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. 2008 Aug;149(8):3970-9.
doi: 10.1210/en.2007-1599. Epub 2008 Apr 24.

Effects of gestational diethylstilbestrol treatment on male and female gonads during early embryonic development

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Effects of gestational diethylstilbestrol treatment on male and female gonads during early embryonic development

Yayoi Ikeda et al. Endocrinology. 2008 Aug.

Abstract

To study the effects of gestational exposure to estrogen on early gonadal differentiation, pregnant mice were treated by sc injection of diethylstilbestrol (DES) or vehicle from embryonic day (E) 8.5 to E14.5, and gonads at E11.5, E12.5, and E14.5 were examined. Quantitative real-time RT-PCR and in situ hybridization revealed that mRNA levels of steroidogenic factor 1 (SF-1), a key regulator of gonadal differentiation, and several male gonad-specific genes, including Müllerian-inhibiting substance (MIS), steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein, were significantly decreased in the DES-treated testis, compared with the control testis at E12.5 and/or E14.5. Immunohistochemistry demonstrated that the staining intensities for SF-1 and MIS in Sertoli cells were apparently reduced in the DES-treated testis, compared with those of the controls, at E12.5 and E14.5. Because MIS, steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein are activated under the regulation of SF-1, the down-regulation of these factors may be due to reduced SF-1 expression. Immunohistochemistry for laminin-1 demonstrated that ovigerous cords in the DES-treated ovary were smaller than those in controls at E14.5. Moreover, the number of 5-bromo-2'deoxyuridine-5-monophosphate-labeled cells in the DES-treated testis was significantly reduced at E12.5 and E14.5, compared with controls, and that in the DES-treated ovary remained higher than that in the control ovary at E14.5. The results suggest that exogenous estrogens can alter sex-specific genetic pathways governing early differentiation and cell proliferation of both male and female gonads.

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Figures

Figure 1
Figure 1
mRNA levels of several regulators of gonadal differentiation. qPCR was performed to analyze gene expression in male (left panels) and female (right panels) gonads at E11.5, E12.5, and E14.5 from mice exposed in utero to vehicle or DES. Graphs show mRNA levels normalized to the levels of 18S rRNA, and each bar shows mean ± sem of three to five embryos. *, P < 0.05 vs. control.
Figure 2
Figure 2
mRNA levels of early markers of testicular differentiation. qRT-PCR was performed to analyze expressions of MIS, P450scc, StAR, and Cbln 1 as early markers of testicular differentiation in developing testis at E11.5, E12.5, and E14.5 from mice exposed in utero to vehicle or DES. Graphs show mRNA levels normalized to the levels of 18S rRNA, and each bar shows mean ± sem of three to five embryos. *, P < 0.05 vs. control.
Figure 3
Figure 3
Localization of mRNAs of SF-1, MIS, and P450scc in DES-treated testis. In situ hybridization using 35S-labeled cRNA probe was performed to analyze localization of mRNAs of SF-1, MIS, and P450scc on sections of fetal mouse testis at E14.5. Left and right panels show testis from mice treated in utero with vehicle and DES, respectively (original magnification, ×33).
Figure 4
Figure 4
Localization of SF-1 protein in DES-treated testis. Immunohistochemistry for SF-1 was performed on sections of fetal mouse testis at E11.5 (A and B), E12. 5 (C and D), and E14.5 (E–H) from mice exposed in utero to vehicle (left panels) or DES (right panels). i, Interstitial region; tc, testicular cords (original magnifications, ×33 in E, F, and ×132 in A–D, G, and H).
Figure 5
Figure 5
Localization of MIS, Sox9, and P450scc proteins in DES-treated testis. IHC for MIS, Sox9, and P450scc proteins was performed on sections of fetal mouse testis at E12.5 (MIS) and E14.5 (MIS, Sox9, and P450scc) from mice exposed in utero to vehicle (left panels) or DES (right panels) (original magnification, ×132).
Figure 6
Figure 6
Localization of laminin-1 in DES-treated gonad. Male and female gonads after in utero exposure to vehicle (left panels) or DES (right panels) were analyzed by immunofluorescence for laminin-1. Laminin-1 is localized to delineate testicular cords in the testis and ovigerous cords in the ovary (original magnification, ×66).
Figure 7
Figure 7
Analyses of cell proliferation in DES-treated gonad. Embryos were labeled transplacentally with BrdU, and BrdU incorporation into gonads was assessed as described in Materials and Methods. Upper panels show representative photomicrographs of sections of testis (left panels) and ovary (right panels) after in utero exposure to vehicle or DES (original magnification, ×66). Lower panels show quantitative analyses of the results of IHC for BrdU. In each graph, the relative number of BrdU-labeled cells is scaled such that the BrdU-labeled somatic cell number of the control group is equal to 100. Three to five gonadal areas from three to five embryos in each treatment were counted, and each bar shows mean ± sem. *, P < 0.05 vs. control.

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