Miki Sakatani

Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells

Abstract Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst

BackgroundThe first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.ResultsA total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.ConclusionAnalysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

Effect of summer heat environment on body temperature, estrous cycles and blood antioxidant levels in Japanese Black cow.

This study investigated the effect of summer heat environment on estrous cycles and blood antioxidant levels in Japanese Black cows. A total of 13 non-lactating Japanese Black cows (summer: 9, winter: 4) were examined. Body temperature was measured rectally and intravaginally using a thermometer and data logger, respectively. Estrous behavior was monitored using a radiotelemetric pedometer that recorded walking activity. Rectal temperatures were higher during summer than winter (P<0.001). There was an acute increase in vaginal temperature at the onset of estrus during winter but such an increase was not observed during summer. Walking activity during estrus decreased dramatically in the summer compared to the winter. Duration of estrous cycle was longer in summer (23.4 days, P<0.05) than winter (21.5 days), and the subsequent rise in progesterone concentrations following estrus tended to be delayed in summer. The level of thiobarbituric acid reactive substances (TBARS) in peripheral blood cells was higher during summer (P<0.05), while the levels of superoixde dismutase (SOD), glutathione peroxidase (GPx) and glutathione were lower (P<0.05). These results indicate that high ambient temperature during summer increases both body temperature and oxidative stress, and also reduces signs of estrus in Japanese Black cows.

Glutamine and hypotaurine improves intracellular oxidative status and in vitro development of porcine preimplantation embryos

We previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and hypotaurine (HT) during in vitro culture improved embryo development. This study examined the effects of Gln and HT on in vitro development, intracellular oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilization (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2 mM Gln and 5 mM HT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control); (2) Gln; (3) HT; or (4) Gln + HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the intracellular GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the intracellular H2O2 content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the development of porcine embryos by improvement of intracellular oxidative status.

Cathepsin B activity is related to the quality of bovine cumulus oocyte complexes and its inhibition can improve their developmental competence.

Recently, the quantity of cathepsin transcripts in cumulus cells was found to be associated with low‐developmental competence of bovine oocytes. In the present study, we investigated (1) the relation between cathepsin B activity and the quality of in vitro‐matured cumulus–oocyte complexes (IVM COCs) and denuded oocytes and (2) the effect of a cathepsin B inhibitor (E‐64) on embryo development and quality. The activity of cathepsin B was evaluated in IVM COCs and denuded oocytes. After maturation of COCs with or without E‐64, followed by in vitro fertilization, zygotes were cultured for 8 days. Cleavage and blastocyst rates were evaluated on days 2 and 8, respectively. Quality of embryos was evaluated by differential staining of day 8 blastocysts. TUNEL staining was conducted on IVM COCs and blastocysts. Cathepsin B activity was clearly detected in the low‐quality oocytes, and in the cumulus cells of both high‐ and low‐quality oocytes. This latter activity was diminished by addition of E‐64. The presence of E‐64 during IVM also significantly increased both the blastocyst rate and the total cell number, and improved blastocyst quality associated with a significant increase of trophoectoderm cells. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in both the cumulus cell layer of matured oocytes and blastocysts. These results indicate that cathepsin B activity can be a useful marker of oocyte quality. Furthermore, inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high‐quality embryos. Mol. Reprod. Dev. 77: 439–448, 2010.

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

BackgroundWhile initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance.ResultsUsing 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT.ConclusionsChanges in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus–oocyte complexes exposed to heat shock during in vitro maturation

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

Intracellular cathepsin B activity is inversely correlated with the quality and developmental competence of bovine preimplantation embryos

Recently, the activity of cathepsins B was found to be correlated inversely with the developmental competence of bovine oocytes. In this study, we investigated (1) the role of intracellular cathepsin B expression and developmental competence as well as the quality of bovine preimplantation embryos, and (2) the effect of cathepsin B inhibitor (E‐64) during in vitro culture (IVC) on the development and quality of bovine embryos. After in vitro fertilization (IVF) followed by IVC for 7 days, good and poor quality embryos classified by morphology and developmental rate on days 2, 4, and 7 were assessed for cathepsin B expression and activity. To investigate the effect of cathepsin B inhibition on embryonic development, putative zygotes were cultured with or without E‐64, followed by evaluation of cleavage and blastocyst rates on days 2 and 7, respectively. Embryonic quality was evaluated by both TUNEL staining and total cell number in day‐7 blastocysts. In each developmental stage, cathepsin B expression and activity were significantly higher in poor quality embryos than good quality ones. Moreover, addition of E‐64 during IVC significantly increased both the blastocyst rate and the total cell number. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in day‐7 blastocysts. These results indicate that cathepsin B activity can be useful as a marker for inferior quality embryos. Moreover, inhibition of cathepsin B greatly improves the developmental competence of preimplantation embryos and increases the number of good quality embryos. Mol. Reprod. Dev. 77:1031–1039, 2010.

Regulation of Pluripotency of Inner Cell Mass and Growth and Differentiation of Trophectoderm of the Bovine Embryo by Colony Stimulating Factor 2

ABSTRACT Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9–16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle.

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.