Transcription factors 3 and 12 regulate non-erythroid uterine hemoglobin biosynthesis and decidualization in mice and humans

Andrea Rochelle Smith

Impaired fecundity affects 7.3 million women in the United States alone, with roughly 75% of pregnancy failures occurring prior to the establishment of the placenta. This corresponds to the time in which the embryo relies heavily on the maternal deciduum for oxygen, nutrients, and endocrine regulation. We previously established that conditional ablation of helix-loop-helix proteins transcription factor (TCF) 3 and TCF12 from the uterus results in complete infertility owing to a post-implantation uterine defect. RNA sequencing analysis of Tcf3/12fl/fl and Tcf3/12d/d uteri revealed differential expression of about 800 genes. Among the down-regulated genes were those associated with hemoglobin biosynthesis. Based on the ablation and RNA sequencing studies, it was hypothesized that TCF3 and/or TCF12 play an essential role in decidualization and in converting the uterus to a non-erythroid hemoglobin biosynthetic organ. Following conditional ablation Tcf3/12 from murine uterus, quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) were used to confirm down-regulation of hemoglobin-associates and characterization of their expression in response to steroid hormones and early gestation. Conditional ablation of Tcf3/12 resulted in a significant decrease in the expression of many of the highly expressed hemoglobin-associated genes at both the mRNA and the protein levels. TCF3 targeting siRNA, RT-qPCR, and IHC were used to determine if the findings observed in mice translate to the human endometrium. In human endometrium, TCF3, HBB, and ALAS2 showed similar expression patterns with mostly weaker stromal staining and limited epithelial staining during the proliferative phase followed by an increase in overall expression in the functionalis layer during the secretory phase. Primary human endometrial stromal cells were found to express TCF3, HBB, ALAS2, among others. siRNA knockdown of TCF3 resulted in decreased expression of several hemoglobin-associated genes, demonstrating its functional role in uterine hemoglobin biosynthesis. Furthermore, TCF3 knockdown resulted in a blunted decidual response. In conclusion, TCF3 regulates endometrial decidualization and uterine hemoglobin biosynthesis in both humans and mice, demonstrating that the uterus functions as a non-erythroid hemoglobin biosynthetic organ in response to TCF3 transcriptional activity. These studies could provide new therapeutic avenues for women who experience impaired fecundity, particularly in an in vitro fertilization setting.

Transcription factors 3 and 12 regulate non-erythroid uterine hemoglobin biosynthesis and decidualization in mice and humans

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