Developmental and sexual differences of T-kininogen levels in rat plasma and liver

T-kininogen levels in plasma and liver microsomes were measured by radioimmunoassay in female and male rats of various ages. High levels of T-kininogen were found in plasma and liver of 1-day to 1-week old male and female rats and their mothers. The levels in newborns gradually decreased along with their development. In mature male rats the levels were as low as 1/5-1/2 of those in mature female rats. Treatment with estradiol increased the plasma and the liver levels of T-kininogen significantly in both sexes, but testosterone decreased the level in female rats and had no effect in male rats. These results suggest that sex hormones may regulate the physiological level of T-kininogen in rats.     

Direct radioimmunoassay for rat T-kininogen

Antibodies raised in rabbits against pure rat T-kininogen did not cross-react with Ile-Ser-Bradykinin, bradykinin, nor with kininogens from other mammalian species. They presented a 1 to 15% cross-reaction with pure rat HMW kininogen, depending on the quantity of HMW kininogen. A direct radioimmunoassay for rat T-kininogen in plasma was developed and it enabled 89 fmol of the protein to be detected. A good correspondence was obtained between the direct RIA and the T-kinin generating assay. By the direct assay, it was found that T-kininogen is increased about ten fold in rats subcutaneously injected with turpentine. These data were confirmed by HPLC analysis of the plasma kinins released by trypsin which demonstrated that only T-kinins are increased, bradykinin being unchanged. It was possible according to the results obtained by the direct RIA and HPLC analysis to estimate that in the normal rat, HMW and LMW kininogen represent about 35% and T-kininogen 65%. In the turpentine-treated rat, T-kininogen reached 95%. This RIA will allow the study of the regulation of T-kininogen in the rat and the synthesis of this protein in cells in culture.     

Characterization of serine proteinases isolated from rat submaxillary gland: with special reference to the degradation of rat kininogens by these enzymes

From the homogenate of rat submaxillary gland, two kinds of serine proteinases, named tentatively proteinases A and B, were isolated and their chemical properties and activities toward rat kininogens were examined, in comparison with those of submaxillary kallikrein. Proteinase A with Mr of 28,200 rapidly cleaved high-molecular-weight (HMW) kininogen into a protein of 67 kDa, which retained thiol-proteinase inhibitory activity, but had lost the correcting activity of HMW kininogen on the prolonged clotting time of Fitzgerald trait plasma. It liberated bradykinin from HMW kininogen but did not liberate kinin from T-kininogen and did not degrade T-kininogen. On the other hand, proteinase B with Mr of 30,400 showed a very weak activity for the liberation of kinin from T-kininogen and the cleavage of T-kininogen at pH 8.0. However, the enzyme extensively degraded T-kininogen at pH 4.5. Proteinase B also degraded HMW kininogen at pH 4.5 and pH 8.0, but liberated bradykinin only at pH 8.0. Thiol-proteinase inhibitory activities of HMW kininogen and T-kininogen were inactivated after the incubation with proteinase B at pH 4.5 but not at pH 8.0, while the correcting activity of HMW kininogen on the Fitzgerald trait plasma was inactivated at pH 4.5 and 8.0. The NH2-terminal amino acid sequences of proteinases A and B were different from each other, and distinguishable with those of serine proteinases in rat submaxillary gland so far reported. These results provide evidence that in addition to the known kallikrein, there exist at least two kinds of serine proteinases in rat submaxillary gland, both of which liberate bradykinin from rat HMW kininogen at pH 8.0 and modulate the functional activities of HMW kininogen and T-kininogen, degrading these proteins at pH 8.0 or 4.5.     

Effect of newborn rat testes on the male sexual behavior of the testosterone-treated adult

Neonatal male rats were castrated either at 0, 6 or 24 hrs. after birth. As adults, testosterone was delivered by subcutaneous implantation of a Silastic capsule containing this hormone. The probability to display mounting behavior in presence of an estrous female was lower when the animals were castrated at 0 hr. than at 6 or 24 hrs. or when they received a subcutaneous injection of 1 microgram of testosterone propionate, at the time of castration at 0 hr. These results suggest that in the rat, during the 6 hrs. following birth, neonatal testes influence the sensitivity of the adult central nervous system to testosterone.     

The response to analgesia testing is affected by gonadal steroids in the rat

The effect of gonadal steroids on the response to analgesia testing was determined in castrated male and female rats and castrated male and female rats treated with testosterone propionate (TP) and estradiol benzoate (EB), respectively. The time to respond to a noxious somatic stimulus in the form of heat was assessed using the tail withdrawal test (tail withdrawal from hot water) and hot plate test (the time to paw lick or jump). In male rats, castration resulted in a significant reduction of the reaction time for tail withdrawal. This effect was reversed by treatment with TP. The time to paw lick or jump in male rats was also diminished by castration. Treatment with TP resulted in a partial reversal of the effect of castration on this response. In castrated female rats, the time required for tail withdrawal was decreased by castration and increased by treatment with EB. The reaction time to the hot plate in female rats was diminished by castration and further reduced by EB administration. These data indicate that gonadal steroids influence the response to a noxious heat stimulus in male and female rats and that the effect may vary according to sex and the way in which the stimulus is applied.     

Sex-differences in adrenocortical responsiveness during development in rats

It is known that the stress hyporesponsive period (SHRP), which seems to be related to an immature hypothalamo-pituitary-adrenal (HPA) regulatory system, occurs during the first 2 weeks after birth in rats. In the present study, we investigated the effects of sex-steroid hormones on adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in neonatal rats. The levels of cyclic adenosine 3',5'-monophosphate (cAMP), corticosterone, and adenylate cyclase activity increased with the dose of ACTH in adrenal cells of males and females in vitro. The ACTH responsiveness in adrenal cells increased with age (7-35 days of age), that is, the loss in responsiveness to ACTH just after birth began to recover in 14-35-day-old rats, but the responsiveness in 14-day-old rats was attenuated in males compared with females. Although castration markedly augmented the responsiveness in male rats, testosterone-replacement in the castrated male rats inhibited the enhancement. Furthermore, the responsiveness in 14-day-intact female rats was suppressed by treatment with testosterone. Expression levels of ACTH receptor mRNA in adrenals increased with age in the female rat, but not in the male. Castration enhanced the level of ACTH receptor mRNA to three-fold of that in intact male rats at 14 days of age, but replacement treatment with testosterone in castrated male rats lowered the elevated levels. Testicular androgens are thought to evoke a gender-specific response in neonates, and the temporal decrease of adrenal ACTH-responsiveness might be due to the topically immature adrenal system as well as the central nervous system in mammals.     

Blood risk factor metabolites associated with heart disease and myocardial fatty acids in copper-deficient male and female rats

Intact and castrated males and intact and ovariectomized female rats were fed a copper-deficient diet in order to establish whether the protection provided in females against cardiovascular pathology and mortality is due to endogenous sex hormones, and different levels of blood lipids and/or myocardial fatty acids. Seventy-three male and female rats were assigned to a copper-deficient diet (0.6 micrograms of copper/g diet) containing 62% fructose for 8 weeks. Twelve of the male rats underwent castration and 12 of the females were ovariectomized. All animals exhibited high levels of plasma cholesterol, triglycerides, and uric acid, which were neither affected by the sex of the rat nor by the surgical treatment. The composition of fatty acids of the myocardium was similar in males and females. Except for those animals that were sacrificed by us, all other male rats died of heart pathology. In contrast, none of the female rats exhibited heart pathology and none died of the deficiency. It is suggested that heart pathology and mortality in copper deficiency are sex related and not due to high levels of plasma cholesterol, triglycerides, and uric acid or to differences in myocardial fatty acid composition.     

Changes in the renin-angiotensin-aldosterone system in rats of both sexes submitted to chronic hypobaric hypoxia

The effect of moderate chronic hypobaric hypoxia (CHH) on the renin-angiotensin-aldosterone system has been analysed in male and female intact and castrated rats. The experimental animals were submitted to a simulated altitude of 4,400 m during ten weeks. Half of the experimental and half of the control animals were castrated at three weeks of age. Arterial pressure (AP) was measured once a week during the whole experimental period. Blood samples were obtained by decapitation at the end of the study. Red cell volume, plasma renin activity (PRA), plasma angiotensinogen (Ao) and aldosterone concentration (ALDO) were determined in the blood samples. Results have shown that the female animals subjected to CHH had lower levels of AP than the control female rats during all the studied periods whereas the AP of male hypoxic rats was only transiently diminished. All these changes were abolished by castration. PRA was not altered in either sex. The enzymatic complex was higher in male than in female control animals and decreased after castration in both hypoxic and control male rats. Ao was decreased by CHH in both sexes of intact rats and in female castrated animals. The renin substrate was higher in male than in female intact rats and decreased after castration in male animals. ALDO was increased after CHH only in male rats. Control female rats have higher levels of ALDO than male animals. Changes in the renin-angiotensin-aldosterone system related to CHH and also significant differences between sexes suggest that adrenal and gonadal corticosteroids may be involved in the main alterations presently observed.     

Sex dimorphism and inflammatory regulation of T-kininogen and T-kininogenase

Studies were carried out in order to better understand hormonal and inflammatory regulation of the T-kininogen and T-kininogenase system. T-kininogen from rat serum and T-kininogenase from rat submandibular gland were purified to homogeneity, and specific antisera to the purified proteins were generated. Simple, sensitive and specific radioimmunoassays were developed for measuring both T-kininogen and T-kininogenase. The assays incorporated a modified poly(ethylene glycol) technique for separating free from antibody-bound forms. Optimal combinations of poly(ethylene glycol) and gamma-globulin were found, yielding low background and high specific binding. The assays can detect a minimum of 160 pg of T-kininogen and 80 pg of T-kininogenase per tube. Serial dilutions of sera from normal and turpentine-treated rats showed complete parallelism with the T-kininogen standard curve. T-kininogen levels in rat serum and rat tissues increased more than 10-fold following turpentine treatment, while T-kininogenase levels in the submandibular gland and other tissues remained unchanged. Through use of a kinin-directed kininogen monoclonal antibody, Western blots of two-dimensional gels of serum following acute inflammation showed increased levels of several kininogens which vary in both molecular weight and isoelectric point. Analysis of serum kininogen levels shows sexual dimorphism, with female rats having 3.9-fold higher levels than males. Contrarily, T-kininogenase levels in the submandibular gland of male rats are 2.4-fold higher than those in females. The studies also showed that the T-kininogen and T-kininogenase system is regulated by sex hormones. T-kininogen is an acute-phase protein whose rapid increase and mobilization following inflammation may provide a primary defense against proteolytic damage during trauma.     

Sexual differences in the expression of copper deficiency in rats

The present investigation was undertaken to establish whether the severity of copper deficiency in rats fed diets containing fructose is affected by the presence and type of endogenous sex hormones. Intact and castrated male rats and intact and ovariectomized females were fed from weaning a copper-deficient diet (0.6 ppm) containing 62% fructose for 8 weeks. Regardless of castration, male rats were anemic, exhibited heart hypertrophy, and died of the deficiency. However, castration ameliorated the anemia and delayed the mortality. In contrast, none of the females died of the deficiency. It is suggested that in addition to the sex of the animal, levels of testosterone in the male may also play a role in the severity of copper deficiency.     

Hormonal regulation of rat renal cytochrome P450s by androgen and the pituitary.

The hormonal regulation of rat renal cytochrome P450s, P450 4A2 (K-5) and K-2, was investigated. The level of P450 4A2 in male rats was five times that in female rats and accounted for some 90% of total cytochrome P450, measured photometrically. Lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes of male rats were also higher than those of female rats. The sex differences in lauric acid hydroxylation activity seemed to arise from the differences in P450 4A2 concentrations, according to an immunochemical study. P450 K-2 was a female-dominant form in rat kidneys. The level of P450 K-2 in renal microsomes of male rats was one-tenth that of P450 4A2. Castration of male rats decreased the levels of P450 4A2 and treatment of castrated male rats with testosterone reversed the decrease. The castration of male rats decreased the lauric acid hydroxylation of the renal microsomes to the level of female rats. The administration of testosterone to castrated male rats reversed the decrease. Hypophysectomy of male rats decreased the level of P450 4A2 and the administration of growth hormone reversed the decrease when intermittent injections mimicking the male secretory pattern were given, although continuous administration mimicking the female secretory pattern did not. Castration of male rats did not affect the level of P450 K-2, but testosterone decreased its level. Hypophysectomy of male rats increased the level of P450 K-2 and growth hormone decreased its level in hypophysectomized rats. These results suggested that the expression of P450 4A2 was regulated by androgen or growth hormone and regulation of P450 4A2 was different from that of P450 K-2. To explore the regulation of renal cytochrome P450 further, testosterone was given to control (intact) or hypophysectomized adult female rats. P450 4A2 was induced in the kidneys of both control and hypophysectomized female rats to close to the level of male rats. Thus, P450 4A2 was directly regulated by testosterone as well as growth hormone, and the regulation of the male-dominant form in rat kidneys was different from that of the male-specific form in the rat liver, which is regulated mostly by growth hormone.     

Sexual hormones terminate in the rat: the significantly enhanced catecholaminergic/serotoninergic tone in the brain characteristic to the post-weaning period

The amount of dopamine released from the striatum, substantia nigra and tuberculum olfactorium, noradrenaline from locus coeruleus and serotonin from the raphe, was significantly higher in four and five weeks old rats than in three month old ones, proving that the catecholaminergic/serotoninergic activity enhancer (CAE/SAE) regulation works unrestrained during developmental longevity and is restricted thereafter. As the dampening of the CAE/SAE regulation (end to the second month of age) coincided temporally with the appearance of sexual hormones, we castrated three weeks old male and female rats and measured at the end of the third month of their life the release of catecholamines and serotonin from selected discrete brain regions. The amount of catecholamines and serotonin released from the neurons was significantly higher in castrated than in untreated or sham operated rats, signalting that sexual hormones inhibit the CAE/SAE regulation in the brain. We therefore treated male and female rats s.c. with oil (0.1 ml/rat), testosterone, (0.1 mg/rat), estrone (0.01 mg/rat) and progesterone (0.5 mg/rat), respectively, and measured their effect on the CAE/SAE regulation. Twenty-four hours after a single injection with the hormones, the release of noradrenaline, dopamine and serotonin was significantly inhibited in the testosterone or estrone treated rats, but remained unchanged after progesteron treatment. In rats treated with a single hormone injection, testosterone in the male and estrone in the female was the significantly more effective inhibitor. Remarkably, the reverse order of potency was found in rats treated with daily hormone injections for 7 or 14 days. After two-week treatment with the hormones estrone was in the male and testosterone in the female the significantly more potent inhibitor of the CAE/SAE regulation. The data indicate that sexual hormones terminate the hyperactive phase of adolescence by dampening the impulse propagation mediated release of catecholamines and serotonin in the brain.     

Differential responsiveness of LH and prolactin to p-tyramine in male and female rats

The effect of p-tyramine, a natural amine which is found in the rat brain in trace amounts, was evaluated for its capacity to influence LH and prolactin secretion in male and female rats under different hormonal conditions. p-Tyramine (40 mg/kg ip) was ineffective in modifying LH levels in either female or male rats which had been gonadectomized for 2 days, but if the animals were injected with 12.5 micrograms of estradiol benzoate (EB) on the day of castration, p-tyramine was able to release LH in female but not in male rats. To evaluate whether early androgenization of brain structures which control LH secretion was involved in the sexual difference observed, p-tyramine was tested in female androgenized rats (200 micrograms of testosterone propionate on the day of birth), and in male rats castrated at birth. The trace amine was ineffective in altering LH levels in both experimental models, even if rats were pretreated with EB as control females. On the other hand, p-tyramine inhibited prolactin secretion in male rats pretreated with EB, and not in similarly treated female rats. The present results suggest that p-tyramine may be involved not only in prolactin regulation as it has been previously shown, but also in LH control, and that the hormonal response to this amine is sexually differentiated in the rat.     

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.     

Mitotic activity of gonadotropes in the anterior pituitary of the castrated male rat

Summary The proliferation of gonadotropes in the anterior pituitary of the castrated male rat was examined immunohistochemically after colchicine treatment. The results show a more than 10-fold increase in mitotic frequency in gonadotropes 1 or 2 weeks after castration, as compared with controls. This result explains the increase in the population of immunoreactive LH cells in castrated male rats. The gonadotropes decreased significantly 1 month after castration. The mitotic activity of gonadotropes was almost completely suppressed in castrates implanted with a silastic tube filled with testosterone.     

Gonadal hormones during puberty organize environment-related social interaction in the male rat

This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood.     

Modulation of aortic vascular reactivity by sex hormones in a male rat model of metabolic syndrome

Modulation by sex hormones of aortic reactivity in rats with the metabolic syndrome (MS) was investigated. The following groups of weanling male Wistar rats were used: control rats (C) received regular tap water while MS rats received 30% sucrose in their drinking water; both had rodent chow for 24 weeks. These two groups were further subdivided into the following four groups: intact (Int), castrated (Cas), castrated plus testosterone (T) and castrated plus estradiol (E). Vascular response of thoracic aortic rings to norepinephrine (NE), acetylcholine (ACh), indomethacin (Indo) and nitro-l-arginine-methyl ester (L-NAME) was investigated. Blood pressure (BP) and serum nitrates and nitrites were measured. BP and serum nitrates and nitrites were modified by castration and treatments with either T or E. Vasoconstriction in Int MS and Cas MS+T aortas was larger than in C and Cas C+T, respectively. Vasodilation in Int MS and Cas MS+T was reduced in comparison with C and Cas C+T, Cas MS and Cas MS+E. Indomethacin decreased vasoconstriction in all groups (P<0.002) but Int C and Cas C+T remained significantly smaller than Int MS and Cas MS+T. l-NAME in NE-contracted vessels induced a significant increase in vasoconstriction, except in Cas C+E rats; the responses of Int MS and Cas MS+T were significantly larger than in Int C and Cas C+T. The results suggest endothelial dysfunction in Int MS and Cas MS+T and a protective effect resulting from castration and castration plus E in MS animals, indicating a sex hormone influence.     

Maturation of gonadotropin function during growth of the male rat: influence of neonatal castration (author's transl)

Evolution during growth of plasma level gonadotropins, was studied in the normal or castrated rat at birth. In the control animals, the plasma level of gonadotropins increased from the 25th day to the 40th day, then declined to the 90th day. Neonatal castration induced a new phenomenon: the increase of plasma gonadotropins in the normal rat corresponded with a decrease in the castrated animals and inversely. Testis hormones affected gonadotropin function; however, an autonomous maturation of this function, independent of gonadal secretions, appeared to exist.     

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17β-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17β-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17β-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17β-HSD3 blocks the proliferation of NRICC. Drug targeting of 17β-HSD3 may be important for managing cholangiopathies.     

Localization of beta-endorphin in the rat heart and modulation by testosterone

Chromatographic analysis and radioimmunoassay were used to identify and quantitate beta-endorphin (BE) and beta-lipotropin (B-LPH) in the hearts (devoid of major blood vessels and atria) from intact male rats, castrated male rats, and castrated male rats treated with testosterone propionate (TP). BE and B-LPH in the plasma of these animals were also identified and measured. In comparison to intact animals, castration resulted in a significant elevation in the content of BE in the heart which was reversed by the administration of TP. The content of B-LPH in the heart was not affected by castration or castration in combination with TP. The ratio of BE to B-LPH in the heart of castrated animals was significantly elevated as compared with intact controls. Treatment of castrates with TP returned the ratio of BE to B-LPH to that observed in intact animals. The concentration of BE in the plasma was greater in castrated rats and castrated rats given TP than in intact males, whereas the concentration of B-LPH was diminished in castrated animals given TP. The ratio of BE to B-LPH was greater in castrated animals treated with TP than in castrated and intact animals. The content of BE and B-LPH, as well as the ratios of the two peptides, varied independently in the cardiac tissue and plasma. The present findings indicated that (i) BE and B-LPH are present in cardiac tissue, (ii) the amount of BE and B-LPH in the heart and the ratio of BE to B-LPH appear to be modulated by TP, and (iii) BE and B-LPH detected in the heart was not simply a reflection of the presence of these peptides in the plasma.