Ryan A. Cabot

Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts

Abstract Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Regulation of Mitogen-Activated Protein Kinase Phosphorylation, Microtubule Organization, Chromatin Behavior, and Cell Cycle Progression by Protein Phosphatases During Pig Oocyte Maturation and Fertilization In Vitro

Abstract We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2. The objective of this study was to determine the expression pattern of Oct4, Nanog, and Sox2 in cleavage stage porcine embryos obtained in vivo or by in vitro fertilization and parthenogenetic activation. We used quantitative, real time PCR to assess the relative amount of each transcript in cleavage stage embryos. We found that Oct4 was transiently activated at the 2‐cell stage (P‐value <0.05) while Nanog and Sox2 were activated at the 4‐cell stage (P‐value <0.05) in in vitro embryos. Embryos derived in vivo showed a similar but not identical pattern of expression of Nanog mRNA been in highest abundance both at the 4 cell and the blastocyst stage. The activation observed at the 4‐cell stage for Nanog and Sox2 was shown to be RNA polymerase II dependent (P‐value <0.05). This study showed that Oct4, Nanog, and Sox2 possess similar, but not identical, patterns of expression between in vitro and in vivo derived porcine embryos. The difference between the amount of transcripts may reflect the reduced developmental potential observed in in vitro cultured embryos. Mol. Reprod. Dev. 75: 1726–1735, 2008.

Differential Remodeling of Mono- and Trimethylated H3K27 During Porcine Embryo Development

Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage porcine embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2‐cell, 4‐cell, 8‐cell, and blastocyst stage embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage embryos produced by fertilization; the signals were faint or absent in nuclei of two‐cell through blastocyst stage embryos. In addition, EED transcripts were increased from the four‐cell stage (P < 0.05) in embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV‐stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four‐cell stage (P < 0.05) in parthenogenetic and in vivo derived embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during porcine embryogenesis. Mol. Reprod. Dev. 76: 1033–1042, 2009.

STIM1 regulates store-operated Ca2+ entry in oocytes.

The single transmembrane-spanning Ca(2+)-binding protein, STIM1, has been proposed to function as a Ca(2+) sensor that links the endoplasmic reticulum to the activation of store-operated Ca(2+) channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca(2+) entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca(2+) stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca(2+) stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca(2+) channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca(2+) levels in the store-depleted oocytes after Ca(2+) add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca(2+) store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca(2+) entry.

KPNA7, an oocyte- and embryo-specific karyopherin ? subtype, is required for porcine embryo development

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherinαsubtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherinα7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherinαsubtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherinαsubtypes and that these karyopherinαsubtypes differentially transport intracellular cargo during cleavage development.

Impact of porcine maternal aerobic exercise training during pregnancy on endothelial cell function of offspring at birth.

The purpose of this investigation was to test the hypothesis that maternal exercise training during pregnancy enhances endothelial function in offspring at birth. Six-month-old gilts (n = 8) were artificially inseminated and randomized into exercise-trained (n = 4) and sedentary groups (n = 4). Exercise training consisted of 15 weeks of treadmill exercise. The thoracic aorta of offspring were harvested within 48 h after birth and vascular responsiveness to cumulative doses of endothelium-dependent (bradykinin: 10-11-10-6 M) and independent (sodium nitroprusside: 10-10-10-4 M) vasodilators were assessed using in vitro wire myography. Female offspring from the exercised-trained gilts had a significantly greater endothelium-dependent relaxation response in the thoracic aorta when compared with the male offspring and female offspring from the sedentary gilts. The results of this investigation demonstrate for the first time that maternal exercise during pregnancy produces an enhanced endothelium-dependent vasorelaxation response in the thoracic aortas of female offspring at birth.

Gene expression and development of early pig embryos produced by serial nuclear transfer.

During nuclear transfer, reprogramming makes the donor nucleus capable of directing development of the reconstructed embryo. In most cases reprogramming is incomplete, which leads to abnormal expression of early embryonic genes and subsequently, to reduced developmental potential. In the present study, we monitored the expression of Oct4, Nanog, and Sox2 in cloned porcine embryos and evaluated whether serial nuclear transfer, the transfer of nuclei of cloned embryos into enucleated oocytes, has the potential to provide a more complete reprogramming of the donor genome. The data suggested that Nanog and Sox2 expression is properly reactivated after nuclear transfer, but the relative abundance of Oct4 transcripts is abnormally low in cloned porcine blastocysts compared to control embryos produced by in vitro fertilization. When the nuclei of 8‐ to 16‐cell stage cloned embryos were introduced into enucleated oocytes to expose the chromosomes repeatedly to the ooplasmic factors, the resulting embryos showed poor developmental potential: a significantly lower percentage of embryos developed to the 4‐cell (12.0% vs. 31.8%), 8‐cell (3.1% vs. 15.0%) and blastocyst (0% vs. 8.7%) stages compared to those produced following a single round of nuclear transfer (P < 0.05). The additional time for reprogramming also did not improve gene expression. By the late 4‐cell stage, Oct4 and Sox2 expression levels were low in serial nuclear transfer embryos compared to those in embryos generated by in vitro fertilization or nuclear transfer. Overall, both developmental and gene expression data indicated that reprogramming of the donor nucleus could not be improved by serial nuclear transfer in the pig. Mol. Reprod. Dev. 76: 555–563, 2009.

Differential developmental requirements for individual histone H3K9 methyltransferases in cleavage-stage porcine embryos

Dimethylated H3K9 is a heritable epigenetic mark that is closely linked with transcriptional silencing and known to undergo global remodelling during cleavage development. Five mammalian histone methyltransferases (HMTases), namely Suv39H1, Suv39H2, SetDB1, EHMT1 and EHMT2, have been shown to mediate the methylation of H3K9. The aim of the present study was to determine the developmental requirements of these HMTases during cleavage development in porcine embryos. We hypothesised that knockdown of the abovementioned HMTases would differentially affect porcine cleavage development. To test this hypothesis, IVM and IVF porcine oocytes were divided into one of three treatment groups, including non-injected controls, oocytes injected with a double-stranded interfering RNA molecule specific for one of the HMTases and oocytes injected with a corresponding mutated (control) double-stranded RNA molecule. Nuclei were counted in all embryos 6 days after fertilisation. Although no significant difference in total cell number was detected in embryos injected with EHMT1 and EHMT2 interfering RNAs (compared with their respective control groups), embryos injected with interfering RNAs that targeted Suv39H1, Suv39H2 and SetDB1had significantly lower cell numbers than their respective control groups (P<0.05). This suggests that individual HMTases differentially affect in vitro developmental potential.