Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men.

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (ß = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.

Pyrethroid insecticide exposure and reproductive hormone levels in healthy Japanese male subjects.

The associations between serum levels of reproductive hormones (follicle-stimulating hormone, luteinizing hormone, testosterone, sex hormone-binding globulin, inhibin B and calculated free testosterone) and urinary metabolite concentration of pyrethroid insecticides [3-phenoxybenzoic acid (3-PBA)] were explored in 322 male university students in suburban Tokyo. The subjects constituted part of a large cross-sectional survey on the reference value of semen quality of Japanese men. Urinary 3-PBA was detectable in 91% of the subjects demonstrating ubiquitous exposure among the general population. However, there were no associations between urinary 3-PBA and serum hormone levels. This result was inconsistent with those reported in China and the USA for subjects who had similar levels of urinary 3-PBA to the present subjects. One of the possible reasons of the inconsistency might be different composition of pyrethroid insecticides to which the subjects were exposed; 3-PBA is a common metabolite of a number of pyrethroids and thus lacks specificity to compounds that may have different potentials of reproductive toxicity. Another reason might be related to the fact that our subjects were university students who were not aware of their own fertility, whereas the previous study subjects were infertility patients. However, the multiple regression models could explain only a limited fraction of total variance in serum levels of hormones. Identification of other contributors is warranted.

Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men.

Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia.

Xenografting of testicular tissue from an infant human donor results in accelerated testicular maturation.

BACKGROUND: Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts. METHODS AND RESULTS: Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development. CONCLUSIONS: Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.

Polybrominated diphenyl ethers in human serum and sperm quality.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants; currently, they are identified as ubiquitous environmental contaminants. Several studies indicate that PBDEs might affect male fertility. We present the results of a pilot study on the relationship between human serum PBDEs and sperm quality. The PBDE levels in Japan are comparable to those found in European countries. Strong inverse correlations were observed between the serum concentration of 2,2',4,4',5,5'-hexabromodiphenyl ether and sperm concentration (r = -0.841, p = 0.002) and testis size (r = -0.764, p = 0.01). Extensive studies on the relationship between PBDEs and sperm quality are required.

Semen quality of 324 fertile Japanese men.

BACKGROUND: A number of studies have indicated regional differences in semen quality. To examine the current status in Japan, we undertook a cross-sectional study on the semen quality of fertile Japanese men for comparison with recent European results. METHODS: Semen parameters of 324 fertile men from the Kawasaki/Yokohama area were investigated. The semen parameters were compared with those published for fertile men from four European cities, Copenhagen, Paris, Edinburgh and Turku. RESULTS: When adjusting for confounders such as ejaculation abstinence period and age, the lowest sperm concentrations were detected in men from Kawasaki/Yokohama followed by men from Copenhagen, Paris, Edinburgh and Turku, but only the differences between men from Kawasaki/Yokohama and men from Edinburgh and Turku were significant (P=0.0008 and P<0.0001, respectively). Total sperm count, percentage of motile sperm and percentage of normal sperm observed in Kawasaki/Yokohama were significantly lower than those from all European centres except for motile sperm in men from Paris. CONCLUSIONS: Japanese fertile men had a semen quality at the level of Danish men, who have been reported to have the lowest among investigated men in Europe. The low level of semen quality of the fertile Japanese men may be due to lifestyle or other environmental factors; however, ethnic differences caused by different genetic variation or combinations cannot be ruled out by this study.

Molecular identification of Trichophyton rubrum isolate from a dog by chitin synthase 1 (CHS1) gene analysis.

A nonsporulating isolate from a dog with dermatophytosis was identified as Trichophyton rubrum by molecular analysis. The nucleotide sequence analysis of the chitin synthase 1 (CHS1) gene from the isolate indicated more than 99% sequence similarity with other human and canine isolates of T rubrum. The molecular typing suggested that isolates of T. rubrum from human and canine sources were genetically identical.

A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker.

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.

Current status of reproductive function in Japanese fertile men: international collaborative project on a study of partners of pregnant women.

The data on reproductive function in 255 Japanese fertile men resident in the Kawasaki/Yokohama area in Japan were described. The sperm concentration was 107.9 +/- 97.4 x 106/mL. The semen volume was 3.2 +/- 1.5 mL and percentage motile spermatozoa (grade A + B in WHO criteria) was 56.8% +/- 14.7%. The evidence for secular changes in semen quality and other changes in male reproductive health is inconclusive, although regional differences would appear to be stronger. The present study is the first large-scaled prospective survey on the reproductive function of Japanese normal men proven fertility, which was planned as an international comparative study.

Spermatogenic ability is different among males in different Y chromosome lineage.

It is a controversial question whether sperm concentrations in humans are changing. Several researchers have reported on environmental factors affecting sperm quality, but the influence of genetic factors is still not fully understood. In this study, we examined the relationship between Y chromosome haplotypes and sperm concentration in fertile males. In addition, we determined the haplotypes of azoospermic patients. The results show that the mean sperm concentration correlates with Y chromosome type. Moreover, the occurrence of azoospermia is related to one particular Y chromosome lineage. Thus, males with a certain haplotype are at a disadvantage for fathering children. The difference of spermatogenic ability among men is important not only in pursuing male competition as in the past but also as relates to the future of modern human males.

Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells.

Human seminal plasma sperm motility inhibitor (SPMI) proteins which are exclusively secreted from seminal vesicles, inhibit sperm motility. It is secreted as biologically active 52 kDa and a mixture of 71 and 76 kDa precursor forms, which are identical to semenogelin-I and II (Sg-I and Sg-II), respectively. To understand the molecular mechanism underlying the inhibition of sperm motility by SPMI proteins, we expressed human Sg-I and Sg-II genes in insect cells using a baculovirus system. The baculoviruses expressing full-size Sg-I and Sg-II proteins that were N-terminally-tagged with a hexahistidine were selected, and were infected with Sf 21 cells. The Sg-I and Sg-II proteins were purified from infected cells by column chromatography using Ni-NTA resin 48 h after infection. The full-size Sg-I and Sg-II proteins were obtained in soluble forms. However, they tended to aggregate to form a gel, as expected from naturally occurring semenogelin. Both the purified recombinant Sg-I and Sg-II proteins showed strong SPMI activities with a complete inhibition of sperm motility at 60 units/mg, equivalent to the natural proteins. This production system that permits the generation of purified Sg-I and Sg-II proteins, as well as mutant derivatives, will be helpful for further study on male infertility.

Oxygenation reaction of methyl valproate with a mono-oxygenase model reagent: implication for stereochemical identification of the mammalian metabolites of valproic acid.

Non-enzymic oxygenation reaction of methyl valproate (2) utilizing a simple model system for mono-oxygenases, Fe(MeCN)2(6+)-H2O2-Ac2O in MeCN, was investigated in connection with stereochemical analyses of the mammalian metabolites of 1. This oxygenation reaction of methyl valproate (2) gave a 92:8 mixture of the anti-isomer 4a and the syn-isomer 4b, together with 5a, and 5b corresponding to the mammalian metabolites of 1. The stereochemistry of 4a, 5a, and 5b was elucidated by spectral analyses of the corresponding beta-lactone 6a, gamma-lactone 7a and 7b prepared from the oxygenation products. The asymmetric synthesis of (+)-7a was also achieved.

Enzymes in intestinal juice from patients with liver diseases and colon polyps: measurement of bilirubin, alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase.

Since the amounts of hepatogenous enzymes discharged into the intestinal tract remain unknown, this study was initiated to evaluate the amounts of the enzymes in the intestinal tract. Whole gut lavage fluid (polyethyleneglycol electrolyte solution) was administered orally to 42 subjects, consisting of 5 patients with hepatoma, 10 with chronic hepatitis, 10 with colon polyps, and 17 control subjects without liver disease. Two hr after the large intestinal lavage, the digestive tract juice was aspirated by colonoscopy, and the bilirubin (Bil), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in the aspirates were measured. A positive correlation between the AST and LDH values was found, and a significant difference in these values between the hepatic disorders and the normal controls was noticed. A significant positive correlation between the ALP and Bil values was found, and a statistical difference in these values between the group of colon polyps and the controls and other groups was observed. This lavage fluid technique enables to estimate the amounts of hepatic enzymes discharged into the intestinal tract, thereby opening a new avenue for future enzyme research.

[A case of advanced esophageal cancer showing good partial response by combination therapy of low dose 5-FU and low dose CDDP].

A 66-year-old man with a complaint of dysphagia was diagnosed as advanced esophageal cancer. Barium swallow examination of the esophagus showed a narrowing 10 cm in length at Ei (type 3), and biopsy specimen from the lesion on endoscopic examination revealed adenosquamous carcinoma. Multiple lymph node metastasis were detected by CT scan. He was treated with a combination of low dose 5-fluorouracil (5-FU) and low dose cisplatin (CDDP). The regimen consisted of 5-FU (300 mg/body/day continuous infusion) and CDDP (10 mg/body/day continuous infusion) for 3 weeks. After 2 courses of this regimen, his symptoms disappeared, and only mild irregularity of the esophageal wall remained on Barium swallow examination. The effect of the therapy was evaluated as a partial response. No side effect was observed. From this case, the possibility that CDDP is able to function as a biochemical modulator for 5-FU was suggested.

Management of acute leukemia during pregnancy: from the results of a nationwide questionnaire survey and literature survey.

In March, 1993, a questionnaire was sent to 362 gynecological and obstetric offices of national, prefectural and municipal hospitals and private university hospitals with 250 beds or more. Answers were collected from 260 institutions. Thus, this study analyzed 39 patients with acute leukemia during pregnancy collected by the questionnaires survey and 64 cases reported in the Japanese literatures during 1975-1993 (total 103 patients). The weeks of pregnancy were defined as the 1st (< 15th week), 2nd (16th-27th week), and 3rd (> 28th week) trimesters. The time of diagnosis of leukemia during pregnancy changed from 25% in the 2nd trimester and 62% in the 3rd trimester during 1975-1984 to 39% and 48% after 1985, respectively. After 1985, the remission rate was 72% in the questionnaire group and 75% in the group from literatures. There was no statistical difference. The 50% survival period was 12 months in the group during 1975-1984, but 25 months in the group after 1985. The survival was significantly longer in the patients whose induction therapy was started before delivery than in those treated after delivery. The results suggest that the treatment for acute leukemia during pregnancy should be initiated as soon as possible after the diagnosis of leukemia, with carefully selected regimens. It is important that the time of delivery should be selected considering the maternal and fetal conditions after consultation with an obstetrician.

Blockade of delta-opioid receptors prevents morphine-induced place preference in mice.

Effects of highly selective delta-opioid receptor antagonists on the morphine-induced place preference in ddY and mu 1-opioid receptor deficient CXBK mice were investigated. Pretreatment with naltrindole (NTI: a non-selective delta-opioid receptor antagonist), 7-benzylidenenaltrexone (BNTX: a selective delta 1-opioid receptor antagonist) or naltriben (NTB: a selective delta 2-opioid receptor antagonist) abolished the morphine-induced place preference in ddY mice in a dose-dependent manner. These findings suggest that the morphine-induced place preference may be mediated by both delta 1- and delta 2-opioid receptors. On the other hand, in mu 1-opioid receptor deficient CXBK mice, pretreatment with these selective delta-opioid receptor antagonists did not affect the morphine-induced place preference, although pretreatment with beta-funaltrexamine (beta-FNA: a selective mu-opioid receptor antagonist) significantly inhibited the morphine-induced place preference. [D-Pen2,D-Pen5]enkephalin (DPDPE: a delta 1-opioid receptor agonist) and [D-Ala2,Glu4]deltorphin (deltorphin II: a delta 2-opioid receptor agonist) induced a significant place preference in ddY mice, but not in CXBK mice. These results suggest that delta 1- and delta 2-opioid receptors in the nucleus accumbens that are related to the DPDPE- and deltorphin II-induced place preference may be dysfunctional and/or poor in CXBK mice. These findings also indicate that delta 1- and delta 2-opioid receptors may be involved in the modulation of the reinforcing effect of morphine.

Potentiation of physical dependence on diazepam by ondansetron in rats.

The effects of ondansetron, a 5-HT3 antagonist, on the development of physical dependence on diazepam were examined in rats using a drug-admixed food method. Rats were treated with diazepam or diazepam in combination with ondansetron for 26 days. After an abrupt withdrawal from diazepam, the incidence of withdrawal signs, such as jerks, tremors and convulsions, and withdrawal scores, were potentiated by co-administration of ondansetron. On the other hand, rats which had been treated with ondansetron alone for 33 days did not show any withdrawal signs after abrupt withdrawal from ondansetron. These findings suggest that ondansetron does not possess physical dependence liability, but does potentiate the development of physical dependence on diazepam. Regulation of serotonergic neurons through 5-HT3 receptors may affect the development of physical dependence on diazepam.

Effect of cyclosporine A on the morphine-induced place preference.

The effect of pretreatment with cyclosporine A, an immunosuppressant, on the morphine-induced place preference was examined in ddY and mu1 opioid receptor-deficient CXBK mice. Morphine produced a significant preference for the drug-associated place in ddY and CXBK mice. Administration of cyclosporine A alone to ddY and CXBK mice did not result in either preference or aversion for either the drug- or vehicle-associated place. On the other hand, pretreatment with cyclosporine A suppressed the place preference induced by morphine in ddY mice in a dose-dependent manner. However, in CXBK mice, pretreatment with cyclosporine A did not affect the morphine-induced place preference. These results suggest that cyclosporine A suppresses the reinforcing effect induced by morphine, and that this suppression by cyclosporine A may be mediated by mu1 opioid receptors.

Effects of Ca2+ channel blockers on physical dependence on diazepam in mice.

The effects of Ca2+ channel blockers on the development of physical dependence on diazepam were examined in mice. Co-administration of flunarizine (T-type Ca2+ channel sensitive blocker), but not of either nifedipine or diltiazem (L-type Ca2+ channel sensitive blockers), with diazepam significantly suppressed the hypersensitivity to FG 7142 following chronic treatment with diazepam. The hypersensitivity to FG 7142 may reflect benzodiazepine withdrawal convulsions. These results suggest that flunarizine, but not nifedipine or diltiazem, may suppress the development of physical dependence on diazepam, and that T-type Ca2+ channels in the brain, rather than L-type Ca2+ channels, may be involved in the development of physical dependence on diazepam.