Manami Urakawa

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts.

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.

Administration of peripheral blood mononuclear cells into the uterine horn to improve pregnancy rate following bovine embryo transfer.

Embryo transfer (ET) has been used to improve reproductive efficiency and genetic make-up in bovine species. However, the success rate of ET has not been improved since its inception. Here we examined whether administration of autologous peripheral blood mononuclear cells (PBMCs) into the uterine horn can improve pregnancy rates following bovine ET. First we determined that the abundance of interleukin (IL)-1alpha, IL-1beta and IL-8 transcripts in PBMCs was greatest after 24h of culture. PBMCs that had been cultured for 24h were gently administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle. On day 7, the ET was carried out and the pregnancy rate in the PBMC-treated group was compared with that in the non-treated group. The pregnancy rate on day 60 in the PBMC-treated group (76.7%, 56/73) was significantly higher than that in the non-treated group (59.7%, 43/72, p<0.05). These results indicate that administration of autologous PBMCs into the uterine horn improves pregnancy rates following bovine ET.

α1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and Cre-expressing adenovirus

Background. Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine &agr;1,3-galactosyltransferase (&agr;1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and &agr;1,3GT-gene knockout cattle. Methods. &agr;1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using &agr;1,3GT−/− fetal fibroblasts, and one &agr;1,3GT knockout calf was generated but died shortly after birth (day 287). Results. Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. Conclusion. The &agr;1,3GT knockout- and antibiotic-resistance gene free (&agr;1,3GT−/−neo-) cells could be cloned normally. Thus, cloned cattle from &agr;1,3GT −/−neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.

Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes

The developmental capacity of bovine oocytes matured and fertilized in vitro was investigated under five different culture systems (IVF-medium, BPM-G and TCM199 with bovine epithelial cells for in vitro culture; and rabbit and sheep oviducts for in vivo culture). Immature bovine oocytes were cultured for 24 h in TCM199 supplemented with 20% estrous cow serum and fresh granulosa cells. Fertilization in vitro was performed using pooled frozen-thawed semen from two bulls after swim-up and heparin treatments. Eighteen to 20 h after in vitro insemination, oocytes were transferred to rabbit or sheep oviducts, or were cultured in the three different media for 6 days. Recovery rates from the rabbit and sheep oviducts were 68.1% and 35.3%, respectively. In the oocytes recovered, the frequency of development to the late morula and blastocyst stages was not significantly different in rabbit (58258: 22.5%) and sheep (849: 16.3%) oviducts. The frequency of in vitro development was higher in TCM199 with added bovine oviduct cells (44361: 12.2%) than in either IVF-medium (5319: 1.6%) or BPM-G (1253: 0.4%). Although the developmental capacity of oocytes in vitro was significantly (P < 0.001) lower than in the rabbit (but not in the sheep) oviduct, the loss of oocytes in vivo counteracted this advantage of the oviduct system. The present results indicate that co-culture of embryos with bovine oviduct epithelial cells is an effective means of supporting the further development of bovine oocytes matured and fertilized in vitro.

Comparison of early development in utero of cloned fetuses derived from bovine fetal fibroblasts at the G1 and G0/G1 phases.

In bovine somatic cell nuclear transfer (NT), embryos are more likely to develop to full term when they are derived from fibroblasts at the G1 phase instead of cells at the G0/G1 phase. To better understand the reason for this difference, we examined morphological development in the early pregnancy of NT embryos using G1 phase cells (G1-NT embryos) and G0/G1 phase cells (G0/G1-NT embryos). Blastocysts derived from G1 and G0/G1-NT embryos were transferred to recipient heifers, and the conceptuses at day 50 of gestation were retrieved non-surgically using prostaglandin F(2alpha) and oxytocin. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The percentages of embryos that developed to the blastocyst stage did not differ between G1 and G0/G1-NT embryos. Pregnancy rates at day 30 of recipient heifers carrying G1-NT, G0/G1-NT, IVF, parthenogenetic and AI embryos were similar (57-100%). Two recipient heifers carrying parthenogenetic embryos returned to estrus between days 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at day 50 of gestation of all experimental groups (83%) were recovered non-surgically by several PGF(2alpha) and oxytocin treatments. Recovery rates of normal fetuses derived from G1-NT embryos (83%), IVF embryos (80%) and AI embryos (88%) were greater than those of G0/G1-NT embryos (33%) and parthenogenetic embryos (0%). Our results suggest that NT embryos reconstructed with cells at the G1 phase have a high developmental competence from the time of embryo transfer to day 50 of gestation.

Donor Cells at the G1 Phase Enhance Homogeneous Gene Expression Among Blastomeres in Bovine Somatic Cell Nuclear Transfer Embryos

The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the β-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.

Heterozygous disruption of the α1,3-galactosyltransferase gene in cattle

Background. Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the &agr;1,3-galactosyltransferase (&agr;1,3-GT) gene in cattle. Methods. The &agr;1,3-GT gene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine &agr;1,3-GT gene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the &agr;1,3-GT locus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer. Results. To establish fetal fibroblasts that were heterozygously disrupted at the &agr;1,3-GT locus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the &agr;1,3-GT gene. Conclusions. Heterozygous knockout of the &agr;1,3-GT gene was performed in JBC, and production of a homozygous &agr;1,3-GT knockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.

Anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, suppresses human to bovine cellular responses

UNLABELLED The effect of an anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, against the human to bovine cellular response was investigated. METHODS Human peripheral mononuclear cells (PBMCs) were cocultured with inactivated self-PBMCs (Self), bovine PBMCs with control antibody (Xeno), or bovine PBMCs with IMMU-114 (IMMU-114). Cellular responses were investigated by thymidine incorporation assay, CFSE (carboxyfluorescein diacetate succinimidyl ester)-mixed lymphocyte reaction, and cytokine production in culture medium. RESULTS Thymidine incorporation rates at a 1:1 responder to stimulator ratio for Xeno + control antibody, Xeno + IMMU-114, Self + control antibody, and Self + IMMU-114 were 14201.3 ± 1968.4, 513.0 ± 49.5, 952.7 ± 128.7, and 423.3 ± 138.8 cpm, respectively (P = 0.032). Those at a 1:2 ratio were 6518.0 ± 690.1, 896.6 ± 92.9, 1051.0 ± 123.6, and 736.0 ± 35.6 cpm, respectively (P = 0.036). CFSE-mixed lymphocyte reaction demonstrated that the frequencies of CFSE-low, CD4(+), and CD25(+) activating T cells in Self, Xeno, and IMMU-114 were 0.27 ± 0.04%, 3.65 ± 0.53%, and 1.23 ± 0.15%, respectively (P = 0.027). Cytokine production in culture medium indicated that IMMU-114 decreased Th1-type cytokines, including interleukin-2, interferon-γ, and tumor necrosis factor-α. CONCLUSION IMMU-114 effectively suppresses human to bovine cellular responses. The mechanism involves direct inhibition of the interaction between class II human leukocyte antigen-DR-positive cells and CD4(+) T cells, and indirect suppression of Th1 cytokine production.