Ablation of Indian hedgehog in the murine uterus results in decreased cell cycle progression, aberrant epidermal growth factor signaling, and increased estrogen signaling - PubMed (original) (raw)

Ablation of Indian hedgehog in the murine uterus results in decreased cell cycle progression, aberrant epidermal growth factor signaling, and increased estrogen signaling

Heather L Franco et al. Biol Reprod. 2010 Apr.

Abstract

Conditional ablation of Indian hedgehog (Ihh) in the murine uterus results in mice that are sterile because of defects in embryo implantation. We performed microarray analysis on these mice at the time point at which the Ihh target genes are induced by the administration of exogenous hormone to mimic Day 3.5 of pregnancy. This analysis identified 863 genes altered by the conditional ablation of Ihh. Of these, genes that regulated the cell cycle were overrepresented. In addition, genes involved in epidermal growth factor (EGF) and estrogen (E2) signaling were found to be deregulated upon Ihh ablation. Furthermore, upon conditional ablation of Ihh, 15-mo-old mice exhibited hallmarks of estrogenized uteri, such as cystically dilated glands and hyalinized stroma. Thus, Ihh regulates embryo implantation by having an impact on the cell cycle, EGF signaling, and E2 signaling.

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Figures

FIG. 1.

FIG. 1.

Schematic of microarray analysis experimental design. Microarray analysis was performed using an abbreviated artificial decidual response protocol in which the uteri were harvested 30 h after the first E2 plus P4 injection. Each vertical bar marks 1 day. E, estrogen injection (100 ng); Rest, no injection; eP, estrogen plus progesterone injection (6.7 ng and 1 mg, respectively).

FIG. 2.

FIG. 2.

Cell cycle genes are deregulated by Ihh ablation. A) Real-time RT-PCR analysis of cell cycle genes (Ccnb1, Cdc2a, Mcm5, Ccnd1). The results represent the mean ± SEM. *P < 0.05; **P < 0.01. B) Immunohistochemical analysis of CCND1 in Pgr+/+ Ihhtm1Blan (left) and Pgrtm2(cre)Lyd/+ Ihhtm1Blan (right) mice. Positive staining is seen by a brown signal. Nuclei were counterstained with hematoxylin. C) Immunohistochemical analysis of MCM3 in Pgr+/+ Ihhtm1Blan (left) and Pgrtm2(cre)Lyd/+ Ihhtm1Blan (right) mice. Positive staining is seen by a brown signal. Nuclei were counterstained with hematoxylin. Original magnification of insets ×40.

FIG. 3.

FIG. 3.

EGF signaling is altered by ablation of Ihh. Real-time RT-PCR analysis of the EGF signaling genes Egfr, Erbb2, Erbb3, Erbb4, and Adam12. The results represent the mean ± SEM. *P < 0.05; ***P < 0.001.

FIG. 4.

FIG. 4.

Estrogen signaling is increased by Ihh ablation. A) Identification of E2- and _Ihh-_regulated genes during embryo implantation. Genes differentially regulated by Ihh were compared to genes regulated by E2 in wild-type mice identified by Hewitt et al. [29]. B) Real-time RT-PCR analysis of E2-regulated genes. The results represent the mean ± SEM. *P < 0.05; **P < 0.01. B) Immunohistochemical analysis of ESR1 in Pgr+/+ Ihhtm1Blan (left) and Pgrtm2(cre)Lyd/+ Ihhtm1Blan (right) mice. Positive staining is seen by a brown signal. Nuclei were counterstained with hematoxylin. Original magnification of insets ×40.

FIG. 5.

FIG. 5.

Examination of 15-mo-old mice. A) Hematoxylin-eosin staining of 15-mo-old Pgr+/+ Ihhtm1Blan (left) and Pgrtm2(cre)Lyd/+ Ihhtm1Blan (right) mice. B) Masson trichrome staining of 15-mo-old Pgr+/+ Ihhtm1Blan (left) and Pgrtm2(cre)Lyd/+ Ihhtm1Blan (right) mice. Blue signal indicates collagen. Red signal indicates muscle. Black signal indicates nuclei. Original magnification of insets ×40.

FIG. 6.

FIG. 6.

Model of Ihh signaling in the uterus during preimplantation. Ihh acts as a mediator of epithelial-stromal communication by mediating multiple signaling pathways in the preimplantation period, such as cell cycle progression, EGF signaling, and E2 signaling.

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