Brucella induces unfolded protein response and inflammatory response via GntR in alveolar macrophages.

Brucella is an intracellular bacterium that causes the zoonosis brucellosis worldwide. Alveolar macrophages (AM) constitute the main cell target of inhaled Brucella. Brucella thwarts immune surveillance and evokes endoplasmic reticulum (ER) stress to replicate in macrophages via virulence factors. The GntR regulators family was concentrated as an important virulence factor in controlling virulence and intracellular survival of Brucella. However, the detailed underlying mechanism for the host-pathogen interaction is poorly understood. In this study the BSS2_II0438 mutant (ΔGntR) was constructed. The type IV secretion system (T4SS) virulence factor genes (VirB2, VirB6, and VirB8) were down-expression in ΔGntR. ΔGntR could infect and proliferate to high titers in GAMs without a significant difference compared with the parental strain. ΔGntR infection increased the expression of ER stress marker genes GRP78, ATF6, and PERK in the early stages of its intracellular cycle but decreased the expression of these genes in the late stages. ΔGntR increased greatly the number of Brucella CFUs in the inactive ER stress state in GAMs. Meanwhile, ΔGntR infection increased the levels of IFN-γ, IL-1ß, and TNF-α, indicating ΔGntR could induce the secretion of inflammatory but not anti-inflammatory cytokines IL-10. Taken together, our results clarified the role of the GntR in B. suis. S2 virulence expression and elucidated that GntR is potentially involved in the signaling pathway of the Brucella-induced UPR and inflammatory response in GAMs.

Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2.

Melatonin and its receptors are found in the testis of many species, where they mediate testicular functions. The present study aimed to investigate the expression of melatonin receptors (MT1 and MT2) in bovine Sertoli cells (SCs), using reverse transcription polymerase chain reaction (RT-PCR) and western blot. In addition, we assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after treatment with various concentrations (0, 80, 160, and 320 pg/mL) of melatonin at different time points (24, 48, or 72 h). We found that bovine SCs express MT1 and MT2 receptors, which were regulated by melatonin in time- and dose-dependent manners after treatment with melatonin. Exogenous melatonin up-regulated the expression of spermatogenesis-related genes, including Cyclin D1, Cyclin E, Pdgfa, Dhh, Occludin, and Claudin, and decreased the mRNA levels of P21 and Kit1 in a time or dose-dependent manner. Meanwhile, melatonin supplementation significantly affected Inhba, Inhbb and Inha mRNA expression. These findings were consistent with inhibin B levels detected in the culture medium. In conclusion, exogenous melatonin acts via its receptors and appears to play regulatory roles in the development and function of bovine SCs.

Tentative identification of sex-specific antibodies and their application for screening bovine sperm proteins for sex-specificity.

Our previous studies indicated that a purified rabbit antiserum against X-sperm contained sex-specific antibodies (SSAbs) which preferentially bound to sex-sorted X-sperm. The specificity of sex-specific antiserum was initially demonstrated using flow cytometry only, which resulted in uncertainty. In this study, the putative SSAbs against bovine X-sperm (XSSAb) were produced by a series of immunological approaches, and the effectiveness of separation of sperm using putative XSSAb was validated. Subsequently, the XSSAb was used to immunoprecipitate sex-specific proteins (SSPs) in bovine sperm, followed by two-dimensional gel electrophoresis. The results showed 7.6, 15.2 and 52.1 % of sex-sorted Y-sperm, sex-sorted X-sperm and unsorted sperm were recognized by the neutralized rabbit antisera against X-sperm, respectively. Also the purity of separation of sperm using putative XSSAb reached 74.3 % when the immunologically separated sperm were injected into oocytes. In addition, three candidate SSP sports about 30 kDa were captured by the XSSAb. Our results confirmed that the putative XSSAb contained SSAbs, and implied that these three protein sports might be SSPs in bovine X-sperm. This provides a potentially more efficient method for sorting sperm and lays a foundation for future search for SSPs.

Effects in cattle of genetic variation within the IGF1R gene on the superovulation performance and pregnancy rates after embryo transfer.

The insulin-like growth factor 1 receptor (IGF1R) is a membrane glycoprotein mediating most biological actions of IGF1 and IGF2, and has an important effect on ovulation, pre-implantation embryo development and pregnancy rate. The objectives of this study were to detect IGF1R gene polymorphisms of cattle and analyze the relationship with superovulation performance and pregnancy rates after embryo transfer (ET), as well as the hormone concentrations at the day of ET. One reported SNP of IGF1R G404T and a novel SNP of IGF1R G399A were analyzed in 170 Chinese Holstein donor cows and 118 Luxi recipients cattle. Statistical analysis revealed that the G404T mutation was associated (p=0.019) with increased ovulation rate and females with this mutation had enhanced performance in producing transferable embryos. For the polymorphic locus G399A, recipients with g.399 GG and g.399 GA genotypes had greater pregnancy rates after ET than that of g.399 AA genotype. Furthermore, the same tendency was observed that the genotype groups with greater pregnancy rates had greater progesterone and lesser estrogen concentrations, but these did not reach statistical significance. Results of the present study showed, for the first time, that the polymorphism in IGF1R is associated with superovulation traits, and indicated that the IGFIR gene can be used as a potential marker for donor selection.

Effects of PGR and ESRα genotypes on the pregnancy rates after embryo transfer in Luxi cattle.

Progesterone receptor (PGR) and estrogen receptor alpha (ESRα), which mediate the biological effects of the steroid hormones progesterone and estrogen, play a central role in the establishment and maintenance of pregnancy. The objectives of this study were to detect bovine PGR and ESRα genes polymorphisms and analyze their relationships with the pregnancy rates after embryo transfer and the hormone concentrations at the day of embryo transfer. One reported SNP of PGR G59752C and a novel SNP of ESRα G75935C were analyzed in 132 recipients of Luxi cattle. For the PGR gene, recipients with g.59752 GG and g.59752 GC genotypes had obviously higher pregnancy rates than g.59752 CC genotype. For the ESRα gene, recipients with g.75935 GC and g.75935 CC genotypes had obviously higher pregnancy rates than g.75935 GG genotype. Furthermore, the same tendency was observed for these two genes that the same genotype groups with high pregnancy rates had high progesterone concentration and low estrogen concentration at the day of embryo transfer. These results showed for the first time that PGR G59752C and ESRα G75935C polymorphisms had obvious effects on the pregnancy rates after embryo transfer, and indicated that PGR G59752C and ESRα G75935C polymorphisms could be potential markers for recipient selection of embryo transfer.

Polymorphisms of the bovine luteinizing hormone/choriogonadotropin receptor (LHCGR) gene and its association with superovulation traits.

The major limitation to the development of embryo transfer technique in cattle is the highly variable between individuals in ovulatory response to FSH-induced superovulation. The objective of this study was to identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, variation in the bovine luteinizing hormone/choriogonadotropin receptor (LHCGR) gene was investigated using PCR-single-strand conformational (PCR-SSCP) and DNA sequencing. Four single nucleotide polymorphisms (SNPs) of G51656T, A51703G, A51726G and G51737A were identified at the intron 9 of the LHCGR gene in 171 Chinese Holstein cows treated for superovulation, and evaluated its associations with superovulatory response. Association analysis showed that these four SNPs had significant effects on the total number of ova (TNO) (P < 0.05). Moreover, the A51703G and A51726G polymorphisms significantly associated with the number of transferable embryos (NTE) (P < 0.05). In addition, significant additive effect on TNO was detected in polymorphisms of G51656T (P < 0.05) and A51703G (P < 0.01), and the A51703G polymorphism also had significant additive effects on NTE (P < 0.01). These results indicate that LHCGR gene is a potential marker for superovulation response and can be used to predict the most appropriate dose of FSH for superovulation in Chinese Holstein cows.

Polymorphisms in follicle stimulation hormone receptor, inhibin alpha, inhibin bata A, and prolactin genes, and their association with sperm quality in Chinese Holstein bulls.

Candidate genes follicle stimulation hormone receptor (FSHR), inhibin alpha (INHA), inhibin bata A (INHBA) and prolactin (PRL) were investigated for their association with sperm quality traits of semen volume per ejaculate (VOL), sperm concentration (SCON), motility (MOT), sperm motility in frozen semen (FMOT), acrosome integrity rate (AIR) and abnormal sperm rate (ASR) in a total of 123 normal mature Holstein bulls. Three reported single nucleotide polymorphisms (SNPs) of FSHR A-234500T (rs43676359), IHNA A192G (rs41257116), and IHNBA C7639T (rs43408735), and 3 novel SNPs (G7550A, C7661T, and T8370C) in exons 4 and 5 of bovine PRL gene (NC 007324) were analyzed. Analysis of variance revealed that FSHR A-234500T and INHBA C7639T polymorphisms significantly associated with VOL (P<0.05) and SCON (P<0.05), and the polymorphism of INHBA C7639T also had significant effects on MOT (P<0.05). Furthermore, the INHA A192G polymorphism significantly associated with AIR (P<0.05). No significant differences were found between the polymorphisms of PRL gene and sperm quality traits.

An MspI polymorphism in the inhibin alpha gene and its associations with superovulation traits in Chinese Holstein cows.

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, the PCR-RFLP method was applied to detect an A>G transition determining an MspI polymorphism at position 192 in the exon I of the bovine inhibin alpha (INHA) gene and evaluate its associations with superovulatory response in 118 Chinese Holstein cows treated for superovulation. Association analysis showed that cows with the GG genotype resulted in a significant increase in the number of ova (TNO) than AG and AA genotypes in the first (P=0.023), second (P=0.004) and third (P=0.002) superovulation treatments and produced more transferable embryos (NTE) than that of AG and AA genotypes in the third (P=0.045) superovulation treatment. Moreover, individuals with GG genotype produced more transferable embryos than AA (P<0.05) genotype in the second superovulation treatment and all cows without superovulation response were mutations with genotypes of AA and AG. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices.

Effects of MboII and BspMI polymorphisms in the gonadotropin releasing hormone receptor (GnRHR) gene on sperm quality in Holstein bulls.

The hypothalamic gonadotropin-releasing hormone receptor (GnRHR) plays an essential physiological role in reproductive function, which triggers the synthesis and release of luteinizing hormone and follicle stimulating hormone in the pituitary. The objective of this study was to investigate the effects of polymorphisms of GnRHR gene on the quality of fresh and frozen semen in Holstein bulls. The PCR-RFLP method was applied to detect G286A and T340C transitions determining MboII and BspMI polymorphisms, respectively, in the exon I of bovine GnRHR gene and evaluated its associations with sperm quality traits in 131 Holstein bulls. In polymorphic locus 286, bulls with the GA genotype had significantly higher sperm motility in frozen semen (FMOT) than GG genotype (P < 0.01). In polymorphic locus 340, bulls with heterozygote CT genotype had significantly higher sperm motility (MOT), semen volume per ejaculate (VOL), and lower abnormal spermatozoa rate (ASR) than homozygote TT genotype (P < 0.05). Bulls contained one A allele or C allele had a favorable, positive effect on sperm quality traits. These results indicate that GnRHR gene can be a potential marker for improving sperm quality traits, and imply that bulls with GA or CT genotype should be selected in breeding program.

Polymorphisms in the 5' upstream region of the FSH receptor gene, and their association with superovulation traits in Chinese Holstein cows.

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, variation in the bovine follicle stimulating hormone receptor (FSHR) gene was investigated using PCR-single-strand conformational (PCR-SSCP) and DNA sequencing. One single nucleotide polymorphism (SNP) of G-278A located in the 5' upstream region of bovine FSHR gene was found in 118 Chinese Holstein cows treated for superovulation. Two SNPs of G-278A (GU253337) and A-320T (rs43676359) were analyzed. In polymorphic locus -278, all cows without superovulation response were mutations with genotypes of CD and DD. Cows with CC genotype had a significant increase in the total number of ova (TNO) (P<0.01), and produced more transferable embryos (NTE) than those of the CD and DD genotypes (P<0.01). At this locus, the additive effects seemed to be highly significant (P<0.01) and allele C was associated with an increase in the TNO and NTE. These results indicated that the FSHR is a potential marker for superovulation response and can be used as a predictor for superovulation in Chinese Holstein cows.