Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1.

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.

[Functional haplotypes of INCENP affect promoter activity and bovine semen quality].

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 G>T, rs 136823901 and g.-692 C>T, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P<0.05) at the SNP site g.-556 G>T, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P<0.05). To further study the possible mechanisms by which g.-556 G>T and g.-692 C>T affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

A comprehensive microRNA expression profile of the backfat tissue from castrated and intact full-sib pair male pigs.

BACKGROUND: It is widely known that castration has a significant effect on the accumulation of adipose tissue. microRNAs (miRNAs) are known to be involved in fat deposition and to be regulated by the androgen-induced androgen receptor (AR). However, there is little understanding of the relationship between miRNAs and fat deposition after castration. In this study, the high-throughput SOLiD sequencing approach was used to identify and characterize miRNA expression in backfat from intact and castrated full-sib male 23-week-old pigs. The patterns of adipogenesis and fat deposition were compared between castrated and intact male pigs. RESULTS: A total of 366 unique miRNA genes were identified, comprising 174 known pre-miRNAs and 192 novel pre-miRNAs. One hundred and sixty-seven pre-miRNAs were common to both castrated (F3) and intact (F4) male pig small RNA libraries. The novel pre-miRNAs encoded 153 miRNAs/miRNA*s and 141 miRNAs/miRNA*s in the F3 and F4 libraries, respectively. One hundred and seventy-seven miRNAs, including 45 up- and 132 down-regulated, had more than 2-fold differential expression between the castrated and intact male pigs (p-value < 0.001). Thirty-five miRNAs were further selected, based on the expression abundance and differentiation between the two libraries, to predict their targets in KEGG pathways. KEGG pathway analyses suggested that miRNAs differentially expressed between the castrated and intact male pigs are involved in proliferation, apoptosis, differentiation, migration, adipose tissue development and other important biological processes. The expression patterns of eight arbitrarily selected miRNAs were validated by stem-loop reverse-transcription quantitative polymerase chain reaction. These data confirmed the expression tendency observed with SOLiD sequencing. miRNA isomiRs and mirtrons were also investigated in this study. Mirtrons are a recently described category of miRNA relying on splicing rather than processing by the microprocessor complex to generate the RNAi pathway. The functions of miRNAs important for regulating fat deposition were also investigated in this study. CONCLUSIONS: This study expands the number of fat-deposition-related miRNAs in pig. The results also indicate that castration can significantly affect the expression patterns of fat-related miRNAs. The differentially expressed miRNAs may play important roles in fat deposition after castration.

[Effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGFb3) mRNA expression and promoter activity in hepatic stellate cells].

OBJECTIVE: To investigate the effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGF b3) mRNA expression and promoter activity in hepatic stellate cells (HSCs). METHODS: Freshly isolated HSCs from rats were divided into six groups: CREB-1 expression plasmid transfected group (C), siRNA-CREB-1 plasmid transfected group (S), negative control group (N), forskolin treated group (F), H-89 treated group (H), and blank group (B). Rats in each group were further sub-divided according to whether (+) or not (-) they were exposed to exogenous TGF b3. TGF b3 mRNA expression was measured by real time quantitative PCR. HSCs of the C, S, N, F, H and B groups were transfected with the TGF b3 promoter luciferase reporter plasmid (PGL3-TGF b3-P; W group), the TGF b3 promoter luciferase reporter plasmid with CRE mutation (PGL3-basic-TGF b3P-mCRE; M group) and the renilla luciferase control plasmid (pRL-SV40; control group). TGF b3 promoter activity was assessed by luciferase reporter assays. RESULTS: Compared to N(-), the TGF b3 mRNA expression was reduced to 0.69+/-0.15 in S(-) (P less than 0.05) and increased to 4.68+/-2.76 in C(-) (P more than 0.05). Compared to B(-), the TGF b3 mRNA expression was reduced to 0.57+/-0.08 in H(-) (P less than 0.05). The differences between N(+) and N(-), S(+) and S(-), B(+) and B(-), and H(+) and H(-) were all significant (P less than 0.05). The values of TGF b3 promoter activity in S(W), N(W), and C(W) were 0.062+/-0.013, 0.122+/-0.011, and 0.165+/-0.016 (P less than 0.05), but the changes of TGF b3 promoter activity in S(M), N(M), and C(M) were not significant (P more than 0.05). The values of TGF b3 promoter activity in H(W), B(W), and F(W) were 0.154+/-0.010, 0.188+/-0.016, and 0.276+/-0.031 (P less than 0.05), but the changes of TGF b3 promoter activity in H(M), B(M), and F(M) were not significant (P more than 0.05). CONCLUSION: Increased levels of CREB-1 mRNA or p-CREB-1 up-regulate the TGF b3 mRNA expression and promoter activity in rat HSCs. The CRE site in the TGF b3 promoter is critical for this effect, and the gene's activity becomes significantly decreased when the site is missing. Exogenous TGF b3 enhances expression of endogenous TGF b3 in rat HSCs.

Effects of p-CREB-1 on transforming growth factor-ß3 auto-regulation in hepatic stellate cells.

Previous studies have demonstrated that transforming growth factor-ß3 (TGF-ß3) protected liver against fibrosis in vivo and vitro, but its regulation is poorly understood. In addition, the cAMP-responsive element (CRE) in TGF-ß3 promoter is recognized as an important regulatory site for TGF-ß3 auto-regulation. Thus, we hypothesize that transcription factor CRE-binding protein-1 (CREB-1) regulates the auto-induction of TGF-ß3 in hepatic stellate cells (HSCs). We used exogenous TGF-ß3 to activate the signal pathway of TGF-ß3 auto-regulation in HSCs, results indicated that exogenous TGF-ß3 could up-regulate the protein and mRNA expressions of TGF-ß3, and provoke the phosphorylation of CREB-1 on Ser-133, besides, it could induce the DNA binding activity of p-CREB-1 and activate TGF-ß3 promoter as well. Additionally, we used pGenesil-1.1-shRNA-CREB-1 and pRSV-CREB-1 expression vector to silence and up-regulate CREB-1 gene expression respectively, and the results indicated that inhibition of CREB-1 suppressed exogenous TGF-ß3 stimulation of TGF-ß3 mRNA and protein expressions in HSCs, whereas up-regulation of CREB-1 induced this stimulation. Our results indicate that exogenous TGF-ß3 up-regulates the activity of TGF-ß3 promoter by activating CREB-1, then induces the mRNA and protein expressions of TGF-ß3. Especially, p-CREB-1 is a critical transcription factor in mediating TGF-ß3 auto-induction.

Oxalate impairs aminophospholipid translocase activity in renal epithelial cells via oxidative stress: implications for calcium oxalate urolithiasis.

PURPOSE: We evaluated the possible involvement of phospholipid transporters and reactive oxygen species in the oxalate induced redistribution of renal epithelial cell phosphatidylserine. MATERIALS AND METHODS: Madin-Darby canine kidney cells were labeled with the fluorescent phospholipid NBD-PS in the inner or outer leaflet of the plasma membrane and then exposed to oxalate in the presence or absence of antioxidant. This probe was tracked using a fluorescent quenching assay to assess the bidirectional transmembrane movement of phosphatidylserine. Surface expressed phosphatidylserine was detected by annexin V binding assay. The cell permeable fluorogenic probe DCFH-DA was used to measure the intracellular reactive oxygen species level. RESULTS: Oxalate produced a time and concentration dependent increase in phosphatidylserine, which may have resulted from impaired aminophospholipid translocase mediated, inward directed phosphatidylserine transport and from enhanced phosphatidylserine outward transport. Adding the antioxidant N-acetyl-L-cysteine significantly attenuated phosphatidylserine externalization by effectively rescuing aminophospholipid translocase activity. CONCLUSIONS: To our knowledge our findings are the first to show that oxalate induced increased reactive oxygen species generation impairs aminophospholipid translocase activity and decreased aminophospholipid translocase activity has a role in hyperoxaluria promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

[Effects of exogenous transforming growth factor-ß3 on the activities of its promoter and cAMP-responsive element binding protein-1 in rat hepatic stellate cell].

OBJECTIVE: To explore the effects of exogenous transforming growth factor-ß3 (TGF-ß3) on the activities of its promoter and cAMP-responsive element binding protein-1 (CREB-1) in rat hepatic stellate cell (HSC-T6). METHODS: HSC-T6 was cultured and treated with or without exogenous TGF-ß3 (10 µg/L). Then cell extracts, total RNA and nuclear proteins were collected at different time points. The specimens were detected by luciferase reporter assay, Western blotting and real-time RT-PCR (reverse transcription-polymerase chain reaction) respectively. RESULTS: After treatment, the activity of TGF-ß3 promoter peaked at 24 h (10.68 ± 0.57 vs 4.83 ± 0.56, 2.2 folds vs control). And the mutational CRE site completely blocked the activity of TGF-ß3 promoter (0.73 ± 0.03, P < 0.05). In addition, exogenous TGF-ß3 increased the expression of phospho-CREB-1 in a time-dependent manner. It peaked at 1 h (2.0 folds vs control) and declined slowly. And exogenous TGF-ß3 had no effect on the mRNA and protein expressions of CREB-1 (P > 0.05). CONCLUSION: The activity of TGF-ß3 promoter is up-regulated by exogenous TGF-ß3. And CRE site in TGF-ß3 promoter region is important for the transcription of TGF-ß3 gene in HSC-T6. While activating CREB-1, exogenous TGF-ß3 has no effect on the expressions of CREB-1 protein and mRNA.

[Polymorphism of POU1F1 gene and PRL gene and their combined effects on milk performance traits in Chinese Holstein cattle].

Three novel SNPs were found by DNA sequencing, PCR-RFLP and CRS-PCR methods were used for genotyping in 979 Chinese Holstein cattle. One SNP, G1178C, was identified in exon 2 of POU1F1 gene. Two novel SNPs, A906G and A1134G, were identified in 5'-flanking regulatory region (5'-UTR) of PRL gene. The association between polymorphisms of the two genes and milk performance traits were analyzed with PROC GLM of SAS. The results showed that GC genotype at 1178 locus of POU1F1 gene was advantageous for milk yield, milk protein yield, and milk fat yield. AG genotype at 906 locus was advantageous for milk yield. There was no significant difference between 1134 locus and milk performance traits of 5'-UTR of PRL gene. Analysis of genotype combination effect on milk production traits showed that the effect of combined genotype was not simple sum of single genotypes and the effects of gene pyramiding seemed to be more important in molecular breeding.

[Clinical research and preliminarily exploration of genetic basis on a hereditary hemochromatosis pedigree].

OBJECTIVE: To describe the clinical features of a hereditary hemochromatosis pedigree and to explore preliminarily the genetic basis of this pedigree. METHODS: Survey was carried out among the family members, including history taking, physical examination, laboratory examination (such as iron-biochemistry indexes, liver function, blood sugar), multiple-organs MRI scan, iron staining of liver biopsy tissues and making the pedigree chart. The blood samples of the pedigree were collected, the genomic DNA was extracted, the PCR amplification was made and finally the sequence analysis for several mutations of common hereditary hemochromatosis genes was done. RESULTS: In this pedigree, 7 members were found with iron overload and clinically diagnosed as hereditary hemochromatosis. The features are transmitted from generation to generation without gender difference and the gene penetrance is about 46%. The common mutations of SLC40A1 and HFE were not found in the family members. CONCLUSION: Cutaneous pigmentation and iron overload in liver and spleen are the most typical characters in this hereditary hemochromatosis pedigree, which corresponds with autosomal dominant inheritance. However, the genetic basis of this pedigree is unknown yet.

[Overview of "tian-yin-yang" acupuncture, an acupuncture school of Huang's Zhuang medicine of Guangxi].

The theory of "tian-yin-yang" of Zhuang medicine was explored and the academic achievements and experience of professor HUANG Jin-ming were summarized to explain the theory and clinical characteristics of tian-yin-yang acupuncture, an acupuncture school of Huang 's Zhuang medicine of Guangxi. This acupuncture method is guided by the theory of "three-qi synchronization" and "tian-yin-yang" of Zhuang medicine, based on the theory of "three channels and two paths", with regulating qi as the method and regulating spirit as the basis. After the patient is calmed and resting, the micro needle shallow needling technique is adopted, mainly at the umbilical ring point, so as to achieve the purpose of regulating the mind and treating the root cause.

[Genetic polymorphism in 5'-flanking region of the lactoferrin gene and its associations with mastitis in Chinese Holstein cows].

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P >0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P<0.01) or AB (P<0.05). In position -478(/G), the cow with genotype CC showed significantly lower SCS in contrast to cow with genotype CD and DD (P < 0.01). In conclusion, genotype AB in position +72(T/C) and genotype CC in position -478(/G) of LF gene were advantageous genotype, which can be used as candidate markers for mastitis resistance selection in dairy cattle.

Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation.

The aim of this study was to test the effects of five different concentrations (0, 10-3, 10-4, 10-5, and 10-6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10-3 M and 10-4 M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10-3 M and 10-4 M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10-4 M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

Correction: Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

[This corrects the article DOI: 10.1371/journal.pone.0159719.].

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

[Umbilical ring acupoints and its clinical application in Zhuang medicine].

The theoretical basis, location, belonging of zang-fu, treatment function and indications, applying principle and manipulation of Umbilical Ring acupoints in Zhuang medicine are explained in this paper. According to Zhuang medicine, umbilicus is an epitome of the body and all the zang-fu and organs in the body have corresponding epitomes like a fetus in front-standing position. The umbilicus is not only a micro-diagnosis system, but also a window for illness treatment that could be divided into superficial, middle and deep layer to respectively communicate different zang-fu and organs. The umbilical inner ring and outer ring are collectively called Umbilical Ring acupoints, they could dredge paths, regulate the balance of qi and blood to regulate qi, expel poison, tonify deficiency and remove stasis to treat many types of diseases in the whole body.

Two novel SNPs in HSF1 gene are associated with thermal tolerance traits in Chinese Holstein cattle.

Heat-shock transcription factors (HSFs) play an important role in regulating heat stress response. The activation of heat-shock protein (HSP) genes is mediated by HSFs, which bind to promoters of HSP genes. In this research, two novel single nucleotide polymorphisms, T909C and G4693T, and their association with thermal tolerance were investigated in 951 Chinese Holstein cattle. Linkage disequilibrium and haplotype construction were analyzed using SHEsis software. Four haplotypes were constructed, and nine haplotype combinations were found. Potassium content in erythrocytes (PCE), decreased rate of milk production (R), rectal temperature (RT), and heat-tolerance coefficient (HTC) were selected for the thermotolerance index. Association analysis showed that thermal tolerance in Chinese Holstein cattle was significantly affected by T909C and G4693T. The PCE of cows with CC or TC genotype was lower than that of TT at the 909 position (p < 0.05). Cows with TT genotype had lower PCE (p < 0.01) and higher HTC (p < 0.05) at the 4693 position. Cows with H2H4 haplotype combination had lower PCE (p < 0.01), R (p < 0.05) and RT (p < 0.05) and higher HTC (p < 0.05) than those with H1H3 haplotype combination. Bioinformatic analysis predicted that the 4693 position was located in the microRNA-binding (bta-miR-484) region. Quantitative reverse transcription-polymerase chain reaction demonstrated that 4693-T mutation caused the disruption of microRNA target binding, resulting in the relief of the transcriptional repression, which, in turn, resulted in increased expression. Thus, the HSF1 gene is useful in dairy cattle thermal tolerant breeding.