Comparison of markers predicting litter size in different pig breeds.

To overcome the limitations of conventional analysis of male fertility in animals and humans, proteomic studies have been performed to develop fertility-related biomarkers for prognosis and diagnosis of male fertility. However, the studies were focused on specific species or breeds. Therefore, a study is required to validate whether fertility-related markers would apply to other breeds in pigs. In this study, previously developed fertility-related biomarkers from Landrace were validated to use for prognosis of male fertility in commercially available breeds. Expression level of eight biomarkers in non-capacitated and capacitated (C) spermatozoa from Yorkshire and Duroc boars was analyzed. And then, to explore the validity of these markers for prognosis of male fertility, i.e. litter size, artificial insemination was performed. Among them, RAB2A (NC) and UQCRC1 (NC) turned out to be highest efficient markers for Yorkshire. RAB2A (C) was most efficient marker for Duroc. Average litter size has increased as much as 1.41 live born after prediction using eight fertility-related biomarkers in Yorkshire. In addition, average 2.52 litter size was increased after prediction using eight fertility-related biomarkers in Duroc. Average litter sizes were especially highly increased after prediction of fertility using RAB2A (NC) in Yorkshire (1.57 piglets) and TPI (NC) in Duroc (3.14 piglets), respectively. As a result, all biomarkers were significantly correlated with litter size. However, overall accuracy to predict litter size in three breeds was different in response with each marker. Average litter size after artificial insemination was also significantly affected by marker selection. Therefore, this study suggests that developed fertility-related markers may be used for prognosis and diagnosis of male fertility irrespective of breed. However, selection of efficient markers for breeds should be considered to obtain more accurate and efficient outcomes.

Actin-related protein 2/3 complex-based actin polymerization is critical for male fertility.

The actin-related protein 2/3 (Arp2/3) complex is critical for regulation of actin polymerization, which is associated with sperm motility and capacitation status. However, the function of the Arp2/3 complex in male fertility has not yet been fully elucidated. Therefore, this study was designed to investigate the role of the Arp2/3 complex in different processes in spermatozoa and its consequences on fertilization and early embryonic development. In this in vitro study, mouse spermatozoa were incubated with different concentrations (10, 100, and 500 µm) of CK-636, an Arp2/3 complex antagonist. Our results demonstrated that inhibition of the Arp2/3 complex by high concentrations (100 and 500 µm) of CK-636 induced hyper-activated motility and acrosomal reaction, whereas intracellular calcium and tyrosine phosphorylation levels in spermatozoa were inhibited. Moreover, exposure of spermatozoa to the highest concentration of CK-636 reduced fertilization and embryo development. Interestingly, fertilization was significantly increased after treatment with 100 µm CK-636, whereas embryonic development was significantly decreased. Therefore, we conclude that the Arp2/3 complex plays a decisive role in regulation of sperm function and male fertility via actin polymerization. We anticipate that the Arp2/3 complex may have clinical application as marker for male fertility and male contraceptive targeting.

Improving litter size by boar spermatozoa: application of combined H33258/CTC staining in field trial with artificial insemination.

Conventional semen analysis offers basic information on infertility; however, its clinical value in predicting fertility status is unclear. To establish an accurate diagnosis of male fertility, semen analysis under capacitation condition is necessary because only capacitated spermatozoa are capable of fertilizing oocytes. The objective of this study was to verify male fertility based on conventional semen analysis before and after capacitation, including the assessment of motility (%), motion kinematics, and capacitation status of spermatozoa. A computer-assisted sperm analysis system and chlortetracycline staining were applied to evaluate the motility parameters and capacitation status, respectively. To enable efficacy of the two methods for predicting fertility, correlation analysis was performed with the historic litter size. Our results showed that sperm motility (%), motion kinematics, and their variations before and after capacitation represented a statistical non-significant correlation with litter size. Litter size showed significant correlation with acrosome reaction (AR) after capacitation (r = 0.375), as well as differences (Δ) in AR (r = 0.333) and capacitated (B) pattern (r = -0.447) before and after capacitation. The overall accuracy of the assay for predicting litter sizes using the AR and differences (Δ) in the AR and B pattern was 70%. On the basis of these results, we propose that capacitation status of spermatozoa is a more reliable indicator for evaluating male fertility status compared to motility parameters. Therefore, we suggest that analysis of capacitation status in company with conventional semen analysis may accept to evaluate more accurate diagnosis or prognosis of male fertility.

Sodium nitroprusside suppresses male fertility in vitro.

Sodium nitroprusside is a nitric oxide donor involved in the regulation of the motility, hyperactivation, capacitation, and acrosome reaction (AR) of spermatozoa. However, the molecular mechanism underlying this regulation has not yet been elucidated. Therefore, this study was designed to evaluate the molecular basis for the effects of sodium nitroprusside on different processes in spermatozoa and its consequences on subsequent oocyte fertilization and embryo development. In this in vitro study, mouse spermatozoa were incubated with various concentrations of sodium nitroprusside (1, 10, and 100 µM) for 90 min. Our results showed that sodium nitroprusside inhibited sperm motility and motion kinematics in a dose-dependent manner by significantly enhancing intracellular iron and reactive oxygen species (ROS), and decreasing Ca(2+), and adenosine triphosphate levels in spermatozoa. Moreover, short-term exposure of spermatozoa to sodium nitroprusside increased the tyrosine phosphorylation of sperm proteins involved in PKA-dependent regulation of intracellular calcium levels, which induced a robust AR. Finally, sodium nitroprusside significantly decreased the rates of fertilization and blastocyst formation during embryo development. Based on these results, we propose that sodium nitroprusside increases ROS production and precocious AR may alter overall sperm physiology, leading to poor fertilization and compromised embryonic development.

Association of the ABCB1 gene polymorphisms 2677G>T/A and 3435C>T with clinical outcomes of paclitaxel monotherapy in metastatic breast cancer patients.

BACKGROUND: ABCB1 is responsible for multidrug resistance, the principal mechanism by which many cancers develop resistance to chemotherapeutic drugs. There is a controversy whether ABCB1 gene polymorphisms correlate with survival and response in cancer patients treated with chemotherapy. We evaluated the association between clinical outcome (safety and efficacy) of paclitaxel monotherapy in metastatic breast cancer patients with ABCB1 gene polymorphisms 2677G>T/A or 3435C>T. PATIENTS AND METHODS: Patients with metastatic breast cancer were treated with 175 mg/m(2) paclitaxel per 3-week cycle. Peripheral blood mononuclear cells from patients were used to genotype ABCB1 2677G>T/A and 3435C>T polymorphisms. Genotypes were investigated for their association with tumor response, survival, toxicity, and chemoresistance. RESULTS: ABCB1 3435 CT showed a significantly lower disease control rate than the CC genotype (P = 0.025). ABCB1 3435 CT was correlated with shorter overall survival (OS) in Cox regression analysis (P = 0.026). The 2677 GG genotype showed a significant association with chemoresistance to paclitaxel and anthracycline (P = 0.04 and 0.04, respectively). None of the ABCB1 genotypes correlated with toxicity. CONCLUSIONS: ABCB1 genotypes may be a predictor of paclitaxel activity as well as a prognostic factor in metastatic breast cancer patients.

Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli.

An advantage of exporting a recombinant protein to the periplasm of Escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm. However, protein degradation in the periplasm also occurs. It has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) fused to alkaline phosphatase leader peptide was used as a model protein. A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm. It was found that the half-life of the barnase variants in vivo increased with their thermodynamic stability in vitro. A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant. The yield of a stabilized mutant was up to 50% higher than that of the wild type. This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.