Effect of Garlic Straw with Silage Corn Stalks on Hu Sheep Rumen Fermentation and Microbial Community In Vitro.

Garlic, an important economic crop, provides nutrient-rich straw. When appropriately balanced with silage corn stalks, it is a high-quality forage resource. However, studies on the impact of garlic straw with silage corn stalks on Hu sheep's digestive metabolism and rumen microbiota are scarce. In this study, different addition ratios of garlic straw and silage corn stalks were utilized for in vitro experiments. We designed six experimental groups (CON, G0, G20, G40, G60, G80, and G100) based on varying ratios of garlic straw to silage corn stalks. Rumen microbiota was analyzed through 16S rRNA sequencing. Nutrient composition analysis indicated that garlic straw's relative feeding value (RFV) closely resembled that of silage corn stalks. After 24 h of fermentation, dry matter digestibility and in vitro gas production significantly increased, reaching peak values at a 60% addition ratio. Furthermore, volatile fatty acids (VFAs) such as acetic, propionic, and butyric acid exhibited elevated contents, with the highest yields observed at 60% inclusion. At the genus level, Prevotella, Rikenellaceae RC9 gut group, and Succiniclasticum were identified as the dominant bacterial groups. The gas production test showed a significant decrease in the G80 group compared to others. Microbial analysis revealed a higher abundance of Prevotella in G80 compared to G20, offering valuable insights for reducing greenhouse gas emissions from ruminant animals. Finally, this study predicted the impact of garlic straw with silage corn stalks' addition on Hu sheep's metabolic pathways and biological functions of the rumen microbiota. This research highlights the potential for effectively utilizing garlic straw as a feed resource for Hu sheep and proposes a rational proportion for combining garlic straw with silage corn stalks.

Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR.

Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.

PLAG1 g.8795C>T Mutation Regulates Early Body Weight in Hu Sheep by Weakening miR-139 Binding.

Sheep birth and weaning weights indicate their growth and survival. Thus, identifying molecular genetic markers for early body weight is important in sheep breeding. Pleomorphic adenoma gene 1 (PLAG1) is important for regulating birth weight and body length in mammals; however, its relationship with sheep body weight remains unknown. Here, the 3'-untranslated region (3'-UTR) of the Hu sheep PLAG1 gene was cloned, single nucleotide polymorphisms (SNPs) were screened, genotype-early body weight relationships were analyzed, and the possible molecular mechanism was explored. PLAG1 3'-UTR sequences with five forms of base sequences plus poly(A) tails were detected in Hu sheep and the g.8795C>T mutation was identified. Luciferase reporter assay indicated that the g.8795C>T mutation influenced PLAG1 post-transcriptional activity. miRBase prediction showed that the g.8795C>T mutation was located in the miR-139 seed sequence binding region, and miR-139 overexpression significantly decreased both PLAG1-CC and PLAG1-TT activities. Moreover, the luciferase activity of PLAG1-CC was significantly lower than that of the PLAG1-TT, but miR-139 inhibition substantially increased both PLAG1-CC and PLAG1-TT luciferase activities, suggesting that PLAG1 is the target gene of miR-139. Thus, the g.8795C>T mutation upregulates PLAG1 expression by weakening its binding with miR-139, promoting PLAG1 expression, and increasing Hu sheep birth and weaning weights.

miR-27a-3p targets NR5A2 to regulate CYP19A1 expression and 17-ß estradiol synthesis in ovine granulosa cells.

Although 17-ß estradiol (E2) synthesis is important in regulating female fertility, we know little regarding the molecular mechanism of miRNA-regulated ovine E2 synthesis. Here, our experiments with granulosa cells (GCs) from Hu sheep revealed miR-27a-3p involvement in E2 synthesis and its association with ovine litter size. First, we showed that miR-27a-3p of sheep and other mammals share a high nucleotide identity. Next, gain- and loss-of-function assays indicated that miR-27a-3p inhibits CYP19A1 expression and E2 synthesis in GCs. Moreover, we demonstrated that NR5A2 is a direct target of miR-27a-3p. Ovine miR-27a-3p suppresses E2 synthesis via the NR5A2 and CYP19A1 axes. We also identified four single nucleotide polymorphisms in the ovine miR-27a gene, and g.-13 G>A and g 0.24 T > G were significantly associated with the first and the second parity litter size, respectively (P < 0.05). In summary, our findings reveal that miR-27a-3p is a novel regulator of E2 synthesis and may predict litter size of Hu sheep, providing insight into mechanisms underlying granulosa cell function and female fertility.

Bta-miR-6531 Regulates Calcium Influx in Bovine Leydig Cells and Is Associated with Sperm Motility.

MicroRNAs (miRNAs) play key roles in sperm as the regulatory factors involved in fertility and subsequent early embryonic development. Bta-miR-6531 is specifically a highly enriched miRNA in low-motility sperms in previous study. To investigate the mechanism of bta-miR-6531, 508 shared target genes of bta-miR-6531 were predicted using two miRNA target databases (TargetScan7 and miRWalk). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the calcium and cAMP signaling pathways were the most enriched of the target genes. A dual-luciferase assay indicated that bta-miR-6531 targeted ATP2A2 mRNA by binding to the coding sequence region. In bovine Leydig cells, bta-miR-6531 overexpression affected the intracellular calcium concentration by restraining ATP2A2 expression. Moreover, we observed high calcium concentrations and high ATP2A2 protein levels in high-motility sperm compared with those in low-motility sperms. Furthermore, high-linkage single-nucleotide polymorphisms (SNPs) of the pre-bta-miR-6531 sequence were identified that related to sperm traits. Genotype TCTC of bta-miR-6531 showed high sperm motility and density and low deformity rate in Holstein bulls. However, the mutation in pre-miR-6531 did not significantly affect mature bta-miR-6531 expression in the sperm or cell models. Our results demonstrate that bta-miR-6531 might involve in sperm motility regulation by targeting ATP2A2 of the calcium signaling pathway in bovine spermatozoa.

Three Complete Linkage SNPs of GDF9 Gene Affect the Litter Size Probably Mediated by OCT1 in Hu Sheep.

Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).

A T > G Mutation in the NR5A2 Gene Is Associated With Litter Size in Hu Sheep Through Upregulation of Promoter Activity by Transcription Factor MTF-1.

Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as LRH-1 or FTF, is an orphan nuclear hormone receptor that is involved in regulating embryonic development, ovarian granulosa cell differentiation, gonadal sex differentiation, and steroidogenesis in mammals. However, little is known about how NR5A2 regulates reproduction in sheep. In this study, we amplified the promoter sequence of NR5A2 and determined that its core promoter region ranged from -721 nt to -281 nt. A T > G polymorphism at -700 nt was detected in the core promoter region. Association analysis found that the litter sizes of Hu ewes at their second and average parities with genotype GG (2.20 ± 0.20 and 1.97 ± 0.06, respectively) were significantly higher than those of ewes with genotype TG (1.68 ± 0.10 and 1.74 ± 0.05, respectively) (p < 0.05) and TT (1.67 ± 0.10 and 1.62 ± 0.06, respectively) (p < 0.05). The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT (1.56 ± 0.12) (p < 0.05). A luciferase assay showed that the -700G allele increased the luciferase activity relative to the -700T allele. Furthermore, the -700T > G polymorphism created a novel binding site for metal-regulatory transcription factor 1 (MTF-1). A competitive electrophoretic mobility shift assay confirmed that MTF-1 specifically bound with the G-type promoter of NR5A2. An overexpression experiment demonstrated that MTF-1 was involved in the alteration of NR5A2 transcription activity and further increased NR5A2 gene mRNA expression. Our findings revealed that the -700T > G polymorphism promoted NR5A2 expression due to the positive effects on NR5A2 gene transcription activity by MTF-1 and thereby increased fecundity in Hu sheep.

MiR-126* is a novel functional target of transcription factor SMAD4 in ovarian granulosa cells.

Both SMAD4 and miR-126* have been proven to be involved in granulosa cell (GC) apoptosis and even follicular atresia, through commonly regulating follicle-stimulating hormone receptor (FSHR), the FSH-specific transmembrane receptor of GCs. However, the regulatory relationship between them in GCs is still unknown. In this study, we report that SMAD4 suppresses the expression of miR-126* and impairs its function in GCs of the porcine ovary by acting as a transcription factor. A classic SMAD4-binding element (SBE) site was found in the promoter of miR-126* by using in silico methods. Luciferase assay, qRT-PCR, and ChIP assay proved that SMAD4 serves as a transcriptional repressor and directly binds to SBE site within miR-126* gene promoter, which further reduces miR-126* gene expression and inhibits its transcriptional activity in GCs. Furthermore, SMAD4 also controls miR-126*-mediated expression of FSHR (a direct target of miR-126* in GCs). In addition, we prove that SMAD4 induces CYP19A1 expression (encodes aromatase, the key enzyme for oestrogen biosynthesis) and inhibits GC apoptosis through the miR-126*/FSHR axis. Taken together, our findings not only established a direct link between SMAD4 and miRNA-126*, two key factors of GC apoptosis, but also revealed an important way in which the SMAD4 regulates GC function, the miRNA-126*/FSHR axis.

Mutation -388 C>G of NR5A1 gene affects litter size and promoter activity in sheep.

The Nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene encodes a nuclear receptor that regulates the transcription of genes involved in steroidogenesis, follicular development and female fertility. Little, however, is known about the relationship of this gene with reproductive performance in sheep. In this study, the transcription initiation site of Hu sheep NR5A1 gene was located 193 nucleotides (i.e., at -193 nt) before the translational start site (ATG). The core promoter region of the NR5A1 gene ranged from -696 nt to -298 nt, and a C>G mutation at -388 nt was detected in this region. Association analysis indicated ewes with the GG genotype had greater litter size at the second and third parity than those with the CC genotype (P < 0.05). The results from the luciferase assay provided evidence that the -388 G allele increased luciferase activity compared with that of the -388 C allele. Furthermore, the -388 C>G mutation lost a CpG site and gained a novel binding site for the transcription factor, SP1, and results from an overexpression experiment and methylation analysis indicated transcription factor SP1 and methylation of the -388 C>G mutation were both involved in alteration of NR5A1 transcription activity. Results of the present study revealed that the -388 C>G mutation lost a CpG site and promoted NR5A1 gene expression, which completely superimposed positive effects on NR5A1 gene transcription activity by transcription factor SP1, resulting in a fecundity increase in Hu sheep.

Different susceptibility to porcine reproductive and respiratory syndrome virus infection among Chinese native pig breeds.

China is rich in native pig breeds, yet information regarding the susceptibility/resistance of local breeds to porcine reproductive and respiratory syndrome virus (PRRSV) infection is lacking. In the present study, an in vitro method based on assessing PRRSV replication in porcine alveolar macrophages (PAMs) was established to evaluate PRRSV susceptibility/resistance in a commercial pig breed (Landrace) and five native pig breeds from Jiangsu and Anhui provinces in China. Expression levels of cytokines (IL-8, IL-10, TNF-α and IFN-γ), Toll-like receptor 3 (TLR3), CD163 (PRRSV receptor), and sialoadhesin (Sn, PRRSV receptor) in infected pigs were determined using real-time PCR, and the association between PRRSV susceptibility/resistance and the abundance of the cytokines and receptors was investigated. The viral replication rate and titer at 0, 6, 12 18, 24 and 36 hours postinfection (hpi) were determined to assess the proliferation dynamics of PRRSV NJGC in PAMs. Based on the PRRSV proliferation dynamics, the results indicated that Dingyuan pigs were the most susceptible to PRRSV infection, whereas Jiangquhai pigs were the least susceptible to PRRSV infection among the six pig breeds tested, as indicated by measuring PRRSV replication and the viral load in PAMs. The different levels of susceptibility to PRRSV infection in PAMs may be associated with differences in the abundance of CD163 (PRRSV receptor), cytokines IL-8, IFN-γ, and TNF-α in Jiangquhai and Dingyuan pig breeds after viral inoculation.

MC1568 Enhances Histone Acetylation During Oocyte Meiosis and Improves Development of Somatic Cell Nuclear Transfer Embryos in Pig.

An increasing number of studies have revealed that histone deacetylase (HDAC) mediated histone deacetylation is important for mammalian oocyte development. However, nonselective HDAC inhibitors (HDACi) were applied in most studies; the precise functions of specific HDAC classes during meiosis are poorly defined. In this study, the class IIa-specific HDACi MC1568 was used to reveal a crucial role of class IIa HDACs in the regulation of histone deacetylation during porcine oocyte meiosis. Besides, the functions of HDACs and histone acetyltransferases in regulating the balance of histone acetylation/deacetylation were also confirmed during oocyte maturation. After the validation of nontoxicity of MC1568 in maturation rate, spindle morphology, and chromosome alignment, effects of MC1568 on developmental competence of porcine somatic cell nuclear transfer (SCNT) embryos were evaluated, and data indicated that treatment with 10 µM MC1568 for 12 hours following electrical activation significantly enhanced the blastocyst rate and cell numbers. Moreover, results showed that optimal MC1568 treatment increased the H4K12 acetylation level in SCNT one cells and two cells. In addition, MC1568 treatment stimulated expression of the development-related genes OCT4, CDX2, SOX2, and NANOG in SCNT blastocysts. Collectively, our investigation uncovered a critical role of class IIa HDACs in the regulation of histone deacetylation during oocyte meiosis. Furthermore, for the first time, we showed that MC1568 can improve the in vitro development of porcine SCNT embryos. These findings provide an alternative HDACi for improving animal cloning efficiency and may shed more light on nuclear reprogramming.

Exploration of mtDNA control region sequences in Chinese Tibetan Mastiffs.

The control region of mitochondrial DNA (mtDNA) was obtained from 40 purebred Chinese Tibetan Mastiffs (TMs). Sequence structure and genetic diversity were analyzed, and a phylogenetic tree was constructed. The TM mtDNA control region was composed of ETAS (extended termination associated sequences), CD (a central domain) and CSBs (conserved sequenced blocks) and sequence length showed some diversity, which was mainly caused by the number of 10 nucleotide repeat units [5'-GTA CAC GT (G/A) C-3'] between CSB I and CSB II, which ranged from 27 to 35 among individuals. Seventy-five polymorphic sites were identified, which defined 37 haplotypes; the haplotype diversity was 0.990, and the nucleotide diversity was 1.201. Based on the control region sequences, Chinese TMs were divided into three categories, which were consistent with the origin and geographical classification of TMs. Phylogenetic analysis of 538-bp HVR-I sequences revealed that TMs were most closely related to Labrador Retrievers.

Effect of inhibin gene immunization on antibody production and reproductive performance in Partridge Shank hens.

To investigate the effect of inhibin gene immunization on antibody production and reproductive performance in broiler breeder females, Partridge Shank hens aged 380 days were immunized with inhibin recombinant plasmid pcISI. One hundred and twenty hens were randomly assigned to four groups and treated intramuscularly with 25, 75, or 125 µg/300-µL inhibin recombinant plasmid pcISI (T1∼T3) or 300-µL saline as control (C), respectively. Booster immunization was given with the same dosage 20 days later. Blood and egg samples were collected to detect the antibody against inhibin by enzyme-linked immunosorbent assay and to evaluate egg performance. The ovaries were collected to classify the follicles and detect the FSH receptor (FSHR) messenger RNA (mRNA) expression by reverse transcription-PCR. The results showed that immunization against pcISI could elicit antibody against inhibin in both plasma and egg yolk compared with the control (P < 0.05), whereas booster immunization did not increase the antibody level in plasma. Vaccination promoted egg lay during the first 30 days after primary vaccination (P < 0.05) with no effect on egg quality and hatching rate. Immunization increased the amounts of dominant, small yellow and large white follicles in the ovary (P < 0.05). Reverse transcription-PCR results showed that immunization increased the FSHR mRNA in the large white follicles, whereas decreased the FSHR mRNA in the small yellow follicles (P < 0.05). In conclusion, inhibin vaccine pcISI can stimulate the production of antibody against inhibin as well as the follicle development and egg laying performance in Partridge Shank hens, which provides a good foundation for the application of inhibin DNA vaccine in avian production.

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat ß-lactoglobulin (ßLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

[Single nuclear polymorphisms in exon 3 of POMC gene and the association with growth traits in Hu sheep and East Friesian x Hu crossbred sheep].

Pro-opiomelancortin (POMC) plays important roles in the regulation of food intake and energy expenditure. The sheep exon 3 of gene POMC was amplified and sequenced by screening the DNA pools to select single nuclear polymorphisms and analyze the association with the growth traits. Two silent SNP mutations (g.273 T/C and g.456 G/A) in Hu sheep were identified. PCR-restriction fragment length polymorphism (RFLP) was used to test the g.273 T/C and the association between the g.273 T/C polymorphism and some growth traits was analyzed in Hu sheep (n = 162) and East Friesian x Hu crossbred sheep (n=130). The results showed that three genotypes, TT, TC and CC, were detected in Hu sheep with the frequencies of 0.469, 0.438 and 0.093, respectively. Two genotypes, TT and TC, were detected in East Friesian x Hu crossbred sheep with the frequencies of 0.754 and 0.246, respectively. The association analysis showed that in Hu sheep the two-month weaning weight, four-month rump height of genotype CC and the four-month body length, cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the four- and six-month weight of genotype CC were significantly higher than those of genotypes TT and TC (P < 0.01); the four-month body height and body length of genotype CC were significantly higher than those of genotypes TT (P < 0.01) and TC (P < 0.05); the four-month cannon circumference of CC genotype was significantly higher than that of TT genotype (P < 0.01). In East Friesian x Hu crossbred sheep the two-month weaning weight, four-month weight, body height, body length, chest depth and cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the six-month weight of genotype TC was significantly higher than that of genotype CC (P < 0.01). In conclusion, the exon 3 of gene POMC was associated with growth traits, and C allele was beneficial to the increase of body weight and body size traits of sheep, which potentially afford a good foundation for further study on POMC gene as aided breeding markers for growth traits in sheep.

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats.

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n=18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 µg (T1, T2 and T3, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P<0.05) in T3 than in either T1 or T2 (0.341±0.123, 0.236±0.068, and 0.251±0.077, respectively, mean±SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P<0.05) in T3, and were higher (P<0.05) in T1 and T2 than in control groups. For antibody-positive rats, there was a correlation (P<0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T3 group (46.00±4.65) was higher (P<0.05) than in two control groups (29.25±3.72 and 27.92±3.48), and also higher (P<0.05) than in T1 and T2 (37.17±4.99 and 38.75±7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75±4.23 vs. 35.60±3.38, P<0.05). Moreover, litter size and number of placentas were increased (P<0.05) in the pcISI immunization groups, except for the T1 group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 µg yielded the best immune response.

VEGF promotes the transcription of the human PRL-3 gene in HUVEC through transcription factor MEF2C.

Phosphatase of regenerating liver 3 (PRL-3) is known to be overexpressed in many tumors, and its transcript level is high in the vasculature and endothelial cells of malignant tumor tissue. However, the mechanism(s) underlying its enhanced expression and its function in endothelial cells remain unknown. Here, we report that vascular endothelial growth factor (VEGF) can induce PRL-3 transcription in human umbilical vein endothelial cells (HUVEC). An analysis of its 5'UTR revealed that PRL-3 transcription is initiated from two distinct sites, which results in the formation of the two transcripts, PRL-3-iso1 and PRL-3-iso2, but only the latter is up-regulated in HUVEC by VEGF. The PRL-3-iso2 promoter region includes two functional MEF2 (myocyte enhancer factor2) binding sites. The over-expression of the constitutively active form of MEF2C promotes the abundance of the PRL-3-iso2 transcript in a number of human cell lines. The siRNA-induced knockdown of MEF2C abolished the stimulative effect of VEGF on PRL-3 transcript in HUVEC, indicating that the VEGF-induced promotion of PRL-3 expression requires the presence of MEF2C. Finally, blocking PRL-3 activity or expression suppresses tube formation by HUVEC. We suggest that PRL-3 functions downstream of the VEGF/MEF2C pathway in endothelial cells and may play an important role in tumor angiogenesis.

Phosphatase of regenerating liver-3 localizes to cyto-membrane and is required for B16F1 melanoma cell metastasis in vitro and in vivo.

BACKGROUND: Phosphatase of regenerating liver-3 (PRL-3) is a member of the novel phosphatases of regenerating liver family, characterized by one protein tyrosine phosphatase active domain and a C-terminal prenylation (CCVM) motif. Though widely proposed to facilitate metastasis in many cancer types, PRL-3's cellular localization and the function of its CCVM motif in metastatic process remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, a series of Myc tagged PRL-3 wild type or mutant plasmids were expressed in B16F1 melanoma cells to investigate the relationship between PRL-3's cellular localization and metastasis. With immuno-fluorescence microcopy and cell adhesion/migration assay in vitro, and an experimental passive metastasis model in vivo, we found that CCVM motif is critical for the localization of PRL-3 on cell plasma membrane and the lung metastasis of melanoma. In particular, Cystine170 is the key site for prenylation in this process. CONCLUSIONS/SIGNIFICANCE: These results suggest that cellular localization of PRL-3 is highly correlated with its function in tumor metastasis, and inhibition of PRL-3 prenylation might be a new approach to cancer therapy.

[Subchronic exposure to acrylamide affects reproduction and testis endocrine function of rats].

OBJECTIVE: To explore the effect of subchronic exposure to acrylamide on the reproduction and testis endocrine function of rats. METHODS: Forty healthy adult male SD rats were randomly divided into 4 groups of equal number, exposed to acrylamide at the dose of 0, 4, 10 and 18 mg/(kg x d) respectively for 9 weeks, and then subjected to the determination of the hindlimb landing foot splay, sperm vitality and morphology, the activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) in the testis homogenate, and the levels of testosterone (T) and estradiol (E2) in the serum and testis homogenate. Based on the primary Leydig cell culture models exposed to acrylamide of 0, 0.1, 0.75, 4 and 8 mmol/L, the activity of Leydig cells was measured by the CCK-8 method. RESULTS: Following acrylamide exposure, the hindlimb landing foot splay increased markedly with dose increase (P < 0.01). The rates of sperm vitality were (6.86 +/- 5.46)%, (65.43 +/- 5.16)%, (60.86 +/- 4.26)% and (46.86 +/- 2.73)% in the exposed groups, significantly lower than in the control (P < 0.01); the rates of abnormal sperm were (39.00 +/- 10.95)%, (35.43 +/- 7.54)%, (45.71 +/- 13.28)% and (56.71 +/- 17.01)%, significantly increased in the 10 and 18 mg/(kg x d) groups (P < 0.05); ACP activities were (82.93 +/- 11.05), (73.52 +/- 8.77), (77.67 +/- 3.04) and (68.56 +/- 3.09) U/g prot, showing a decreasing tendency, while ALP activities were (0.96 +/- 0.15), (1.07 +/- 0.22), (1.12 +/- 0.22) and (0.74 +/- 0.10) U/g prot, displaying a tendency of first increasing and then decreasing. Both ACP and ALP activities were inhibited significantly in the 18 mg/(kg x d) group as compared with the control (P < 0.05). A marked reduction was noted in T levels in the serum, (13.44 +/- 4.76), (7.69 +/- 3.84), (5.23 +/- 1.42) and (1.36 +/- 0.86) ng/ml, as well as in the testis homogenate, (4.95 +/- 1.64), (3.01 +/- 0.76), (2.44 +/- 0.91) and (0.85 +/- 0.49) ng/mg prot, (P < 0.01), but no significant changes were observed in 17beta-E2 levels. After 24 hours exposure to acrylamide, the optical densities were 0.82 +/- 0.06, 0.56 +/- 0.07, 0.44 +/- 0.06, 0.26 +/- 0.03 and 0.45 +/- 0.21, showing an evident inhibition of the activity of Leydig cells at the dose of 0.1, 0.75, 4 and 8 mmol/L (P < 0.01). CONCLUSION: Subchronic exposure to acrylamide could affect the normal development of sperm, cause changes of the activity of some enzymes in the testis and significantly influence hindlimb motor coordination. Acrylamide directly damages Leydig cells and affects the endocrine function of the testis.

Construction and evaluation of the eukaryotic expression plasmid encoding two copies of somatostatin genes fused with hepatitis B surface antigen gene S.

The aim of current study was to evaluate the prospects of somatostatin DNA vaccine. Two copies of somatostatin (SS) genes were fused with the hepatitis B surface antigen (HBsAg) S gene using genetic engineering methods, the identified recombinant plasmid designated as pcS/2SS was transfected into HeLa cells to detect expression and antigenicity of target fusion protein, and its immunoreaction as well as safety was evaluated with animal experiments. The expressed target protein had a specific reaction with somatostatin antibody and showed a single strip result. A single injection of this vector stimulated long-term antigen-specific antibody responses in rats, and peak antibody levels occurred at the 2nd week of the initial injection. Additionally, the 50 microg immunized group resulted in a 13.5% increase in growth rate as compared with control group (111.7 g vs. 98.4 g). The genomic DNA was assayed for integrated plasmid using a sensitive PCR method, and the risk of mutation due to integration of pcS/2SS plasmid following intramuscular injection in mice was negligible. The successful construction of pcS/2SS DNA vaccine with good immunogenicity and safety has prospects to promote growth of animals.