(157) Role of Iroquois-class Homeodomain Protein IRX-3 in the Uterus during Early Pregnancy

Adarsh Ram¹, Athilakshmi Kannan¹, Alaina Houghton-Chamberlain³, Milan K. Bagchi², Joan S. Jorgensen³, and Indrani C. Bagchi¹

1.Departments of Comparative Biosciences,  University of Illinois at Urbana-Champaign, Urbana, IL
2.Mol & Integrative Physiology,  University of Illinois at Urbana-Champaign, Urbana, IL
3.Department of Comparative Biosciences³, University of Wisconsin-Madison, Madison, WI

 


Abstract Text:

Role of Iroquois-class homeodomain protein IRX-3 in the uterus during early pregnancy

Adarsh Ram¹, Athilakshmi Kannan¹, Alaina Houghton-Chamberlain³, Milan K. Bagchi², Joan S. Jorgensen³, and Indrani C. Bagchi¹

Departments of Comparative Biosciences¹, Mol & Integrative Physiology², University of Illinois at Urbana-Champaign, Urbana, IL, Department of Comparative Biosciences³, University of Wisconsin-Madison, Madison, WI

Two critical factors contributing to female infertility are the premature depletion of ovarian reserve and early implantation or placentation failure. Canonical Wnt/β-catenin signaling has been shown to directly activate the transcription of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, which are essential for oocyte and follicle survival during ovarian development. In female mice, Irx3 ablation leads to follicular atresia due to disorganized gap junction assembly and compromised oocyte-granulosa cell interactions. Interestingly, recent studies indicate that Irx3 is induced in the uterus during early pregnancy as endometrial stromal cells differentiate into decidual cells in a process known as decidualization. We and others have previously reported that BMP2, a morphogen belonging to the TGFβ superfamily, and its downstream target Wnt4 play a crucial role in regulating uterine decidualization during early pregnancy. In a primary culture system where undifferentiated stromal cells isolated from pregnant mouse uteri undergo decidualization in vitro, we discovered that Irx3 transcripts were significantly upregulated within 12 hours after Wnt4 was induced in response to exogenous BMP2. Consistent with this observation, we found that siRNA-mediated silencing of β-catenin expression in these cells resulted in a dramatic reduction in the expression of IRX3 protein, indicating that it functions downstream of the Wnt4/β-catenin signaling pathway in the uterus during decidualization. Although IRX3 functions as a transcription factor, a significant portion of this protein is intriguingly localized in the cytoplasm of endometrial stromal cells, with punctate dots organized in a donut-shaped appearance, suggesting IRX3’s presence on the surface of cytoplasmic vesicles. Further studies using proximity ligase assays indicated the interaction of IRX3 with Septin 7 (SEPT7), a GTP-binding protein that plays a crucial role in various cellular processes, including vesicular trafficking. To investigate the function of Irx3 in the uterus, we next conditionally deleted the Irx3 gene in the uterine stromal cells of adult mice by using progesterone receptor (PR)-driven Cre. A six-month breeding study was performed by crossing mice harboring the “floxed” Irx3 (Irx3^f/f) or Irx3-ablated (Irx3^d/d) females with wild-type males of proven fertility. This breeding scheme was employed to ensure that the Irx3 gene locus is intact in the embryos implanting in Irx3-deficient uteri. In this study, we noted more than a 50% reduction in the total number of pups born to Irx3^d/d dams compared with control Irx3^f/f females. These results indicated a severe fertility defect due to the lack of IRX3 expression in uterine stromal cells of Irx3^d/d females. We next investigated whether the subfertility of Irx3^d/d females was due to a defect in embryo implantation. Our results showed that the ablation of Irx3 expression in stromal cells did not impact the attachment of the embryo to the uterine epithelium. However, it led to impaired decidualization, angiogenesis, and trophoblast differentiation, resulting in a significant decline in fertility. Future studies will investigate how IRX3 operates in the uterus to coordinate cell-cell interactions that are essential for the success of early pregnancy.

Supported by NIH R01 HD107846 (ICB, JSJ)