טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHarduf Haggar
SubjectEmbryonic and Maternal Signaling Mechanisms in Endometrial
Cell Lines with High and Low Receptivity
(RL95-2 and HEC-1A)
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Eliezer Shalev
Full Thesis textFull thesis text - English Version


Abstract

Introduction:

Implantation in humans involves cross talk between an active blastocyst and receptive endometrium. The role of the endometrial receptors in this complex embryo-maternal interaction is still unclear.

Aims:

To determine gene and protein expression of endometrial receptors [Plexin-B1 (PB1), c-Met, Ron, Progesterone-receptor (PR) and Estrogen-receptor (ER)] in high and low receptive endometrial cells. Study the effect of the specific endometrial receptors in endometrial receptivity.

Methods:

Two endometrial cell lines were used: HEC-1A and RL95-2 considered as being of low and high receptivity, respectively, as well as primary endometrial cells. Western blot and RT-PCR analysis were utilized to study the receptor expression profile.

The role of endometrial receptors in endometrial receptivity was studied by attachment and invasion assays of JAR spheroids (made of trophoblast cell line) on endometrial cells. Different manipulations of inhibition and stimulation of the receptors were used.

Results:

Different protein expression patterns of endometrial receptors were observed between the tested endometrial cells. The expression levels of PB1, Ron, 50kDa c-Met isoform and the ratio PRA/PRB were significantly lower in HEC-1A as compared with RL95-2.

Attachment rates and growth of JAR spheroids into HEC-1A were significantly lower as compared with RL95-2. Inhibition of PB1 decreased attachment rates of JAR spheroids whereas it did not affect spheroid growth. Overexpression of PB1 in HEC-1A cells (HEC-1A-2) by transfection of PB1 gene increased attachment rates of spheroids. c-Met inhibition decreased attachment rates only to HEC-1A-2. Immunoprecipitation studies revealed that c-Met and PB1 associate in complexes. Inhibition of Ron did not affect attachment rates. Stimulation of PR with progesterone altered attachment rates to HEC-1A. Inhibition of PR with RU-486 mildly increased attachment rate to HEC-1A whereas it slightly decreased attachment rate to RL95-2.

Attachment rates of JAR spheroids to the primary endometrial cultures were significantly higher in cells obtained from window of implantation stage as compared with cells before window of implantation.

Conclusion:

Differential endometrial receptor profiles are expressed during the receptivity period.

The attachment and invasion processes are separately regulated and we suggest a biologically role for epithelial endometrial receptors in the attachment process. PB1 plays the most important role; PRA is most probably involved in endometrial receptivity and in the attachment process. c-Met contribution seems to be minor and related with creation of a complex with PB1.

We hypothesize that before cell attachment, an interaction between the endometrial receptors and the ligands of the blastocyst occurs. Consequently cell-cell attachment occurs.