The involvement of nitric oxide in corpus luteum regression in the rat: feedback mechanism between prostaglandin F(2alpha) and nitric oxide.

In the corpus luteum (CL), prostaglandin F(2alpha) (PGF(2alpha)) is a physiological agent with luteolytic actions. Nitric oxide (NO) is a messenger molecule capable of modulating diverse pathophysiological processes. The aim of the present study was to investigate the role of ovarian NO in PGE (a luteotrophic prostanoid) and PGF(2alpha) (a luteolytic prostanoid) production and in progesterone synthesis during CL regression in the rat. To obtain a longer functional CL, we used a pseudopregnant (PSP) rat model. By means of intrabursa ovarian sac treatment of two competitive nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine (L-NMMA, 1 mg/kg) and N(W)-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), and sodium nitroprusside (SNP, 0.05 mg/kg) as a NO generator, we found that NO, produced by the ovarian tissue during the last 2 days of CL development (days 8 and 9), increased PGF(2alpha) production in the ovary and diminished serum progesterone concentrations leading to CL involution. We also proposed a positive feedback mechanism between PGF(2alpha) and NO, to ensure luteal regression. Thus, we injected intraperitoneally a luteolytic dose (3 microg/kg) of a synthetic PGF(2alpha) during the mid and late phase of CL development. Ovarian NOS activity was evaluated. The results confirmed our hypothesis; we did not see any effect in the mid-stage of CL development, but increased ovarian NOS activity was found in PGF(2alpha)-injected late pseudopregnant rats.

Low chronic ethanol consumption affects ovulation and PGE synthesis by the cumulus cell masses in mice.

Central and gonadal function can be affected by chronic consumption of high and moderate doses of ethanol. Few studies have been conducted to determine the effect of ethanol intake at ovarian and gamete level. Previously, we showed that fertilization rates of low chronic ethanol treated female mice were diminished. Also, our recent results indicated that moderate chronic intake of ethanol by immature females could alter the ovulatory quantity and produce morphological alterations in the superovulated oocytes. Furthermore, PGE production by oocyte cumulus complexes (OCCs) was reduced in the females treated with 10% (w/v) ethanol. In the present investigation, we studied the effects of 5% ethanol treatment given to immature mice for 30 days on the quality and quantity of oocytes superovulated at 16 h posthuman chronic gonadotrophin. Treated females had impaired ovulation rates (P < 0.05) as compared to the controls. The percentage of activated and morphologically abnormal oocytes was elevated in the ethanol-treated females (P < 0.05). PGE synthesis by the OCCs was higher than in the controls (P < 0.01). In summary, the administration of long-term ethanol at a relatively low dose to immature females produces decreased ovulation rates, abnormal oocyte morphology with high spontaneous activation and altered levels of PGE production by the oocytes' cumulus complexes. The relationship between the oocyte quality and abnormal synthesis of PGE is discussed.

Regulation by nitric oxide of prostaglandin E synthesis and spontaneous motility in rat uterine tissue.

We explored the role of endogenous nitric oxide (NO) in the spontaneous motility of uterine tissue from pseudopregnant (psp) rats and the correlation between this action and the uterotonic prostaglandin (PG) E production. We worked in the early psp (on day 5 of psp), and in late psp (on day 8 and day 9). Treatment with N(G)-monomethyl-L-arginine L-NMMA (300 microM), a competitive nitric oxide synthase (NOS) inhibitor, did not modify isometric developed tension (IDT) and frequency of contractions (FC) on day 5 of psp; on day 8, tissue pretreated with L-NMMA showed an increase in the IDT and FC compared with controls, while on day 9 of psp, both IDT and FC showed a lower stability after treatment with the inhibitor. These data suggest that NO modulates uterine motility on day 8 (decreasing it) and on day 9 of psp (enhancing it). We also evaluated the total NOS activity and that of its isoforms at the three times mentioned, demonstrating that total NOS activity was higher on day 5 of psp and decreased with psp development. On day 5 of psp, calcium-dependent and calcium-independent NOS each forms around 50% of total NOS activity. On day 8 of psp, the calcium-dependent was the predominant NOS form, while on day 9 of psp, the uterine tissue showed a higher calcium-independent form of the enzyme. In view of the fact that we found an inhibitor effect of the endogenous NO in uterine contractility on day 8 of psp and an inverse action on day 9 of psp (enhancing uterine contractility), we suggest that the NOS calcium-dependent form could be responsible for uterine contractility in psp rats. Finally, we evaluated the relationship between endogenous NO and PGE production. We observed that on days 5 and 8 of psp, the L-NMMA (300 microM) treatment did not affect PGE production, but on day 9 of psp, the preincubation with the NOS inhibitor diminished PGE synthesis, suggesting that at this time endogenous NO can upregulate uterine PGE production. These results confirm that NO can modulate uterine motility by means of PGE production. In summary, we suggest that in uterine tissue from psp rats, the NO system can alternatively decrease or increase uterine contractions, this last effect by enhancing uterine PGE synthesis.

Mouse oocyte quality and prostaglandin synthesis by cumulus oocyte complex after moderate chronic ethanol intake.

Chronic ingestion of ethanol produces a variety of effects on female reproductive function, depending on the dose and the exposure time but the mechanism of alcohol-induced ovarian failure has been little studied. Also the effects of chronic ethanol consumption on the oocyte quality in relation to morphological alterations and PGE synthesis by the oocyte cumulus complexes (OCCs) have not been described. In this study, immature female mice were treated with 10% ethanol in drinking water for 30 days. Then they were induced to superovulate, and at 14, 16 and 20 h post-hCG the quality of the ovarian and oviductal oocytes and PGE production by OCC was determined. At 14 h post-hCG, the percentage of oviductal immature oocytes was increased in the ethanol-treated females (P < 0.05). At 16 h post-hCG, the percent of oviductal activated oocytes was higher in the treated females (P < 0.05), and the ovarian immature oocytes were decreased as compared to the control females (P < 0.05). At 20 h post-hCG, the ethanol-treated females had higher percents of activated oocytes in the oviducts and in the ovaries (P < 0.05) with respect to the controls. PGE synthesis by OCCs, assessed by RIA, was decreased in the treated female mice (P < 0.001). In summary, moderate chronic ethanol treatment in immature female mice can produce morphologic abnormalities in the oocytes (high parthenogenetic activated rates) and altered PGE production in the OCCs.

Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

Analysis of the effect of nitric oxide synthase inhibition on mouse sperm employing a modified staining method for assessment of the acrosome reaction.

A commercially available staining kit (Spermac) was combined with swelling in a hypoosmotic medium (HOS) for simultaneous assessment of viability and acrosome reaction in mouse spermatozoa. We compared the results obtained with the combined technique (HOS-Spermac) with those obtained with currently used techniques: the chlortetracycline fluorescence assay and eosin exclusion. The results obtained with HOS-Spermac were the same as those obtained with the chlortetracycline fluorescence assay. Viability assessment with HOS-Spermac showed a good correlation with the percentage of spermatozoa showing eosin dye exclusion. Using this novel technique, we studied the effect of a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) on the acrosome reaction. L-NAME produced a dose-dependent inhibition of spontaneous acrosome reaction and its inhibitory effect was specifically counteracted by L-arginine. We conclude that HOS-Spermac provides a simple and reliable tool for assessment of the acrosome reaction and that nitric oxide synthase participates in this important function of the spermatozoon.

Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa.

The localization of nitric oxide synthase was studied in mouse epididymal spermatozoa and freshly ejaculated human sperm. A rabbit antiserum against the neuronal isoform of the enzyme was used, and antibody binding was detected with a fluorescein isothiocyanate-conjugated polyclonal antibody specific for rabbit IgG. In mouse spermatozoa, the percentage of cells staining specifically ranged from 88% to 98%. Samples were examined after 0-, 90- and 150-min incubations in vitro. Three different patterns of staining were observed: (a) Pattern I, intense fluorescent staining localized in the acrosome and in a segment of the tail; (b) Pattern II, fluorescent staining localized only in the tail; and (c) Pattern III, faint fluorescent staining localized in the acrosomal cap and in the tail. The potential physiological significance of these patterns is discussed. Nitric oxide synthase was also localized in the acrosome of freshly ejaculated human sperm.

Effects of nitric oxide synthase inhibitors on the outcome of in vitro fertilization in the mouse.

The effect of three nitric oxide (NO) synthase inhibitors, L-NG-nitro-arginine (NO2Arg), NG-Nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on in vitro fertilization in the mouse was examined. Mouse epididymal spermatozoa were capacitated in a medium with or without NO synthase inhibitors. Oocytes were inseminated and the percentage of oocytes with two pronuclei was scored after an 8-h incubation. NO2Arg and L-NAME, but not aminoguanidine, inhibited fertilization. L-NAME inhibited fertilization in a dose-dependent manner, and its effects were stereospecific. The inhibitory effect was neutralized by L-arginine but not by D-arginine. Moreover, D-NAME did not inhibit fertilization. The results suggest that NO synthase activity (presumably of the constitutive type is necessary for spermatozoa to display their full fertilizing ability.

Prostaglandin synthesis by cumulus-oocyte complexes: effects on in vitro fertilization in mice.

Cumulus-oocyte complexes, obtained from superovulated Balb/C virgin female mice, released to the incubation media significant amounts of PGE1, PGE2 and PGF2 alpha, as estimated by bioassay. Fertilization rates in vitro decreased sharply when cumulus-oocyte complexes were treated with indomethacin (10(-6) M) and then inseminated with 5000 sperm per oocyte. In order to explore if the reduced prostaglandin (PG) concentration was responsible for diminished fertilization rates, PGE1, PGE2 and PGF2 alpha (10(-9) M) were added to the fertilization media of treated oocytes. PGE1 and PGE2 but not PGF2 alpha returned fertilization rates to control levels. Besides, PGE1 (10(-9) M) enhanced fertilization rates with reduced sperm numbers (1000 sperm per oocyte) of untreated cumulus-oocyte complexes. In conclusion, PG synthesis and release of mouse cumulus-oocyte complexes affects fertilization in vitro, and it is suggested that PGs of the E series modulate sperm function at the moment of fertilization.

Is tubulin involved in the electrically-induced mechanical activity of the isolated rat sciatic nerve?

The electrically-induced mechanical activity of isolated segments of rat sciatic nerves remains unaffected following incubation with 10(-4) M colchicine, vinblastine or melatonin. Vinblastine depressed tubulin levels in incubated nerves. These results suggest that microtubules are not involved in nerve mechanical activity in vitro.