Imprinting of male sex tissues by neonatal endogenous androgens in mice. Molecular alterations following exposure to cyproterone acetate

This study was conducted to evaluate the growth and biochemical responsiveness of the epididymis, vas deferens and seminal vesicles of adult mice exposed to cyproterone acetate during the first 10 days of life. Results indicate that the weight and protein content of sex accessory organs were significantly depressed, testosterone and dihydrotestosterone concentrations were unaffected or increased, the number of cytosolic androgen-binding sites was slightly or significantly reduced. The efficiency of exogenous testosterone in promoting growth and protein synthesis in target organs of castrated adult males was significantly lowered by neonatal cyproterone acetate treatment. It is concluded that a deficient androgenic stimulation during neonatal life induces a limited response of sex target organs to endogenous or exogenous androgens in adulthood.     

In-vitro prostanoid production by the rat vas deferens

Prostaglandins (PGs), E-2, F-2 alpha, 6-keto-F-1 alpha and thromboxane B-2 (TXB-2), produced in vitro by the vas deferens of rats at different stages of sexual maturation (10, 35 and 100 days of age), were estimated by radioimmunoassay. Reversed-phase high-performance liquid chromatography was used to evaluate the RIA and to give a more complete profile, after incubation of the vas deferens with [14C]arachidonic acid. The amount of PGE-2 released into the medium after incubation at 37 degrees C for 5 min increased with age, and was the predominant prostanoid in the adult vas deferens. In prepubertal organs, 6-keto-PGF-1 alpha predominated. TXB-2 was always a minor product. The addition of exogenous arachidonic acid (10 micrograms/ml/20 mg tissue), provoked a higher production of the four compounds. PGE-2 and PGF-2 alpha levels were reduced after castration or hypophysectomy and were re-instated after treatment with testosterone propionate. PGE-2 was much more sensitive to hormonal deprivation than PGF-2 alpha. The production of 6-keto-PGF-1 alpha was not significantly affected by the above treatments.     

Preliminary characterization, androgen-dependence and ontogeny of an abundant protein from mouse vas deferens

Polyacrylamide gel electrophoresis analysis revealed that the vas deferens of adult mouse contains a major protein. Mouse vas deferens protein is a basic glycoprotein with a molecular weight of 34,800 +/- 300. The protein represents 17 +/- 0.7% and 42 +/- 2.4% of soluble proteins from homogenate and luminal fluid respectively, an estimate based on densitometric scanning of polyacrylamide gels. The protein originated from the vas deferens since it was not detected in blood plasma or in sexual organs and it was still present after ligation of the epididymis. Changes in androgen status of the animal markedly affected the vas deferens protein. After castration a progressive decrease in the protein was observed and its relative percentage dropped to 2 +/- 0.4% after 45 days. The concentration of the protein returned to precastration levels after 2 weeks of testosterone treatment but oestradiol, progesterone and corticosterone were ineffective in this respect. The vas deferens protein was not synthesized in significant amounts until animals were 20 days old and its concentration increased rapidly from 20 to 30 days in concert with the pubertal increase of androgens in the vas deferens.     

Endogenous prostaglandin formation in the field-stimulated guinea-pig vas deferens: comparison of the inhibitory effects of indomethacin and NS-398

Prostaglandin (PG) E2 inhibited both phases of contraction produced by electrical field stimulation of the guinea-pig vas deferens. PGF2alpha and PGD2 were without effect on this preparation. Carbacyclin (a PGI2) analogue inhibited the first phase of contraction at higher concentrations, whereas U46619 (a thromboxane mimetic) potentiated both phases of contraction. As exogenous arachidonic acid inhibits both phases of contraction of the electrically field-stimulated guinea-pig vas deferens, it is likely that the arachidonic acid is converted to PGE2 in the vas deferens. Indomethacin, a non-specific inhibitor of prostaglandin H synthase (PGHS), attenuated the inhibitory actions of exogenous arachidonic acid when examined on the first phase of contraction. NS-398, a relatively specific inhibitor of PGHS-2, also prevented the inhibitory action of exogenous arachidonic acid. However, NS-398 was much less effective than indomethacin in this respect even though NS-398 and indomethacin inhibit PGHS-2 with similar potencies. Consequently, the findings suggest that exogenous arachidonic acid is converted to PGE2 in the guinea-pig vas deferens by the actions of PGHS-1 and, to a lesser extent, by PGHS-2.     

Effects of ketoacidosis and puberty on basal and TRH-stimulated thyroid hormones and TSH in children with diabetes mellitus

Basal and TRH-stimulated thyroid hormones and TSH were evaluated in two groups of prepubertal and pubertal diabetics: group B - 45 children without ketoacidosis; group C - 16 children with ketoacidosis. The diabetic patients showed no signs of diabetic microangiopathy. Fifty-three healthy subjects served as controls (group A). T4, T3, FT4 and FT3 serum levels were reduced in diabetics, particularly in ketotic ones; T4 and T3 values were lower in pubertal than in prepubertal non-ketotic diabetics and in pubertal than in prepubertal controls, while no significant difference was observed between pubertal and prepubertal ketotic patients. Moreover, no difference in rT3 serum concentrations was found between group A, B and C, but non-ketotic and ketotic pubertals showed a significant rT3 reduction if compared with non-ketotic and ketotic prepubertals and with healthy pubertals. TBG was lower in group B and group C diabetics than in controls. After TRH stimulus, T3 levels showed a significant increase both in controls and in non-ketotic diabetics, while no variation was observed in ketotic children; furthermore, at 120 minutes T3 values were lower in diabetic than in healthy children, particularly in ketotic ones. Basal TSH serum concentrations were reduced in ketotic diabetics, while no difference was found between nonketotic and control subjects. After TRH stimulus, TSH peak was higher in pubertal non-ketotic diabetics than in pubertal controls, while no difference was found between prepubertal and pubertal diabetics, both in non-ketotic and in ketotic status.(ABSTRACT TRUNCATED AT 250 WORDS)     

The androgenic effect of norethisterone and 5alpha-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.     

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.     

Stimulation of anterior pituitary prostaglandin E content and somatotropin (growth hormone) synthesis by phospholipase A.

The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.     

Do endogenous prostaglandins modulate noradrenergic transmission in the rat isolated perfused vas deferens?

The effects of the prostaglandin synthetase inhibitors aspirin, indomethacin and meclofenamic acid have been studied on the response of the rat isolated perfused vas deferens. None of these drugs, up to a concentration of 5 x 10(-5) M affected either phase of the biphasic constrictor response to 30 s periods of field stimulation. In the same preparations an inhibition of the response to field stimulation was seen in the presence of prostaglandin E1 at concentrations of 1 to 5 ng ml-1. All three prostaglandin synthetase inhibitors, at 5 x 10(-5) M, caused significant reduction of prostaglandin biosynthesis by homogenates of rat vas deferens. It is, therefore, suggested that stimuli which activate directly the noradrenergic nerves in the rat vas do not activate simultaneously a release of endogenous prostaglandins.     

Zinc and copper content in rat epididymis and vas deferens

The zinc and copper content in the different epididymal segments and vas deferens of castrated rats were investigated with the help of atomic absorption spectrophotometer. The vas deferens showed maximum zinc content as compared to that of different parts of epididymis in all groups whether castrated unilaterally, bilaterally or in the intact control. Zinc content was reduced in the epididymis and vas deferens ipsilateral to the castrated side as compared to that of contralateral control and intake animals. Lowest zinc content was observed in the epididymis and vas deferens of bilaterally castrated animals from that of other groups. Absence of sperms was observed in all segments of epididymis and vas in bilaterally castrated animals and from the unilaterally castrated side. Copper content was unaltered in all epididymal segments and vas deferens. There appears to be a correlation between the absence of sperms in the male genital tract and the decrease in zinc content.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

The occurrence of GABA in vas deferens, prostate, epididymis, seminal vesicle and testicle of the rat

The concentration of gamma-aminobutyric acid (GABA) was determined in vas deferens, prostate, epididymis, seminal vesicle and testicle of the adult rat. Among the organs examined, vas deferens was found to be the richest in GABA and the lowest concentration was measured in testicle. Although the GABA levels appear to be 10-50 times lower in the sex organs examined than in the brain tissue, even the low GABA contents are suggestive of a role of this amino acid in the reproductive organs of the male rat.     

Prostanoid synthesis in peripheral nerve

The transformation of [1-14C]arachidonic acid into radiolabeled prostanoids was studied with homogenates and desheathed sciatic nerves of rats and frogs. All of the preparations studied were shown to synthesize prostaglandins; the specific prostanoids made were characterized by their migration on thin-layer chromatograms in three separate solvent systems. Both desheathed rat nerve and homogenates synthesize prostaglandin E2, prostaglandin F2 alpha, prostaglandin D2, 6-ketoprostaglandin F1 alpha and thromboxane B2. With preparations from frog nerve, prostaglandin E2 was the major prostanoid product formed. Several conditions were able to modulate the production of prostaglandin E2 with desheathed frog nerve. Electrical stimulation at high frequency (100 Hz) for 30 min increased the formation of labeled prostaglandin E2. Inclusion of glutathione also affected prostaglandin E2 formation. A lower concentration (0.1 mM) stimulated prostaglandin synthesis, while 1 mM glutathione was partially inhibitory. In both the rat and frog system, prostanoid synthesis was suppressed by indomethacin and aspirin.     

Effect of castration on prostaglandin-mediated changes in membrane potential and prostaglandin synthesis in guinea-pig seminal vesicle tissue

Arachidonic acid stimulated an increase in transmural electrical potential difference (p.d.) in guinea-pig seminal vesicle tissue in vitro. Pretreatment with indomethacin abolished this response. Indomethacin pretreatment did not prevent the p.d. from increasing in response to theophylline. Changes in p.d. in response to arachidonic acid were greatly attenuated, and the response to theophylline was abolished in seminal vesicle tissue taken from castrated guinea-pigs. Seminal vesicles, aorta and ileum taken from castrated guinea-pigs synthesized and released more prostaglandins than did those from control animals. It is concluded that the effects of arachidonic acid on p.d. are mediated by its metabolism to prostaglandins; the inability of seminal vesicles from castrated animals to respond to arachidonic acid is not a result of a decrease in prostaglandin production, but is more likely a result of other degenerative changes attendant upon castration; and androgens appear to have some regulatory function on prostaglandin synthesis in a variety of tissues.     

Activities of prostaglandins and prostaglandin endoperoxides at adrenergic neuroeffector junctions.

The results presented in this paper indicate that: 1. The prostaglandin synthesis inhibitor, indomethacin, increases noradrenaline turnover in a variety of rat organs. This observation increases the probability that prostaglandins are involved in the control of adrenergic neurotransmission in vivo. 2. Administration of endoperoxides inhibits the release of noradrenaline from adrenergic nerve terminals. The effect can be explained, however, at least in part, by formation of degradation products, presumably mainly prostaglandin E2. 3. Prostaglandin F2 alpha enhances smooth muscle responses to adrenergic nerve stimulation in rabbit heart and guinea pig vas deferens. These actions must be considered prostjunctional, since the release of noradrenaline is unchanged or depressed.     

Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats

Norepinephrine levels and tyrosine hydroxylase and dopamine-beta-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult vas deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatment. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-beta-hydroxylase activity. The results suggest that in rat vas deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-beta-hydroxylase activity rather than a modulation of tyrosine hydroxylase.     

Interferon-γ and Nitric Oxide Down-Regulate Lipopolysaccharide-Induced Prostanoid Production in Cultured Rat Microglial Cells by Inhibiting Cyclooxygenase-2 Expression

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down-regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l -arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor N^G-monomethyl-l -arginine, led to a remarkable increase in lipopolysaccharide-induced prostanoid release. Moreover, the nitric oxide-generating compound 3-morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon-γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide-evoked production of prostaglandin E₂, prostaglandin D₂, and thromboxane. It is interesting that the inhibitory effect of interferon-γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon-γ and nitric oxide act mainly by down-regulating the lipopolysaccharide-induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase-2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon-γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.     

Gonadal hormones and conspecific marking in male rats

Urine deposited by a rat on a conspecific was quantified with injections of sodium fluorescein, a substance that changes the color of urine. The hypothesis examined in experiment 1 was that marking the environment and a conspecific would be similarly androgen-sensitive behaviors during each of three stages--before castration, after castration, and with restorative therapy with testosterone propionate. Findings were that castration reduced both forms of marking, and testosterone therapy to castrated males restored environmental marking in a dose-response fashion. However, the findings for social marking were more complex; for example, a physiological 200-micrograms testosterone dosage to castrated males was unable to elevate conspecific marking over the rates of marking by castrates without testosterone replacement. In experiment 2 the ontogeny conspecific marking in juvenile males was examined in relation to the pubertal surge of androgens. Results suggested that juvenile male marking of an adult male decreased despite a pubertal increase of androgens. Conclusions were that testicular hormones influenced both forms of marking but were less important in the social setting. Moreover, conspecific marking is not simply an extension of marking the environment.     

The role of arachidonic acid in rat heart cell metabolism

Although it is known that arachidonic acid accumulates in the ischemic myocardium and that cardiac prostaglandin formation from the precursor arachidonic acid is altered during disease states, the role of arachidonic acid in the myocyte itself is not yet clear. Using isolated Ca-tolerant adult rat heart muscle cells, we were able to study cardiac metabolism of arachidonic acid without the effects induced by endothelial or other non-muscle tissue. Myocytes rapidly incorporate arachidonic acid as well as other fatty acids into their lipid pools, the predominant acceptor being the triacylglycerols at an extracellular fatty acid concentration of 20 microM. As exogenous arachidonic acid is decreased, the distribution pattern shifts to favor phospholipid esterification. Cardiocyte prostaglandin production from arachidonic acid added to the incubation medium was limited (less than 1% conversion of added arachidonic acid) and lipoxygenase pathway activity was not detected. Oxidation rates of arachidonic acid were 3-fold lower than for palmitic acid, indicating that it is of secondary importance in energy-yielding reactions. Our results suggest that arachidonic acid serves primarily as a structural component of myocardial membranes and that its release during ischemia would permit its use as a substrate for prostaglandin production by coronary vascular tissue.     

Developmental and hormonal regulation of specific proteins in mouse vas deferens and seminal vesicle.

This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.