Epithelial cell protein milk fat globule–epidermal growth factor 8 and human chorionic gonadotropin regulate stromal cell apoptosis in the human endometrium

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Presentation transcript:

Epithelial cell protein milk fat globule–epidermal growth factor 8 and human chorionic gonadotropin regulate stromal cell apoptosis in the human endometrium Ryan M. Riggs, M.D., Silvina Bocca, M.D., Ph.D., Sandra Anderson, B.S., Anahi Franchi, Ph.D., Bhaskara S. Rhavi, M.Sc., Sergio Oehninger, M.D., Ph.D. Fertility and Sterility Volume 98, Issue 6, Pages 1549-1556.e3 (December 2012) DOI: 10.1016/j.fertnstert.2012.07.1127 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Characterization of cultured EEC and ESC. Immunocytochemical analysis of human EEC (A) and ESC (B) using markers of epithelial (cytokeratin) and stromal (vimentin) phenotypes, confirming purity of cultures (original magnification, ×200). (C) Milk fat globule–E8 protein immunolocation (fluorescein isothiocyanate conjugate) in primary EEC and ESC in culture by immunofluorescence (MFG-E8 green, 6-diamino-2-phenylindole counterstain blue, and green and blue merged; original magnification, ×400; NC = negative control). The EEC showed strong immunoreactivity, whereas ESC (merged) were negative. (D) Immunolocation of hCG-R in ESC cells by immunofluorescence (MFG-E8 green, 6-diamino-2-phenylindole counterstain blue, and green and blue merged; original magnification, ×250; NC = negative control). Fertility and Sterility 2012 98, 1549-1556.e3DOI: (10.1016/j.fertnstert.2012.07.1127) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of recombinant MFG-E8 (2 μg/mL) on caspase activation in ESC (*P<.04 vs. controls without treatment). Positive controls: cells treated with staurosporin. Milk fat globule–E8 significantly increased caspase-3 activity in endometrial stromal cells compared with controls. Fertility and Sterility 2012 98, 1549-1556.e3DOI: (10.1016/j.fertnstert.2012.07.1127) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of recombinant MFG-E8 (2 μg/mL) on DNA fragmentation (TUNEL) in ESC (∗P<.0001 vs. controls without treatment). Milk fat globule–E8 significantly increased ESC DNA fragmentation in a dose-dependent manner. Merged photographs of PI (red fluorescence) and TUNEL (green fluorescence at ×200). (A) Positive control (DNAase). (B) Negative control (absence of enzyme). (C) Untreated cells. (D) Cells treated with MFG-E8; dose-dependent effect of MFG-E8 (P=.0001). Fertility and Sterility 2012 98, 1549-1556.e3DOI: (10.1016/j.fertnstert.2012.07.1127) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of hCG on gene expression of Bax and Bcl-2 in cultured cells. In EEC, hCG did not have any effect at the doses and time points examined (A: 6 hours of culture; B: 24 hours of culture). In ESC, hCG had no effect on Bcl-2 and did not affect Bax expression at 6 hours (C); on the other hand, Bax mRNA levels significantly decreased (*P<0.05) in ESC upon hCG treatment at the higher doses (50 and 500 mIU/mL) for the longest treatment duration (24 hours) (D). Fertility and Sterility 2012 98, 1549-1556.e3DOI: (10.1016/j.fertnstert.2012.07.1127) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions