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ANSC 555

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by

Kelly Tippmann

on 28 April 2010

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Transcript of ANSC 555

lkjlkjlkjlj kjljjj DADF JLKJ KJ Important Factors for Implantation
and Embryo Survival Gap Junction Communication De Novo Estrogen Synthesis Implantation Decidualization Embryo invades stromal compartment
Stromal cells differentiate into decidua human and mouse Induced by progesterone and other factors
Decidua forms implantation chamber Linked with stromal differentiation in rodents
Steroid pathway is unknown Uterine Angiogenesis Used delayed implantation mouse
Ovariectomized pregnant mice
Pregnancy maintained with Progesterone
Implantation induced by Estrogen injection
Microarray and immunohistochemical analysis
shows mouse had increased Connexin 43 gene expression Cx43 is a gap junction protein Cx43 expression pattern followed
the progression of decidualization Examine loss of Cx43 function Cre-LoxP strategy Progesterone Receptor promoter-Cre Mouse 1 Mouse 2 Floxed Cx43 gene X Progeny Have Cx43 gene deleted
in uterine cells expressing PR Cx43 null mice had fertility defects Reduction in litter size
At day 8 of pregnancy showed embryo loss
At day 8 showed abnormally small embryos
Reduced Pecam staining
Reduced Ki67 staining
Pecam is a marker for endothelial cells. Loss of Cx43 expression=Reduction in endothelial cell proliferation Ki57 is a marker for cell proliferation How does loss of Cx43 affect decidualization? Examine decidualization markers:
Prolactin-related protein (PRP)
Prolactin-like protein (PLP) Decidualization Impaired Does Cx43 control decidualization independently of embryonic development? Artificially decidualize mice Cx43 null mice have reduced response reduced uterine weight
reduced BMP2 and Hoxa10
still have reduced Pecam staining Conceptus has no effect Examine Vegf expression Paracrine factor needed for angiogenesis Vegf expression reduced to only a few layers around embryo Angiogenic factors also reduced Relation to Human Decidualization? Use strand of human endometrial stromal cells Use siRNA to target Cx43 RNA Reduced # of CX43 mRNA
Reduced gap junctions
No decidualization
VEGF expression decreased Gap Junctions important for VEGF production
and decidualization Summary Gap Junction communciation is important for implantation Cx43 is regulated by estrogen
Cx43 expression is associated with decidualization
Loss of Cx43 expression results in reduction of angiogenic factors (Vegf, angiopoietins 2 and 4)
Estrogen plays essential role in blastocyst attachment to uterine lining Implantation does not occur with absence of ovarian E Decidualization can occur
without ovarian E Mice ovariectomized on day 5
Implantation occurs on day 4 of pregnancy
Treated with Progesterone Ovarian E not needed for maintenance of Decidualization The decidual uterus produces E Monitor expression of steroid biosynthetic enzymes in early pregnancy uterus
Laser capture decidual cells and measure aromatase mRNA expression
Immunohistochemical analysis to detect aromatase expression Decidualization Expression of P450 is increased at decidualization Aromatase expression increases during decidualization Day 4 before attachment Day 5 Day 6 Stromal cells in culture subjected to steroid-induced decidualization Uterine aromatase activity needed for implantation Mouse ovariectomized on d 5
Daily injections of P
Treated with or without letrozole (aromatase inhibitor) AI administration reduces decidual mass and causes embryo resorption. Blocked aromatase activity blocks stromal differentiation Local E synthesis important for uterine angiogenesis Summary Artificially induce decidualization
Treat with P+letrozole (AI)
Or P without AI
Assess decidualization response Uterine weight decreased Reduced expression of stromal differentiation markers Artificially induce decidualization
Treat with P+AI or just P
Immunostain stromal compartment for PECAM, and endothelial-cell specific marker
Perform gene expression analysis of uterine tissue for angiogenic factors

PECAM staining reduced in AI treated animals Downregulation of factors involved in angiogenesis Uterine produced E is important for decidualization
This promotes uterine angiogenesis
Questions?
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