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BACKGROUND: In vitro culture of spermatogonial stem cells (SSCs) is important for exploration of SSCs self-renewal, differentiation, and manipulation. There are several reports on rodent SSC cultures; however, data on SSC cultures in domestic animals are limited. To provide basic scientific information on canine SSC cultures, we report canine testes development, and the development of spermatogonia-derived colonies (SDCs) for in vitro cultures. METHODOLOGY/PRINCIPAL FINDINGS: Testes from 2-, 3-, and 12-month-old beagles were used for histology, immunohistochemistry, in vitro culture, immunocytochemistry, and PCR. Protein gene product 9.5 (PGP9.5)-positive spermatogonia, both single and paired, were found to be abundant in the testes of 2-month-old beagles. stempro-34 and Dulbecco's modified Eagle medium with 5% fetal bovine serum provided as useful substrates for culture of SDCs, and fibroblast growth factor (FGF) played a key role in colony formation. Colonies were positive for alkaline phosphatase and anti-PGP9.5 staining. The early spermatogonia and stem cell markers such as octamer binding protein 4 (Oct4), Nanog homeobox (Nanog), promyelocytic leukemia zinc finger (PLZF), PGP9.5, and GDNF family receptor alpha-1 (GFRα-1) were expressed in the colonies at higher levels than in the testis tissue. CONCLUSIONS: Testes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of SDCs, and FGF was important for colony formation.

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs).

Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi) that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152). Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos.

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division. [ABSTRACT FROM AUTHOR]

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected Xlinked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects. [ABSTRACT FROM AUTHOR]

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation. [ABSTRACT FROM AUTHOR]

The transgenic chicken has been considered as a prospective bioreactor for large-scale production of costly pharmaceutical proteins. In the present study, we report successful generation of transgenic hens that lay eggs containing a high concentration of human erythropoietin (hEPO) in the ovalbumin. Using a feline immunodeficiency virus (FIV)-based pseudotyped lentivirus vector enveloped with G glycoproteins of the vesicular stomatitis virus, the replication-defective vector virus carrying the hEPO gene under the control of the chicken ovalbumin promoter was microinjected to the subgerminal cavity of freshly laid chicken eggs (stage X). Stable germline transmission of the hEPO transgene to the G1 progeny, which were non-mosaic and hemizygous for the hEPO gene under the ovalbumin promoter, was confirmed by mating of a G0 rooster with non-transgenic hens. Quantitative analysis of hEPO in the egg whites and in the blood samples taken from G1 transgenic chickens showed 4,810 ~ 6,600 IU/ml (40.1 ~ 55.0 μg/ml) and almost no detectable concentration, respectively, indicating tightly regulated oviduct-specific expression of the hEPO transgene. In terms of biological activity, there was no difference between the recombinant hEPO contained in the transgenic egg white and the commercially available counterpart, in vitro. We suggest that these results imply an important step toward efficient production of human cytokines from a transgenic animal bioreactor.

Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 μM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs.

To identify putative biomarkers of porcine spermatogonial stem cells (pSSCs), total RNA sequencing (RNA-seq) analysis was performed on 5- and 180-day-old porcine testes and on pSSC colonies that were established under low temperature culture conditions as reported previously. In total, 10,184 genes were selected using Cufflink software, followed by a logarithm and quantile normalization of the pairwise scatter plot. The correlation rates of pSSCs compared to 5- and 180-day-old testes were 0.869 and 0.529, respectively and that between 5- and 180-day-old testes was 0.580. Hierarchical clustering data revealed that gene expression patterns of pSSCs were similar to 5-day-old testis. By applying a differential expression filter of four fold or greater, 607 genes were identified between pSSCs and 5-day-old testis, and 2118 genes were identified between the 5- and 180-day-old testes. Among these differentially expressed genes, 293 genes were upregulated and 314 genes were downregulated in the 5-day-old testis compared to pSSCs, and 1106 genes were upregulated and 1012 genes were downregulated in the 180-day-old testis compared to the 5-day-old testis. The following genes upregulated in pSSCs compared to 5-day-old testes were selected for additional analysis: matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 1 (MMP1), glutathione peroxidase 1 (GPX1), chemokine receptor 1 (CCR1), insulin-like growth factor binding protein 3 (IGFBP3), CD14, CD209, and Kruppel-like factor 9 (KLF9). Expression levels of these genes were evaluated in pSSCs and in 5- and 180-day-old porcine testes. In addition, immunohistochemistry analysis confirmed their germ cell-specific expression in 5- and 180-day-old testes. These finding may not only be useful in facilitating the enrichment and sorting of porcine spermatogonia, but may also be useful in the study of the early stages of spermatogenic meiosis.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37-50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

Iron is an important trace element involved in several biological processes. The role of iron in porcine early embryonic development remains unknown. In the present study, we depleted iron (III, Fe3+) with deferoxamine (DFM), a specific Fe3+ chelator, in cultured porcine parthenotes and monitored embryonic development, apoptosis, mitochondrial membrane potential, and ATP production. Results showed biphasic function of Fe3+ in porcine embryo development. 0.5 μM DFM obviously increased blastocyst formation (57.49 ± 2.18% vs. control, 43.99 ± 1.72%, P < 0.05) via reduced (P < 0.05) production of reactive oxygen species (ROS), further increased mitochondrial membrane potential and ATP production in blastocysts (P < 0.05). 0.5 μM DFM decreased mRNA expression of Caspase 3 (Casp3) and increased Bcl-xL. However, results showed a significant reduction in blastocyst formation in the presence of 5.0 μM DFM compared with the control group (DFM, 21.62 ± 3.92% vs. control, 43.99 ± 1.73%, P < 0.05). Fe3+ depletion reduced the total (DFM, 21.10 ± 8.78 vs. control, 44.09 ± 13.65, P < 0.05) and increased apoptotic cell number (DFM, 11.10 ± 5.24 vs. control, 2.64 ± 1.43, P < 0.05) in the blastocyst. An obvious reduction in mitochondrial membrane potential and ATP level after 5.0 μM DFM treatment was observed. Co-localization between mitochondria and cytochrome c was reduced after high concentration of DFM treatment. In conclusion, Fe3+ is essential for porcine embryonic development via mitochondrial function maintenance, but redundant Fe3+ impairs the function of mitochondria.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.

Melamine is a nitrogen heterocyclic triazine compound which is widely used as an industrial chemical. Although melamine is not considered to be acutely toxic with a high LD50 in animals, food contaminated with melamine expose risks to the human health. Melamine has been reported to be responsible for the renal impairment in mammals, its toxicity on the reproductive system, however, has not been adequately assessed. In the present study, we examined the effect of melamine on the follicle development and ovary formation. The data showed that melamine increased reactive oxygen species (ROS) levels, and induced granulosa cell apoptosis as well as follicle atresia. To further analyze the mechanism by which melamine induces oxidative stress, the expression and activities of two key antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) were analyzed, and the concentration of malondialdehyde (MDA) were compared between control and melamine-treated ovaries. The result revealed that melamine changed the expression and activities of SOD and GPX in the melamine-treated mice. Therefore, we demonstrate that melamine causes damage to the ovaries via oxidative stress pathway.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P

Zinc is an extremely important trace element that plays important roles in several biological processes. However, the function of zinc in meiotic division of porcine oocytes is unknown. In this study, we investigated the role of zinc during meiotic resumption in in vitro matured porcine oocytes. During meiotic division, a massive release of zinc was observed. The level of free zinc in the cytoplasm significantly increased during maturation. Depletion of zinc using N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+ chelator, blocked meiotic resumption in a dose dependent manner. The level of phosphorylated mitogen activated protein kinase (MAPK) and p34cdc2 kinase activity were reduced when zinc was depleted. Moreover, zinc depletion reduced the levels of phosphorylated protein kinase C (PKC) substrates in a dose dependent manner. Real-time PCR analysis showed that expression of the MAPK- and maturation promoting factor related genes C-mos, CyclinB1, and Cdc2 was downregulated following zinc depletion. Treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) increased phosphorylation of PKC substrates and MAPK and increased p34cdc2 kinase activity. This rescued the meiotic arrest, even in the presence of TPEN. Activation of PKC by PMA increased the level of zinc in the cytoplasm. These data demonstrate that zinc is required for meiotic resumption in porcine oocytes, and this appears to be regulated via a PKC related pathway.

Junction-mediating and regulatory protein(JMY) is a multifunctional protein with roles in the transcriptional co-activation of p53 and the regulation of actin nucleation promoting factors and, hence, cell migration; however, its role in the maturation of porcine oocytes is unclear. In the current study, we investigated functional roles of JMY in porcine oocytes. Porcine oocytes expressed JMY mRNA and protein, and the mRNA expression level decreased during oocyte maturation. Knockdown of JMY by RNA interference decreased the rate of polar body extrusion, validating its role in the asymmetric division of porcine oocytes. JMY knockdown also down-regulated the mRNA and protein levels of actin and Arp2/3. Furthermore, JMY accumulated in the nucleus in response to DNA damage, and JMY knockdown suppressed DNA damage-mediated p53 activation. In conclusion, our results show that JMY has important roles in oocyte maturation as a regulator of actin nucleation-promoting factors and an activator of p53 during DNA damage during DNA damages in porcine oocytes.

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.

Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities. Although each component has been shown to cause liver toxicity and oxidative stress in several species, there is no evidence regarding the effect of naturally contained multiple mycotoxins on tissue toxicity and oxidative stress in vivo. In the present study, mycotoxins-contaminated maize (AF 597 µg/kg, ZEN 729 µg/kg, DON 3.1 mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice, and blood and tissue samples were collected to examine the oxidative stress related indexes. The results showed that the indexes of liver, kidney and spleen were all increased and the liver and kidney morphologies changed in the mycotoxin-treated mice. Also, the treatment resulted in the elevated glutathione peroxidase (GPx) activity and malondialdehyde (MDA) level in the serum and liver, indicating the presence of the oxidative stress. Moreover, the decrease of catalase (CAT) activity in the serum, liver and kidney as well as superoxide dismutase (SOD) activity in the liver and kidney tissue further confirmed the occurrence of oxidative stress. In conclusion, our data indicate that the naturally contained mycotoxins are toxic in vivo and able to induce the oxidant stress in the mouse.