Kyu-Chan Hwang

Serial Cloning of Pigs by Somatic Cell Nuclear Transfer: Restoration of Phenotypic Normality During Serial Cloning

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 clones cells during its gestational development. Developmental Dynamics 236:3369–3382, 2007.

Cloning, sequencing, and characterization of the murine nm23-M5 gene during mouse spermatogenesis and spermiogenesis

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 0.7kb, 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.

Nm23-M5 mediates round and elongated spermatid survival by regulating GPX-5 levels

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.

Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death.

BackgroundSomatic 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.ResultsMicroscopic 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.ConclusionThese 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.

Modulation of autophagy influences development and apoptosis in mouse embryos developing in vitro

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. Mol. Reprod. Dev. 78:498–509, 2011.

Depigmentation of Skin and Hair Color in the Somatic Cell Cloned Pig

Previously, we have successfully produced nine cloned piglets using Duroc donor cells. Among these clones, one showed distinct depigmentation of the skin and hair color during puberty. In this study, we selected a clone with depigmentation to investigate the etiology of the anomaly in somatic cell nuclear transfer. We hypothesized that genes related to Waardenburg syndrome (Mitf, Pax‐3, Sox‐10, Slug, and Kit) are closely associated with the depigmentation of pig, which was derived from somatic cell nuclear transfer (scNT). Total RNA was extracted from the ear tissue of affected and unaffected scNT‐derived pigs, and the transcripts encoding Mitf, Pax‐3, Sox‐10, and Slug, together with the Kit gene, were amplified by reverse transcription‐polymerase chain reaction, sequenced, and analyzed. The cDNA sequences from the scNT pig that showed progressive depigmentation did not reveal a mutation in these genes. Although we did not find any mutations in these genes, expression of the genes implicated in Waardenburg syndrome was severely down‐regulated in the affected scNT pig when compared with unaffected scNT pigs. This down‐regulation of gene expression may result in a previously undescribed phenotype that shows melanocyte instability, leading to progressive loss of pigmentation. Developmental Dynamics 238:1701–1708, 2009.

Identification and characterization of a novel mouse and human MOPT gene containing MORN-motif protein in testis.

A novel testis-derived membrane occupation and recognition nexus (MORN)-motif protein was identified in mouse testis (MOPT) by subtraction screening methods and found to be localized on chromosome 17E3, spanning approximately 7kb. Sequence analysis showed that MOPT contains 669 base pair nucleotides of open reading frame and the corresponding 79 amino acids. The protein is predicted to have theoretical molecular mass of 9000 Da and an expected isoelectric point of 5.8 and seems to have unique sequences except for MORN-motif domain. The transcript of MOPT is highly and specifically expressed in adult testis as well as skeletal muscle. Moreover, MOPT transcript and protein are confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increase in abundance at subsequent stages. MOPT first appeared in the proacrosomic vesicles of the early Golgi phase spermatids and was translocated from the head cap of elongated spermatid to the nucleus of mature spermatozoa at the final stage of spermiogenesis. Our study suggests that MOPT may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis.

Developmental arrest of scNT-derived fetuses by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness

Somatic cell nuclear transfer (scNT)‐derived pig placenta tissues of gestational day 30 displayed avascularization and hypovascularization. Most of the cytotrophoblast‐like cells of the developing scNT‐derived placenta villi were improperly localized or exhibited impaired migration to their targeting loci. Id‐2, Met, MMP‐9, and MCM‐7 were barely detectable in the cytotrophoblast cells of the scNT‐derived placenta villi. Active MMP‐2 and MMP‐9 expression was significantly down‐regulated in the scNT‐embryo transferred recipient uteri. scNT clones exhibited a hypermethylated pattern within the pig MMP‐9 promoter region and the significance of GC box in the regulation of MMP‐9 promoter activity. Marked apoptosis was observed in the developing endometrial gland of scNT‐embryo transferred recipient uteri. Collectively, our data strongly indicated that early gestational death of scNT clones is caused, at least in part, by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness due to the down‐regulation of active MMP‐9 expression. Developmental Dynamics 240:627–639, 2011.

Long-term follow-up of porcine male germ cells transplanted into mouse testes.

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.

Comparative Gene Expression Analysis of Somatic Cell Nuclear Transfer-Derived Cloned Pigs with Normal and Abnormal Umbilical Cords

Gene expression profiling of compromised umbilical cords (CUCs) derived from somatic cell nuclear transfer (scNT) clones was performed to determine why scNT-derived clones often exhibit malformed umbilical arteries. Umbilical cord samples were obtained from 65 scNT piglets, and of these, nine displayed a CUC. Microscopic analyses of the scNT clones with CUCs (scNT-CUCs) revealed complete occlusive thrombi that were not detected in the arteries of scNT clones with normal umbilical cords (scNT-Ns). Moreover, whereas the allantoic ducts of the scNT-Ns contained columnar epithelium, the scNT-CUCs lacked this epithelial layer. Compared to scNT-Ns, the scNT-CUCs exhibited severe histological damage, including tissue swelling and vein and arterial damage with complete occlusive thrombi. To investigate functional abnormality, gene expression profiles were created in duplicate using the Platinum Pig 13K oligonucleotide microarray, which contains 13 610 probes of 70 bp in length and is capable of interrogating 13 297 targets with up to one probe per target. Probe sets were selected according to a 2-fold or greater increase or decrease of gene expression in scNT-CUCs compared to scNT-Ns. Most genes expressed in scNT-Ns were also expressed by scNT-CUCs. However, most genes involved in transcriptional regulation, such as JUN, JUNB, and FOSL2, showed a significant decrease in expression in the scNT-CUCs, which may produce a ripple effect capable of altering the transcriptomes of many other cellular processes, including angiogenesis, antioxidation, and apoptosis. The scNT-CUCs with thrombosis showed extensive apoptosis leading to placental insufficiency and related pathology. Considering that the umbilical cord plays a role in the transportation of metabolites to the fetus, placental insufficiency in scNT-CUCs may be caused by an increase in apoptotic protein expression from scNT-derived umbilical cords with hypoplastic arteries, and our results provide evidence that porcine oligonucleotide microarray analysis is a useful tool for screening scNT-derived abnormalities in pigs.