Influence of pathogenic bacteria species present in the postpartum bovine uterus on proteome profiles.

In the first 2-3 weeks after parturition >90% of dairy cows will have some form of uterine infection. Uterine contamination with pathogens, such as Trueperella (formerly Arcanobacterium) pyogenes increases the risk of developing more severe endometritis, which can reduce conception rates. In this study, we compared the uterine proteome of cows infected with Trueperella pyogenes with that of uninfected cows, using 2D gel electrophoresis, and identified annexins A1 and A2 (ANXA1 and ANXA2), apolipoprotein A-1, calprotectin (S100A9), cathelicidin, enolase 1 (ENO1), peptidoglycan recognition protein 1 (PGLYRP1), phosphoglycerate mutase 1 (PGAM1), serine dehydratase (SDS) and serine protease inhibitors (SERPIN) B1, B3 and B4 proteins as differing in abundance in endometritis. Subsequently, levels of ten of these proteins were monitored in uterine samples collected from a herd of lactating, dairy cows at 15 and 42 days post-partum (DPP). The levels were compared with the cytology scores of the samples and the bacterial species isolated from the uterus. Cathelicidin, PGLYRP1, SERPINB1 and S100A9 levels at 15DPP showed strong positive correlations (r=0.78, 0.80, 0.79, and 0.68 respectively; P<0.001) with % of polymorphonuclear neutrophils (PMN). When compared with other bacterial pathogens identified, Streptococcus agalactiae and Truperella pyogenes induced increased expression of the indicator proteins, suggesting that these organisms may adversely affect the subsequent ability of the cow to conceive. Interestingly, there was no difference in the proportion of cows pregnant at 6 and 17 weeks after start of mating between the cows with high or low %PMN.

Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer.

Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta.

Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus.

The precise mechanism of placentation in the bovine species where a restricted trophoblast invasion occurs to form the synepitheliochorial placenta is not fully understood. This study initially investigated the conceptus-maternal interactions in the peri-attachment period by comparing the proteins present at Days 16 and 18 in uterine luminal fluid (ULF) of pregnant with nonpregnant cows using 2-D gel electrophoresis. Nine protein spots were identified that were present in greater amounts in pregnant compared to nonpregnant ULF: carbonic anhydrase, ezrin, heat shock protein 70, isocitrate dehydrogenase, nucleoside diphosphate kinase, peroxiredoxin 1, purine nucleoside phosphorylase, thioredoxin and triosephosphate isomerase and four proteins that were less abundant in ULF from the gravid compared to the nongravid horns or nonpregnant uteri: cystatin E/M, legumain, retinol-binding protein (RBP) and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). Successful placentation requires the remodelling of the endometrial surface therefore uterine mRNA and protein expression of legumain, a protease activator, and TIMP-2, a protease inhibitor, was examined in detail during the oestrous cycle and from Days 13 to 31 of pregnancy. Both mRNAs were up-regulated in the endometrium during the luteal phase of the oestrous cycle and during early pregnancy. Although legumain and TIMP-2 mRNA expression levels were similar between uterine horns at the same day of pregnancy, the amount of protein differed between gravid and nongravid horns possibly modulated by interferon-tau or by other factors produced by the conceptus. These events at the conceptus-maternal interface may provide localised control of protease activity necessary for controlling trophoblast invasion of the endometrium.

Expression of genes associated with allantois emergence in ovine and bovine conceptuses.

In the development of ruminant embryos, the emergence and growth of the allantois is critical for the establishment of the chorioallantoic placenta. The allantoic membrane contributes to all the vasculature that perfuses the placental tissues and the fetal membranes. Using suppressive subtractive hybridization to compare mRNA from Day 13 ovine preimplantation conceptuses (prior to allantoic emergence) with Day 17 allantoic membrane, we identified nine genes whose expression was associated with the emergence of the allantoic sac. Collagen alpha 1 type XII, collagen alpha 2 type I, collagen alpha 2 type V, epsilon 4 beta-globin, osteonectin, and uroplakin were expressed at significantly greater levels in ovine Day 17 allantois compared to Day 13 conceptuses. These genes are associated with the extracellular matrix and most likely are involved in establishing and strengthening the structural integrity of the allantoic sac and in the development of the blood vessels. RalB expression increased with development although at significantly greater levels in the allantois only at Day 19. Hoxa-10 and RhoA showed no differential expression during this period. All these genes showed a similar temporal pattern of expression in bovine conceptuses at equivalent stages of development with significantly greater expression of all these genes, except for Hoxa-10, found in Day 24 allantois compared to Day 14 conceptuses. This suggests that the role they play in allantoic emergence, growth and function is conserved in both ruminant species and that their expression is regulated in a similar manner. The interactions and regulation of this process remains to be fully explained.

Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies.

Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

Improving successful pregnancies after embryo transfer.

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo. We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7-10 days after transfer.