Peroxiredoxin 4 directly affects the male fertility outcome in porcine.

Peroxiredoxins (Prdxs) are known to play a critical role in regulating male fertility as antioxidant enzymes. Although several studies have suggested a close association between Prdxs and male fertility, few studies have explored the efficacy of Prdxs to predict male fertility. Therefore, the current study was designed to discover the most efficient biomarkers among the Prdxs with six isoforms. Our study showed a significant positive correlation between the litter size and the levels of PRDX 4 among all isoforms in spermatozoa. Subsequently, a regression analysis using a combination of markers was conducted to increase efficacy for fertility prediction. Nevertheless, PRDX4 had the highest efficacy compared to other combination models to predict litter size. The prediction accuracy of male fertility was further evaluated through receiver operating characteristic curve analysis, which showed that PRDX 4 could predict the litter size with high overall accuracy of 95%. Moreover, litter size was increased by 1.55 piglets after predicting high litter size using PRDX 4. This is the first study to comprehensively elucidate the role of all isoforms of PRDXs on male fertility to the best of our knowledge. PRDX 4 was tested and evaluated up to a practical level. Data here reported suggesting PRDX 4 marker allowed the highest accuracy for male fertility prediction and diagnosis, leading to a measurable improvement in the male fertility outcome.

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.

Stage-specific embryonic antigen-1 expression by undifferentiated spermatogonia in the prepubertal boar testis.

The objective of this study was to use fluorescence-activated cell sorting (FACS) and spermatogonial stem cell (SSC) xenotransplantation to identify cell surface markers of putative porcine SSC. Analysis of porcine testis cells enriched for spermatogonia using FACS indicated that nearly half of stage-specific embryonic antigen-1 (SSEA-1) expressing testis cells expressed the undifferentiated spermatogonia marker protein gene product 9.5 (PGP 9.5) whereas significantly fewer (P < 0.05) cells selected for thymus cell antigen-1 (Thy-1), also known as cluster of differentiation 90 (CD90), cluster of differentiation 9 (CD9), or other SSC markers expressed PGP 9.5. Immunocytochemical analysis indicated that promyelocytic leukemia zinc finger (PLZF) protein and germ cell lineage marker VASA homolog (VASA), also known as DEAD box protein 4 (DDX4), were expressed by SSEA-1 expressing germ cells. Spermatogonial stem cell xenotransplantation of testis cell populations enriched for cells expressing SSEA-1 generated significantly (P < 0.05; greater than 15-fold) more colonies of donor derived germ cells than unselected testis cells. In conclusion, these data indicate that SSC markers identified in rodents are likely not entirely conserved in pigs and that SSEA-1 is a marker for porcine undifferentiated spermatogonia including SSC in prepubertal boars and its expression may serve as a target for the further study of porcine germ cells.

Cloning of avian G(0)/G(1) switch gene 2 genes and developmental and nutritional regulation of G(0)/G(1) switch gene 2 in chicken adipose tissue.

Adipose triglyceride lipase (ATGL), a newly identified lipase, is a rate-limiting enzyme for triglyceride hydrolysis in adipocytes. The regulatory proteins involved in ATGL-mediated lipolysis in fat tissue are not fully identified and understood. The G(0)/G(1) switch gene 2 (G0S2) is an inhibitor of ATGL activity by interacting with ATGL through the hydrophobic domain of G0S2. Here, for the first time, we have cloned the coding sequence of G0S2 cDNA for the chicken, turkey, and quail. Sequence comparisons with mammals revealed that the avian G0S2 also have a conserved hydrophobic domain. Avian G0S2 is predominantly expressed in adipose tissues relative to other tested tissues. Within the adipose tissue, G0S2 is expressed 20-fold greater in the adipocyte than in the stromal-vascular (SV) fraction (P < 0.001). Expression of G0S2 mRNA gradually increased during differentiation of chicken adipocytes in culture (P < 0.05). However, there is G0S2 expression in embryonic adipose tissue, SV fraction, and primary preadipocytes before confluence that generally have an increased capacity of cell proliferation, which indicates it has an important role in adipocyte differentiation rather than proliferation. For a better understanding of how G0S2 responds to environmental stimuli, chickens were fasted for 24 h and then refed. Expression of G0S2 in adipose tissue was dramatically decreased (P < 0.05) in the chickens and quail after a 24-h fasting period, and increased to the control level after refeeding. In contrast to G0S2 expression, ATGL expression was induced (P < 0.05) after the 24-h fasting period and rapidly returned to the control level during the refeeding period. These data indicate that changes in lipolytic activities of adipose tissue in vivo can be regulated by G0S2 expression, as an inhibitor of ATGL.

Capacitation status of stored boar spermatozoa is related to litter size of sows.

Semen parameters can be considered useful predictors of sperm fertility. The objective of this study was to address the question of whether differences in in vivo fertility after the use of different ejaculates could be predicted using sperm kinematics, capacitation status, and sperm penetration ability under commercial pig production conditions. The percentage of capacitated sperm, as assessed by chlortetracycline (CTC) staining, was positively correlated with litter size (p<0.01). Our data suggest that litter size increases in proportion to the number of capacitated spermatozoa. When all semen parameters (kinematics, sperm capacitation status, and sperm penetration ability) and litter size were included in a multiple linear regression analysis, significant associations were found between the percentage of capacitated sperm (B-type), the sperm fertility index as assessed by a sperm penetration assay (SPA), and litter size. This relationship between capacitated sperm and litter size, however, was more predictive for smaller litter groups than larger ones. We found that the percentage of B-type sperm was significantly correlated with historic average litter size. However, there was no significant correlation between the percentage of B-type sperm and historic farrowing rates. To determine the normal range for B-type sperm, the lower limits were established as 30% for small litters (<8 piglets) and 35% for large litters. The overall accuracy of the assay was 92% and 83% for small and large litters, respectively. These results indicate that capacitation status as measured by CTC staining is a useful predictor of sperm fertility, equivalent to SPA. Moreover, original capacitation status exhibited better predictive ability for small litters than for large ones. Therefore, subfertile boars can be identified primarily by capacitation status.

A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double-density gradient system.

OBJECTIVES: To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield. METHODS: Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer-Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X-Y probes. RESULTS: For single-density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H(2)O with 1.077 g/mL Percoll and 520 mOsm/kg H(2)O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double-Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus. CONCLUSION: The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods.

Extraordinarily high rates of male circumcision in South Korea: history and underlying causes.

OBJECTIVE: To investigate the high circumcision rate in South Korea and its rapid increase in the short period since its introduction. SUBJECTS AND METHODS: From January to December 2000, 5434 South Korean males (or their parents) aged 0-92 years were interviewed in detail about their circumcision status, their age at circumcision, and the possible effect of circumcision on their sexuality. In addition, 267 practising medical doctors were surveyed about their basic understanding of circumcision and phimosis. RESULTS: Currently the circumcision rate for high-school boys is > 90% and for those > 70 years old is < 10%. The circumcision rate in 1945 was < 0.1%. When averaged over the whole population, the present South Korean circumcision rate is approximately 60%; the rate has increased dramatically with time and particularly in the past 20 years, when the estimated number of male circumcisions has exceeded the number of male births. Although circumcision in South Korea has been strongly influenced by American culture, it has never been predominantly neonatal. The age at circumcision has continued to decrease and boys are now circumcised at approximately 12 years old. Of those who were circumcised long after they had been sexually active, > 80% reported no noticeable difference in sexuality, but a man was twice as likely to have experienced diminished sexuality than improved sexuality. Of the doctors who were surveyed, 41% carried out circumcision but, unlike in America, gynaecologists and paediatricians rarely did so. Among the doctors, basic knowledge on circumcision and phimosis was generally lacking, regardless of whether they practised circumcision or not. Amongst the factors contributing to the high circumcision rate was the mistaken notion held by both doctors and the general public that circumcision is directly correlated with industrialization and general progress of living standards. Many doctors believe the out-dated and sometimes controversial benefits of circumcision, i.e. prevention of cervical cancer and sexually transmitted diseases, and improved sexuality. Thus the vast majority of doctors recommend circumcision regardless of the patient's age. Peer pressure was also an important contributing factor. CONCLUSION: South Korea has an unusual history of circumcision. The mistaken and out-dated notions about circumcision and lack of knowledge of phimosis by physicians seem to be a leading contributory factor to the extraordinarily high circumcision rate.

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat.

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 microg/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN: Prospective study. SETTING: Infertility clinic and molecular genetics laboratory. PATIENT(S): Ten patients with severe OAT. INTERVENTION(S): Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S): Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S): In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S): The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.

Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.

Cytogenetics of somatic cells and sperm from a 46,XY/45,X mosaic male with moderate oligoasthenoteratozoospermia.

OBJECTIVE: To determine aneuploidy frequencies in sperm from a patient with normal phenotype and 46,XY/45,X mosaicism in somatic cells (peripheral lymphocytes). DESIGN: Case report. SETTING: Infertility clinic and genetics laboratory. PATIENT: A 30-year-old male with primary infertility and moderate oligoasthenoteratozoospermia. INTERVENTION(S): Cytogenetic analysis of somatic cells and determination by fluorescence in situ hybridization of aneuploidy frequencies for the gonosomes (sex chromosomes) and chromosome 18 in sperm from whole and Percoll-separated semen. MAIN OUTCOME MEASURE(S): Somatic and gametic aneuploidy were scored. RESULT(S): Analysis of lymphocyte metaphase cells showed a mosaic 46,XY (90%)/ 45,X (10%) karyotype. Significantly higher frequencies of gonosomal (semen, 1.92% versus 0.70%; Percoll, 1.12% versus 0.46%), and chromosome 18 (semen, 0.89% versus 0.28%; Percoll, 0.26% versus 0.10%) disomy were detected in the sperm of the patient compared with those observed in spermatozoa from a proved fertile control. CONCLUSION(S): Significantly higher frequencies of aneuploid sperm suggest that the patient is at elevated risk of producing offspring with numerical chromosome abnormalities.

Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes.

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

Assignment of the hypoxia-inducible factor 1alpha gene to a region of conserved synteny on mouse chromosome 12 and human chromosome 14q.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that mediates homeostatic responses to hypoxia. HIF-1 is a heterodimer consisting of HIF-1alpha, which is encoded by the HIF1A gene, complexed with HIF-1beta, which is encoded by the ARNT gene. In this paper we report the assignment of Hif1a and HIF1A to mouse chromosome 12 and human chromosome 14, respectively. HIF1A was assigned to human chromosome 14q21-q24 by analysis of somatic cell hybrids and by fluorescence in situ hybridization. Hif1a was localized by interspecific backcross analysis within a region of mouse chromosome 12 encompassing >30 cM that demonstrates conservation of synteny with a region of human chromosome 14 extending from PAX9 at 14q12-q13 to IGHC at 14q32.33.

Alcohol, personality traits, and high risk driving: a comparison of young, drinking driver groups.

Four types of drinking driver groups were compared with each other and also with two nondrinking driver groups on sensation seeking, social responsibility, and hostility. Groups were also compared on traffic violations, accidents, alcohol consumption, frequency of driving after drinking, frequency of driving impaired, and perception of driving risk taking after drinking. Drivers under the influence apprehended in conjunction with an accident or moving violation had significantly greater alcohol consumption, frequency of driving after drinking, frequency of driving impaired, traffic violations, accidents, and self rating of risk taking after drinking in comparison with other groups.