Y chromosome gr/gr subdeletion is associated with lower semen quality in young men from the general Japanese population but not in fertile Japanese Men.

Several case-control studies have investigated whether Y chromosome haplogroups or deletions are associated with spermatogenic failure. However, the relationships between Y chromosome haplogroups or deletions and semen quality in general population have not been elucidated. In this study, we assessed relationships between Y chromosome haplogroups or deletions and semen parameters in 791 fertile Japanese men and 1221 young men from the general Japanese population. We found that the haplogroup D2 (M55 lineage) was significantly associated with lower semen parameters, especially total motile sperm count (P = 0.00051, beta = -0.097), in men from the general population but not in fertile men. In addition, we found that the gr/gr subdeletion was associated with semen quality and in particular, strongly associated with decreased sperm motility (P = 0.00041, beta = -3.14) and total motile sperm count (P = 0.00031, beta = -0.099) in men from the general population but not in fertile men. The combined analysis of fertile Japanese men and men from the general Japanese population showed that the haplogroup D2 (M55 lineage) and the gr/gr subdeletion were strongly associated with reduced sperm motility (P = 0.00056, beta = -2.71, and P = 7.7 × 10(-5), beta = -3.05, respectively) and that haplogroup O2b1 was strongly associated with elevated sperm motility (P = 0.00089, beta = 2.94). These observations add further support for the view that the gr/gr subdeletion diminishes sperm motility that consequently may result in male infertility.

Y chromosome haplogroup d2* lineage is associated with azoospermia in Japanese males.

Several studies have investigated whether particular Y chromosome haplogroups are associated with spermatogenic failure in Japanese males; however, they produced differing results. In this study, to investigate the association of Y chromosome haplogroup with spermatogenic failure, we recruited 451 infertile patients and 730 fertile men from a Japanese population and typed their Y chromosome haplogroups. The infertile patients were suffering from varicocele, azoospermia, oligozoospermia, asthenozoospermia, obstructive azoospermia, karyotype abnormalities, microdeletions of the long arm of the Y chromosome, or other conditions that affect fertility. The frequency of haplogroup D2* was significantly higher (odds ratio = 2.28, 95% confidence interval = 1.44-3.61, P = 0.00034 using chi-square test) among the men with azoospermia than among the fertile men. None of the other Y haplogroups displayed associations with particular types of infertility. In conclusion, Y chromosome haplogroup D2* is associated with spermatogenic failure in Japanese males, suggesting that the Y chromosome lineage can have significant effects on spermatogenesis.

Impact of annual body mass index gain on obesity development in Japanese 6-year-old non-obese children.

BACKGROUND: Effective timing of preventive intervention for adolescent obesity in non-obese school-aged children remains unclear. The aim of this study was to examine the impact of annual body mass index (BMI) gain on the development of adolescent obesity in 6-year-old non-obese Japanese children. METHODS: Longitudinal weight and height data were collected annually from 9723 children aged 6-14 years, and individual per-year BMI gains were calculated. The BMI ≥ the 95th percentile for each age and sex defined obesity. In 6-year-old non-obese children, logistic regression analyses were applied to correlate the annual BMI gain at each age with obesity at a final survey. RESULTS: The 6-year-old non-obese children who became obese at a final survey showed larger annual BMI gains at any age compared with their peers with respect to baseline BMI. Increases in annual BMI gain, even in early school age, raised the risk of adolescent obesity. Categorical analysis also showed that children aged 6-7 years with higher annual BMI gains than 1-SD above the mean had a significant risk for adolescent obesity (OR: 4.39 [95%CI: 2.98-6.46] in boys and 3.83 [95%CI: 2.60-5.63] in girls, respectively). CONCLUSIONS: A larger annual BMI gain at any school age is a risk for adolescent obesity in 6-year-old non-obese children with no critical period. This suggests the need for earlier and continuous school-based surveillance using annual BMI gain for preventive intervention of adolescent obesity development.

Climatic influence on the reproductive characteristics of Japanese males.

We previously performed a survey of the sperm characteristics of the partners of pregnant women in four cities in Japan. In the present study, we analyzed the sperm characteristics of these subjects and the correlations between these sperm characteristics and climatic changes or Y chromosome haplogroups. Our results showed that more haplogroup D2a1 males than O2b1 males were born in the first half of the year (January to June), whereas more O2b1 males were born in the last half of the year (July to December) (P<0.05). This was agreed and correlated with the seasonal variations in their mean sperm concentrations. The haplogroup C, D* and D2a1 males displayed lower sperm concentrations from March to May, followed by an increase in their sperm concentrations starting in June or July, while the O2b1 males displayed higher sperm concentrations in the first half of the year followed by a sudden decrease from July to August (P<0.05). We hypothesize that the Japanese climate has different effects on the sperm characteristics and reproductive seasonality of males from different lineages; and therefore, has influenced the modern population of Japan.

SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

The male-determining gene SRY is a hybrid of DGCR8 and SOX3, and is regulated by the transcription factor CP2.

In mammals, sex is determined by the presence or absence of the Y chromosome that bears a male-dominant sex-determining gene SRY, which switches the differentiation of gonads into male testes. The molecular signaling mechanism turning on the switch, however, has remained unclear for 18 years since the identification of the gene. Here, we describe how this gene emerged and started to work. From amino acid homology, we realized that SRY is a hybrid gene between a portion of the first exon of DiGeorge syndrome critical region gene 8 (DGCR8) and the high-mobility group (HMG) box of SRY box-3 (SOX3) gene. We identified the regulatory sequence in the SRY promotor region by searching for a common motif shared with DGCR8 mRNA. From the motif search between DGCR8 mRNA and the SRY upstream sequence, we found that the transcription factor CP2 (TFCP2) binding motif is present in both. TFCP2 overexpression did not show a significant increase of SRY mRNA expression, and TFCP2 suppression by RNA interference (RNAi) significantly reduced SRY mRNA expression. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that TFCP2 acts as a regulator by directly binding to the SRY promoter. We conclude that SRY is a hybrid gene composed of two genes, DGCR8 and SOX3; and TFCP2 is an essential transcription factor for SRY expression regulation.

Proteasome subunits are regulated and expressed in comparable concentrations as a functional cluster.

The proteasome is the main proteolytic enzyme that functions in the ubiquitin-proteasome system. The 26S proteasome has multi-subunit protease complexes consisting of 20S subunits composed of four seven-numbered rings with two outer rings containing alpha subunits and two central rings composed of beta subunits, and 19S caps of 6 ATPase and 11 non-ATPase subunits; however, it is unclear how these subunits are regulated and the 26S proteasomes assembled. To verify whether each subunit's mRNA expression is associated with the mRNA expression of other proteasome subunits, we carried out expression analysis of 34 proteasome subunits mRNA on peripheral blood from 75 subjects. The expression of proteasome subunits mRNA was comparable in each individual of the studied population and the mRNA expression has been investigated in each 20S or 19S proteasome. Our results suggest that each type of subunit is regulated by respectively common factors, and that the 20S and 19S proteasomes are regulated by different systems.

Proteomics and transcriptome approaches to investigate the mechanism of human sex determination.

The SRY gene (sex-determining region on the Y chromosome) was isolated in 1990 and is known as the testis-determining factor on the Y chromosome. The SRY has been considered as a transcription factor since it contains an HMG box, which functions as a DNA-binding domain. However, a direct target for SRY remains to be identified. We have investigated the function of SRY through proteomics and transcriptome approaches, and by using two stable SRY-overexpressing cell lines (SRY1 and SRY2) in NT2/D1 cells derived from human testicular embryonal cell carcinoma. The results of 2-dimensional gel electrophoresis show that SRY overexpression causes a considerable downregulation of many chaperone proteins. SRY also upregulates laminin, which is important for Sertoli cell differentiation. Additionally, transcriptome analysis shows that SRY overexpression upregulates many zinc finger proteins and downregulates cellular growth factors with S or G(2)/M arrest of the cell cycle and inhibition of cellular proliferation.

[An approach to prevent lifestyle-related diseases of children in collaboration with various organizations in Tokushima].

OBJECTIVE: To support the goal of "Lifetime health promotion from childhood", a Committee for Strategies to Prevent Lifestyle-related Diseases was established as part of the Tokushima Prefecture Medical Association in 2000. In this report, we present the activities of this committee, in collaboration with various organizations such as schools, a medical association, health administrators and universities. ACTIVITIES: In 2000, a physical survey was performed for all students in primary and junior high schools in Tokushima prefecture. Subsequently, a software program for determining the degree of obesity using the standard body weight of Tokushima children was produced. In 2001, the committee conducted a survey concerning measures taken against lifestyle-related diseases by each organization. In 2003, a "Health management system for obesity in children" and a "School urine examination system" were established to identify high-risk students who should be taken to consult primary physicians. These medical intervention systems have allowed continuous calculation of real numbers and actual status of problems with overweight and obese children. Moreover, we performed lifestyle habit surveys among about 3000 students and produced manuals for population-based approaches. RESULTS: Compared with nationwide values, there was no difference in height, but the weight and BMI (Body Mass Index) of Tokushima students were larger. The survey concerning measures against lifestyle-related diseases clarified the present status of school health committees, staffing available to provide individual/nutritional guidance and the execution rate of collaborative projects in each organization. The intervention systems for visits to primary physicians have continued to show almost constant consultation rates. Approximately 80% of severely obese children had at least one medical problem. The lifestyle habits survey did not identify any marked differences in children of Tokushima Prefecture compared with nationwide values, except for a slightly earlier waking-up time. However, this survey demonstrated differences in lifestyle habits according to the body physique, and a relationship between eating meals with the family and other lifestyle habits. The numbers of overweight and severely obese children in Tokushima have been decreasing since the peaks of 2001 and 2002. CONCLUSIONS: Activities to prevent lifestyle-related diseases from childhood have continued in collaboration with various organizations in Tokushima. The prefecture-wide physical surveys and high-risk intervention strategies might have had good social effects in Tokushima. As a result, the number of obese children may be decreasing.

Effects of lifestyle habits and eating meals together with the family on the prevalence of obesity among school children in Tokushima, Japan: a cross-sectional questionnaire-based survey.

Obesity in children has become a major global public health concern. The prevention of obesity must start from early childhood in order to establish sound lifestyle habits and promote healthy adulthood. In this study, we evaluated factors associated with the prevention of obesity and the development of healthy lifestyle habits in children. A cross-sectional, questionnaire-based survey was performed in elementary and junior high school students in Tokushima Prefecture, Japan, during the summer of 2004. The questionnaire consisted of 30 items such as physique, sleep, eating habits, diet, exercise, free time, and attending after-school lessons. Our study revealed that eating meals as a family every day is associated with a lower rate of obesity as well as getting good lifestyle habits such as eating balanced meals and getting enough sleep. Of the 3,291 students who responded to the questionnaire, 2,688 (81.7%) reported that they eat meals with their family every day. The percentage of students who eat meals with their family every day decreased with increasing school grade, with the lowest percent in the junior high school students. However, the results regarding female junior high school students revealed a marked association between eating meals with the family every day and good lifestyle habits. We recommend that parents and school teaching staff encourage the establishment of sound, healthy lifestyle habits in children from early childhood as an effective measure for the prevention of obesity.

Overexpression of SOX15 inhibits proliferation of NT2/D1 cells derived from a testicular embryonal cell carcinoma.

SOX (Sry-related HMG box) family proteins, which have an evolutionarily conserved DNA binding domain, have crucial roles in cell differentiation. However, their target genes remain enigmatic. Some members of the SOX family may have roles in regulation of cell proliferation. We established stable NT2/D1 cell lines overexpressing SOX15 (SOX15-NT2/D1), and a modified 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the SOX15-NT2/D1 cells exhibited significantly slower growth than the controls. Flow cytometry analysis revealed that an increased fraction of the SOX15-NT2/D1 cells were in G1-G0. In addition, a microarray analysis identified 26 genes that were up-regulated in the SOX15-NT2/D1 cells, but none that were down-regulated genes. Among the up-regulated genes, IGFBP5, S100A4, ID2, FABP5, MTSS1, PDCD4 have been shown to be related to cell proliferation and/or the cell cycle.

Correlation of month and season of birth with height, weight and degree of obesity of rural Japanese children.

Month and season of birth are thought to influence height, weight and degree of obesity in schoolchildren. A cross-sectional study was designed to measure the height and weight of all children aged 6-15 years attending primary and junior high schools in Tokushima Prefecture, Japan. Data were standardized (z-scores) and analysed separately by gender and age. The mean z-score for height and weight were the highest in subjects born during the months of spring and the lowest in those born during the months of winter (p < 0.0001), whereas the means were significantly higher in children born during the months of summer than in those born during the months of autumn (p < 0.0001). A gradually decreasing trend of height and weight was observed in children of both genders born between May and Mar (from spring to winter). There was no significant difference in degree of obesity among the four seasons of birth for boys and girls. The highest prevalence of obese boys have born during spring (among 6-year-old boys) and summer (among 7-year-old boys), whereas the highest prevalence of obese girls have born during spring (among 6-year-old girls) and winter (among 10-year-old girls). Our findings suggest that month and season of birth influence height and weight of schoolchildren in Tokushima but not their degree of obesity.

Y chromosomal STRs haplotypes in two populations from Bolivia.

In the present study, we typed our previously reported two microsatellite markers, DXYS241 and DXYS266 together with a basic set of nine Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413) on Y chromosomes from two Bolivian populations. Unrelated males from communities living at high- (N=59) and low- (N=142) altitude, were studied. Combining the alleles into 11 Y-STRs haplotypes revealed that the high-altitude population is significantly less diverse than the low-altitude population. Haplotype diversities of 0.927+/-0.029 and 0.996+/-0.002 were found within the high-altitude, and the low-altitude populations, respectively. Within the high-altitude population 40 haplotypes were detected, whereas in the low-altitude population 113 haplotypes were found. Only three haplotypes were shared between both populations. Haplotyping-based discrimination using the 11 Y-STRs including our new two microsatellite markers DXYS241 and DXYS266 was shown to be powerful than using the conventional 9 Y-STRs, especially for the low-altitude Bolivian population. This 11 Y-STRs-based haplotyping system shows a very high potential for discrimination and could provide an ideal tool for forensic analysis and population studies. Moreover, this study includes data about two Bolivian populations which were not previously reported, this will help in building a world-wide database for future use in forensic and legal studies.

A rapid and simple system of detecting deletions on the Y chromosome related with male infertility using multiplex PCR.

Around 10% of males with idiopathic azoospermia or oligozoospermia, which are important causes of male infertility, have partial deletions on the long arm of the Y chromosome. To develop a rapid and accurate detection system for screening major Y deletions found in infertile men, we developed a multiplex PCR system that can simultaneously amplify five loci on the Y chromosome, SRY, AMELY, DBY, RBMY, DAZ and one locus on the X chromosome, AMELX. The size of the PCR products was designed to increase gradually from the distal Yp to the distal Yq. Our system could detect deletions of three major candidate regions for the azoospermic factor, AZFa, AZFb and AZFc on the Y chromosome together with sex. The gradual increase in the size of the PCR products was convenient for imaging the location of deletions on the Y chromosome. Moreover, the multiplex PCR system was combined with microchip-based electrophoresis, and the PCR products derived from each locus were separated within 4 min. Our system is useful for screening Y chromosomes bearing the structural anomalies including three major AZF deletions found among azoospermic or oligozoospermic males.

A/G heterozygote of the A-3826G polymorphism in the UCP-1 gene has higher BMI than A/A and G/G homozygote in young Japanese males.

UCP-1 is suggested to have important roles for thermogenesis and energy expenditure. To elucidate whether the A-3826G polymorphism that is located in the 5' flanking region of the UCP-1 gene has roles in healthy young people, the polymorphism was genotyped among 251 young Japanese men whose mean age is 22.7 years old. We analyzed relationship between the A-3826G polymorphism and body mass index (BMI) or six biochemical parameters, serum concentration of total cholesterol (TC), high density lipoprotein (HDL) cholesterol, triglyceride (TG), asparatate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose. The genotype frequencies were observed at the frequencies of 24.3% for AA, 48.2% for AG and 27.5% for GG, respectively. When BMI and the biochemical parameters were compared by ANOVA among individuals with each genotype, the statistical difference was observed only for BMI (P=0.016). Bonferroni's test demonstrated that the men with the AG genotype have higher BMI than those with the AA genotype (22.4+/-2.8 vs. 21.4+/-2.2) (P=0.04). The individuals with the AG genotype also showed trend to have higher BMI than those with the GG, although the difference was not statistically apparent (22.4+/-2.8 vs. 21.5+/-2.3) (P=0.07). Our results indicated that the young healthy Japanese men with the AG heterozygote showed higher BMI than those with other genotypes.

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome.

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.

[An evaluation of teaching objectives for seminars and clerkship of social medicine in medical schools in Japan].

OBJECTIVES: The purpose of this study was to evaluate the curriculum of seminars and clerkship for social medicine in medical schools in Japan, with special reference to teaching objectives. METHODS: A survey was conducted in December, 2002 by sending questionnaires to all the member departments of the Conference for Hygiene and Public Health Teaching in Medical Schools in Japan. Teaching objectives for seminars and clerkship of social medicine stated in their curricula were analyzed by frequencies of key words related to learner's "Knowledge, Attitude, and Behavior". Also, five professors of public health independently rated the stated teaching objectives using nine evaluation criteria divided into three levels and mean ratings were obtained. RESULTS: Although 80% of the schools described their General Instructional Objectives (GIOs), only 63% of the universities stated Specific Behavioral Objectives (SBOs). Evaluation of the contents of the descriptions revealed that, although many courses described the GIOs with the student as subjective, only a small number of courses mentioned "Attitude and Behavior" in SBO. Also, many courses did not make any apparent distinction between GIOs and SBOs. CONCLUSIONS: Practical training is a crucial component in medical education and seminars and clerkship play an important role in teaching social medicine to medical students. However, the present study revealed that many medical schools in Japan do not have adequately defined teaching objectives. Improvement of the curricula in courses of social medicine is required with particular reference to specific behavioral objectives and goals for seminars and clerkship.

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis.

We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.

Ultrafast diagnosis of the genetic-related disorders using the combined technologies of multiplex PCR and multichannel microchip electrophoresis.

For the diagnosis of unexplained male infertility a multiplex PCR for 6 markers, which are well-known as candidate genes for studying male infertility and located on the human Y-chromosome, has been designed. The multiplex PCR products have been separated on a 12 channel microchip electrophoresis system, which can analyze different samples simultaneously. By combining the technologies of multiplex PCR with multichannel microchip electrophoresis, the number of the DNA markers that can be screened simultaneously is increased to be 72 marker (12 x 6) in a single run while the electrophoresis analysis time is reduced to be only 180 s.

Multiplex PCR with multichannel microchip electrophoresis: an ultrafast analysis for genetic diseases.

A Y chromosomal polymorphic markers screening strategy using a multiplex polymerase chain reaction (PCR) and DNA microchip electrophoresis technology has recently been developed. It is a part of the human Y chromosome haplotyping system for studying Japanese population genetics and its relationship with male spermatogenic failure. This strategy is based on optimizing and modifying the primer set concentrations while keeping all other components of the PCR mixtures and conditions similar to those of a singleplex PCR. Well-balanced PCR products are obtained without changing even the DNA oligomer melting temperatures. Here, a panel of primer sets are used to amplify two groups of Y chromosome markers. The first consists of five markers and the second consists of seven markers. Both are possibly deleted in infertile men. The microchip electrophoresis technology is fast and sensitive, enables direct molecular typing of several Y chromosomal markers, and is separated by a difference of as many as six base pairs.