Radiocompetition of secretory pregnancy-associated glycoproteins as chorionic ligands with luteal and uterine gonadotrophin receptors of pregnant pigs

Porcine pregnancy-associated glycoprotein (pPAG) family is very promiscuous and its role(s) remains unknown. The objective of this study was to identify whether secretory placental proteins (including pPAGs), produced in vitro by porcine chorionic explants, may interact with other proteins/targets, i.e. luteal and uterine binding sites of pregnant pigs. Trophoblast (TRF) and trophectoderm (TRD) were harvested during peri-implantation and placentation periods (14-61 dpc-day post coitum). In vitro-produced TRF/TRD proteins were isolated from media by ultrafractionation (>10 kDa MWCO) or precipitation with 20-75% saturation of (NH4)2SO4 and pPAG proteins were monitored by Western blotting. Secretory TRF/TRD ligands (including PAGs) were serially diluted (0.78-25 μg/ligand) and examined by radioreceptor assay (RRA). Luteal and uterine membrane receptors of pregnant pigs (pRc) were isolated from corpora lutea (pCLRc), myometrium (pMYORc) and endometrium (pENDRc). The three pRc types were harvested during three periods of pregnancy: 14 dpc (14Rc), 21-26 dpc (21-26Rc) and 31 dpc (31Rc). The RRA competitions of individual TRF or TRD ligands were performed with 125I-hCG as tracer and different pRc types. The RRA results of TRF/TRD were compared to hCG/pLH ligands - as positive controls (0.39-50 ng/ml), and endometrial (END) proteins (0.78-25 μg/ml) produced in vitro by END explants of cyclic, pseudopregnant and pregnant gilts (cEND, PsEND and pEND, respectively) - as negative control ligands. Results indicated that secretory TRF/TRD proteins (+pPAGs) were able to compete with 125I-hCG for binding with other proteins/targets, i.e. luteal and uterine receptors of pregnant pigs (pCLRc, pMYORc and pENDRc) in a concentration- and pregnancy stage-dependent manner. This study indicated that porcine secretory 14-15 dpc TRF (pPAG; 30-73 kDa) ligands, effectively displaced 125I-hCG tracer from pCL14Rc (up to P ≤ 0.01), corresponding to displacement by hCG and porcine LH. During the early stage of pregnancy, some competition tendency (P ≤ 0.01) was also detected for TRF ligands (14-15 dpc) with pEND14Rc. As pregnancy advanced, significant 125I-hCG competition (at least P ≤ 0.05) with secretory semi-purified TRD ligands (30-42 dpc) was determined for all types of examined receptors pCL31Rc, pMYO31Rc and pEND31Rc, mainly with TRD fractions precipitated by 20% saturation of (NH4)2SO4. It seems that chorionic pPAG family can be involved in luteoprotective mechanism during implantation and placentation, according to the binding-interaction with luteal and uterine gonadotropin receptors of pregnant pigs.