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Abstract

Identification of Integrins Expressed on Apical Membranes of Endometrial and Trophectodermal Cells of Pigs during Early Pregnancy

Wendy Browning, Dana Massuto, Laurie JaegerWendy Browning Picture

Abstract

 

Objective – To determine which specific integrin heterodimers are present on the luminal surface of the endometrial mucosa and the apical surface of the trophectoderm (i.e., at the maternal-conceptus interface) in pigs during early pregnancy.  This is a subset of a larger project that will ultimately determine if the integrins that are found on the surface of the endometrium are actually bound by a particular extracellular matrix protein, the latency-associated peptide (LAP) of transforming growth factor beta, and are therefore part of the implantation and placentation process of the conceptus.

Animals or Sample population – Three pregnant crossbred gilts and a cultured porcine trophectoderm cells (pTr2) originally derived from porcine conceptuses at day 12 of gestation.

Procedure – Apical cell surfaces of uterine luminal epithelium and trophectoderm cells were labeled with biotin. Antibodies to the candidate integrin proteins, as well as control antibodies, were used to perform immunoprecipitation of candidate integrins.   Identification of the precipitated integrins was performed by electrophoresis, labeling with horseradish peroxidase linked to avidin (A-HRP), blotting on to nitrocellulose, and determination of relative molecular weights.  Western Blotting (WB) with integrin-specific antibodies will be used to more definitively identify the proteins. Identification of biotinylated integrins versus non-biotinylated integrins will determine whether or not specific integrins are present on cell surfaces at the maternal-conceptus interface and thus available for binding with the transforming growth factor’s (TGFβ’s) latency associated peptide (LAP) or other extracellular matrix proteins.  

Results – Fluorescence microscopy and blotting verified that cell surface molecules were biotinylated.   Immunoprecipitation of pTr2 cells yielded a band at the expected relative molecular weight of the integrin beta 1 subunit.  Immunoprecipitation of endometrial extracts resulted in excessive unexplained background signal in the region of the anticipated band(s); a higher molecular weight band, consistent with the size of an integrin beta 1 dimer, was present in the labeled endometrial extract immunoprecipitated with antibody to integrin beta 1 but not in that similarly precipitated with control immunoglobulins.   Immunofluorescence supported the presence of integrin beta on the endometrial luminal epithelium.

Conclusions and Clinical Relevance – Based on relative molecular weight, integrin beta 1 is expressed on apical membranes of trophectoderm cells. Immunoprecipitation suggests that integrin beta 1 is also present on the luminal surface of endometrial epithelium, but additional experimentation is needed to verify these results.  Further steps will need to be taken to adjust the general protocol for the immunoprecipitation on the endometrial extracts, but the success with the trophectoderm cells suggests that these cells may be able to serve as useful purpose when working up protocols for additional antibodies to assess the presences of other integrins in the trophectoderm and endometrium. 

Impact for Human Medicine - Many embryonic losses occur during early pregnancy in domestic animals species as well as humans.  Further mechanistic studies based on the data presented will be relevant to human infertility in addition to embryonic losses in domestic livestock.