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Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and β-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P

Autophagyis, the bulk degradation of proteins and organelles, is essential for cellular maintenance, cell viability, and development, and is often involved in type II programmed cell death in mammals. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on the in vitro development and apoptosis of mouse embryos. LC3, which is essential for the formation of autophagosomes, was widely expressed in mouse embryos, and high levels of transcript were present from 1 to 4 cells but gradually decreased through the morula and blastocyst stages. 3-MA-treated embryos exhibited significantly reduced developmental rates and total cell numbers, but increased rates of apoptosis. Furthermore, both the expression of Lc3, Gabarap, Atg4A, and Atg4B, and the synthesis of LC3 were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect developmental rates, cell numbers decreased, and the apoptosis rate increased. Expression of Lc3, Gabarap, Atg4A, and Atg4B, and synthesis of LC3 increased as well. Modulation of Lc3 mRNA and LC3 protein levels using 3-MA or rapamycin significantly increased apoptotic cell death through the disruption of mitochondrial morphology and reduction of mtDNA copy number at the blastocyst stage. Interestingly, the inner cell mass, detected by immunostaining with POU5F1 (OCT3/4) after 3-MA or rapamycin treatment of embryos, was significantly increased compared to controls. These results suggest that autophagy influences developmental patterning and apoptosis, and may play a role in early mouse embryogenesis.

Nucleoside diphosphate (NDP) kinases are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Previously, we cloned a new member of the NDPK family from mouse, Nm23-M5, which encodes a 211-amino acid protein and has 86% identity to the human Nm23-H5 [Hwang, K.C., Ok, D.W., Hong, J.C., Kim, M.O. and Kim, J.H. (2003) Cloning, sequencing, and characterization of the murine Nm23-M5 gene during mouse spermatogenesis and spermiogenesis. Biochem. Biophys. Res. Commun. 306, 198–207]. To better understand Nm23-M5 function, we generated transgenic mice with reduced Nm23-M5 levels in vivo using a short hairpin RNA (shRNA) knock-down system. Nm23-M5 expression was markedly reduced, as indicated by Northern and Western blot analysis. Nm23-M5 shRNA transgenic mice exhibited reduced numbers of haploid cells. Furthermore, the antioxidant enzyme glutathione peroxidase 5 (GPX-5) is regulated by Nm23-M5 at the level of both expression and activity. These results reveal that expression of Nm23-M5 plays a critical role in spermiogenesis by increasing the cellular levels of GPX-5 to eliminate reactive oxygen species.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.

We used annealing control primer (ACP)-based differential display reverse transcription polymerase chain reaction (DDRT-PCR) to isolate differentially expressed amplicons in bovine germinal vesicle (GV) stage oocytes, 8-cell stage embryos produced in vitro, and blastocyst stage embryos produced in vitro. Four expressed sequence tags (ESTs) of genes that were specifically and predominantly expressed in GV oocytes were cloned and sequenced. We have used a fluorescence monitored real-time quantitative PCR (qPCR) to quantify and analyzed the temporal expression of the target differentially expressed transcripts throughout the preimplantation stages from oocytes to blastocysts. The cloned genes or ESTs all exhibited significant sequence similarity with known bovine genes (98%–100%; DNCL1 and ZP2) or ESTs (81%–97%; FANK1 and GTL3) of other species. As revealed by real-time qRT-PCR, DNCL1, FANK1, GTL3, and ZP2 transcripts were observed in the GV stage oocytes and expression gradually decreased up to the 8-cell stage embryo and the transcripts were not detected in later stages. Similarly, upregulation was observed in GV stage mouse oocytes and metaphase II, suggesting that these four differentially expressed orthologous genes play important roles in early preimplantation, as maternally-derived transcripts. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.

Successful embryonic develop- ment is dependent on the temporal and stage-specific expression of appropriate genes. Currently, information on specific gene expression during early cleavage-stage embryos before zygotic gene activation (ZGA) is limit- ed. In the present study, we compare gene expression between porcine 2-cell and blastocyst stage parthe- notes to identify genes that are specifically or pre- dominantly expressed by employing annealing control primer (ACP)-based GeneFishing PCR. Using 60 ACPs, we identified and sequenced nine differentially ex- pressed genes (DEGs). A BLAST search revealed that cloned genes or ESTs (GDI-2, MTMR3, MKLN1, NUP88, ePAD, CIRHIM, UPF3B, ITGA2, and CGI- 140) had significant sequence similarities with known genes (78-95%) of other species in the GenBank/ EMBL database. Real-time reverse transcriptase-poly- merase chain reaction (RT-PCR) data disclosed that these genes were regulated upstream in metaphase II (MII) oocyte, 1-cell, and 2-cell stage embryos during early pre-implantation. Similarly, upregulation was ob- served in MII mouse oocytes and 1-cell stage embryos before ZGA, suggesting that these nine differentially expressed orthologous genes play important roles during early cleavage before ZGA. Further analysis of the differentially expressed genes identified in this re- port should provide the basis for research on early cleavage and activation of the embryonic genome. Mol. Reprod. Dev. 70: 314-323, 2005. 2005 Wiley-Liss, Inc.

Nucleoside diphosphate kinases (NDPKs) are conserved throughout evolution and have been shown to be involved in various biological phenomena. By functional screening in yeast, we identified a new member of the NDPK family, nm23-M5, which encodes a 211-amino acid protein with 86% identity to the human homolog Nm23-H5. Northern blot analysis revealed that nm23-M5 encodes two transcripts of 0.8 and 0.7 kb, which are highly and specifically expressed in adult testis. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that nm23-M5 transcripts first appear in pachytene spermatocytes and increase in abundance in subsequent stages. However, a low level of nm23-M5 mRNA was detected by RT-PCR in other tissues, such as ovary, brain, heart, and kidney. In situ hybridization studies showed that testicular nm23-M5 transcripts are localized in stage 12 to stage 16 spermatids in the neighboring lumen of seminiferous tubules. This distribution contrasts with that of Nm23-H5 transcripts, which are specifically found in spermatogonia and early spermatocytes. The heterologous expression of nm23-M5 in yeast cells confers protection from cell death induced by Bax, which is due to the generation of reactive oxygen species. Furthermore, overexpression of nm23-M5 in fibroblasts altered the cellular levels of several antioxidant enzymes, particularly glutathione peroxidase 5. Thus, we believe that the murine nm23-M5 gene plays an important role in late spermiogenesis by elevating the ability of late-stage spermatids to eliminate reactive oxygen species.

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.

Background Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. Results Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. Conclusion These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development.