37 PREGNANCY-ASSOCIATED GLYCOPROTEIN (PAG) PROFILES DURING THE PERI-IMPLANTATION PERIOD IN RECIPIENTS CARRYING BOVINE SOMATIC CLONES: PRELIMINARY RESULTS

Abstract

Bovine pregnancy-associated glycoproteins (bPAGs) are secreted by binucleate cells of the placenta and can be assayed in maternal plasma as indicators of pregnancy and markers for the functional status of the trophoblast. The concentrations of PAGs vary differentially during the peri-implantation period. Large offspring syndrome (LOS) and abnormal placentation have been associated in cloned fetuses with abnormally increased pregnancy-specific protein 60 (PSP60). The aim of this study was to investigate the evolution of plasma levels of PAGs measured by the use of three different RIA systems in heifers after transfer of somatic cloned embryos and to compare that to plasma levels of control recipients during early pregnancy to distinguish which ones vary differentially. Cloned embryos were derived from nuclear transfer from fibroblasts of an adult cell line to in vitro-matured bovine oocytes according to Vignon et al. (1998 Theriogenology 49, 392). Control embryos were obtained in vitro after IVF and cultured in serum-free medium up to the blastocyst stage. By Day 7, cloned (n = 29) and control embryos (n = 10) were transferred to synchronous recipient heifers of the Normande breed (one embryo per recipient). Blood samples were taken every 2–3 days between Days 25 and 50 of pregnancy from recipients that had a positive progesterone test on Day 21, and the plasma was stored frozen until assay. Pregnancy status was monitored by repeated ultrasound scanning from Day 35 to Day 90. Concentrations of PAGs were determined by validated RIA assays for the different forms (PAGI67, PAG55+62, PAG55+59) using 3 antisera (AS 497, AS 706, AS 708, respectively) (Perenyi et al. 2002 Reprod. Domest. Anim. 37, 100–104). profiles of only the recipients that were confirmed pregnant by scanning over Day 50 (n = 18 clones, n = 4 controls) were analyzed and compared. Concentrations of PAGs measured by AS 497 and AS 706 were significantly higher in clones than in control recipients during the second month of pregnancy, indicating that the placenta of clones secreted these forms in a different manner compared to controls. Moreover, we found that concentrations of PAGs as determined with the two same antisera were higher for recipients with a cloned fetus that developed to term compared to those which had fetal loss before 3 months. For instance, using the AS 706, respective values of PAGs at Day 45 were 17.64 ± 7.59 and 10.96 ± 6.38 ng/mL for pregnancies with cloned and control fetuses, respectively, (P < 0.05) and 21.29 ± 4.77 and 12.88 ± 8.90 ng/mL in pregnancies with cloned fetuses that developed normally to term or died before 3 months of age, respectively, (P < 0.05). We conclude that our assays using two antisera could be a predictive test for fetal loss in clone pregnancies. Until now, recipients carrying cloned embryos that develop LOS during late pregnancy could not be detected by conventional assays in maternal serum or by scanning during the period of 35–50 days. This study provides a new diagnostic tool to detect them. Investigations are in progress to check the localization of these different forms of PAG in placentomes removed from recipients carrying somatic clones.