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Luiz G B Siqueira and Peter J Hansen

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

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Amber M Brad, Katherine E M Hendricks and Peter J Hansen

The capacity of the preimplantation embryo to undergo apoptosis in response to external stimuli is developmentally regulated. Acquisition of apoptosis does not occur in the cow embryo until between the 8- and 16-cell stages. The purpose of the present experiments was to determine the mechanism by which apoptosis is blocked in the bovine two-cell embryo. Heat shock (41 °C for 15 h) did not increase activity of caspase-9 or group II caspases (caspase-2, -3, and -7) in two-cell embryos but did in day 5 embryos. Exposure of embryos to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to depolarize mitochondria resulted in activation of caspase-9 and group II caspases at both stages of development. For day 5 embryos, CCCP also increased the proportion of blastomeres that underwent DNA fragmentation as determined by the TUNEL assay. In contrast, CCCP did not increase TUNEL labeling when applied at the two-cell stage. In conclusion, failure of heat shock to increase caspase-9 and group II caspase activity in the two-cell embryo indicates that the signaling pathway leading to mitochondrial depolarization and caspase activation is inhibited at this stage of development. The fact that CCCP treatment of two-cell embryos induced caspase-9 and group II-caspase activity indicates that caspase activation is possible following mitochondrial depolarization. However, since CCCP did not increase TUNEL labeling of two-cell embryos, actions of group II-caspases to activate DNases is inhibited.

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Verónica M Negrón-Pérez, Yanping Zhang and Peter J Hansen

The first two differentiation events in the embryo result in three cell types – epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2. Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17, while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN. The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype.

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Kun Zhang, Peter J Hansen and Alan D Ealy

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus–oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8–16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent factor.

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Paula Tribulo, James I Moss, Manabu Ozawa, Zongliang Jiang, Xiuchun (Cindy) Tian and Peter J Hansen

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1. Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1.

Free access

Barbara Loureiro, Jeremy Block, Mauricio G Favoreto, Silvia Carambula, Kathleen A Pennington, Alan D Ealy and Peter J Hansen

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Barbara Loureiro, Jeremy Block, Mauricio G Favoreto, Silvia Carambula, Kathleen A Pennington, Alan D Ealy and Peter J Hansen

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage 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 days 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 versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (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=0.08) 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.

Free access

Rocío Melissa Rivera, Gabriella M Dahlgren, Luiz Augusto de Castro e Paula, Robert T Kennedy and Peter J Hansen

The mechanism by which heat shock disrupts development of the two-cell bovine embryo was examined. The reduction in the proportion of embryos that became blastocysts caused by heat shock was not exacerbated when embryos were cultured in air (20.95% O2) as compared with 5% O2. In addition, heat shock did not reduce embryonic content of glutathione, cause a significant alteration in oxygen consumption, or change embryonic ATP content. When embryos were heat-shocked at the two-cell stage and allowed to continue development until 72 h post insemination, heat-shocked embryos had fewer total nuclei and a higher percentage of them were condensed. Moreover, embryos became blocked in development at the eight-cell stage. The lack of effect of the oxygen environment on the survival of embryos exposed to heat shock, as well as the unchanged content of glutathione, suggest that free radical production is not a major cause for the inhibition in development caused by heat shock at the two-cell stage. In addition, heat shock appears to have no immediate effect on oxidative phosphorylation since no differences in ATP content were observed. Finally, the finding that heat shock causes a block to development at the eight-cell stage implies that previously reported mitochondrial damage caused by heat shock or other heat shock-induced alterations in cellular physiology render the embryo unable to proceed past the eight-cell stage.