Project description:An increase in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our objective was to ascertain differential effects of elevated P4 concentrations following conception on endometrial gene expression in beef heifers on Days 5, 7, 13 and 16 of pregnancy, corresponding to the morula, blastocyst, elongation and maternal recognition of pregnancy stages, respectively. Estrus was synchronized in beef heifers (N=263). Two-thirds (N=140) were inseminated (Day 0), and all animals were randomly assigned to one of the following treatments: (i) pregnant, high P4; (ii) pregnant, normal P4; (iii) cycling, high P4; (iv) and cycling, normal P4. All high P4 groups received a P4 release intravaginal device (PRID) on Day 3 post-estrus/mating. Tissue was collected on Days 5, 7, 13 or 16 of the cycle or pregnancy, and pregnancy was confirmed by the presence of an appropriately developed embryo/conceptus. PRID insertion elevated (P<0.05) P4 concentrations from Day 3.5 to 8 compared with untreated animals and conceptus size was larger (P<0.05) in animals with elevated P4 on Days 13 and 16 compared with normal P4. Total RNA was extracted from predominantly intercaruncular endometria from the ipsilateral uterine horn. Samples from individual heifers were selected on the basis of their P4 profiles and gene expression was analyzed using bovine Affymetrix microarrays (N=5 per treatment per time point). Microarray data from analyses using Bioconductor GCRMA and Limma packages were subjected to a modified t-test and P-values were adjusted for multiple testing using the Benjamin and Hochberg false discovery rate method. Differentially expressed genes were selected on the basis of an adjusted P-value of <0.01. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7 and 13 post-estrus, but, the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). On Days 5 and 7, elevated P4 in pregnant heifers, altered the expression of 36 and 124 genes respectively but on Days 13 and 16 there were relatively few DEG between high and normal P4 heifers (15 and 25). Of the genes that were differentially regulated by P4, the majority were unique to a specific day of the estrous cycle/early pregnancy. In conclusion, gene expression in endometria did not differ between pregnant and cycling heifers until Day 16 of pregnancy (i.e. the time of maternal recognition of pregnancy and production of interferon tau by conceptus trophectoderm); however, elevating P4 in early pregnancy programmed changes in gene expression in endometria that are hypothesized to impact early conceptus growth and development. Thus, on Days 5, 7 and 13 differential gene expression was affected by P4, but on Day 16 the conceptus primarily influenced gene expression in uterine endometria of heifers.

Project description:An increase in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our objective was to ascertain differential effects of elevated P4 concentrations following conception on endometrial gene expression in beef heifers on Days 5, 7, 13 and 16 of pregnancy, corresponding to the morula, blastocyst, elongation and maternal recognition of pregnancy stages, respectively. Estrus was synchronized in beef heifers (N=263). Two-thirds (N=140) were inseminated (Day 0), and all animals were randomly assigned to one of the following treatments: (i) pregnant, high P4; (ii) pregnant, normal P4; (iii) cycling, high P4; (iv) and cycling, normal P4. All high P4 groups received a P4 release intravaginal device (PRID) on Day 3 post-estrus/mating. Tissue was collected on Days 5, 7, 13 or 16 of the cycle or pregnancy, and pregnancy was confirmed by the presence of an appropriately developed embryo/conceptus. PRID insertion elevated (P<0.05) P4 concentrations from Day 3.5 to 8 compared with untreated animals and conceptus size was larger (P<0.05) in animals with elevated P4 on Days 13 and 16 compared with normal P4. Total RNA was extracted from predominantly intercaruncular endometria from the ipsilateral uterine horn. Samples from individual heifers were selected on the basis of their P4 profiles and gene expression was analyzed using bovine Affymetrix microarrays (N=5 per treatment per time point). Microarray data from analyses using Bioconductor GCRMA and Limma packages were subjected to a modified t-test and P-values were adjusted for multiple testing using the Benjamin and Hochberg false discovery rate method. Differentially expressed genes were selected on the basis of an adjusted P-value of <0.01. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7 and 13 post-estrus, but, the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). On Days 5 and 7, elevated P4 in pregnant heifers, altered the expression of 36 and 124 genes respectively but on Days 13 and 16 there were relatively few DEG between high and normal P4 heifers (15 and 25). Of the genes that were differentially regulated by P4, the majority were unique to a specific day of the estrous cycle/early pregnancy. In conclusion, gene expression in endometria did not differ between pregnant and cycling heifers until Day 16 of pregnancy (i.e. the time of maternal recognition of pregnancy and production of interferon tau by conceptus trophectoderm); however, elevating P4 in early pregnancy programmed changes in gene expression in endometria that are hypothesized to impact early conceptus growth and development. Thus, on Days 5, 7 and 13 differential gene expression was affected by P4, but on Day 16 the conceptus primarily influenced gene expression in uterine endometria of heifers. Endometiral samples were taken from estrous synchronized beef heifers that were either cyclic or confirmed pregnant with either normal or high concentrations of progesterone on Day 5, 7, 13 and 16 of the estrous cycle/early pregnancy.

Project description:Background: With the rapid development of assisted reproductive technology (ART), although ovulation induction has made great progress, the accompanying health risks should not be ignored. Epigenetic modifications play an important role during the development of gametes and embryos. Recently, the epigenetic abnormalities caused by superovulation have attracted increasing attention. The follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation and appropriate concentration of them might be necessary. However, there is still no systematic study on the similarities and differences of DNA methylation alterations induced by superovulation with different dosage of FSH/hMG at the single cell levels. Methods: In the current study, the different doses of FSH/hMG combined with (add hcg full name here) hCG were used in C57BL/6 strain female mice to get experimental group oocytes while naturally matured oocytes and superovulated oocytes with only hCG were respectively set as controls. Single-cell level DNA methylation sequencing was carried out to all the mature oocytes to investigate the effect of superovulation with different dosage of FSH/hMG on DNA methylation during oocyte maturation. Results: In this study, we found that the hypo differentially methylated regions (DMRs) number of FSH/hMG 200 IU group both increased significantly although the whole genome methylation pattern of mature oocytes derived from superovulation were unaffected as compared with the control matured oocytes, suggesting that high dosage of FSH/hMG might lead to increased genomic methylation instability. At the same time, some of the DMRs annotated genes related to oocyte quality and embryonic development potential were disturbed in all dosage of FSH/hMG group (including Twist2, Cdk1, Ntrk2, Slc22a4, Sstr2, Wnt2, Cntnap5a, Gm2087, Slc16a7, and so on) as well as the certain ones (containing Abcc3, Zfp532, Atp1a1, Zfp804a, and so on) were impaired after high dosage of gonadotropins. We speculated that the effect of superovulation on DNA methylation during oocyte maturation might be dose-dependent to a certain extent, and such effect might affect oocyte quality and embryonic development competency. Conclusions: The current study firstly analyzed the differential impacts of different doses of gonadotropins on single-cell level DNA methylation of mouse oocytes, indicating a dose-dependent effect of superovulation with FSH/hMG and hCG on the epigenetic variations in mouse oocytes. Furthermore, some important genes that play a critical role in oocyte quality and embryo development were indicated to form the possible molecular basis of the corresponding regulation. This study laid a foundation for evaluating the safety of superovulation and highlighted the need for more and further research into ART.

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Embryo response to different progesterone concentrations during superovulation of Holstein heifers

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