Localization of the Inhibitor of Differentiation 1-4 (Id1-4) Transcription Factors in the Human Corpus Luteum Across the Luteal Phase

The Inhibitor of Differentiation (Id) proteins are a subfamily of dimeric basic loop helix (bHLH) transcription factors. There are four mammalian isoforms (Id1-4). They act during embryogenesis and development to repress transcription of genes required for lineage commitment and promote cell growth. Their expression can be regulated by members of the transforming growth factor-β (TGFβ) superfamily, notably the bone morphogenic proteins (BMPs) and activin A. We have previously shown that the Ids are differentially expressed during follicle development in the sheep in vivo, and in ovine granulosa cells in vitro their expression was stimulated by BMP6 and inhibited by activin A. This suggested a role for Ids in the physiological regulation of mammalian ovarian structure and function. The human corpus luteum (CL) undergoes dynamic tissue remodeling across the luteal phase. Luteinization is associated with terminal differentiation of the granulosa-lutein cells, with arrest of cell division, and intense neovascularization involving marked endothelial cell proliferation. In addition, although the paracrine regulation of the human corpus luteum is not fully understood, it is likely that members of the TGFβ family have a major role. We therefore hypothesized that the Ids are important transcription factors involved in the tissue and vascular remodeling seen across the luteal phase. We aimed to A) determine if Id1-4 were expressed in the human CL; B) determine which cell types express Id1-4 in the human CL; C) assess if there was differential localization of Id-1-4 across the luteal phase. We studied human CL from normally cycling women collected at the time of hysterectomy for benign gynecological conditions, and dated on the basis of the urinary luteinizing hormone (LH) surge. We studied CL from the early (LH+1 to LH+5) (n=6), mid (LH+6 to LH+10) (n=6) and late (LH+11 to LH+14) (n=6) luteal phases. Id1, Id2, Id3 and Id4, and specific cellular markers, were localized in serial sections using immunohistochemistry. A) Id1, Id2, Id3 and Id4 transcription factors are expressed, at the protein level, in human CL. B) We could detect specific immunostaining for Id1-4 in multiple cell types including endothelial cells of capillaries within the steroidogenic cell layer, endothelial cells of larger vessels in the stromal cell layer, granulosa-lutein cells, theca-lutein cells and stromal cells. C) Within the endothelial cells of the steroidogenic cell layer Id immunostaining decreased as the luteal phase progressed (P<0.05) and this was most marked for Id3 and Id4. As the steroidogenic cells are terminally differentiated, and activin A seems to have a luteolytic role, we suspected that Id expression would decrease from the early to late luteal phase. However nuclear Id1-4 localization in granulosa-lutein cells and theca-lutein cells was maintained across the luteal phase. There was no reduction of immunostaining in the late-luteal phase despite likely increased local activin A activity at this time. Although Ids may have a role in luteal steroidogenic cells the lack of detectable alterations in immunolocalization across the luteal phase means that their function is not yet clear. However as endothelial Id expression was highest in the early luteal phase, at the time of marked neovascularization, Id proteins may have a role in the regulation of endothelial cell proliferation during luteal angiogenesis.Supported by The Cunningham Trust.