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Seventies'
In 1978, when we began our research on placental endocrinology in cows, knowledge of the endocrine function of the placenta was limited to placental lactogen (PL) hormones. The subject of my thesis for the habilitation of higher education degree in the University of Liège focused on the purification, characterization and development of the dosage of this placental protein in cattle (Beckers 1983). According to the literature, in humans, the concentration of hPL in maternal blood rises rapidly from the beginning of pregnancy while in the fetus, the concentration is always low, much lower than that found in the mother and this regardless of the stage of development. In sheep, the oPL concentration rises regularly in the mother while it decreases regularly in the fetus; around day 100 of gestation, the two concentrations cross.
Unpredictably, in bovine species, placental lactogen (bPL) is released in low concentration into the maternal circulation. The fetal concentration decreases steadily during fetal life but always remains higher than that of the mother (Beckers et al., 1982).
Eighties'
A few years later, when we were writing the discussion of the manuscript entitled “Trinucleate cells and ultrastructural localization of bovine placental lactogen” in which we described the migration of binucleated cells, then their crossing of the “feto-maternal” border and their fusion with epithelial cells of the endometrium, we noted that the majority of cells did not deliver their contents until after the end of migration. Furthermore, we did not find any forms of exocytosis as long as the cells were localized on the fetal side or were migrating. This did not correspond to the characteristics of the concentrations measured concerning placental lactogen in the fetus and in the mother and this indirectly suggested that the binucleated cells were capable of synthesizing and delivering into the maternal compartment other molecules still unknown at that time …
The PAS positive character of the binucleated and trinucleated cells and the suspicion of the existence of a chorionic gonadotropic hormone equivalent to hCG or eCG (PMSG) oriented our research on the purification of glycoproteins associated with gestation and secreted into the maternal bloodstream. The first steps of our research consisted of carrying out extraction and fractionation, followed by purification by affinity chromatography and finally by the production of antisera. The affinity purifications consisted essentially of removing the bovine albumin, adult and fetal hemoglobin, bPL and immunoglobulins from the solutions (due to the large quantity of blood that usually contaminates placental extracts). This approach, combined with radial immunodiffusion and immunoelectrophoresis, made it possible to highlight two placental proteins.
PAG-1 family
The first placental protein was found to be devoid of any hormonal activity (neither FSH, nor LH, nor PRL, nor GH, nor TSH, nor ACTH, etc.) and whose plasma concentration increased in the mother’s blood as the gestation progressed (Beckers et al. 1988a). We called this protein Pregnancy-Associated Glycoprotein (PAG) because its presence was also detected in testicular extracts and in ovarian extracts of ruminants (protein not strictly specific to pregnancy).
Bovine PAG was purified to homogeneity and characterized in our laboratory at the University of Liege. We were quickly able to develop a sensitive radioimmunoassay (RIA) that allowed us to detect and quantify the protein in maternal blood from the beginning of gestation. At that time, a collaboration was developed with the University of Missouri-Columbia in the USA, allowing us to isolate the cDNAs coding for bovine and ovine PAGs, to sequence them and to show their membership in the aspartic protease family. In this family, PAGs coexist with human, porcine and simian pepsinogens, with cathepsins D and E, chymosins and renin, etc.
PAG-2 family
The second identified placental protein have been shown to inhibit the binding of lutropin (LH) and follitropin (FSH) to specific membrane receptors. We considered this molecule as a bovine Chorionic Gonadotropin (bCG) and although it did not show any cross-reaction with PMSG or hCG, we considered it as an equivalent of these hormones in the cow. However, we were also surprised that the binding of this bCG to bovine and porcine anti-lutropin antisera was weak and inconsistent (Beckers et al., 1988b).
Surprisingly, this protein, initially designed as a bovine Chorionic Gonadotrophin, was later characterized as also belonging to the aspartic proteinase family. Indeed, the availability of semi-purified protein fractions and production of polyclonal antisera allowed the cloning of an additional cDNA from the placenta, which exhibited 58% homology with PAG-1. This outcome allowed us to designate this protein PAG-2 (Protein Associated with Pregnancy Class II), clarifying the question whether placental gonadotropic hormone exists or not in bovine species (Xie et al., 1994). Our results were presented to the Royal Academy of Belgium, whose presentation was entitled: “Molecules of the aspartic proteinase family in the placenta of ruminants: hormones or proteins?” (Beckers et al., 1994). Because of its structural similarity with aspartic proteases and important dissimilarities with pituitary gonadotropins (sequence, subunit structure), the term bovine chorionic gonadotropic hormone was no more adopted even if an LH activity is linked to this molecule.
Nineties and Noughties
Thanks to our expertise, we could successfully purify PAGs molecules in different ruminant species (sheep, goats, buffalo, bison, etc.). Preliminary studies in order to purify PAGs from porcine species were also carried out in our laboratory.
Development of different sensitive radioimmunoassays allowed PAG measurement and pregnancy diagnosis in different ruminant species. For instance, in sheep and goats, the development of specific and sensitive RIA allows an early pregnancy diagnosis (from the 3rd-4th week of gestation). In these species, however, the profiles appear different from those of cattle; the increase in concentrations at the beginning of gestation is much more pronounced.
In parallel, we carried out different studies in order to purify and develop radioimmunoassays for gastric aspartic proteinases (Prochymosin, Pepsinogens A and C) in porcine and bovine species. Despite they are secreted in mucosal cells from stomach, these zymogens are released into the peripheral circulation. Therefore, measuring their serum or plasma concentrations can help to diagnose lesions of the gastric mucosa (infestations by the larvae of different worms, displacement of abomasum, gastritis, ulcer, cancer). In cattle, it is expected that the measurement of the two forms of pepsinogens in defined environmental conditions can assist vets and breeders in management of anti-parasitic treatments.