Noelita Melo de Sousa

The glycosylation of pregnancy-associated glycoproteins and prolactin-related protein-I in bovine binucleate trophoblast giant cells changes before parturition.

Binucleate trophoblast giant cells (BNC) in the bovine placenta produce glycoproteins, which are delivered into the mother after fusion of BNC with uterine epithelial cells. During most time of pregnancy, BNC produce pregnancy-associated glycoproteins (PAGs) and prolactin-related protein-I (PRP-I) with asparagine-linked lactosamine-type glycans terminating with N-acetyl-galactosamine. We show by lectin histochemistry that terminal N-acetyl-galactosamine (detected by Dolichos biflorus agglutinin, DBA) in placentomal BNC is greatly reduced prior to parturition, while lactosamine-type N-glycans (detected by Phaseolus vulgaris leucoagglutinin, PHA-L) remain unaltered. The change in DBA-staining showed no statistically significant differences between placentomes of cows with and without retention of fetal membranes. Western blots revealed that, at parturition the apparent molecular mass of PAGs and PRP-I is 1-2 kDa lower than in late pregnancy. These changes are due to alterations of asparagine-linked glycans, since the molecular weight of the peptide backbones after enzymatical release of asparagine-linked glycans is identical at late pregnancy and parturition. Lectin western blots showed a reduction of terminal N-acetyl-galactosamine on PAGs at parturition. A lectin sandwich-ELISAwas used to differentiate DBA- and PHA-L-binding PAGs in sera of pregnant and non-pregnant cows. The values for DBA-binding PAGs at parturition were not significantly different from non-pregnancy, while the values for PHA-L-binding PAGs were significantly higher at parturition. The peripartal changes of PAG- and PRP-I-glycosylation could alter functional properties of these proteins and might therefore be considered for functional studies. The differentiation of PAG glycoforms in maternal serum could be valuable for a further optimization of PAG-based pregnancy diagnosis in cattle.

Pregnancy-associated glycoprotein concentrations during pregnancy and the postpartum period in Azawak Zebu cattle

Specific RIA systems were developed and used to measure pregnancy-associated glycoprotein (PAG) concentrations during gestation and the postpartum period in Azawak Zebu cows. Twelve females were palpated per rectum and diagnosed as pregnant. Blood samples were taken at 5-10-day intervals from approximately Week 8 of gestation until Week 10 postpartum (pp). One Zebu cow (Z15) initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow (ZSand) showed abnormally high PAG concentrations during gestation and was excluded from the general PAG profile. The 10 other Zebu cows exhibited a very similar PAG profile. In these animals, concentrations increased progressively from Week 8 to 35 of gestation (from 6.0+/-4.2 to 196.0+/-34.8 ng/ml), remaining relatively constant until Week 39 (210.8+/-74.8 ng/ml), when they increased sharply to reach their highest level (1095.6+/-607.2 ng/ml) at around parturition. After delivery, PAG concentrations declined significantly (P<0.05) until Week 2 postpartum (348.4+/-85.6 ng/ml) and slowly until Week 10 postpartum. Our results revealed that the PAG pattern in Zebu cattle was similar to those of taurine breeds during the first two trimesters of pregnancy, but differed in the peripartum period.

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle.

The present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) and bovine pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) for diagnosing pregnancy at Day 28 after insemination in dairy cows. Transrectal ultrasonography (TRUS) was performed in 100 Holstein-Friesian cows at Day 28 after artificial insemination (AI; Day 0) to diagnose pregnancy. After TRUS examination, blood sample was collected from the coccygeal vessels of each cow to measure the concentrations of bPAGs by PAG-RIA test and Bovine Preg Test 29. Milk samples were collected at Days 0, 21 and 28 for measurement of progesterone (P4) by ELISA test. The cows were re-examined by TRUS at Day 42 to confirm the pregnancy diagnoses. The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAG-RIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42. Out of 100 inseminated cows, 41 were confirmed pregnant at Day 28 after AI. Based on the actual gold standard, the sensitivity of TRUS, PAG-ELISA and PAG-RIA tests for diagnosing pregnant cows at Day 28 were 92.7%, 90.2% and 100%, while the specificity of the three tests for diagnosing non-pregnant cows were 91.5%, 98.3% and 94.4%, respectively. The overall accuracy of the three tests were 92%, 95% and 97%, respectively. The degree of agreement (Kappa±S.E.) between PAG-RIA and PAG-ELISA test was 0.90 ±0.04. The degrees of agreement between PAG-RIA and PAG-ELISA and TRUS at Day 28 were 0.80±0.05 and 0.76±0.06, respectively. In conclusion, the commercial PAG-ELISA test is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test.

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes.

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.

Correlation of five radioimmunoassay systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancy period

The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them ( > or = 0.81; P<0.001). PAG concentrations increased significantly from Day 21 (n=27) to 30 (n=37) after AI by use of all PAG-RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B(0) binding to the tracer. Our results suggest that all tested PAG-RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.

Pregnancy-associated Glycoprotein Profile during the First Trimester of Pregnancy in Egyptian Buffalo Cows

Pregnancy-associated glycoprotein (PAG) concentrations were measured in buffalo cows starting from day 28 after breeding. Oestrus was synchronized in 10 buffaloes using two injections of 25 mg prostraglandin (PG)F(2alpha) (Lutalyse) at a 11-day interval. Blood sampling was conducted nearly twice weekly. Results indicated that plasma PAG concentrations in non-pregnant buffaloes were low (<0.20 ng/ml) during the whole experimental period (day 28 to 103), while in pregnant animals plasma PAG levels increased from day 28 (4.48 +/- 0.92 ng/ml) until day 41 (27.27 +/- 6.74 ng/ml), remaining high (20.71 +/- 9.20 ng/ml) until day 103. Progesterone levels were significantly (p < 0.0001) higher in pregnant (3.51-4.80 ng/ml) than in non-pregnant buffaloes (0.28-1.52 ng/ml). A significant difference (p < 0.0001) in plasma PAG concentrations between pregnant and non-pregnant animals starting at day 28 after breeding suggests that PAG-radioimmunoassay could be suitable for pregnancy diagnosis in buffaloes during this period. In conclusion, PAG test offers the advantages that it requires a single plasma sample for early pregnancy diagnosis as well as the accuracy of the test for the detection of pregnancy as early as day 28.

Double radial immunodiffusion as a tool to identify pregnancy-associated glycoproteins in ruminant and nonruminant placentae.

Pregnancy-associated glycoproteins (PAGs) are antigens synthesized in the superficial layers of the ruminant trophoblast. Initially, they were identified either as proteins released into the maternal bloodstream (where they have applications in pregnancy diagnosis) (PAG1) or as molecules binding to the LH receptor (PAG2). In this study, double radial immunodiffusion was used to test the ability of antisera raised against different PAG molecules (bovine, ovine and caprine) to react with placental extracts from nonruminants (rabbit, cat, mouse, pig, and wild pig) and ruminants (cow, ewe, and goat). Placental extracts from all nonruminants tested except rabbit reacted with anti bovine PAG2 (anti-boPAG2). Extracts of ruminant placentas reacted with different antisera, confirming the expression of various PAG molecules. According to the time at which the placentas were collected (early or middle pregnancy), the reaction differed as regards the thickness, position, and number of precipitation lines, suggesting that PAG expression varies as pregnancy progresses. Bos indicus and Bos taurus placental extracts exhibited different reactions with anti-boPAG2: a single precipitation line in the former case and two lines in the latter. This suggests differential expression of boPAG2 related glycoproteins in these two subspecies.

Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

BackgroundThis paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing.ResultsFour distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues.ConclusionOur results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family.

Native and recombinant bovine placental lactogens.

The bovine placenta produces a wide variety of proteins that are structurally and functionally similar to the pituitary proteins from the GH/PRL gene family. Bovine placental lactogen (bPL) is a 200-amino acid long glycoprotein hormone that exhibits both lactogenic and somatogenic properties. The apparent molecular masses of purified native (n) bPL molecules (31-33 kDa) exceed 23 041 Da, which is the theoretical molecular mass of the protein core. At least six isoelectric variants (pI: 4.85-6.3) of bPL were described in cotyledonary extracts and three different bPL isoforms (pI: 4.85-5.25) were found in fetal sera. The bPL molecules that are detected in higher concentrations in peripheral circulation exhibit a more acidic pI than those present in placental homogenates. This may reflect an important glycosylation process occurring just prior to the bPL secretion. The bPL mRNA is transcribed in trophectoderm binucleate cells starting from Day 30 of pregnancy until the end of gestation. In mothers, bPL is involved in the regulation of ovarian function, mammogenesis, lactogenesis, and pregnancy stage-dependent adaptation of nutrient supplies to the fetus. Due to the higher fetal, compared to maternal concentrations of circulating hormone, it has been suggested that bPL primarily targets fetal tissues.