S. Oshio

Application of liquid chromatography-mass spectrometry to the quantification of bisphenol A in human semen.

The potential risks to human health and reproduction from the xenoestrogen bisphenol A (BPA) have not been well established. This is due in part to the absence of accurate analytical methods to quantify BPA in biological samples. In this study we establish an accurate, sensitive and selective analytical method for the quantification of BPA in human semen. To quantify BPA we compared the techniques of liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). In addition we have taken steps to eliminate BPA contamination during sample extraction and preparation. Results show that the ELISA method gives an over-estimate of BPA concentration, which may be due, at least in part, to non-specific interactions with the BPA-antibodies. LC-MS gave much more accurate results and proved to be more sensitive with a detection limit of 0.5 ng ml(-1) compared to 2.0 ng ml(-1) by ELISA.

Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

Antisera to calreticulin inhibits sperm motility in mice

Mouse sperm were rapidly immobilized when exposed to rabbit antisera against rat calreticulin. The inhibition of sperm motility was concentration dependent at dilutions 1:50-350. The velocity of sperm did not change significantly as long as they were motile. Neither motility nor velocity of sperm was affected by adding sheep antisera to bovine calmodulin. The antisera to calreticulin also inhibited in vitro fertilization of mouse eggs. Using the avidin-biotin-peroxidase complex method, the antigen was found to be localized in the acrosome of sperm. The results indicate that calreticulin is present in the acrosome of mouse sperm and may play an important role in sperm motility.

BASIC ARGININE ESTERASE FROM HUMAN SEMINAL PLASMA: PURIFICATION AND SOME PROPERTIES

Basic arginine esterase (amidase) with a specific activity of 3.2 mumol N-alpha-tosyl-L-arginine methyl ester (Tos-Arg-Me) esterolysis per A280 was purified about 230-fold from a CM-cellulose absorbed preparation of human seminal plasma. The purified enzyme was a single band with an apparent molecular weight of 3.4-4.1 x 10(4). The amidolytic activity of this enzyme was suppressed by aprotinin, soybean trypsin inhibitor (SBTI), leupeptin, and antipain, while alpha 1-antitrypsin, ovomucoid trypsin inhibitor (OTI), EDTA, and chymostatin had no or weak effect. This enzyme hydrolyzed synthetic basic amino acid derivatives and N-alpha-tosyl-glycyl-L-prolyl-arginine-p-nitroanilide (Tos-Gly-Pro-Arg-pNA) and N-alpha-tert-butyloxycarbonyl-L-leucyl-L-prolyl-L-arginine-p-nitroanilid e (Boc-Leu-Pro-Arg-pNA) were the best substrates. The enzymatic characteristics of present enzyme were clearly different from tissue kallikrein, acrosin, and seminin in human semen.

INDIVIDUAL VARIATION IN SEMEN PARAMETERS OF HEALTHY YOUNG VOLUNTEERS

Individual variation in semen parameters was investigated in healthy young volunteers. Semen samples were collected approximately once a month over a one-year period for a total of 93 samples (5 to 10 samples per subject) from 12 volunteers in their twenties. Semen analysis was carried out according to the WHO Manual. The amount of variation in each semen variable was calculated for each subject by dividing the maximum value by the minimum value. The results showed that the semen volume varied by 1.9 ± 0.8 fold (1.3 to 4.2 fold), the sperm concentration by 4.8 ± 4.3 fold (1.5 to 17.2 fold), the percentage of sperm with forward progression by 2.8 ± 1.4 fold (1.6 to 6.4 fold), the percentage of sperm with rapid linear progression by 3.4 ± 2.6 fold (1.7 to 10.9 fold), the percentage of sperm with normal morphology by 1.9 ± 0.4 fold (1.3 to 2.4 fold), and the percentage of live sperm by 1.5 ± 0.4 fold (1.1 to 2.6 fold). A between-group comparison showed significant differences in all of the variables except the percentage of sperm with normal morphology. These results suggest multiple and considerable semen analyses are needed when evaluating semen parameters.

BLOOD COAGULATION FACTOR X (FX) IN HUMAN SEMINAL PLASMA

The active form of human blood coagulation factor X (FXa, EC 3.4.21.6) showing N - f -Benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine- p -nitroanilide (S-2222) hydrolyzing activity was first detected in human semen (seminal plasma) by affinity chromatography using anti-human coagulation factor X, and this enzyme activity was inhibited by anti-human FX. This enzyme has been associated with the human coagulation factor X (FX) in human semen (seminal plasma) by Western blot analysis, and the molecular mass of mature FX was also estimated to be 59 KDa by SDS-PAGE and Western blot analysis.

XX-male syndrome bearing the sex-determining region Y

The case of a 25-year-old man who presented for evaluation of infertility is described. The physical examination revealed testicular atrophy without gynecomastia. Repeated seminal analyses showed azoospermia, and serum hormonal levels suggested a state of a hypergonadotropic hypogonadism. Chromosomal analysis demonstrated 46XX. Polymerase chain reaction revealed the existence of a sex-determining region Y. The etiology of this rare sex reversal syndrome is discussed and cases reported in Japan are reviewed.

Gonadal Function in Patients with Testicular Germ Cell Tumors

The gonadal function of 18 patients with testicular germ cell tumors was evaluated. Seminal parameters after orchiectomy were examined in 15 patients. Six of them were available for follow-up observation after 2 or 3 courses of adjuvant chemotherapy. Serum gonadal hormones before and after orchiectomy were evaluated in 7 patients (testosterone and PRL were not examined in one patient). Five of 15 (33.3%), 8 of 15 (53.3%), 13 of 15 (86.7%), 7 of 13 (53.8%), and 9 of 12 (75.0%) had abnormal values in seminal volume, sperm concentration, motility, morphology, and vitality, respectively. The sperm concentration gradually improved after chemotherapy following orchiectomy in 5 of 6 (83.3%) patients. In all the patients examined, serum levels of follicular stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) increased after orchiectomy. Serum levels of testosterone increased in 4 patients, but decreased in 2 after orchiectomy. These findings suggest that several factors, including preexisting intrinsic defect and disturbance of the hypothalamus-pituitary-gonadal axis, are involved in the deterioration of gonadal function in patients with testicular germ cell tumors.

Two Forms of Basic Trypsin-Like Arginine Amidases in Boar Sperm

Acrosin and newly detected basic arginine amidase were separated from boar sperm by affinity adsorption using lima bean trypsin inhibitor (LBTI) and aprotinin columns, respectively. These enzymes differed in various respects, including response against calcium chloride, amidolytic substrate specificity, and reaction against the inhibitor. They also differed widely in the affinity to LBTI. The difference appears to be expressed as the difference in ability of affinity of the enzymes with LBTI, thus leading to their separation.

Detection and Separation of Two Kinds of Acidic Arginine Amidases from Boar Sperm Using Lima Bean Trypsin Inhibitor and Aprotinin Affinity Adsorptions

Two kinds of acidic arginine amidase activity were found in boar sperm. One enzyme was separated by a treatment consisting of lima bean trypsin inhibitor (LBTI) affinity adsorption and elution. The other enzyme was separated by aprotinin affinity adsorption and elution through the same solutions as those used for first enzyme; the two enzymes provisionally named boar sperm acidic arginine amidases 1 (BSAA-1) and 2 (BSAA-2), respectively. The amidolytic activity of BSAA-1 was increased by high concentrations of calcium chloride, while the activity of BSAA-2 was independent of calcium chloride. Their behavior with LBTI and aprotinin, and profiles of their substrate specificities, were also different. The affinity of LBTI to BSAA-1 was approximately 14 times higher than that to BSAA-2.