Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos.

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos.

Sphingosine-1-phosphate (S1P) analog phytosphingosine-1-phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis.

Phytosphingosine-1-phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM).

Mammary alveolar cell as evaluation system for casein gene expression involved in glucose level.

Objective: Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar) epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of analyzing models.

Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes.

Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos.

Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-α or erythropoietin for a bovine mammary bioreactor.

Interferon α (IFN-α) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia.

Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines.

Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies.

A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.

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.

Altered gene expression profiles in mouse tetraploid blastocysts.

In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (P<0.

Electrical activation enhances pre-implantation embryo development following sperm injection into in vitro matured pig oocytes.

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.

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.

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.

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).

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

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.

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.

Effect of vanadate on the chromatin configuration in pig GV-oocytes.

Vanadate, an inhibitor of tyrosine phosphatases, has been reported to prevent germinal vesicle breakdown in mammalian oocytes. We examined the effect of vanadate on the chromatin configuration of fully grown pig oocytes. In the presence of human menopausal gonadotropin (hMG), vanadate (0.

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.

Production of transgenic pigs harboring the human erythropoietin (hEPO) gene using somatic cell nuclear transfer.

The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells in vitro and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector.

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.

Comparative proteomic analysis associated with term placental insufficiency in cloned pig.

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.

Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods.

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.

Identification of maternal mRNAs in porcine parthenotes at the 2-cell stage: a comparison with the blastocyst stage.

Successful embryonic development 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 limited. In the present study, we compare gene expression between porcine 2-cell and blastocyst stage parthenotes to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based GeneFishing PCR.

Identification of differentially regulated genes in bovine blastocysts using an annealing control primer system.

The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) technology that involves annealing control primers (ACPs) to identify the genes that are specifically or prominently expressed in bovine early blastocysts and hatched blastocysts produced in vitro.

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.