Effect of lactation on conceptus-maternal interactions at the initiation of implantation in cattle: Effects on the conceptus transcriptome
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ABSTRACT: The hypothesis tested was that the uterine environment of lactating cows would affect conceptus gene expression. Approximately 65-75 days post-partum (dpp) the estrous cycles of non-lactating (dried off immediately post partum: n=12) and lactating (n=13) cows were synchronized and on Day 7 a high quality blastocyst derived from superovulated heifers was transferred. A control group of maiden heifers (n=8) were synchronized, inseminated to a standing heat and slaughtered on the same day as non-lactating and lactating recipients (Day 19; estrus=Day 0). The ipsilateral uterine horn was flushed with 10 ml PBS and the conceptus, when present, and uterine luminal fluid (ULF) snap frozen in liquid nitrogen prior to analysis. Gene expression analysis of the conceptus was performed by RNA sequencing analysis while amino acid (aa) composition of ULF was determined by High Performance Liquid Chromatography (HPLC). Eight differentially expressed genes (DEGs) were identified between conceptuses recovered from non-lactating cows versus heifers while 269 DEGs (100 up-regulated and 169 down-regulated) were identified between conceptuses recovered from lactating cows compared to heifers. The aa, alanine, glycine, serine and threonine, arginine, leucine and valine, were significantly lower in abundance in ULF recovered from heifers compared to both non-lactating or lactating cows. Glutamic acid, glutamine and lysine concentrations were lowest in heifers compared to both cow groups. This study demonstrates that exposure of a grade one embryo to a uterine environment that has been exposed to the metabolic stresses associated with lactation modifies the transcriptome of the conceptus and aa composition of the ULF.
Project description:The objective is to investigate changes in metabolomics of uterine lumen content of lactating dairy cows associated with the onset of conceptus (embryo and associated membranes) elongation. Lactating dairy cows had estrous cycles synchronized and were subjected to induced ovulation and timed artificial insemination (AI). The day of AI was considered study d 0. On d 15, uteri were flushed by transcervical catheterization and infusion of 20 mL of phosphate buffered solution with 0.1% of polyvinyl acetate. Recovered conceptuses were classified based on morphology/length as ovoid (OV; 1-4 mm), tubular (TUB; 5-19 mm) and filamentous (FIL; 20-85 mm). The first 20 mL infused in the uterus were recovered, placed in conical tubes and centrifuged at 2,000 × g at 4ᵒC. The supernatant was collect, aliquoted and stored at -80ᵒC for later analyses of fluid composition, including measurement of IFN-τ concentration. Cows with no conceptus recovered and no detection of IFN-τ in uterine flushing were considered as nonpregnant (NPREG). The experimental design was then considered a prospective cohort study with 4 independent groups (NPREG, OV, TUB, and FIL). The additional 5th group represents a specific physiological condition of cows within the study and it will be compared to TUB and FIL groups combined, working as a pilot study for future research.
Project description:Exposure of bovine conceptuses to colony stimulating factor 2 (CSF2) from Day 5 to 7 of development can increase the percent of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from Day 5 to 7 after insemination, transferred into cows, and flushed from the uterus at Day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2 treated conceptus (35% for control vs. 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-{tau} (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.06) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at Day 15 of pregnancy.
Project description:Exposure of bovine conceptuses to colony stimulating factor 2 (CSF2) from Day 5 to 7 of development can increase the percent of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from Day 5 to 7 after insemination, transferred into cows, and flushed from the uterus at Day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2 treated conceptus (35% for control vs. 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-{tau} (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.06) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at Day 15 of pregnancy. Experimental conditions: CSF2 treated vs. CSF2 nontreated bovine in vitro produced preimplantation embryos were tranfered to a receptor cow and recovered at Day 15 of embryo development. The embryonic disc (ED) and the trophectoderm (Tr) were used for the expression analysis separately. Biological replicates: CSF2 treated vs. nontreated bovine preimplantation embryos were used in a dye switch two-color microarray experimental design.
Project description:The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy. Transcriptional profiles from day 14 conceptus extracellular vesicles isolated from 24 hour conceptus-conditioned culture media (n=3) were generated by sequencing on the Illumina HiSeq 2500 platform.
Project description:The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy.
Project description:The aim of this study was to examine the effect of sire fertility status on conceptus-induced changes in the endometrial transcriptome. Holstein Friesian bulls (3 High fertility, HF, 3 Low fertility, LF) were selected from the Irish national population of AI bulls (minimum of 500 inseminations/bull) based on adjusted fertility scores (HF: +4.37% and LF: -12.7%; mean = 0%). To generate elongated conceptuses, Day 7 blastocysts produced in vitro using sperm from these six bulls were transferred in groups of 5-10 to synchronized heifers (n=7 heifers per bull; total 42 heifers). Conceptuses were recovered following slaughter on Day 15 (recovery rate: HF 59.4% vs. LF 45.0%; P<0.05). In parallel, Day 15 endometrial explants were recovered from synchronized cyclic heifers (n=4). Explants from each heifer were co-cultured for 6 h in RPMI medium with (i) nothing, control (ii) 100 ng/ml ovine recombinant interferon tau (IFNT) (iii) a single conceptus from each high fertility bull, or (iv) a single conceptus from each low fertility bull. To minimize variation, explants from the same uterus were used across all treatments, replicated across 4 heifers. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq (Illumina NextSeq 500) and the resulting data were analyzed through a bioinformatic pipeline with R software.
Project description:A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 (IL6) supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine IL6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n=5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA-sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from IL6-treated embryos. The IL6 treatment generated 591 differentially expressed genes (DEG) in conceptuses (n=9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few DEG were associated with extraembryonic development, but several DEG were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that IL6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.
Project description:Embryo mortality (EM) contributes to infertility, but its exact mechanism is poorly understood. It was hypothesized that bovine EM pregnancies have impaired conceptus-derived interferon-tau (IFNT) release and action and are associated with altered transcriptome responses. The objective was to discover transcriptome response in EM tissues (endometrium [ENDO], corpus luteum [CL] and peripheral blood mononuclear cells [PBMC]) in lactating Holstein-Friesian cows. Two experiments (E1, E2) in day 16 pregnant (exposed to semen; E1:n=13, E2:n=15) or non-pregnant (NP; not exposed to semen; E1:n=7, E2:n=7) cows were completed. Uterine flushings (UF) and tissues were collected. Pregnant cows were also classified based on conceptus morphology and appearance for EM (E1:n=5, E2:n=6) or normal (N) conceptuses (E1:n=8, E2:n=9). Conceptuses and tissues were RNA sequenced and analyzed. The N conceptuses were longer (P<0.05) than EM conceptuses. The IFNT protein concentrations in UF were greater in N compared to EM and NP cows in E1 but not for E2. The ENDO conjugated ISG15 concentrations were greater in N (E1) than EM and NP cows but N (E2) were only greater than NP not EM cows. The major up-regulated canonical pathway in EM conceptuses was T helper 1 (Th1) and Th2. The ENDO had a massive increase in interferon stimulated genes in N and EM compared to NP cows. Estradiol-associated genes were up-regulated in EM compared to N ENDO for E2. The PBMC in E1 reflected up-regulation of genes associated Th1 immune responses in EM compared to N cows. Luteolytic pathways were upregulated in EM CL compared to N cows. This disruption of maternal recognition of pregnancy in EM pregnancies entails a massive T helper immune response within the conceptus, estradiol re-modelling of the ENDO, abnormal immune system in PBMC, luteolysis cascade in CL and, potentially, loss of pregnancy.
Project description:Conceptus implantation to the uterine endometrium is required for pregnancy establishment, during which non-invasive trophoblasts attach and adhere to the uterine endometrium or invasive trophoblasts invade into the uterine stroma, followed by placental formation in most mammalian species. During peri-implantation period, conceptuses must communicate with the uterine endometrium if they are to survive and proceed to attachment to the uterine epithelium. Despite numerous studies performed on the bovine species, molecular mechanisms associated with their attachment processes, particularly the initial attachment to the endometrial epithelium, have not been well characterized.
Project description:The bovine conceptus elongates near Day 16 of development and releases interferon-tau (IFNT), disrupting the endometrial luteolytic mechanism to sustain luteal P4 and pregnancy. Conceptus factors other than IFNT modify local endometrial activities in support of pregnancy; however, the microenvironment is largely uncharacterized. We utilized a bovine conceptus-endometrial culture system to elucidate the microenvironment in the form of RNA and protein. Estrus synchronized heifers remained cyclic (13) or were inseminated (9) to produce Day 16 cyclic endometrium and elongating conceptuses, respectively. Conceptus sections and cyclic endometrium were then used to generate tissue cultures in 1 mL of medium: (1) no tissue (Control Med; n=7), (2) mono-cultured conceptus (Conceptus; n=9), (3) mono-cultured endometrium (Endo; n=13), or (4) Endo-Conceptus co-culture (n=15). After 12 h, tissue RNA was sequenced (RNA-Seq) and media underwent proteomic analysis (LC-MS/MS). Compared to Conceptus and Endo, co-cultured conceptus and endometrial tissue contained 3,400 and 4,575 differentially expressed genes (DEG), respectively (P ≤ 0.01). Upregulated endometrial DEG, independent of IFNT, were associated with transcription whereas upregulated conceptus DEG were associated with protein homeostasis and metabolism (P ≤ 0.001).