Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy. Endometrial biopsies were subjected to RNA sequencing from high fertile (HF; n=5), subfertile (SF; n=5) and infertile (IF; n=5) classified heifers on day 14 of pregnancy.

Project description:Purpouse: Aims of this study were (1) to characterize the endometrial transcriptome, (2) to identify functional pathways, and (3) to molecularly characterize selected pathways overrepresented in the endometrium of day 7 post-ovulation induction of cows treated to ovulate larger versus smaller follicles Methods: Seventy-four multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D?10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N = 35) or not (small follicle-small CL group; SF-SCL; N = 39) on D?10. Progesterone devices were withdrawn and cloprostenol administered 42 to 54 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. Transcriptional profiling of the endometrial tissue was performed by RNA-seq and protein markers for proliferation and apoptosis were identified by immunohistochemistry. Results: Functional enrichment data indicated that LF-LCL endometrium expressed greater abundance of genes associated with biosynthetic and metabolic processes, whereas SF-SCL endometrium .gene expression profile was biased towards cell proliferation. Extracellular matrix-related genes were also upregulated in the SF-SCL endometrium suggesting reorganization of the ECM towards a proliferation permissive phenotype. Immunohistochemistry data confirmed the greater proliferative activity observed in SF-SCL endometrium I comparison to LF-LCL counterparts, and indicated a time-related transition within experimental group. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial gene expression. Furthermore, timing and amplitude of ovarian steroid secretion pattern seem to determine the transition from a proliferation permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment. Conclusion: the periovulatory endocrine milieu affected D7 endometrial molecular signature. Main pathways affected were related to cell proliferation, ECM composition and remodeling, biosynthetic and metabolic processes. Reported data further suggest that the endometrial tissue from LF-LCL cows experienced an early proliferative phase (D4), whereas the SF-SCL endometrium, exposed to a distinct periovulatory endocrine environment, expressed a delayed onset of the proliferative activity (D7). endometrial mRNA profiles of endocrine manipulated cows were generated by deep sequencing using Illumina HiScanSQ platform

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 Î¼L 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.

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Transcriptome profile form oviduct samples of endocrine manipulated cows

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