Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

The pregnancy-associated glycoproteins (PAG) form a multigenic family of aspartic peptidases specifically expressed within the trophoblast of the ruminant placenta. In Bos taurus, this family is composed of 21 members segregated into 2 phylogenetic groups. The PAG are mainly produced by the cotyledons, which are discrete areas of massive interdigitations between the maternal endometrium and the foetal trophoblast. Cotyledons are separated by the flat cell layer of intercotyledonary trophoblast areas. According to former studies, modern PAG (PAG I) are produced by binucleate trophoblastic cells of the cotyledons, whereas ancient PAG (PAG II) are reported to be synthesized by both mononucleate and binucleate trophoblastic cells. Binucleate cells migrate into the maternal endometrium and to pour their intracellular content, allowing modern PAG as PAG1 to reach maternal blood circulation where they can be assayed as gestation diagnosis. Investigations about the PAG family are often restricted to a few molecules used for the diagnosis of gestation in blood circulation or in cotyledonary extracts. The aim of this study was to inform PAG I and PAG II expression during gestation in cotyledonary and intercotyledonary tissues. To build strong and innovative results in spite of the high identity sequence between PAG, we designed very specific tools such as RT-PCR primers for the transcripts analysis and antibodies for the proteins studies. Using real-time reverse-transcription PCR, we described the transcript expression of 16 of the 21 bovine PAG. Overall, they showed an increasing expression during gestation. However, we demonstrated a segregation of modern PAG in cotyledon and ancient PAG in the intercotyledonary trophoblast. Belonging to the ancient group, PAG2 was expressed in the cotyledon as a modern PAG. We raised specific antibodies against PAG1 (I), PAG11 (II), and PAG2 (II) that allowed to us confirm transcript data at the protein level using Western blot analysis. Three glycosylation variants of PAG11 were detected along with the gestation: a 45-kDa PAG11 is synthesized from 50 to 220 days of gestation, whereas a 78-kDa and a 70-kDa PAG11 were synthesized from 100 days of gestation. Immunolocalization described specific populations of binucleate cells producing PAG1 or PAG11 or PAG2. The PAG1 is mainly detected in binucleate cells of the whole cotyledon and in a lesser extent in the intercotyledonary trophoblast. The PAG11 was mainly localized in binucleate cells of the intercotyledonary trophoblast but also in a restricted population of binucleate cells of the cotyledon. The PAG2 was only synthesized in the villi of the cotyledon. To conclude, we demonstrated modern and ancient PAG have specific anatomical localization according to their phylogenetic origin. Ancient PAG are specific of nonvillous tissues, whereas modern PAG are mainly synthesized within the villous part of the cotyledon. The PAG2 seem to be an exception, belonging to the ancient group but expressed as a modern PAG and synthesized only in the cotyledon.