Maternal-fetal transplacental leakage of mitochondrial DNA in bovine nuclear transfer pregnancies: potential implications for offspring and recipients.

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus-recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected.

Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle.

During the oestrous cycle, the bovine endometrium exhibits characteristic morphological and functional changes, which are mainly induced by progesterone (P(4)), oestrogens and oxytocin. We studied the response of the endometrium to this changing hormonal environment at the transcriptome level using a custom-made cDNA microarray. Endometrium samples were recovered from Simmental heifers on days 0 (oestrus), 3.5 (metoestrus), 12 (dioestrus) and 18. The latter group was divided into animals with high (late dioestrus) and low P(4) levels (preoestrus). Significance analysis of microarrays revealed 269 genes exhibiting significant changes in their transcript levels during the oestrous cycle in distinct temporal patterns. Two major types of expression profiles were observed, which showed the highest mRNA levels during the oestrus phase or the highest levels during the luteal phase respectively. A minor group of genes exhibited the highest mRNA levels on day 3.5. Gene ontology (GO) analyses revealed GO categories related to extracellular matrix remodelling, transport, and cell growth and morphogenesis enriched at oestrus, whereas immune response and particular metabolic pathways were overrepresented at dioestrus. Generation of gene interaction networks uncovered the genes possibly involved in endometrial remodelling (e.g. collagen genes, TNC, SPARC, MMP2, MEP1B, TIMP1, TIMP2, HTRA1), regulation of angiogenesis (e.g. ANGPTL2, TEK, NPY, AGT, EPAS1, KLF5 ), regulation of invasive growth (e.g. PCSK5, tight junction proteins, GRP, LGALS1, ANXA2, NOV, PLAT, MET, TDGF1, CST6, ITGB4), cell adhesion (e.g. MUC16, LGALS3BP) and embryo feeding (e.g. SLC1A1, SLC11A2, SLC16A1, SEPP1, ENPP1). Localisation of mRNA expression in the endometrium was analysed for CLDN4, CLDN10, TJP1, PCSK5, MAGED1, and LGALS1.

Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses.

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity.

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. This study investigated transcriptome profiles of endometrium samples from day 18 pregnant vs non-pregnant heifers to get insight into the molecular mechanisms involved in conditioning the endometrium for embryo attachment and implantation. Using a combination of subtracted cDNA libraries and cDNA array hybridisation, 109 mRNAs with at least twofold higher abundance in endometrium of pregnant animals and 70 mRNAs with higher levels in the control group were identified. Among the mRNAs with higher abundance in pregnant animals, at least 41 are already described as induced by interferons. In addition, transcript levels of many new candidate genes involved in the regulation of transcription, cell adhesion, modulation of the maternal immune system and endometrial remodelling were found to be increased. The different expression level was confirmed with real-time PCR for nine genes. Localisation of mRNA expression in the endometrium was shown by in situ hybridisation for AGRN, LGALS3BP, LGALS9, USP18, PARP12 and BST2. A comparison with similar studies in humans, mice, and revealed species-specific and common molecular markers of uterine receptivity.

Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach.

We studied differential gene expression in ipsilateral and contralateral bovine oviduct epithelial cells using a combination of subtracted cDNA libraries and cDNA array hybridization. Four Simmental heifers were synchronized and slaughtered 3.5 days after they entered standing heat. Epithelial cells were isolated from ipsilateral and contralateral oviducts. To identify genes that are differentially regulated in ipsilateral and contralateral epithelium, subtracted cDNA libraries were produced by suppression subtractive hybridization and analyzed by cDNA array hybridization. Sequencing of cDNAs showing differential expression levels in ipsilateral and contralateral epithelium revealed 35 different cDNAs, 30 of which matched genes with known functions and 5 of which matched genes without a known function. The majority of genes (n = 27) were expressed at a higher level in the ipsilateral oviduct, but for some genes (n = 8), mRNA abundance was higher in the contralateral oviduct. The regulated genes or their products include a variety of functional classes such as cell-surface proteins, cell-cell interaction proteins, members of signal transduction pathways, immune-related proteins, and enzymes. Identification of genes differentially regulated in ipsilateral and contralateral oviduct epithelial cells is the first step toward a systematic analysis of local mechanisms that regulate the function of the bovine oviduct epithelium in the postovulation period.

Generation of transgenic cattle by lentiviral gene transfer into oocytes.

The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.

Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques.

Epigenetic perturbations are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques. We have designed an experiment with bovine Day 80 fetuses generated by somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF), and artificial insemination (AI) to determine the relationship between fetal phenotype and genome-wide 5-methylcytosine (5mC) content. When compared with AI controls, SCNT and IVF fetuses displayed significantly increased body weight (61% and 28%), liver weight (100% and 36%), and thorax circumference (20% and 11%). A reduced crown-rump length:thorax circumference ratio (1.175 +/- 0.017 in SCNT and 1.292 +/- 0.018 in IVF vs. 1.390 +/- 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of this disproportionate overgrowth phenotype. The SCNT fetuses showed significant hypermethylation of liver DNA in comparison with AI controls (3.46% +/- 0.08% vs. 3.17% +/- 0.09% 5mC, P < 0.03), and the cytosine methylation levels for IVF fetuses (3.34% +/- 0.09%) were, as observed for phenotypic parameters, intermediate to the other groups. Regressions of fetal body and liver weight and thorax circumference on 5mC content of liver DNA were positive (P < 0.073-0.079). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length:thorax circumference ratio on liver 5mC was observed. The 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among groups. These data are in striking contrast with the recently reported hypomethylation of DNA from SCNT fetuses and indicate that hypermethylation of fetal tissue, but not placenta, is linked to the overgrowth phenotype in bovine SCNT and IVF fetuses.

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses.

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.

Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donors.

The efficiency of bovine nuclear transfer using recipient oocytes recovered by ultrasound-guided follicle aspiration (ovum pick-up [OPU]) was investigated. Oocyte donors were selected from 2 distinct maternal lineages (A and B) differing in 11 nucleotide positions of the mitochondrial DNA control region. A total of 1342 cumulus-oocyte complexes (COCs) were recovered. The numbers of total COCs and class I/II COCs recovered from donors of lineage A were higher (P < 0.001) than those obtained from lineage B. Follicle aspiration once per week yielded a higher (P < 0.001) total number of COCs per session than aspiration twice per week, whereas the reproduction status of donors (heifer vs. cow) had no effect on OPU results. Of the 1342 oocytes recovered, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) karyoplast-cytoplast complexes (KCCs), resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 84 transferable embryos (15% based on fused KCCs) were obtained. After 38 transfers (10 single, 22 double, and 6 triple transfers), 9 recipients (8 double and 1 triple transfer) were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. The proportion of transferable embryos on Day 7 was significantly (P < 0.05) influenced by maternal lineage of oocyte donors and by the frequency of follicle aspiration. Our study demonstrates the feasibility of generating nuclear transfer embryos with defined cytoplasmic background. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic components on embryonic, fetal, and postnatal development.

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood.

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.

Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing.

Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing.