Naomichi Okamura

A PORCINE HOMOLOG OF THE MAJOR SECRETORY PROTEIN OF HUMAN EPIDIDYMIS, HE1,SPECIFICALLY BINDS CHOLESTEROL

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

The activating effects of bicarbonate on sperm motility and respiration at ejaculation.

Mature porcine sperm preserved in the cauda epididymis are quiescent. At ejaculation, they are mixed with the seminal vesicle fluid containing HCO3- and are rapidly activated. The role of HCO3- on the sperm activation process at ejaculation was studied in vitro. HCO3- quickly increased the motility, respiration rate and cAMP content of the porcine epididymal sperm. The extent of activation was proportional to the pCO2 in the medium. The activating effect of HCO3- on the motility was observed even in the absence of fructose as well as in the presence of KCN. 8-Bromoadenosine 3,5-cyclic monophosphate and theophylline showed similar activating effects to that of HCO3-. However, HCO3(-)-free seminal plasma, Ca2+, amino acids, intermediates of the Krebs cycle, substrates of respiration and increases in the intracellular pH, extracellular pH or ionic strength of the medium had no effect. Fructose sustained the active state of the sperm and gradually increased both the motility and respiration rate when the dose of HCO3- was low. The anion channel blocker enhanced the activating effect of HCO3-. These results suggest that, upon ejaculation, HCO3- is a unique activator in vivo which makes the quiescent sperm motile via the HCO3(-)-adenylate cyclase-cAMP system, to which an endogenous HCO3- derived from metabolic CO2 may be related.

Promoting Effect of β-Mercaptoethanol on In Vitro Development under Oxidative Stress and Cystine Uptake of Bovine Embryos

Abstract The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O2 supplemented with β-ME. Addition of β-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O2, to almost the same rates when they were cultured in 5% O2. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by β-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.

Decrease in bicarbonate transport activities during epididymal maturation of porcine sperm

Kinetic studies of [14C]HCO3- and [35S]SO4(2-) transport show the existence of a common transport system for the two anions on the plasma membranes of porcine epididymal sperm. Km value for bicarbonate (8.3mM) is within the range of physiological concentration of bicarbonate. The anion transport activity is dependent on the temperature but independent of co-existence of Na+. Its pH dependency and sensitivity to the several anion transport inhibitors are similar to those observed in erythrocyte. Anion transport activity decreases during sperm maturation in epididymis.

Lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients

Both the adenylate cyclase activity and the motility of human sperm were stimulated by bicarbonate with the same concentration dependency. The correlation between bicarbonate levels in semen and the motility of sperm from the patients with male infertility was investigated. Bicarbonate in semen was found to originate mainly from the seminal vesicles, and a significant positive correlation was observed between bicarbonate levels and volume of semen. The motility of infertile sperm was also found to correlate positively to the seminal levels of bicarbonate. These results suggest that the lowered levels of bicarbonate in semen are at least in part responsible for the poor sperm motility in infertile patients, as a result of the failure in the activation of sperm adenylate cyclase.

Positive inotropic effect of calcitonin gene-related peptide mediated by cyclic AMP in guinea pig heart.

The mechanism of cardiac actions of rat calcitonin gene-related peptide (CGRP) was analyzed on isolated guinea pig hearts. CGRP exerted a positive inotropic effect in a dose-dependent manner on the electrically driven left atria bat not on the ventricles. Immunohistochemical studies demonstrated that CGRP-like immunoreactive nerves were distributed densely in the myocardia of the atria but only sparsely in those of the ventricles. The CGRP-induced augmentation of the contraction was accompanied by the shortening of the time to peak force and the increase in the relaxation velocity. The positive inotropic response to CGRP was significantly enhanced by isobutylmethylxanthine and was attenuated by adenosine. CGRP increased the action potential amplitude and prolonged action potential duration at the level of 50% repolarization in the left atria. In the preparations, which were partially depolarized with an increase in extracellular potassium, CGRP induced slow response action potentials. These electrophysiological results indicate that CGRP causes an increase in the slow inward Ca2+ current. The cyclic AMP content in the left atria significantly increased following the addition of CGRP, the time course of which was nearly consistent with that of the augmentation of the contractile force. In the membrane preparation of the atria, the activity of adenylate cydase was enhanced by CGRP in a dose-dependent manner. These effects of CGRP are qualitatively similar to those of β-adrenoceptor stimulation. It is concluded that the CGRP-induced response in the guinea pig atria is attributed to the activation of adenylate cyclase via stimulation of its specific receptor and the subsequent increase in the intracellular cyclic AMP level.

Characterization of autofluorescence in oral squamous cell carcinoma

This study was carried out to evaluate the clinical characteristics of autofluorescence in oral squamous cell carcinoma (SCC) and analyze the fluorescent substances using high-performance liquid chromatography (HPLC). Fifty of 55 oral SCCs (91%) emitted orange or red fluorescence, which was recorded by fluorescence photography. The intensity of the fluorescence significantly correlated with the T and N categories of the cancers, but did not show statistical difference for the types of clinical appearance and primary sites. Protoporphyrin and coproporphyrin were identified as the fluorescent substance in the SCC samples, and the elution patterns on HPLC revealed some porphyrin compounds as specific to oral cancer. These results suggest that the autofluorescence in oral SCC correlates with the progression of lesions, and that fluorescent substances such as protoporphyrin are produced in association with the cancerous tissue.

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid

The epididymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymis. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA library of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the cDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit cDNA, similarly to the results previously obtained for cDNAs encoding the epididymis-specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20 microM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosomal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantly retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

Effects of calcitonin gene-related peptide (CGRP) and isoproterenolon the contractility and adenylate cyclase activity in the rat heart

The occurrence of calcitonin gene-related peptide (CGRP) has been predicted by the study of the calcitonin gene of rats [1]. Recently CGRP was isolated from porcine spinal cord [3], and thus, CGRP is assumed to be a neuropeptide. It has been shown that CGRP exhibits potent positive chronotropic and inotropic effects on the atrial muscles of rats and guinea-pigs [5, 9, 11] . We have previously demonstrated the presence of nonadrenergic noncholinergic (NANC) nerves in the atria of rats and guinea-pigs [10] and have suggested that CGRP is a neurotransmitter of the NANC nerves in the guinea-pig atria [9, 11]. It is supposed that the positive inotropic response mediated by the beta-adrenoceptor results from a stimulation of the adenylate cyclase (AC) activity and subsequent elevation of cyclic AMP (cAMP) content. In the present study, the effects of calcitonin gene-related peptide (CGRP) and isoproterenol (ISO) on the myocardial contractility and adenylate cyclase (AC) activity were compared in cardiac muscles of the rat. In atrial muscles, CGRP exerted a positive inotropic effect and stimulated the AC activity at the similar dose range. On the contrary, the concentrations of ISO needed to stimulate the AC activity were nearly 10-fold higher than those needed for eliciting positive inotropic responses. In ventricular muscles, CGRP produced neither a positive inotropic responses nor a stimulation of AC activity, while ISO induced both responses. Inasmuch as CGRP-like immunoreactive (CGRP-I) nerves were found to be present in the atria but rarely in the ventricles, CGRP was assumed to be physiologically important in the atria rather than in the ventricles.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS‐mediated oxidant injury. Cystine/glutamate transporter, designated as system xc– and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian‐cultured cells. In the present paper, we have investigated system xc– in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc– nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc– activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT‐deficient (xCT−/−) mice had lower cysteine content than those from the wild‐type mice. GSH levels in the xCT−/−cells decreased rapidly when they were cultured, whereas those in the wild‐type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT−/−cells compared with the wild‐type cells. These results suggest that system xc– plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.