ovary, results of previous in vitro studies have suggested that high concentrations of estrogens stimulate the growth of primordial germ cells in culture [37
], and several previous studies have reported effects of EDCs on male germ cell development. Exposure to phthalates in vivo has been reported to decrease the number of gonocytes in the fetal rat testis [38
], and a similar decrease has been reported from in vitro exposures of fetal rat [39
], mouse [40
], and human [41
] testes. Furthermore, recent in vitro studies of human testicular seminoma cells revealed that very low concentrations of BPA stimulated cell proliferation by inducing rapid, nongenomic, membrane-initiated activation of the cAMP-dependent protein kinase and cGMP-dependent protein kinase signaling pathways. From this, it was postulated that the high affinity of BPA for G protein-coupled, nonclassical membrane-associated receptors might interfere with germ cell proliferation and differentiation in the developing fetus [43
Summary and Future Directions
The present study demonstrates a large number of subtle changes in gene expression in the fetal ovary following low-dose (20-ng/g) BPA exposure. Because we have recently found that increasing doses of BPA are associated with increased levels of both recombination and synaptic defects (Lawson and Hunt, unpublished data), these relatively modest changes in gene expression likely reflect the low BPA dose used in the present study. Thus, in future studies, it will be important to re-examine those genes we have identified as being affected by BPA and ask how their expression levels change over a range of BPA exposures. This will provide a means to validate and refine a list of candidate genes responsible for the meiotic disturbances induced by prenatal BPA exposure and should aid in translating our studies of mice to humans. Importantly, in addition to supporting the conclusion from our previous meiotic studies that BPA acts by several different mechanisms to perturb the early stages of oogenesis, our expression studies provide evidence that BPA may also affect primordial germ cells, influencing mitotic proliferation and the timing of meiotic entry.