Multiple Pregnancy-Associated Glycoproteins are Secreted by Day 100 Ovine Placental Tissue1

Abstract

Pregnancy-associated glycoprotein (PAG)-1 (PAG1) and pregnancy-specific protein B are either identical or closely related antigens released by trophoblast binucleate cells of placentas of cattle. Sheep and other ruminants produce similar products. There is evidence, however, that these antigens, which are related structurally to the pepsinogens and other aspartic proteinases, are not single gene products but members of an extensive family. Here, the sequential use of ammonium sulfate precipitation and Sepharose blue, anion-exchange, and cation-exchange chromatographies, as well as isoelectric elution from a Mono P column, has allowed several PAG1-related molecules to be purified from the medium after culture of explants from Day 100 sheep placentas. Each of these PAGs cross-reacted to a varying extent with a panel of three different anti-PAG1 antisera. Four of them, all of which were major secretory products of the placenta, were subjected to amino-terminal microsequencing. Although each was related to ovine (ov) PAG1, none was identical. Reverse transcription-polymerase chain reaction was then used to amplify PAG1-related cDNA from Day 100 placental RNA. Seven novel full-length cDNA, all distinct from ovPAG1, were identified from 25 cDNA selected for sequencing. Only two of these (ovPAG3 and ovPAG7) encoded polypeptides identical in sequence at their inferred amino termini to one of the PAGs (ovPAG65) purified from explant cultures. Even so, they were only 84% identical in overall sequence. The remaining five cDNA were unique. In situ hybridization analysis revealed that expression of ovPAG3 and ovPAG7, like that of ovPAG1, is confined to trophoblast binucleate cells. The data confirm that at Day 100 of pregnancy the ovine placenta produces many different PAGs, which differ considerably in sequence and immunological cross-reactivity.

Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation

“…This highlighted at an early date the surprising size and diversity of this glycoprotein family in sheep. Xie et al [9] confirmed this diversity when, using a series of chromatographic procedures, they purified four additional molecules from the medium after culture of explants from day-100 ovine placenta. These molecules were named according to their molecular masses: ovPAG 55 , ovPAG 60 , ovPAG 61 , and ovPAG 65 .…”

“…The stage of pregnancy was assessed by measuring the crown-rump length of the foetuses [15]. As in previous works, the placentae were collected after day 100 [9,10]; in this study the period was focused on days 60 to 100. Total protein (TP) concentrations of all fractions were determined by the Lowry method [16].…”

“…Secondly, PAG fall into two groups: PAG expressed predominantly in binucleate cells (ovPAG-1, -3, -4, -5, -6, -7, -8 and -9) and a PAG (ovPAG-2) whose mRNA is detected throughout the trophectoderm [13]. So far, only one molecule (ovPAG 65 ) of the nine identified cDNA has been successfully column-purified [9].…”

Evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep

“…Bovine and ovine cDNA encoding PAG-1 have 86% similarity in nucleotide sequences and results in encoded proteins which differ in only 2 amino acids (XIE et al, 1991). There are numerous isoforms of PAGs produced from ovine placenta (e.g., ovPAG-55, 57, 59, 60, 61 and 65), which were identified at random stages of pregnancy and used to develop PAGs-based assays with placental antigens from ewes (XIE et al, 1997;EL AMIRI et al, 2003;EL AMIRI et al, 2004). These studies involving sheep PAGs were based on radioimmunoassay (RIA), a radioactive technique (DE SOUSA et al, 2003;KAREN et al, 2003).…”

Autoimmunization of ewes against pregnancy-associated glycoproteins does not interfere with the establishment and maintenance of pregnancy

“…This feature of the modern/ BNC PAGs has been exploited as the basis for pregnancy diagnosis in cattle and related ruminants. Not surprisingly, the modern PAGs have received the bulk of attention from animal scientists and have been the subject of several biochemical, molecular and functional studies (Del Vecchio et al, 1996;Xie et al, 1997;Green et al, 2000;Hughes et al, 2003;Klisch and Leiser, 2003;Weems et al, 2003;Wooding et al, 2005;Klisch et al, 2008). However, despite such efforts, the functional role of the PAGs remains a mystery.…”