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

Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n = 22) or with KLH alone (n = 9), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.6 ± 2.2 of pregnancy. Those ewes immunized against PAGs (n = 7), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.9 ± 1.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n = 15), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1 ± 15.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.     

Influence of different isoforms of recombinant trophoblastic interferons on prostaglandin production in cultured bovine endometrial cells

In ruminants, interferon produced by the trophectoderm (IFN-tau) is recognized as the embryonic signal responsible for maternal recognition of pregnancy. IFN-tau is believed to act by down-regulating estrogen receptors, thus preventing appearance of oxytocin receptors responsible for the release of prostaglandin F(2alpha) (PGF(2alpha)) by the endometrium. The present study was undertaken to determine in vitro the biological activities of different IFN-tau isoforms and document putative alternate luteotrophic mechanisms. Endometrial cells in primary cultures were treated with five different rIFN-tau isoforms: two ovine isoforms (ro-4 and ro-11) and three bovine isoforms (rb-1a, rb-2b and rb-3b). Their effect was quantified by measurement of PGE(2) and PGF(2alpha) production by ELISA and induction of cyclooxygenase (COX-2) by Western and Northern analysis and correlated with antiviral activity previously reported. The overall pattern of response to the IFNs tested suggests that low concentrations (<1 microg/ml) reduced the production of both PGs and higher concentrations (>1 microg/ml) stimulated preferentially PGE(2); however, exceptions were noted. Isoform rb-2b with high antiviral activity inhibited PG production in both cell types at all concentrations tested. IFNs rb-1a and ro-11 had similar antiviral activities, inhibiting PG at low concentrations and stimulating them at high concentrations. Isoform rb-3b stands out relative to the other IFNs tested because it induced a variable non-dose-dependent effect on PG production and low antiviral activity. An increase in COX-2 protein expression and messenger was correlated with increased PG production. The results showing two distinct responses to IFN-tau depending on its concentration and/or isoform and the absence of correlation with antiviral activity suggest that complex transduction mechanisms are involved.     

A cloning and expression analysis of pregnancy-associated glycoproteins expressed in trophoblasts of the white-tail deer placenta

The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction.     

Evolution of the relaxin-like peptide family

Background  

The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.     

Seasonal changes in the response of winter wheat to elevated atmospheric CO2 concentration grown in Open-Top Chambers and field tracking enclosures

Winter wheat was grown at ambient and elevated (ambient plus 350 μL L^–1) CO₂ concentrations in open top chambers and in field-tracking sun-lit climatized enclosures (elevated is 718 μL L^–1). There was no significant effect of CO₂ concentration on sheath, leaf and root biomass and leaf area in the early spring (January to April). 24-h canopy CO₂ exchange rate (CCER) was not significantly affected either. However, elevated CO₂ concentration increased CCER at midday, decreased evapotranspiration rate and increased instantaneous water-use-efficiency during early spring. Leaf, sheath and root nitrogen concentration per unit dry weight decreased and nonstructural carbohydrate concentration increased under elevated CO₂, and N-uptake per unit ground area decreased significantly (– 22%) towards the end of this period. These results contrast with results from the final harvest, when grain yield and biomass were increased by 19% under elevated CO₂. N concentration per dry weight was reduced by 5%, but N-uptake per unit ground area was significantly higher (+ 11%) for the elevated CO₂ treatment. 24-h and midday-CCER increased significantly more in late spring (period of 21 April to 30 May) (respectively by + 40% and 53%) than in the early spring (respectively 5% and 19%) in response to elevated CO₂. Midday evapotranspiration rate was reduced less by elevated CO₂ in the late spring (– 13%) than in early spring (– 21%). The CO₂ response of midday and 24-h CCER decreased again (+ 27% and + 23% resp.) towards the end of the growing season. We conclude that the low response to CO₂ concentration during the early spring was associated with a growth-restriction, caused by low temperature and irradiance levels. The reduction of nitrogen concentration, the increase of nonstructural carbohydrate, and the lower evapotranspiration indicated that CO₂ did have an effect towards the end of early spring, but not on biomass accumulation. Regression analysis showed that both irradiance and temperature affected the response to CO₂.     

Ca2+, annexins, and GTP modulate exocytosis from maize root cap protoplasts

Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.     

Effects of climate change on productivity of cereals and legumes; model evaluation of observed year-to-year variability of the CO2 response

The effect of elevated [CO₂] on the productivity of spring wheat, winter wheat and faba bean was studied in experiments in climatized crop enclosures in the Wageningen Rhizolab in 1991–93. Simulation models for crop growth were used to explore possible causes for the observed differences in the CO₂ response. Measurements of the canopy gas exchange (CO₂ and water vapour) were made continuously from emergence until harvest. At an external [CO₂] of 700 μmol mol^?1 Maximum Canopy CO₂ Exchange Rate (CCERmax) at canopy closure was stimulated by 51% for spring wheat and by 71% for faba bean. At the end of the growing season, above ground biomass increase at 700 μmol mol^?1 was 58% (faba bean), 35% (spring wheat) and 19% (winter wheat) and the harvest index did not change. For model exploration, weather data sets for the period 1975-88 and 1991–93 were used, assuming adequate water supply and [CO₂] at 350 and 700 μmol mol^?1. For spring wheat the simulated responses (35–50%) were at the upper end of the experimental results. In agreement with experiments, simulations showed smaller responses for winter wheat and larger responses for faba bean. Further model explorations showed that this differential effect in the CO₂ response may not be primarily due to fundamental physiological differences between the crops, but may be at least partly due to differences in the daily air temperatures during comparable stages of growth of these crops. Simulations also showed that variations between years in CO₂ response can be largely explained by differences in weather conditions (especially temperature) between growing seasons.