Identification of the pregnancy-associated glycoprotein family (PAGs) and some aspects of placenta development in the European moose (Alces alces L.)

Abstract

This study describes the identification and a broad-based characterization of the pregnancy-associated glycoprotein (PAG) genes expressed in the synepitheliochorial placenta of the Alces alces (Aa; N = 51). We used: (1) both size measurements (cm) of various Aa embryos/fetuses (crown-rump length) and placentomes (PLCs); (2) PCR, Southern and sequencing; (3) Western-blot for total placental glycoproteins; (4) deglycosylation of total cotyledonary proteins; and (5) double heterologous IHC for cellular immune-localization of the PAGs as pregnancy advanced (50–200 days post coitum). The crown-rump length and PLC size measurements permitted a novel pattern estimation of various pregnancy stages in wild Aa. The PLC number varied (5–21) and was the greatest at the mid and late stages of gestation in females bearing singletons or twins. The genomic existence of the identified PAG-like family was named AaPAG-L. Amplicon profiles of the AaPAG-L varied in the number and length (118–2000 bp). Southern with porcine cDNA probes confirmed specificity and revealed dominant AaPAG-L amplicons in males and females. Nucleotide sequences of the AaPAG-L amplicons shared 86.27% homology with the bovine PAG1 (bPAG1) gene. Amino acid AaPAG sequences revealed in silico 88.23% to 100% homology with the bPAG1 precursor. Western-blots revealed a dominant mature 55 kDa AaPAG fraction, and the major ∼48 kDa glycosylated form that was deglycosylated to ∼44 kDa. The AaPAG-Ls was immuno-localized to mono- and bi-nucleated trophectodermal cells (TRD–chorionic epithelium), where signal intensity resembled intense TRD proliferation within developing PLCs as pregnancy advanced. This is the first study identifying the AaPAG-L family in the largest representative among the Cervidae.