Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

Different levels of autophagy induced by transient serum starvation regulate metabolism and differentiation of porcine skeletal muscle satellite cells.

This study aimed to investigate the effects of different levels of autophagy induced by transient serum starvation on the metabolism, lipid metabolism, and differentiation of porcine skeletal muscle satellite cells (SMSCs) to preliminary elucidate the role and function of autophagy in the regulatory network of skeletal muscle development. Different levels of autophagy were induced by controlling the serum concentration in the culture system for 24 h. Apoptosis, membrane potential, reactive oxygen species (ROS), ATP, and myogenic and lipogenic differentiation markers were monitored to determine if autophagy affected the metabolism and differentiation of SMSCs. Autophagy was induced in SMSCs via serum starvation (5%, 15%), as evidenced by decreased p62 and mTOR phosphorylation levels and increased LC3B lipidation and AMPK phosphorylation levels. Transmission electron microscopy revealed the presence of autophagosomes, and the rates of morphologically abnormal nuclei and mitochondria gradually increased with the decrease in serum concentration, the number of autophagic lysosomes also increased, indicating that 5% serum starvation induced severe autophagy, while 15% serum starvation induced mild autophagy. Compared with the control group and 15% serum-starved SMSCs, SMSCs undergoing 5% serum starvation had the highest intracellular ATP and ROS levels, the highest percentage of apoptotic cells, and the lowest membrane potential. The 15% serum-starved SMSCs had the highest membrane potential, but the percentage of apoptotic cells did not change significantly compared with the control group. The levels of the myogenic markers MyoD1 and MHC were significantly higher in 15% serum-starved SMSCs than in serum-sufficient SMSCs and the lowest in the 5% serum-starved SMSCs. The lipid contents (measured by Oil Red O staining and quantification of triglycerides) and lipogenic markers Peroxisome Proliferators-activated Receptors γ and Lipoprotein Lipase were also significantly higher in SMSCs undergoing 15% serum starvation than in the control group, and the lowest in the 5% serum-starved SMSCs. Different levels of starvation stress induce different levels of autophagy. Mild autophagy induced by moderate serum starvation promotes the metabolism and differentiation of SMSCs, while severe autophagy renders SMSCs more apoptotic, abnormal metabolism and suppresses SMSC differentiation into adipocytes or myocytes, and reduces lipid metabolisms. Our study suggests that autophagy plays a role in skeletal muscle development and may help design strategies for improving meat production traits in domestic pigs.

Antioxidants and Oxidants in Boar Spermatozoa and Their Surrounding Environment Are Associated with AMPK Activation during Liquid Storage.

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 µM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm's total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Effect of the Porcine STC-1 Gene on Autophagy and Mitochondrial Function as Induced by Serum Starvation.

Stanniocalcin-1 (STC-1) is a glycoprotein hormone involved in calcium/phosphorus metabolism and direct inhibition of bone and muscle growth. The aim of this study was to investigate the STC-1 gene with respect to the regulatory mechanisms of porcine growth metabolic pathways involving autophagy. Western blotting was used to detect the expression of autophagy and mitochondrial function-related proteins, and flow cytometry was used to detect mitochondrial function-related. Changes in the autophagosome and mitochondrial were observed by electron microscopy. The expression of the autophagy-related proteins was detected by confocal microscopy. The results showed that Pink1, Parkin and LC3B expression was increased; SQSTM1/P62 expression was reduced. Electron microscopy revealed that the cells in the serum starvation group all produced autophagosomes. The fluorescence intensity of GFP-LC3B and GFP-Parkin increased. The Bax/Bcl-2 ratio, Pink1 and Parkin protein levels were profoundly reduced in the STC-KO. In addition, the increase in Mfn2, OPA1, DRP1 and LC3B proteins was attenuated; the increase in the apoptosis rate and amount of active oxygen was attenuated; the decrease in membrane potential; the decrease in ATP was reversed; the fluorescence intensity of GFP-LC3B and GFP-Parkin was increased. These results indicate that autophagy can be caused by serum starvation. Knocking out the porcine STC-1 gene had an obvious antiapoptotic effect on cells, the inhibition of serum starvation-induced autophagy. This is the first study to show that the porcine STC-1 gene confers self-protection in the absence of nutrients. To provide a theoretical basis for studying the effect of STC-1 on pig growth and development.

The effect of the PLIN1 gene on the metabolism and mitochondria of porcine skeletal muscle satellite cells.

BACKGROUND: Perilipin 1 (PLIN1) is a lipid droplet scaffolding protein that plays a regulatory role in fat decomposition and mitochondrial function. OBJECTIVE: In this study, the effects of PLIN1 gene knockout (PLIN1-KO) and PLIN1 gene overexpression (PLIN1-EX) on cell metabolism and mitochondrial function in porcine skeletal muscle satellite cells were assessed. METHODS: Porcine skeletal muscle satellite cells were used as the control group (NC). The expression of mitochondrial function-related proteins was detected by western blot. Apoptosis, cell cycle, mitochondrial function-related indices, mitochondrial structure, and morphology were measured by flow cytometry. RESULTS: Our results demonstrated that stable expression of the PLIN1 gene in skeletal muscle satellite cells is critical to maintaining cell metabolism and mitochondrial function. After knockout and overexpression of the PLIN1 gene, the anti-apoptotic ability of cells was enhanced, and the metabolic activity of the cells was accelerated, but at the cost of mitochondrial structural damage, reduction in the number of mitochondria, and decreased mitochondrial function. CONCLUSION: This study explored the effect of the PLIN1 gene on the mitochondria and metabolism of porcine skeletal muscle satellite cells and provided a theoretical basis for the subsequent study of the effects of PLIN1 on muscle tissue development and meat quality.

Effects of porcine STC-1 on cell metabolism and mitochondrial function.

Stanniocalcin (STC-1), a kind of glycoprotein hormone, was first found in fish and mainly regulates calcium/phosphorus metabolism in the body. To explore the biological function of the porcine STC-1 gene, the effects of changes in stanniocalcin expression on cellular metabolism and mitochondrial function were studied. A vector overexpressing the STC-1 gene and an siRNA silencer of the STC-1 gene were transfected into porcine kidney epithelial PK15 cells. After the STC-1 gene expression level was induced to change, STC-1 protein- and mitochondrial function-related proteins such as PMP70, OPA, DRP, Mfn and STC-1-related acetylated protein were detected by Western blotting. Cell apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP were detected using flow cytometry methods. Transmission electron microscopy was used to observe the changes in mitochondrial structure and morphology. The results showed that overexpression of the STC-1 gene could significantly upregulate the levels of PMP70, OPA, DRP and Mfn. STC-1 gene expression, which could decrease the apoptosis rate and reactive oxygen species production to significantly increase the cell membrane potential and reduce the formation of intracellular ATP, which also affected the morphology and number of mitochondria. The results were reversed when the STC-1 gene expression was silenced. The results suggested that the porcine STC-1 gene is closely related to cell growth metabolism and mitochondrial function, which influence the mitochondrial function-related proteins. The present study is useful for further understanding STC-1 gene function and provides a theoretical basis for improving the production characteristics of domestic pigs.

MicroRNA-378 promotes myogenic differentiation by targeting BMP4.

MicroRNA-378 (miRNA-378) has been reported to have a crucial role in skeletal muscle differentiation; however, the underlying mechanisms have largely remained to be elucidated. The present study employed high­throughput RNA sequencing to investigate the transcriptome following transfection of miRNA­378 mimics or control RNAs into C2C12 myoblast cells. By sequencing and annotation, 2,802 transcripts that were changed by >1.5 fold were obtained and then subjected to signaling pathway enrichment and gene ontology analysis. Eight genes associated with development were subsequently selected for validation by quantitative qPCR, the results of which were highly consistent with those of the high­throughput RNA sequencing. The protein levels of bone morphogenetic protein 4 (BMP4), which was among the differentially expressed genes, were decreased following ectopic expression of miRNA­378. BMP4 was further confirmed to be a direct target of miRNA­378 by using a dual luciferase assay. Finally, treatment with miRNA­378 or small interfering RNA against BMP4 induced myogenic differentiation in C2C12 cells. In conclusion, the present study suggested that miRNA­378 is critical for the promotion of myoblast differentiation by targeting BMP4.

Stanniocalcin-1 inhibits thrombin-induced signaling and protects from bleomycin-induced lung injury.

Thrombin-induced and proteinase-activated receptor 1 (PAR1)-mediated signaling increases ROS production, activates ERK, and promotes inflammation and fibroblast proliferation in bleomycin-induced lung injury. Stanniocalcin-1 (STC1) activates anti-oxidant pathways, inhibits inflammation and provides cytoprotection; hence, we hypothesized that STC1 will inhibit thrombin/PAR1 signaling and protect from bleomycin-induced pneumonitis. We determined thrombin level and activity, thrombin-induced PAR-1-mediated signaling, superoxide generation and lung pathology after intra-tracheal administration of bleomycin to WT and STC1 Tg mice. Lungs of bleomycin-treated WT mice display: severe pneumonitis; increased generation of superoxide; vascular leak; increased thrombin protein abundance and activity; activation of ERK; greater cytokine/chemokine release and infiltration with T-cells and macrophages. Lungs of STC1 Tg mice displayed none of the above changes. Mechanistic analysis in cultured pulmonary epithelial cells (A549) suggests that STC1 inhibits thrombin-induced and PAR1-mediated ERK activation through suppression of superoxide. In conclusion, STC1 blunts bleomycin-induced rise in thrombin protein and activity, diminishes thrombin-induced signaling through PAR1 to ERK, and inhibits bleomycin-induced pneumonitis. Moreover, our study identifies a new set of cytokines/chemokines, which play a role in the pathogenesis of bleomycin-induced lung injury. These findings broaden the array of potential therapeutic targets for the treatment of lung diseases characterized by thrombin activation, oxidant stress and inflammation.

Severe Nephrotoxic Nephritis following Conditional and Kidney-Specific Knockdown of Stanniocalcin-1.

BACKGROUND: Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1. METHODS: We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis. RESULTS: Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C3 and IgG in the kidney, and antibody response to sheep IgG were equal. CONCLUSIONS: nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium.

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production.

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased.

Role of microRNAs in skeletal muscle development and rhabdomyosarcoma (review).

Skeletal muscle accounts for ~40% of total body mass. The principle functions of skeletal muscle include supporting the body structure, controlling motor movements and storing energy. Rhabdomyosarcoma (RMS) is a skeletal muscle­derived soft tissue tumor widely occurring in the pediatric population. In previous years, microRNAs (miRNAs) have been demonstrated to be important in skeletal muscle development, function and the pathogenesis of various diseases, including RMS. The present review provided an overview of current knowledge on the muscle­specific and ubiquitously­expressed miRNAs involved in skeletal muscle differentiation and their dysregulation in RMS. Additionally, the potential use and challenges of miRNAs as therapeutic targets in this soft­tissue sarcoma were examined and the future prospects for miRNAs in muscle biology and muscle disorders were discussed.

Stanniocalcin-1 inhibits renal ischemia/reperfusion injury via an AMP-activated protein kinase-dependent pathway.

AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgenic overexpression of STC1 inhibits reactive oxygen species and protects from ischemia/reperfusion (I/R) kidney injury. Our observations revealed high AMP-activated protein kinase (AMPK) activity in STC1 transgenic kidneys relative to wild-type (WT) kidneys; thus, we hypothesized that STC1 protects from I/R kidney injury through activation of AMPK. Baseline activity of AMPK in the kidney correlated with the expression of STCs, such that the highest activity was observed in STC1 transgenic mice followed (in decreasing order) by WT, STC1 knockout, and STC1/STC2 double-knockout mice. I/R in WT kidneys increased AMPK activity and the expression of STC1, UCP2, and sirtuin 3. Inhibition of AMPK by administration of compound C before I/R abolished the activation of AMPK, diminished the expression of UCP2 and sirtuin 3, and aggravated kidney injury but did not affect STC1 expression. Treatment of cultured HEK cells with recombinant STC1 activated AMPK and increased the expression of UCP2 and sirtuin 3, and concomitant treatment with compound C abolished these responses. STC1 knockout mice displayed high susceptibility to I/R, whereas pretreatment of STC1 transgenic mice with compound C restored the susceptibility to I/R kidney injury. These data suggest that STC1 is important for activation of AMPK in the kidney, which mediates STC1-induced expression of UCP2 and sirtuin 3 and protection from I/R.

AKI after conditional and kidney-specific knockdown of stanniocalcin-1.

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre-transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions.

Discovery of MicroRNAs associated with myogenesis by deep sequencing of serial developmental skeletal muscles in pigs.

MicroRNAs (miRNAs) are short, single-stranded non-coding RNAs that repress their target genes by binding their 3' UTRs. These RNAs play critical roles in myogenesis. To gain knowledge about miRNAs involved in the regulation of myogenesis, porcine longissimus muscles were collected from 18 developmental stages (33-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100- and 105-day post-gestation fetuses, 0 and 10-day postnatal piglets and adult pigs) to identify miRNAs using Solexa sequencing technology. We detected 197 known miRNAs and 78 novel miRNAs according to comparison with known miRNAs in the miRBase (release 17.0) database. Moreover, variations in sequence length and single nucleotide polymorphisms were also observed in 110 known miRNAs. Expression analysis of the 11 most abundant miRNAs were conducted using quantitative PCR (qPCR) in eleven tissues (longissimus muscles, leg muscles, heart, liver, spleen, lung, kidney, stomach, small intestine and colon), and the results revealed that ssc-miR-378, ssc-miR-1 and ssc-miR-206 were abundantly expressed in skeletal muscles. During skeletal muscle development, the expression level of ssc-miR-378 was low at 33 days post-coitus (dpc), increased at 65 and 90 dpc, peaked at postnatal day 0, and finally declined and maintained a comparatively stable level. This expression profile suggested that ssc-miR-378 was a new candidate miRNA for myogenesis and participated in skeletal muscle development in pigs. Target prediction and KEGG pathway analysis suggested that bone morphogenetic protein 2 (BMP2) and mitogen-activated protein kinase 1 (MAPK1), both of which were relevant to proliferation and differentiation, might be the potential targets of miR-378. Luciferase activities of report vectors containing the 3'UTR of porcine BMP2 or MAPK1 were downregulated by miR-378, which suggested that miR-378 probably regulated myogenesis though the regulation of these two genes.

Identification and tissue distribution of a novel rat glucocorticoid receptor splice variant.

Glucocorticoid receptor (GR) is a steroid hormone receptor that has been shown to play important roles in diverse cellular and physiological processes. More and more evidence has revealed that the effects of glucocorticoids are mediated by the glucocorticoid receptor through genomic or nongenomic mechanisms. A growing number of glucocorticoid receptor splice variants have been identified in human tissues, but few are known in rat tissues. In this work, a novel rGR cDNA, called rGRbeta, was cloned from Sprague Dawlay (SD) rat liver. Sequence analysis revealed that the rGRbeta mRNA was 39 base pairs (bp) shorter than the rGR mRNA reported earlier. The deleted segment is located in exon 1 and encodes 13 repeated glutamine residues. Both the rGR and rGRbeta mRNAs were quantitated by Northern blot hybridization using non-homologous glucocorticoid cDNA probes. Results showed that the rGR and rGRbeta mRNAs were most abundant in the lung, the least abundant in the heart, and there were more rGR and rGRbeta mRNAs in the kidney than in the liver. The identification of rGRbeta may contribute to the understanding of the genomic or nongenomic effects of glucocorticoids.

[Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice].

OBJECTIVE: To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. METHODS: A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. RESULTS: PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. CONCLUSION: HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.

[Construction and identification of HBD-2 transgenic mice].

Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.

[Cloned goats produced from the somatic cells of an adult transgenic goat].

This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.