Involvement of TLR7 and TLR8 in conceptus development and establishment of pregnancy in sheep.

Toll-like receptors (TLRs) belong to the innate immune system and regulate inflammatory events that affect mammalian reproduction. In Study 1, we demonstrated that abundance of ovine TLR1-TLR9 mRNAs in the uterus differs due to reproductive status (TLR2, TLR3, TLR7, and TLR8) and the day of the estrous cycle and pregnancy (TLR1-TLR3, TLR5-TLR7, and TLR9). Expression of TLR7 and TLR8 proteins was localized primarily to uterine epithelia and stroma and regulated in a temporal manner. In Study 2, we determined that ovine conceptuses express TLR7 and TLR8 on all days studied and that expression of the envelope protein of ovine endogenous retrovirus (enJSRV-Env) declined in conceptus trophectoderm from Day 13 to Day 16 of pregnancy. In Study 3, loss-of-function experiments were conducted in vivo using morpholino antisense oligonucleotides (MAOs) injected into the uterine lumen to block synthesis of TLR7 and TLR8 proteins, individually and jointly. Conceptuses were recovered on Day 16 to assess their morphology. MAO-treated conceptuses were developmentally retarded, produced less interferon tau (IFNT), and had fewer binucleate cells (BNCs) compared with MAO-Controls. Moreover, expression of enJSRV-Env mRNA in MAO-TLR7 conceptuses was greater than that for MAO-Control and MAO-TLR8 conceptuses, but similar to MAO-TLR7/TLR8 conceptuses. Results of this study indicated differences in TLR1-TLR9 expression due to reproductive status and the day of the estrous cycle and pregnancy. TLR7 and TLR8 also influence development, enJSRV-Env abundance, secretion of IFNT, and formation of BNCs by conceptuses. These findings corroborate our hypothesis that TLR7 and TLR8 mediate pathways whereby enJSRV-Env regulates key peri-implantation events in conceptus development and differentiated functions of trophectoderm cells.

Effects of fertility on gene expression and function of the bovine endometrium.

Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy.

Effects of low progesterone on the endometrial transcriptome in cattle.

The objective of the present study was to determine how low progesterone (P4) affects the endometrial transcriptome, with specific emphasis on those changes that may impact conceptus elongation. Following estrous synchronization and detection (estrus = Day 0, n = 40), heifers were randomly assigned to a control group (n = 12) or a low P4 group (n = 28). Heifers in the low P4 group had consistently lower P4 concentrations compared to controls (P < 0.05). Microarray analysis of endometrial gene expression revealed low P4 altered the expression of 498 differentially expressed genes (DEGs; 215 up- and 283 down-regulated) on Day 7 and 351 DEGs (272 up- and 79 down-regulated) on Day 13. A similar number of temporal changes occurred between Day 7 and Day 13 in both groups (2212 in heifers with normal P4 compared with 2247 in heifers with low P4); of these DEGs, 1278 were common to both groups. Little overlap in the number of DEGs affected by high or low P4 was observed across days. Comparison of the temporal changes that occur during normal estrous cycle progression (i.e., from Day 7 to Day 13) to those affected by altered P4 found significant numbers of genes were modulated by elevated (4157) and decreased (809) P4 alone. Analysis of selected genes by quantitative real-time PCR and in situ hybridization revealed that expression of MEP1B, NID2, and PRSS23 increased on Day 13 compared to Day 7 (P < 0.05) and that the magnitude of increase was significantly diminished in heifers with low P4 compared to controls. MEP1B predominantly localized to the both the superficial and deep glandular epithelium (GE), NID2 localized to the deep GE, whereas PRSS23 localized only to the luminal epithelium. In conclusion, we have determined the global changes in the endometrial transcriptome induced by decreasing the output of P4 from the corpus luteum in vivo using a unique animal model. Placing these data into context with previous data in which P4 was supplemented or elevated after ovulation, we have identified a panel of genes that are truly regulated in the endometrium by circulating concentrations of P4 in vivo and that likely impact conceptus elongation.

Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium.

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

Placental development during early pregnancy in sheep: cell proliferation, global methylation, and angiogenesis in the fetal placenta.

To characterize early fetal placental development, gravid uterine tissues were collected from pregnant ewes every other day from day 16 to 30 after mating. Determination of 1) cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA methyltransferases (DNMTs) 1, 3a, and 3b; and 3) vascular development was based on smooth muscle cell actin immunolocalization and on mRNA expression of several factors involved in the regulation of angiogenesis in fetal membranes (FMs). Throughout early pregnancy, the labeling index (proportion of proliferating cells) was very high (21%) and did not change. Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a increased. Blood vessels were detected in FM on days 18-30 of pregnancy, and their number per tissue area did not change. The patterns of mRNA expression for placental growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO receptor GUCY13B; and hypoxia inducing factor 1 α changed in FM during early pregnancy. These data demonstrate high cellular proliferation rates, and changes in global methylation and mRNA expression of factors involved in the regulation of DNA methylation and angiogenesis in FM during early pregnancy. This description of cellular and molecular changes in FM during early pregnancy will provide the foundation for determining the basis of altered placental development in pregnancies compromised by environmental, genetic, or other factors.

Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in maternal placenta.

Placental vascular development (angiogenesis) is critical for placental function and thus for normal embryonic/fetal growth and development. Specific environmental factors or use of assisted reproductive techniques may result in poor placental angiogenesis, which may contribute to embryonic losses and/or fetal growth retardation. Uterine tissues were collected on days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after mating and on day 10 after estrus (nonpregnant controls) to determine vascular development and expression of several factors involved in the regulation of angiogenesis in the endometrium. Compared with controls, several measurements of endometrial vascularity increased (P<0.001) including vascular labeling index (LI; proportion of proliferating cells), the tissue area occupied by capillaries, area per capillary (capillary size), total capillary circumference per unit of tissue area, and expression of factor VIII (marker of endothelial cells), but capillary number decreased (P<0.001). Compared with controls, mRNA for placental growth factor, vascular endothelial growth factor receptors, angiopoietins (ANGPT) 1 and 2, ANGPT receptor TEK, endothelial nitric oxide synthase, and hypoxia-inducible factor 1alpha increased (P<0.05) during early pregnancy. Vascular LI was positively correlated (P<0.05) with several measurements of vascularity and with mRNA expression of angiogenic factors. These data indicate that endometrial angiogenesis, manifested by increased vascularity and increased expression of several factors involved in the regulation of angiogenesis, is initiated very early in pregnancy. This more complete description of early placental angiogenesis may provide the foundation for determining whether placental vascular development is altered in compromised pregnancies.

The endometrium responds differently to cloned versus fertilized embryos.

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.