Project description:A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients, prior to or during the implantation window. We investigated this problem by using RNA-seq to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at d18 (pre-implantation) and d34 (post-implantation) of gestation. In addition, endometrium was profiled in order to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality prior to or during the implantation window. At d18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d34, ~36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a bottleneck model for cloned conceptus survival during the peri-implantation period determined by gene expression levels in extra-embryonic tissues and the endometrial response to altered signaling from clones.
Project description:This study relates to embryo-maternal interaction. The aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to Day 14. Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between Days 7 and 14.
Project description:Implantation is crucial for placental development whose quality will directly impact fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas at implantation in three types of pregnancies, namely artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially regulated genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the issue of pregnancy. Keywords: Fluorescence Microarray 30 samples
Project description:In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus gene expression and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes were differently expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid with male compared to female conceptuses, while female conceptuses had higher expression of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in gene expression and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different response in the endometrium. Following a synchronized estrous cycle, all heifers observed in standing estrus (=Day 0, n=30) were inseminated with semen from a proven sire. All samples were recovered at slaughter on Day 19 following estrus corresponding to the initiation of implantation in cattle, flushed with 10 ml of PBS and the presence of a conceptus was observed under a stereo-microscope (n=24). Each conceptus was dissected into 4 pieces, 3 containing only trophectoderm cells and one containing the embryonic disc along with associated trophectoderm cells, and immediately snap-frozen in liquid nitrogen along with the corresponding intercaruncular endometrium from the uterine horn ipsilateral to the corpus luteum. DNA was extracted from each conceptus with phenol/chloroform treatment and finally re-suspended in 200 μL of milliQ water. Two microliters of each sample were used to perform embryo sexing by PCR amplification of sex-specific polymorphic fragments in the amelogenin gene. N=5 samples of intercaruncular endometirum and the corresponding trophectoderm only sample were anaylsed for gene expression.
Project description:In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus gene expression and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes were differently expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid with male compared to female conceptuses, while female conceptuses had higher expression of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in gene expression and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different response in the endometrium.
Project description:Implantation is crucial for placental development which directly impacts fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas during the critical period of implantation in three types of pregnancies: artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially expressed genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that the endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the pregnancy outlet. Keywords: Fluorescence Microarray - Dye switch loop design 44 samples
Project description:Effect of lactation on conceptus-maternal interactions at the initiation of implantation in cattle: Effects on the conceptus transcriptome
Project description:Implantation is crucial for placental development which directly impacts fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas during the critical period of implantation in three types of pregnancies: artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially expressed genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that the endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the pregnancy outlet. Keywords: Fluorescence Microarray - Dye switch loop design
Project description:Implantation is crucial for placental development whose quality will directly impact fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas at implantation in three types of pregnancies, namely artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially regulated genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the issue of pregnancy. Keywords: Fluorescence Microarray
Project description:Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatchingembryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22?700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P < 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n = 236) and 6 (n = 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy. mRNA-seq study of bovine conceptuses at 5 stages of development (days 7, 10, 13, 16 and 19) post fertilization.