Kenneth L. White

Establishment of Pregnancy after the Transfer of Nuclear Transfer Embryos Produced from the Fusion of Argali (Ovis ammon) Nuclei into Domestic Sheep (Ovis aries) Enucleated Oocytes

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the Peoples Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbeccos modified Eagles medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.

In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi.

Plasmodium falciparum and Trypanosoma cruzi were killed by two novel lytic peptides (SB‐37 and Shiva‐1) in vitro. Human erythrocytes infected with P. falciparum, and Vero cells infected with T. cruzi, were exposed to these peptides. The result, in both cases, was a significant decrease in the level of parasite infection. Furthermore, the peptides had a marked cytocidal effect on trypomastigote stages of T cruzi in media, whereas host eukaryotic cells were unaffected by the treatments. In view of the worldwide prevalence of these protozoan diseases and the lack of completely suitable treatments, lytic peptides may provide new and unique chemother‐apeutic agents for the treatment of these infections.—Jaynes, J. M.; Burton, C. A.; Barr, S. B.; Jeffers, G. W.; Julian, G. R.; White, K. L.; Enright, F. M.; Klei, T. R.; Laine, R. A. In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi. FASEB J. 2: 2878‐2883; 1988.

Somatic Cell Cloning in Buffalo (Bubalus bubalis): Effects of Interspecies Cytoplasmic Recipients and Activation Procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.

Ability of Integrins to Mediate Fertilization, Intracellular Calcium Release, and Parthenogenetic Development in Bovine Oocytes

Abstract The ability of arginine-glycine-aspartic acid (RGD; a sequence recognized by integrins) or non-RGD-containing peptides to block fertilization, induce intracellular Ca2+ oscillations, and initiate parthenogenetic development in bovine oocytes was investigated. Addition of a soluble RGD peptide during fertilization at concentrations ranging from 10 to 1000 μg/ml significantly decreased (P < 0.05) fertilization as compared to the in vitro-fertilized controls. The addition of non-RGD peptide had no effect on fertilization. Two intracellular Ca2+ transients 21.5 ± 1.9 min apart were observed in 56 of 60 oocytes incubated in RGD peptide concentrations ranging from 20 to 1000 μg/ml. No intracellular Ca2+ transients were observed in medium alone, non-RGD treatment groups or in the RGD peptide at 10 μg/ml. The percentage of oocytes activated with ionomycin and 6-dimethylaminopurine (63% cleavage and 34% blastocyst development) was significantly higher (P < 0.05) than those activated with the RGD peptide and 6-dimethylaminopurine (35% cleavage and 19% blastocyst development). These groups were significantly higher (P < 0.05) than either peptide alone, 6-dimethylaminopurine alone, or the non-RGD peptide and 6-dimethylaminopurine treatment groups. These data provide evidence that ligation of an integrin on bovine oocytes with a soluble RGD peptide is capable of blocking fertilization, inducing intracellular Ca2+ transients, and initiating parthenogenetic development.

Review of Enucleation Methods and Procedures Used in Animal Cloning: State of the Art

Enucleation of a recipient oocyte is a crucially important process for nuclear transfer efficiency. Several procedures have been developed and used in the production of nuclear transfer embryos. Although the use of excitable fluorochromes and ultraviolet (UV) light are commonly used for complete enucleation, they also pose the risk of damaging the maternal cytoplast. Telophase and chemically assisted enucleation have also been used for cloning, but the quality and quantity of the recipient cytoplasm varies with the procedure used. This paper reviews various methods used for enucleation, and discusses their benefits and limitations with respect to cloning efficiency.

Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos

Monoclonal antibodies to histocompatibility (H-Y) antigen, of IgM subclass, were used to immunologically determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Two experiments were performed, one using cytolysis of H-Y positive embryos and the other using binding of a Fluorescein Isothiocyanate-(FITC) labeled second antibody. Eight- to 16-cell embryos used in the cytolytic assay were cultured in Whittens Medium without bovine serum albumin (WM), to which monoclonal antibody and normal guinea pig serum were added. Embryos were classified as affected or unaffected, based on morphology of the embryo and its blastomeres. A total of 550 embryos were cultured; 294 (53.5%) were scored as unaffected and 263 of these were transferred to recipients. Forty-three (81.1%) of 53 pups born were female. Morulae and early blastocysts were used in the FITC-labeled second antibody assay. Embryos were cultured in WM containing monoclonal antibody, washed and placed in drops of WM containing FITC-labeled anti-IgM. Following another wash embryos were individually evaluated at 200X for fluorescence. Fifty-five percent (169 of 305) of the embryos displayed cell-specific fluorescence. A total of twenty-three pups, 18 males (78.3%) and five females (21.7%), were born following transfer of 156 fluorescing embryos. Four male (17.4%) and nineteen female (82.6%) pups resulted from embryos classified as non-fluorescing.

Culture of in vitro fertilized bovine embryos with bovine oviductal epithelial cells, Buffalo rat liver (BRL) cells, or BRL-cell-conditioned medium.

Co-culture with various cell types can enhance development of bovine embryos, especially through the transition from maternal to embryonic mRNA utilization, a stage of growth refractory to most in vitro methods. Bovine oviductal epithelial (BOE) cells have been particularly successful for culturing embryos through the refractory stage; however, Buffalo rat liver (BRL) cells are a readily available, long-lived, easy-to-care-for alternative. This study compared the embryotrophic activity of BOE to BRL cells with particular emphasis on the transition stage of growth. A total of 7158 immature bovine oocytes, matured and fertilized in vitro, were divided into 4 different culture treatments: Treatment 1: BRL conditioned medium for 72 h then BRL co-culture; Treatment 2: BRL co-culture; Treatment 3: BOE co-culture for 72 h in 5% oxygen then BRL co-culture; and Treatment 4: BOE co-culture for 72 h in 5% oxygen followed by BOE co-culture in air. Those same treatments were used to evaluate embryotrophic differences of early (4 to 5) versus late (14 to 15) passage BRL cells maintained in M-199 medium with 10% serum. Two bulls were also evaluated to determine if there exists a bull-by-culture system interaction. Treatment 3 resulted in the best development after 9 d; 9.1% of selected immature oocytes developed to expanded blastocyst. Early passage BRL cells were significantly more embryotrophic than later passage cells; this was most pronounced for Treatment 2. There was a treatment-by-bull interaction, which should be considered when comparing results among similar studies.

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera☆

Abstract Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P

Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropinclass lytic peptide in transgenic mice and subsequent resistance to Brucella abortus

The addition of an antimicrobial that can be synthesized by the mammalian immune system at the point of challenge may enhance disease resistance. A possible group of agents are cecropins, broad-spectrum antimicrobial peptides, which have been described and characterized. They are relatively non-toxic to normal cells from multicellular organisms but are toxic to a wide range of bacteria, protozoa and fungi, as well as infected and abnormal cells. Twenty-six lines of transgenic mice were produced by pronuclear injection of DNA consisting of the 5′-flanking region from −593 to +110 of the mouse interleukin 2 (IL-2) gene, Shiva 1a (a synthetic cecropin-class lytic peptide), and the SV40 polyadenylation/splice signal. A reverse-transcription PCR assay determined that two lines of transgenic mice were produced whose spleen-derived lymphocytes could be induced to transcribe and mature mRNA for Shiva 1a by exposure to 3.25 mg ml−1 of Con A. Two lines were challenged with an inoculation of 5 × 104 Brucella abortus strain 2308. After four weeks, there were significantly fewer B. abortus organisms in the spleens of transgenic mice than in non-transgenic control mice of the same strain (p < 0.05). Since the controlling regions of the IL-2 enhancer and the amino acid sequence of the signal peptide are highly conserved among several species, it is likely that this recombinant gene will function in other mammals