Abnormal oviduct histology after neonatal Gen treatment. As a first step in determining why preimplantation embryos from neonatal Gen-treated mice had poor survival in the oviduct after the two-cell stage, we analyzed the histology of oviducts collected on GD2. Oviducts from Gen-treated mice retained the expected regional differences between the infundibulum, ampulla, and isthmus (); however, significant abnormalities were present in tissue and cellular morphology compared with controls. The stromal and muscle compartments were abnormally thickened in all oviduct regions, reminiscent of uterine myometrium and stroma (). The thickened areas mainly consisted of additional muscle tissue, and many areas did not have the normal clearly defined outer and inner smooth muscle layers (). In some areas of the ampulla, pseudoglands were near the outer epithelial surface of the oviduct, with no intervening muscle layers (). There were localized areas of excessive epithelial proliferation, and many regions had areas of epithelial disorganization that were particularly apparent after cell borders were labeled by immunostaining for E-cadherin (). This staining also revealed that the epithelial cells had enlarged, rounded nuclei and were cuboidal rather than the predominant tall columnar epithelial cells observed in controls.
Figure 1 Oviduct histology on GD2 in controls (A,E,I,C,G,K) and after Gen treatment (B,F,J,D,H,L). (A and B) Low-power view of H&E-stained oviducts; arrows highlight muscle/stroma thickness, and embryos are indicated (e). (C–H) H&E-stained (more ...) Alterations in oviduct gene expression during neonatal Gen treatment.
We hypothesized that the changes in oviduct morphology were a result of Gen-induced alterations in expression of genes that influence postnatal FRT morphogenesis. The effect of Gen treatment on oviduct expression of secreted signaling proteins and transcription factors previously documented to affect FRT development was examined by real-time PCR at the completion of Gen treatment on PND5 (). Wnt genes required for FRT development include Wnt4
, and Wnt7a
(Masse et al. 2009
). There was no difference in Wnt4
expression, whereas Wnt5a
was up-regulated and Wnt7a
was almost completely repressed in Gen-treated mice compared with controls. Follistatin-like-1 (Fstl1), a diffusible oviduct mesenchymal factor, which promotes formation of ciliated epithelial cells (Yamanouchi et al. 2010
), was significantly reduced in Gen-treated mice compared with controls. Secreted proteins in the hedgehog signaling pathway can regulate postnatal FRT morphogenesis and adult FRT function, and Wnt genes can be targets of hedgehog signaling (Franco et al. 2010
). Indian hedgehog (Ihh
) and hedgehog interacting protein (Hhip
) were highly up-regulated, and desert hedgehog (Dhh
) was up-regulated 2-fold, after Gen treatment compared with controls, whereas sonic hedgehog (Shh
) was not detected (data not shown). GLI-Kruppel family member 1 (Gli1
), a common downstream mediator of hedgehog signaling, was also up-regulated, indicating that Gen treatment caused activation of hedgehog signaling in the neonatal oviduct.
Expression of genes important for FRT development on PND5 after Gen treatment. Data are mean ± SE relative oviduct mRNA expression plotted for indicated genes. *p < 0.05 compared with control.
Transcription factors that regulate patterning and differentiation of the FRT include forkhead box a2 (Foxa2
) and the posterior Hoxa genes (Jeong et al. 2010
expression was up-regulated on PND5 by Gen treatment. Expression of Hoxa9
, and Hoxa11
was decreased in oviducts of Gen-treated mice compared with controls, whereas Hoxa13
was highly up-regulated. This Hoxa gene expression pattern indicated that the PND5 oviduct was posteriorized; that is, the oviduct expressed Hoxa genes in a pattern similar to that normally observed in the posterior cervical and vaginal tissues (Ma et al. 1998
Alterations in oviduct gene expression on GD2.
We hypothesized that genes important for FRT morphogenesis and perturbed by neonatal Gen treatment would continue to be altered in adult oviducts and that additional oviduct genes would be misexpressed around the time of preimplantation embryo loss. Rather than choosing candidate genes, we performed a microarray-based gene expression analysis of oviducts from control and Gen-treated mice on GD2. We validated selected probes that covered a range of intensity values and fold changes by real-time RT-PCR [see Supplemental Material, (http://dx.doi.org/10.1289/ehp.1104018
)]. Principal component analysis showed sample separation into distinct groups based on treatment; this was confirmed using hierarchical cluster analysis (see Supplemental Material, ).
Most highly up- and down-regulated oviduct genes.
Of the > 41,000 probes on the array, 1,125 probes were significantly altered (analysis of variance, p
< 0.01) in Gen-treated mice compared with controls. Duplicate probes and probes with average intensity < 100 in both treatment groups were removed from the analysis data set. Within the remaining probes, 335 genes were up- or down-regulated ≥ 2-fold; the most highly altered genes are shown in . Significantly altered Ingenuity biological function categories included development, immune response, and cell proliferation [see Supplemental Material, Table 2 (http://dx.doi.org/10.1289/ehp.1104018
)]. Four of the six secreted signaling proteins (Wnt7a
, and Hhip
) and one transcription factor (Foxa2
) that were altered on PND5 after Gen treatment by PCR analysis were also significantly altered on GD2 by microarray analysis. Taken together, these findings indicate that neonatal Gen treatment permanently altered gene expression in the adult oviduct.
Three of the most highly up-regulated genes on GD2 were the homeobox transcription factors Pitx1, Nkx3-1 [NK-3 transcription factor, locus 1 (Drosophila)], and Six1. These genes all have critical roles in embryonic development but are not reported to be expressed in FRT tissues. We hypothesized that misexpression of these early development transcription factors contributed to abnormal oviduct morphogenesis in Gen-treated mice. To test this idea and to further verify the microarray data, we performed real-time RT-PCR on independently collected biological replicates. Oviducts were collected on PND5, on PND22 (prepubertal) from superovulated adult mice before and after ovulation, and on GD2 and GD4. Pitx1 and Nkx3-1 were highly up-regulated in Gen-treated mice compared with controls at all time points (). Pitx1 protein was not detected in control oviducts on PND5 (). However, Gen treatment induced Pitx1 expression in nuclei of sporadic oviduct epithelial cells in all areas of the PND5 oviduct (). As on PND5, Pitx1 was not observed in adult control oviducts (), but it was abundantly expressed in focal areas throughout the oviduct epithelium of adult Gen-treated mice (). Like Pitx1, Six1 mRNA was highly up-regulated at all time points, and Six1 protein was significantly increased in the oviducts of Gen-treated mice compared with controls ().
Figure 3 Expression of Pitx1, Nkx3-1, and Six1 in oviduct after neonatal Gen treatment. (A and B) Relative expression (mean ± SE) of Pitx1 (A) and Nkx3-1 (B). (C–J) Pitx1 immunohistochemistry in PND5 isthmus (C and D) and ampulla/infundibulum (more ...)
Broadly posteriorized oviduct gene expression after neonatal Gen treatment. Because oviduct Hoxa gene expression was posteriorized in Gen-treated mice, we tested the idea that aberrant expression of Pitx1, Six1, and Nkx3-1 was also related to FRT posteriorization. To do this, we first needed to determine the timing of Hoxa gene pattern establishment in controls. Hoxa gene patterning in PND1 controls resembled that in adult controls; that is, the highest levels of Hoxa9 were in the oviduct, Hoxa10 and Hoxa11 in the uterus, and Hoxa13 in the cervix/vagina (). These findings, together with alterations in the oviduct Hoxa pattern in Gen-treated mice on PND5 (), indicate that Gen exposure disrupted maintenance of previously established Hoxa gene patterning.
Figure 4 Expression of homeobox transcription factors in FRT tissues of control mice. (A and B) Relative expression (mean ± SE) of genes in tissues from PND1 (A) and adult (B) mice. (C–E) Pitx1 immunohistochemistry in adult uterus (C), cervix ( (more ...)
To determine if genes highly up-regulated in the PND5 oviduct by Gen treatment are normally expressed in the posterior FRT, we compared gene expression levels in the oviduct, uterus, cervix, and vagina of control mice. Pitx1, Six1, and Nkx3-1, like Hoxa13, were expressed exclusively in the cervix/vagina and not in the uterus or oviduct on PND1 () and PND5 (data not shown). This expression pattern was still observed in control adults, when Pitx1, Six1, Nkx3-1, and Hoxa13 were highly expressed in the vagina and cervix but had minimal expression in the uterus or oviduct (). There was no Pitx1 protein in the adult uterus (). However, beginning in the cervix at the squamocolumnar junction, Pitx1 was detected in the nuclei of squamous epithelial cells (), and the staining intensity increased in vaginal epithelium (). Expression of the secreted signaling factors Wnt4, Wnt5a, Wnt7a, and Fstl1 was similar in all regions of the FRT on PND1 and PND5 and in adults (data not shown). In contrast, several hedgehog pathway genes and Foxa2 were more highly expressed on PND1 in the cervix/vagina compared with both the oviduct (Ihh, 11.0-fold; Dhh, 5.8-fold; Hhip, 42.1-fold; and Foxa2, 6.1-fold increased) and uterus (Ihh, 9.4-fold; Dhh, 6.2-fold; Hhip, 63.1-fold; and Foxa2, 651.7-fold increased), suggesting that these genes are also normally restricted to the posterior FRT. Together, these data demonstrate that neonatal Gen treatment blocked the normal process of transcriptional silencing of many posterior developmental genes in the anterior FRT.
To determine if the effects of neonatal Gen treatment on the oviduct are mediated by its estrogenic actions (Jefferson et al. 2002
), we examined whether the estrogen receptor (ER) antagonist ICI (Wakeling and Bowler 1992
) blocked these effects. Neonatal mice treated with both Gen and ICI did not have altered expression of Hoxa9
, nor did they have altered expression of the three most highly up-regulated homeobox transcription factors (Pitx1
, and Nkx3-1
) observed in mice treated with Gen alone (). These findings indicate that molecular posteriorization of the oviduct in response to Gen treatment is mediated by its estrogenic action.
Inhibition of posteriorized gene expression (mean ± SE) by ER antagonist in the oviduct on PND5. *p < 0.05 compared with control or Gen + ICI.