Exceptional material requirement for reproduction in mouse oocytes.

Limited information on oocytes and fertilization prevents the efficient therapy of patients with infertility. The most important reason for this lack of understanding is a deficiency in research dedicated to oocytes and fertilization. Currently, we are concerned with the role of nutrition in the process of oocyte development to better understand the relationship between nutrition and infertility. The aim of this study was to explore the relationship between some exceptional materials and infertility to elucidate the role of these materials in oocyte development. We used proteomic analysis to identify numerous nutrition-related proteins in three developmental stages: the germinal vesicle stage, the metaphase II-arrested stage, and the fertilized oocyte-zygote stage. Specific proteins were abundantly expressed during the three stages. These proteins included astacin-like metalloendopeptidase, selenium-binding proteins, and other proteins involved in metabolic and signaling pathways. Other proteins were involved in the citrate cycle, the electron transport chain, the urea cycle, fatty acid metabolism, and the insulin signaling pathway. Almost all these proteins exhibited different expression levels in the three stages. The results of the present study provide a better understanding of the molecular mechanisms of early embryonic development and suggest new treatment methods for infertility.

The NMDA receptor antagonist MK-801 attenuates c-Fos expression in the lumbosacral spinal cord following repetitive noxious and non-noxious colorectal distention.

The effects of pretreatment with an NMDA receptor antagonist, MK-801, on c-Fos (Fos) expression in the lumbosacral spinal cord following repetitive, noxious (80 mmHg) or non-noxious (20 mmHg) colorectal distention (CRD) was examined immunocytochemically in awake and urethane anesthetized rats. In awake rats, noxious CRD induced Fos expression in the lumbosacral spinal cord. Pretreatment with MK-801 (0.1-1.0 mg/kg, i.p.) produced no change or an increase in noxious CRD induced-Fos expression and caused aversive side effects. In order to examine greater doses of MK-801, further experiments were performed in rats anesthetized with urethane. Both noxious and non-noxious CRD induced Fos in the lumbosacral spinal cord. Pretreatment with MK-801 (0.5, 1.0, 5.0 mg/kg, i.p.) dose-dependently attenuated noxious CRD-induced Fos by 20-40%. Five mg/kg MK-801 attenuated non-noxious CRD-induced Fos by 20%. Lesser doses did not significantly attenuate Fos expression. The laminar distribution of Fos following MK-801 pretreatment revealed a tendency towards the deeper laminae showing the greatest attenuation at the highest dose of MK-801. Protein plasma extravasation in the colon measured with Evan's blue dye showed no difference between rats without balloons, rats with balloons that were not distended and non-noxious CRD. There was significantly more extravasation following noxious CRD. Pretreatment with systemic MK-801 had no effect on plasma extravasation produced by noxious CRD. These data suggest that the induction of Fos in the lumbosacral spinal cord by noxious and non-noxious CRD is partially NMDA receptor mediated. However, NMDA receptor activation contributes significantly more to noxious than non-noxious CRD-induced Fos. Inflammation of the colon following noxious CRD likely contributes to sensitization of colonic afferents which may contribute to the increased NMDA receptor-mediated Fos following the noxious stimulus.

Chronic administration of morphine decreases level of dynorphin A in the rat nucleus accumbens.

The effect of chronically administered morphine on the levels of dynorphin A in distinct regions of the brain (including medial frontal cortex, olfactory tubercule, nucleus accumbens, dorsal and medial striatum), was determined in male Sprague-Dawley rats. The drug was delivered through a subcutaneously implanted Azlet miniosmotic pump over a period of 5 days. The concentration of peptide was probed by radioimmunoassay, following pre-separation of tissue extracts by reversed phase separation on a SepPak C-18 cartridge. The result showed that the level of dynorphin A remained unaltered in all regions studied immediately before (tolerance) and 20 hr after (withdrawal) the pump was removed. A significant decrease in the level of dynorphin was found in the n. accumbens 48 hr (abstinence) after removal of the pump. It is suggested that previously observed changes in the reward system during abstinence may be connected with dynorphinergic neurones in the limbic system.

Intrathecal injection of an oxytocin-receptor antagonist attenuated postnephrectomy natriuresis in the male rat.

We have previously shown that oxytocin (OT) is a major humoral mediator in postnephrectomy natriuresis. As immunoassayable OT has been demonstrated in the spinal cord, the aim of this investigation was to determine whether OT receptors in the spinal cord are also involved in this natriuresis. The experiments were performed on anesthetized male rats. Before acute unilateral nephrectomy, an oxytocin-receptor antagonist was injected intrathecally in the thoracolumbar region in rats. Postnephrectomy natriuresis was attenuated by this injection but not by intrathecal injection of artificial cerebrospinal fluid. Our results suggest that OT receptors within the spinal cord may influence the autonomic nervous regulation of renal function. In an additional experiment, intravenously infused hexamethonium did not prevent the adaptive natriuresis in the remaining kidney. We conclude that OT receptors in the spinal cord are involved in the postnephrectomy natriuresis, possibly as a component in the afferent signal pathway.

Decreased binding of growth hormone in the rat hypothalamus and choroid plexus following morphine treatment.

Male Sprague-Dawley rats were continuously infused with morphine through subcutaneously implanted mini-osmotic pumps over a period of 5 days. The binding of rat growth hormone (rGH) to specific sites in choroid plexus, cortex, hypothalamus, hippocampus and striatum was determined. It was found that in the acute phase of morphine administration the density of growth hormone-binding sites was significantly decreased in choroid plexus and in hypothalamus, but not in any other of the tissues studied. When tolerance to morphine was developed, the level of growth hormone-binding was restored to control level. In the acute phase, the plasma levels of GH, as measured by radioimmunoassay, correlated negatively with the density of the binding sites in choroid plexus and hypothalamus. The decrease in growth hormone-binding in these regions of the rat brain was also confirmed by SDS-polyacrylamide gel electrophoresis of cross-linked complexes of the binding entities to 125I-labelled rGH as visualized by autoradiography. In experiments, where morphine was administrated by intermittent injections, a similar decrease in rGH-binding was observed. However, the time-course of this decrease seemed to be dependent upon the route of administration. Following intracerebroventricular (i.c.v.) injections, the binding of the hormone was already affected after 30 min, whereas the binding of rGH in brain areas after subcutaneous (s.c.) injections was affected at a later stage.

Simultaneous determination of lanthanum and cerium in mixed rare earths with p-acetylarsenazo by spectrophotometry.

A new method has been developed for the simultaneous spectrophotometric determination of small amounts of lanthanum and cerium in the presence of large amounts of rare earth elements. Lanthanum (III) and cerium (III) were determined spectrophotometrically with p-acetylarsenazo as the color reagent in the chloroacetic acid medium at pH 3.1 by measuring the absorbances of the complexes at 670 nm. The remained rare earths were masked with ethylenediaminetetracetic acid and ethylenediaminetetracetic acid-zinc during the analysis. The optimum conditions for the simultaneous determination of lanthanum and cerium have been defined. The individual content of lanthanum (III) and cerium (III) were determined by varying the amounts of EDTA and EDTA-Zn used in the analysis and solving the simultaneous absorbance equations based on the Beer's law. The proposed method has been successfully applied to the determination of lanthanum and cerium in Longnan mixed rare earth oxides and other heavy rare earths without preliminary separation with satisfactory results. The relative errors of all analytical results of the method were not more than 2% with good precision. The procedure does not require separation of lanthanum, cerium and the other rare earth elements.

Effects of rat brain kappa 1- and kappa 2-opioid receptors after chronic treatment with non-peptide kappa-agonists.

Injection of kappa-agonist dynorphins and non-peptide kappa-agonists into the hippocampus induces a reduction in blood pressure. It has been postulated that kappa-opioid agonists and kappa-receptors are important in one mechanism of antihypertension and might have clinical potential for the treatment of hypertension. We have investigated whether chronic treatment with U-50488H and U-62066E, two non-peptide kappa-agonists, effects brain kappa 1- or kappa 2-receptor numbers or affinities in areas that might correlate with changes in blood pressure. kappa 1- and kappa 2-Opioid receptor affinities and densities were determined in cortex, hippocampus, hypothalamus, midbrain and pons after 14 days subcutaneous infusion of two non-peptide kappa-agonists, U-50488H and U-62066E, 9.6 mg kg day-1, by means of osmotic minipumps, to spontaneously hypertensive rats (SHR) and to Wistar-Kyoto (WKY) rats. This infusion significantly reduced blood pressure. Brains were removed within 48 h of the end of drug infusion and kappa-receptor binding studies were performed on homogenates from each brain area using [3H]U-69593 to assay kappa 1-receptors and [3H]bremazocine to assay kappa 2-receptors. U-62066E treatment seemed to cause a slight decrease in the number of [3H]bremazocine binding sites (kappa 2-receptors) from 98.2 +/- 9 to 74.9 +/- 8 fmol (mg protein)-1 in the hippocampus when compared with SHR controls. A small decrease in kappa 2-receptor density in the pons of WKY rats was also observed after U-50488H treatment (control, 51.2 +/- 5; U-50488H-treated, 24.3 +/- 9 fmol (mg protein)-1. Although SHR blood pressure values were consistently reduced by treatment with kappa-agonists, there was little if any significant change in apparent numbers of kappa 1- or kappa 2-receptors or their affinities in any of the brain regions examined. These data indicate that although chronic treatment with kappa-agonists reduces blood pressure in SHR, the treatment does not elicit major changes in brain kappa-receptors either in SHR or in WKY rats. The potential use of kappa-agonists for treating hypertension might not cause receptor changes in the brain and might, therefore, result in fewer side effects or negligible rebound hypertension.

[A study on serum suppressive factor(s) on lymphocyte proliferation in rats under restraint stress].

In order to study the effect of stress on lymphocyte proliferation, SD rats were restrained with four limbs tied on a frame in supine position at room temperature (20 degrees C) for 20 h, and control animals were not disturbed in home cage. The blood was then collected from the heart under light ether anesthesia. The peripheral blood lymphocytes were separated from heparinized whole blood by density gradient (d 1.077) centrifugation, or the serum was obtained after the blood coagulated at 4 degrees C for about 6h. It was found that the blood lymphocyte proliferation induced by Con A was significantly inhibited in the stressed group as compared with the control (P less than 0.01, n = 8, ANOVA). The result was in accordance with our earlier study in which the animals were stressed with electric shock. In the present study, it was also found that the serum of the stressed animals was capable of suppressing Con A-induced lymphocyte proliferation of normal mice (P less than 0.01, n = 8, ANOVA) to a significant extent. Thus the present experiment suggests that there is some substance with suppressive activity on lymphocyte proliferation in the serum of the stressed rats. The serum lost its suppressive activity when it was heated to 100 degrees C (3 min), treated with 60% methanol or incubated with trypsin (64 micrograms/ml), thus suggesting that the suppressive factor(s) most likely is a kind of protein.

Comparative hypotensive actions of three nonpeptide kappa opioid agonists on hippocampus of SHRs and normotensive WKY rats.

Comparative centrally mediated hypotensive effects of three nonpeptide kappa opioid agonist drugs (bremazocine, spiradoline, and U-50,488H) were evaluated in chloralose-anesthetized male spontaneously hypertensive rats (SHRs) and in normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. The drugs were administered unilaterally into previously established active hypotensive sites in the dorsal hippocampus at doses of 12, 24, and 48 nmol. Each drug produced dose-related decreases in mean arterial pressure, ranging from -5 to -40% of predrug control values, with bremazocine being somewhat more effective than spiradoline, which was in turn slightly more active than U-50,488H. The effects were only marginally greater in SHRs than in normotensive controls. Each drug caused a modest decrease in heart rate, but except for the highest dose of bremazocine, the effects were not statistically significant. The onset of hypotension after intrahippocampal injection of each agent was approximately 2 min and lasted approximately 30 min with U-50,488H and spiradoline and >60 min with bremazocine. The responses to all three drugs were completely blocked by prior injection of the active hippocampal sites with nor-binaltorphimine (nor-BNI), a selective kappa-receptor antagonist. Because bremazocine is more selective for kappa-2 opioid receptors, whereas U-50,488H and spiradoline favor the kappa-1 subtype, the results suggest that drugs active on each of these subtypes should be investigated as potential antihypertensives.

Sustained antihypertensive effects of chronic administration of two kappa-opioid agonists in spontaneously hypertensive rats.

The ability of two nonpeptide kappa-opioid receptor agonists, U-50, 488H and U-62,O66E (spiradoline), to lower arterial pressure on chronic administration to 12-week-old male spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar Kyoto (WKY) rats was assessed. Each drug was infused subcutaneously at approximately 9.6 mg/kg/d, over a 14-day period with Alzet osmotic pumps in each strain of rat. Arterial pressures were determined daily in each rat by photoelectric tail-cuff plethysmography. Control rats received similar infusions of 0.9% NaCl. Body weights and water consumption were also recorded daily. Both drugs effected a 10% to 20% sustained lowering of arterial pressure beginning on the second day of infusion, until explantation of the pumps on day 14. Heart rates similarly were decreased by 10% to 15% in SHRs. By contrast, neither drug caused significant decrements in arterial pressure or heart rate in the normotensive WKY group. Control infusions of 0.9% NaCl had no significant effects on arterial pressure or heart rate in either SHRs or WKY rats. U-62,066E, but not U-50,488H, caused sustained increased water consumption in both SHRs and WKY rats, with a greater effect in the SHR strain. This likely was accompanied by a water diuresis. Body weight gains over the 14-day period were similar for both strains of rats treated with U-50,488H, compared with saline-treated controls, but rats of both strains infused with U-62,066E gained significantly less weight than saline controls over the 14-day period. The results are supportive of further experimental evaluations of the potential antihypertensive use of nonpeptide kappa-opioid agonist drugs.