Expression of Peroxiredoxins and Pulmonary Surfactant Protein A Induced by Silica in Rat Lung Tissue.

Silicosis is one of the most serious occupational diseases in China and dates back to centuries ago. In this study, we successfully established a rat model of silicosis by intratracheal silica injection for 28 days and determined hydroxyproline levels to evaluate collagen metabolism in lung homogenates. Oxidative stress status was evaluated by detecting catalase and glutathione peroxidase activities. Expression levels of peroxiredoxins (Prx I and Prx VI) were detected by Western blotting. Pulmonary surfactant protein A (SP-A) levels in rat serum and lung tissue were analyzed by ELISA, and SP-A and Prx expression levels in lung tissues were detected by immunohistochemistry. The results suggest that Prx proteins may be involved in pulmonary fibrosis induced by silica. Downregulation of SP-A expression caused due to silica is an important factor in the occurrence and development of silicosis.

[Research progress in G protein-coupled receptor 40].

GPR40 is one of the G protein-coupled receptors, which has 7 transmembrance spanning helical bundles. GPR40 distributes in pancreas and central nervous system. It can be bound by medium and long chain fatty acid and activate the intracellular signal pathways, which in turn regulates the function of cells. In b-cell, intracellular calcium concentration elevates when GPR40 is binding to fatty acid, thereby promoting the release of insulin. According to the theory, new drugs, the agonist of GPR40, have been used for prepreventing and treating the diabete as. In the nervous system, GPR40 is involved in neurogenesis, but the mechanism how GPR40 works is unclear until now. In this review, the research progress of GPR40 was reviewed, especially about the structure and characteristics of GPR40 gene, ligands and function. We focused on the problems encountered in the current research and the hot points in the future.

[Isolation and biological characterization of mesenchymal stem cells from Beijing fatty chicken fetal liver].

Mesenchymal stem cells (MSCS) from chicken fetal liver are multipotent stem cells that can differentiate in vitro into various terminally differentiated cells. The majority of studies have focused on rats and mice now. Reports from other animals are less and analyses on domestic animals are few. In this study, chicken liver-derived MSCs were isolated from 7-day-old embryo of Beijing fatty chickens. Primary liver-derived MSCs were subcultured to passage 15. The surface markers of liver-derived MSCs, CD29, and CD44 were detected by immunofluorescence and the surface markers CD34 and CK19 of hematopoietic progenitor cells/hepatic oval cells were not detected. RT-PCR analysis detected positive expression of CD29, CD44, CD71, and CD73. The growth curves were typically sigmoidal. Liver-derived MSCs of different passages were successfully induced and differentiated into neuronal and osteoblast cells. The results suggest that the MSCs isolated from chicken fetal liver possess similar biological characteristics with those derived from mice, and their multilineage differentiation provides many potential applications.

Neural progenitor cells derived from fibroblasts induced by small molecule compounds under hypoxia for treatment of Parkinson's disease in rats.

Neural progenitor cells (NPCs) capable of self-renewal and differentiation into neural cell lineages offer broad prospects for cell therapy for neurodegenerative diseases. However, cell therapy based on NPC transplantation is limited by the inability to acquire sufficient quantities of NPCs. Previous studies have found that a chemical cocktail of valproic acid, CHIR99021, and Repsox (VCR) promotes mouse fibroblasts to differentiate into NPCs under hypoxic conditions. Therefore, we used VCR (0.5 mM valproic acid, 3 µM CHIR99021, and 1 µM Repsox) to induce the reprogramming of rat embryonic fibroblasts into NPCs under a hypoxic condition (5%). These NPCs exhibited typical neurosphere-like structures that can express NPC markers, such as Nestin, SRY-box transcription factor 2, and paired box 6 (Pax6), and could also differentiate into multiple types of functional neurons and astrocytes in vitro. They had similar gene expression profiles to those of rat brain-derived neural stem cells. Subsequently, the chemically-induced NPCs (ciNPCs) were stereotactically transplanted into the substantia nigra of 6-hydroxydopamine-lesioned parkinsonian rats. We found that the ciNPCs exhibited long-term survival, migrated long distances, and differentiated into multiple types of functional neurons and glial cells in vivo. Moreover, the parkinsonian behavioral defects of the parkinsonian model rats grafted with ciNPCs showed remarkable functional recovery. These findings suggest that rat fibroblasts can be directly transformed into NPCs using a chemical cocktail of VCR without introducing exogenous factors, which may be an attractive donor material for transplantation therapy for Parkinson's disease.

[The change of pulmonary surfactant protein of rat following silica exposure].

OBJECTIVE: To investigate the change of lung surfactant protein (SP) A,B,C,D of rats following silica dust exposure in order to provide the evidences for the early diagnosis indices or therapy of silicosis. METHODS: 60 male SD rats were randomly divided into silica group, and corresponding controls group. Rats in silica group were administrated 1 ml silica solution by intratracheal instillation at dose of 50 mg/ml. Rats in control group were administrated the same amount saline. At 3rd, 7th, 14th, 21st, 28th after silica exposure, serum and bronchoalveolar lavage fluid (BALF) samples were obtained. The concentration of SP-A, SP-B, SP-C, SP-D in serum and BALF were measured by using enzyme immunoassay (ELISA). Meanwhile the levels of total anti-oxidative activity (T-AOC) and hydroxyproline (HYP) in lung tissue were also detected. The pathology of lung tissue was conducted. RESULTS: Compared with control group, SP-A concentration in BALF of silica exposed rat for 3, 14, 21, 28d was significant lower and SP-D concentration in BALF of silica exposed rat for all time points was also lower. The differences were significant (P < 0.05). Meanwhile SP-B level in 7, 14, 21, 28 d silica exposed rats BALF and SP-C level in 14, 21, 28 d silica exposed rats markedly decreased (P < 0.05). In addition compared with control group, SP-A, SP-B and SP-C concentration in serum of silica exposed rat were higher when SP-A for 14, 21, 28 d silica exposure, SP-B for 7, 14, 21 d silica exposure and Sp-C for 7, 14, 21, 28 d exposure. And all difference were significant (P < 0.05). As silica exposure time increased, SP-C concentration in serum showed an increase trend, which showed a time-response relationship (r = 0.618, P = 0.042). However, SP-D concentration in serum of rat for 7, 14, 21, 28d silica exposure were significant lower than that of control group (P < 0.005). And there was a decrease trend with time point exposure regarding of SP-D (r = -0.731, P = 0.016). The HYP content in lung tissue of experiment rats increased at 3rd, 7th, 14th, 21st and 28th day time point and The T-AOC activity in lung tissue decrease at, 7th, 14th, 21st and 28th day time point. The differences were significant (P < 0.05). There was a positive correlation (P = 0.803, P = 0.045) between SP-C in BALF and HYP of silica exposed rats and a negative correlation between SP-D in BALF and HYP (r = -0.867, P = 0.033). No significant correlation were seen between SP-A, SP-B BALF and HYP (y = 0.416, P = 0.28; r = 0.592, P = 0.071). SP-C concentration in BALF and serum all showed an increased trend and a positive correlation was seen (r = 0.539, P = 0.046). The same decrease trend was seen between SP-D in BALF and serum and correlation value was 0.870 (P = 0.034). CONCLUSION: The silica exposure did cause the change of SP content both in BALF and serum. The SP-C and SP-D content in serum might be served as an early effective biomarker of silicosis.

Directed neural differentiation of duck embryonic germ cells.

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.

[Regulatory effect of GDNF on the proliferation and differentiation of mammalian spermatogonial stem cells].

The glial cell-derived neurotrophic factor (GDNF) is a member of the transforming growth factor beta (Tgf-beta) superfamily, which is produced by Sertoli cells and plays an important role in the proliferation and differentiation of spermatogonial stem cells (SSC). The addition of proper amount of GDNF to the culture media can promote SSC proliferation in vitro. Besides, GDNF regulates the self-renewal and differentiation of SSCs through various signaling pathways. This review focuses on the effects of GDNF on the proliferation and differentiation of mammalian SSCs and GDNF-mediated signaling pathways.

[Relationship between urinary 1-hydroxypyrene level and peripheral blood lymphocyte chromosomal damage among coke oven workers].

OBJECTIVE: To investigate the relationship between the urinary 1-hydroxypyrene level and cytokinesis-block micronucleus in peripheral blood lymphocyte in coke oven workers. METHODS: One hundred and fifty-eight workers from a coke plant and 158 referents without occupational PAHs exposure were recruited in this study. Urnary level of 1-hydroxypyrene was measured by alkaline hydrolysis combined with high performance liquid chromatography as an internal exposure dose, and the chromosomal damage of peripheral blood lymphocyte were evaluated with cytokinesis-block micronucleus (CBMN) method. Personal information including occupational history, age, sex, smoking and alcohol drinking, was collected by questionnaire. RESULTS: The lymphocyte chromosomal damage level expressed as frequency of CBMN in coke oven workers was significantly higher than that of controls (3.32 ± 2.90 vs 0.57 ± 0.88, P < 0.01) after adjusting for sex, age, smoking and alcohol drinking, and correlation between urinary 1-hydroxypyrene concentrations and frequency of CBMN was found (Spearman Partial correlation coefficient = 0.28, P < 0.05) in coke oven workers. Three hundreds and sixteen subjects were divided into three groups by their urine 1-hydroxypyrene level (expressed as 0.11 ∼ 0.70, 0.71 ∼ 4.09 and 4.10 ∼ 24.74 µmol/mol Cr). After adjusting for age, sex, smoking and alcohol drinking by multiple nonparametric analysis of covariance, the frequency of CBMN in the groups of 0.71 ∼ 4.09 and 4.10 ∼ 24.74 µmol/mol C were 1.89 ± 2.37 and 3.29 ± 2.36, significantly higher than that in the group of 0 ∼ 0.70 µmol/mol Cr (0.56 ± 0.89). CONCLUSIONS: Under present PAHs exposure levels, the Cytokinesis-block micronucleus test could detect PAHs-induced genotoxicity in coke oven workers.

Establishment and characterization of a fibroblast line from Simmental cattle.

A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2n=60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N(3), pEYFP-N(1) and pDsRed1-N(1)) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.

[Characterization of expression of alpha1-acid glycoprotein gene in Beijing fatty chicken (Gallus gallus)].

The specific expression of alpha1-AGP gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR. The full-length cDNA of alpha1-AGP was inserted into pEGFP-C1 multi-cloning sites to construct recombinant eukaryotic expression vector pEGFP-alpha1-AGP. The lipofectin method was used to transfect the pEGFP-alpha1-AGP into Beijing fatty chicken fibroblast cells. The open reading frame of Beijing fatty chicken alpha1-AGP gene was 612 base pairs in length, which was expressed higher in liver and lung than in muscle. This gene did not express in heart and kidney. The expression efficiency ranged from 31.3% to 47.6% in 24, 48, and 72 h after transformation. The green fluorescence mainly concentrated in the nucleus. With the increase of the expression of green fluorescence, granula was observed in the nucleus. RT-PCR and Western blotting analyses showed that pEGFP-alpha1-AGP had been integrated into the genome of Beijing chicken fibroblast cell with normal expression level. In optimized condition, there was no significant effect (P>0.05) on apoptosis ratio, positive cell shape, growth and reduplication state comparing with the control group. This research established the foundation for further function research of alpha1-AGP gene and application in transgenic animal cloning.

[Study on single nucleotide polymorphisms of H-FABP gene in sheep].

PCR-SSCP and DNA sequencing approaches were applied to assess the single nucleotide polymorphisms (SNPs) and analyze the genetic polymorphisms at partial exon 2 and intron 2 of H-FABP(Heart fatty acid-binding protein)gene, and five sheep populations that comprised of Small-Tailed Han sheep (SH, 48), Ningxia Tan sheep (Tan, 121), Tan x SH F1 (23), Poll Dorset (48) and Suffolk (24) sheep were screened in this study. The result showed: (1)four SNPs at 981(G/A), 1014(A/C) 1019(T/C) and 1058 (-/G ) and nine genotypes (AA, BB, CC, AB, AC, BC, AD, CD and BD) were detected using primer 2, the AA was the predominant genotype. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium except the Tan and Suffolk populations. Statistical analysis revealed a low polymorphism information content (PIC) in the Suffolk and Tan x SH F1 populations (PIC amp; 0.25) but an intermediate PIC in the remaining three populations (0.25 amp; PIC amp;0.50). It meant that the fragment of H-FABP had polymorphisms, which could be used as a candidate gene associated gene with phenotypic traits like intramuscular fat content in different sheep populations. (2)Three genotypes (HH, Hh and hh) determined by a SNP at 2407(T-C) were detected using primer 4. The genotype frequencies were in the order of HHHhhh. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium in the Tan and Poll Dorset populations, and the PIC values were low (PIC amp; 0.25). However, there was no polymorphisms in SH, Tan x SH F1 and Suffolk populations.

[Effect of different choices and treatments with donor cells on reprogramming].

The donor nucleus must experienced the epigenetic modification of the process reprogramming and went back to the initial state after the donor cell was injected into the oocytes. If the reprogramming is not completed, the efficiency of cloning will be reduced. However, reprogramming of nucleus muct was not only embodied in its ability after it was transferred into the oocytes. It was different in the potential if the cell type was not identical. In addition, different treatment to the donor cells resulted in different ability and the level of reprogramming. This paper described different effects of the type, algebra, cycles, age, and species of the donor cells after nuclear transplantation on the reprogramming. An overview of the exposition and analysis through the donor cell cryopreservation, serum starvation, and different reagent treatments were discussed.

[Variation of MSTN gene UTR in eleven sheep breeds].

PCR-RFLP was applied to analyze the polymorphism of MSTN gene UTR in 345 sheep that comprised of eleven sheep breeds, namely Texel sheep, Charolais sheep, Small-tailed Han sheep, Monggolian sheep, Ujumqin sheep, Altay Fat-rumped sheep, Hulunbeir sheep, Tashikurgan sheep, Duolang sheep, Hu sheep, and Gangba sheep. A 271 bp and a 1 003 bp long PCR products were digested with Mboand Bsato demonstrate polymorphism in the eleven sheep breeds, which were all at Hardy-Weinberg equilibrium (P>0.05). The distribution of 3 genotypes in 11 sheep breeds was significantly different (P<0.01). Digestion of the PCR products with HpyCH4 proved that 9 domestic local sheep breeds were different from Texel sheep in the SNP site that was associated with muscularity. The individual mutation base could generate the motifs for miRNA in the 3'UTR, and sequencing analysis demonstrated high frequency of mutation in the 3'UTR region.

Expression profiling and functional characterization of miR-192 throughout sheep skeletal muscle development.

MicroRNAs (miRNAs) are evolutionarily conserved, small, non-coding RNAs that have emerged as key regulators of myogenesis. Here, we examined the miRNA expression profiles of developing sheep skeletal muscle using a deep sequencing approach. We detected 2,396 miRNAs in the sheep skeletal muscle tissues. Of these, miR-192 was found to be up-regulated in prenatal skeletal muscle, but was down-regulated postnatally. MiR-192 expression also decreased during the myogenic differentiation of sheep satellite cells (SCs). MiR-192 overexpression significantly attenuated SCs myogenic differentiation but promoted SCs proliferation, whereas miR-192 inhibition enhanced SCs differentiation but suppressed SCs proliferation. We found that miR-192 targeted retinoblastoma 1 (RB1), a known regulator of myogenesis. Furthermore, knockdown of RB1 in cultured cells significantly inhibited SCs myogenic differentiation but accelerated SCs proliferation, confirming the role of RB1 in myogenesis. Taken together, our findings enrich the ovine miRNA database, and outline the miRNA transcriptome of sheep during skeletal muscle development. Moreover, we show that miR-192 affects SCs proliferation and myogenic differentiation via down-regulation of RB1.

Betaine Effects on Morphology, Proliferation, and p53-induced Apoptosis of HeLa Cervical Carcinoma Cells in Vitro.

OBJECTIVES: To investigate the effects of betaine on HeLa cell growth and apoptosis and molecular mechanisms. MATERIALS AND METHODS: Concentrations of 0.1, 1.0, 5.0, 20.0, 100.0 mg/ml of betaine were used to evaluate the anticancer efficacy for HeLa cells respectively, and MCF-10A was also detected as a normal diploid cell control. RESULTS: We found that proliferation of HeLa cells was inhibited significantly upon exposure to increasing betaine levels with the MTT test (p<0.05). The percentage of S phase cells in the low dose groups (< 5mg/ml) were distinctly higher than in high dose groups, and the rates of Sub-G1 phase were the opposite (p<0.01); A high concentration of betaine (>5.0mg/ml) significantly promoted the apoptosis of HeLa cells (p<0.01). SOD activities of the low dose groups were slightly higher than the control group (p<0.05) and there were obvious synchronicity and correlation among the expression of promoting apoptosis genes Bax, P53, Caspase 3 and apoptosis suppression gene Bcl-2. In response to an apoptosis-inducing stimulus, p53 and cyclin D1 could be activated with blockage of the cell cycle at G1/S or S/G2 checkpoints. CONCLUSIONS: Our data showed that betaine could promote HeLa cells proliferation in vitro at low concentrations.In contrast, high concentrations could significantly inhibit cell growth and migration, and induce apoptosis of HeLa cells through caspase 3 signaling and further promoted necrosis. This might imply that betaine exhibits tumoricidal effects and acts as a biological response modifier in cancer treatment by inducing apoptosis and cell cycle arrest in a dose and time-dependent manner.

In vitro study of nucleostemin as a potential therapeutic target in human breast carcinoma SKBR-3 cells.

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group (12.1-15.4 ± 3.8%) was significantly higher than those of pcDNA3.1-NS (7.2-12.0 ± 1.7%) and non-transfection groups (4.1-6.5 ± 1.8%, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down- regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

Establishment and characterization of a fibroblast cell line derived from Mongolian sheep.

The Mongolian sheep ear marginal tissue fibroblast cell line (MSF32) from 32 samples was successfully established by using primary explants technique and cell cryoconservation technology. MSF32 cells were adherent, with a population doubling time of 28.2 h. Chromosome analysis showed that >90.2% of cells were diploid (2n=54) prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the MSF22 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses and mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1 and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 12.3% and 63.3%. Every index of the MSF32 cell line meets all the standard quality controls of American Type Culture Collection (ATCC). Not only has the genetic resources of the Mongolian sheep been preserved at the cell level, but also valuable materials had been provided for genome, postgenome and somacloning research.

Cultivation and Biological Characterization of Chicken Primordial Germ Cells

The purpose of this work was to investigate the isolation, culture process of chicken gonadal primordial germ cells (PGCs) and study their biological characterization. PGCs were harvested from 5.5-day-old chicken embryonic genital ridges and explanted onto chicken embryonic fibroblasts (CEFs). The results showed that the primary cultivation of chicken PGCs on their own gonadal stroma cells were better than CEFs at first two days for reproduction. The conditioned media supported the growth and colony formation of PGCs for a prolonged time in vitro and maintained a normal diploid karyotype, which were positively stained by alkaline phosphatase (AKP), periodic acid Schiff (PAS) and reacted with anti-SSEA-1, SSEA-3, Oct4, Blimp1 and Sox2. Real-time PCR showed that they expressed the stage specific genes CVH, Blimp1 and Dazl, the stem cell specific genes Sox2, Pouv and Nanog. They also formed the embryoid bodies (EBs). These results suggested that the chicken PGCs cultured in vitro not only had strong self-renewal ability, but also had the potential capability of multi-lineage differentiation.

Dynamic analysis of Ca²+ level during bovine oocytes maturation and early embryonic development.

Mammalian oocyte maturation and early embryo development processes are Ca(2+)-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca(2+) and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca(2+) was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca(2+) was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca(2+) was present throughout the blastomere. In PA embryos, Ca(2+) was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca(2+) in the SCNT embryos. However, Ca(2+) was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca(2+) showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca(2+) location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.