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Placental abnormalities account for a high proportion of pregnancy loss after transfer of cloned (SCNT) bovine embryos. The high rate of pregnancy failure has been linked to the finding of abnormal placental formation and function. Because bovine SCNT embryos were shown to exhibit altered trophoblast differentiation (Arnold DR et al.2006 Reproduction 132, 279–290), we hypothesized that placental abnormalities in bovine clone pregnancies may originate from disturbed embryo-maternal communication in the pre-implantation period. To test this hypothesis, we evaluated the response of the endometrium to SCNT v. IVF embryos. The SCNT embryos were produced from fibroblast cultures derived from 4 different fetuses to exclude specific effects of a particular donor cell culture. After SCNT or IVF, embryos were cultured under identical conditions. Two SCNT or IVF blastocysts (grade 1) were transferred per recipient heifer (Day 8 of estrous cycle). Ten days later the recipients were slaughtered, the uteri were recovered, and pregnancy was verified by the presence of at least one normally developed embryo. Endometrium samples of 9 SCNT and 10 IVF pregnancies were used for transcriptome profiling with a custom cDNA microarray (BOE array; Bauersachs S et al.2007 J. Dairy Sci. 90, 4420–4423). In total, 58 transcripts were found to be differently abundant between endometrium samples from SCNT v. IVF pregnancies (SAM, FDR 5.24%). Interestingly, for some of them an important role in implantation and/or placentation has already been shown. NR2F2, encoding the orphan nuclear receptor superfamily member NR2F2 (COUP-TFII), was downregulated in endometrium from SCNT pregnancies (1.5-fold; P< 0.01). Uterine-specific Nr2f2mutant mice are infertile due to implantation failure (Kurihara I et al.2007 PLoS Genetics 3, e102). Another interesting candidate is GJA1, encoding connexin 43 (Cx43), with 1.8-fold (P< 0.001) downregulated transcript levels in SCNT pregnancies. A striking increase of stromal Cx43 has been observed in the ovine intercaruncular and caruncular endometrium during intensification of the feto-maternal contact (Gabriel et al.2005 Placenta 25, 287–296), suggesting that reduced GJA1mRNA expression in bovine clone pregnancies may negatively affect placentation. In view of the well-orchestrated spectrum of transcriptome changes in endometrium during the peri-attachment period (Bauersachs S et al.2006 Reproduction 132, 319–331), these findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period.This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478). [ABSTRACT FROM AUTHOR]

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17? (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 > 2 ng mL-1 and E2 < 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 < 1.5 ng mL-1 and E2 > 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17?, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17? appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication.This work was supported by the DFG FOR 478. [ABSTRACT FROM AUTHOR]

The endometrium provides the optimal conditions for the transport of sperm to the oviduct, to the site of fertilization, and later on for the reception of the embryo. This is reflected by specific morphological and functional changes during the estrous cycle, which are mainly regulated by the hormones progesterone, estradiol, and oxytocin. To study these changes on the level of messenger RNA, a microarray analysis of endometrial tissue samples was performed. Tissue samples were collected from 20 cyclic heifers (Deutsches Fleckvieh, between 17 and 31 months of age) after slaughter at Days 0, 3.5, 12, and 18 of the estrous cycle. The Day 18 group split into two subgroups, one with high and one with low progesterone levels in peripheral blood. Altogether there were 4 heifers in each experimental group. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array (Bauersachs et al. 2007 J. Dairy Sci. 90, in press). The cDNAs present on the array were derived from several previously conducted differential gene expression studies of bovine endometrium between different stages of the estrous cycle, during early pregnancy, and from studies of bovine oviduct epithelial cells. In all of these studies, cDNAs of differentially expressed genes were identified using a combination of subtracted cDNA libraries and cDNA array hybridization. Redundant cDNA clones were removed, resulting in 1440 cDNA fragments on the array, representing 950 different genes. Twenty radioactively (33P) labeled cDNA samples (n = 4 for each cycle stage) were hybridized with the BOE array. After normalization of raw data (using BioConductor open source software) and significance analysis (SAM, FDR 1%), 272 mRNAs were identified that showed alterations of their concentration during the estrous cycle. Expression data from cDNAs with significant changes during the estrous cycle were used for cluster analyses with MultiExperiment Viewer 4.0 (Saeed et al. 2003 Biotechniques 34, 374–378). The main clusters represented genes upregulated either during the estrus or during the diestrus phase. Quantitatively enriched Gene Ontology (GO) categories were identified to find relevant functional groups and prominent biological processes. At estrus, e.g., GO categories extracellular matrix, cytoskeleton organization, and growth factor activity indicate changes in the composition of the endometrium during the estrous cycle. At diestrus, only a few overrepresented GO categories were found, mostly related to immune response and metabolism. The genes of known function were further analyzed in the context of interaction and regulatory networks. One of a number of central factors was TGF-β, which controls the expression of 12 genes upregulated at estrus and 8 at diestrus. In conclusion, the present study extended the results of our previously conducted analysis of bovine endometrium between the estrus and diestrus stages (Bauersachs et al. 2005 J. Mol. Endocrinol. 32, 449–466), revealed distinct temporal expression profiles, and identified additional genes differentially expressed during the estrous cycle.This work was supported by Grant BMBF FUGATO-Fertilink. [ABSTRACT FROM AUTHOR]

Major problems in breeding of modern dairy cows include increasing rates of stillbirth and dystocia, associated with elevated costs for veterinary intervention (caesarean section) and loss of production animals (cows and calves). Rates of stillbirth as high as 12% have been reported, primarily caused by fetal overgrowth and increased birth weight. In order to discover molecular markers for selection against stillbirth, we developed a model for the inheritance of fetal growth traits. A Fleckvieh bull segregating for paternal stillbirth was used for insemination of 36 cows that were slaughtered at Day 150 of pregnancy in order to recover and phenotype the fetuses. The mode of inheritance indicated involvement of imprinting. Mapping results suggested an imprinted region on chromosome 9 as candidate for the stillbirth QTL. Due to the complexity of the trait, we opted for a holistic approach that is not restricted to the QTL candidate region but allows identification of genes and networks that influence fetal growth. Transcriptome profiles of liver and cotyledon samples from the fetuses with the highest (n = 10) and the lowest total weight n = 10) were analyzed using Affymetrix Bovine GeneChips (Affymetrix, Inc., Santa Clara, CA, USA). Analysis with the program SAM showed 41 up- and 4 down-regulated genes in liver samples of the heavy-weight group. Most of these genes are involved in immune response. Interestingly, many of these genes are reported to be regulated by vitamin D. Furthermore, vitamin D is closely connected to the IGF1 system and thus the most important fetal growth regulation circuit. Seasonal effects on vitamin D levels could mostly be excluded by the experimental design and did not correlate with growth traits. Most likely, the mRNA levels of our candidate genes were influenced by alterations in the IGF1/vitamin D circuit and did not cause the observed weight differences. The imprinted candidate genes showed no correlation with fetal weight. Gene set enrichment analysis indicated enhanced metabolic activity in the liver of heavy-weight fetuses. Genes from the QTL region showed a clear enrichment in correlation with fetal weight, confirming their involvement in fetal growth. The gene with the best correlation, GHITM (growth hormone inducible transmembrane protein), could give an explanation for the enhanced metabolic activity, as it is reported to function as a metabolic regulator. Simultaneous analysis of the data sets for liver and placenta in a linear model (R-package LIMMA; Smyth 2004 Stat. Applic. Genet. Mol. Biol. 3, art. 3) yielded essentially the same differentially expressed genes for liver and a higher number of differentially expressed genes for placenta (89 up- and 114 down-regulated), with little overlap between the two tissues.This work was supported by Grant BMBF FUGATO-Fertilink. [ABSTRACT FROM AUTHOR]