Notch1 mediates uterine stromal differentiation and is critical for complete decidualization in the mouse

Abstract

Uterine receptivity implies a dialogue between the hormonally primed maternal endometrium and the free-floating blastocyst. Endometrial stromal cells proliferate, avert apoptosis, and undergo decidualization in preparation for implantation; however, the molecular mechanisms that underlie differentiation into the decidual phenotype remain largely undefined. The Notch family of transmembrane receptors transduce extracellular signals responsible for cell survival, cell-to-cell communication, and differentiation, all fundamental processes for decidualization and pregnancy. Using a murine artificial decidualization model, pharmacological inhibition of Notch signaling by γ-secretase inhibition resulted in a significantly decreased deciduoma. Furthermore, a progesterone receptor (PR)-Cre Notch1 bigenic (Notch1(d/d)) confirmed a Notch1-dependent hypomorphic decidual phenotype. Microarray and pathway analysis, following Notch1 ablation, demonstrated significantly altered signaling repertoire. Concomitantly, hierarchical clustering demonstrated Notch1-dependent differences in gene expression. Uteri deprived of Notch1 signaling demonstrated decreased cellular proliferation; namely, reduced proliferation-specific antigen, Ki67, altered p21, cdk6, and cyclinD activity and an increased apoptotic-profile, cleaved caspase-3, Bad, and attenuated Bcl2. The results demonstrate that the preimplantation uterus relies on Notch signaling to inhibit apoptosis of stromal fibroblasts and regulate cell cycle progression, which together promotes successful decidualization. In summary, Notch1 signaling modulates multiple signaling mechanisms crucial for decidualization and these studies provide additional perspectives to the coordination of multiple signaling modalities required during decidualization.

Figure 1.

Notch1 protein expression profile during normal pregnancy. A, B) Notch1 protein levels are low in undifferentiated stromal cells during the preimplantation period, at d 1.5 (A) and 4.5 (B). C) However, with the initiation of implantation, a marked induction in Notch1 protein expression, which was predominantly in the nucleus, was observed in stromal cells of the primary decidual zone (C). D, E). As pregnancy progressed, Notch1 protein expression was decreased in the primary decidual zone (D), but the decidual cells in the secondary decidual zone continued to stain intensely (E). F) Negative control from d 6 of pregnancy that was treated with nonimmune IgG at the same concentration as the C-20 Notch1 antibody. Immunohistochemical images are at ×40.

Figure 2.

Decidualization defect with pharmacological inhibition of Notch1. A) Uterine section from wild-type (WT) vs. GSI-fed mice demonstrated an absence of Notch1 immunoreactivity for the cleaved Notch1-IC, using an antibody Notch1 Val1744, that specifically recognizes Notch1-IC. B) Gross morphology of the deciduoma in WT and GSI-fed mouse uteri, after decidual stimulus, shows a decidualization defect in the GSI mice. Right uterine horn was stimulated; left horn was maintained as an internal control. C) Uterine wet weight ratio of the decidual horn to the control horn demonstrated a statistically significant decrease in uterine weight in the absence of Notch1 *P < 0.035; n = 9.

Figure 3.

Loss of Notch1 expression in uterus of conditional-knockout mice, PR^Cre/+Notch1^flox/flox (Notch1^d/d). A) Mice carrying a Cre recombinase gene controlled by a PR promoter were crossed with mice carrying a floxed allele of the Notch1 signal peptide (exon1, Notch1^f/f). The resultant bigenic, Notch1^d/d, inactivates Notch1 in PR-positive tissues. B) Ovariectomized mice were subject to artificial decidualization. At d 5 following the decidual reaction, uteri were snap-frozen, and RNA was extracted and assessed by RT-PCR for Notch1. Notch1 (exon1) transcript is absent in Notch1^d/d uteri. C) Protein lysate was extracted from snap-frozen d-5 uterine horns immunobloted with Notch1-FL and Notch1-TM. Western blotting demonstrated attenuated and efficient protein reduction of Notch1 in Notch1^d/d uteri. Likewise, immunoblotting for Notch1-IC demonstrated decreased transcriptionally competent Notch1 in Notch1^d/d uteri. D) Immunohistochemical staining of Notch1 shows efficient protein ablation in uterine stromal cells from Notch1^d/d mouse uteri obtained at d 4 following the decidual stimulus (view: ×100).

Figure 4.

Decidualization defect in PR^Cre/+Notch1^flox/flox (Notch1^d/d) mice. A) Gross morphological evidence of the significant defect of a decidual response in Notch1d/d. In both groups, the left uterine horn was stimulated; the right horn was left untreated and served as an internal control. B) Quantification of uterine wet weight ratio of the decidual horn to the control horn in Notch1^d/d vs. Notch1^f/f uteri demonstrates a significant decrease in uterine weight in the absence of Notch1 that is apparent at 3 d after decidual reaction. **P < 0.01; ***P < 0.001.

Figure 5.

Diminished expression of decidualization-specific genes associated with Notch ablation Notch1-dependent decidualization defects parallel diminished transcription of two decidualization-specific genes, Bmp2 (A) and Wnt4 (B), in the Notch1^d/d uteri. Data represent mean values in tissues pooled from 3 animals/group.

Figure 6.

Heat maps and dendrograms. Hierarchical clustering was applied to the expression values of the cell growth- and apoptosis-related genes in each heat map. Both Notch1^d/d and Notch1^f/f were tested for biological enrichment using DAVID bioinformatics. The expression level of these genes was clustered (hierarchical clustering) across the 6 samples. A heat map for each group (and its subclusters) was obtained to show the variation in expression levels. Dendrograms at left mark the intercluster distances between the different clusters. Genes with similar expression patterns across samples cluster together.

Figure 7.

Reduced stromal cell proliferation in the absence of Notch1 signaling. A) Markedly attenuated stromal cell proliferation in Notch1^d/d mice, as evident by reduced immunohistochemical positivity to proliferation-specific antigen Ki67, at both d 0 and 4 following artificial decidualization. B) Schematic model of the G1/S transition complex. Real-time RT-PCR, presented as fold change, demonstrated down-regulation of cyclinD2, which correlates with the cessation of proliferation. Transcript levels of the regulator of cell cycle G1 progression, p21, the cyclin-dependent kinase inhibitor 1A, is diminished in Notch1^d/d uteri, implying a failure to undergo growth arrest and differentiate into the decidual phenotype. In addition, the transcript of the cyclin-dependent kinase, cdk6, is also reduced with Notch1 abrogation.

Figure 8.

Functional Notch1 is necessary to avert stromal apoptosis. A) Stromal fibroblasts undergo apoptosis, rather than decidualization, in the absence of Notch1 signaling after artificial decidualization, as demonstrated by Western blot for the executer of apoptosis, caspase-3. B) Quantification of the caspase-3-positive cells (arrowhead: positive caspase-3) from 3 independent experiments that confirm increased caspase-3 activity in Notch1^d/d mice. This finding implies that cells are undergoing apoptosis in the absence of Notch1 signaling. C) Bcl2 serves an antiapoptotic function. Bcl2 expression is markedly decreased in uteri from Notch1^d/d mice that are undergoing apoptosis. Bcl2 transcript levels are 2.4-fold (P=0.0023) lower in the Notch1^d/d uteri. D) In contrast, the proapoptotic gene, Bad, is 1.6-fold higher (P=0.0058) in Notch1^d/d uteri that are undergoing apoptosis.

Figure 9.

Working model: Notch1 regulates cell fate in endometrial stromal fibroblasts and is essential for successful decidualization and the aversion of apoptosis, which are critical for maintaining endometrial integrity for the successful establishment of pregnancy.