Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep.

Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of neonatal castration on sex steroid receptor levels in the hypophysis of male rats

The content of estradiol and testosterone cytosolic and nuclear receptors has been studied in the pituitary body of adult male rats gonadectomized on day 1-3 after birth (long-term castrates) or in adulthood (short-term castrates). Intact male rats and long- and short-term castrates had the same level of cytosolic and nuclear estrogen receptors. The number of cytoplasmic and nuclear testosterone-binding sites was identical in the pituitary body of adult intact mice and long-term castrates. Contrastingly, the concentrations of androgen cytosolic and nuclear receptors were significantly lower in neonatally castrated males compared to intact adult animals. The results obtained indicate that nuclear testosterone receptors in the pituitary body mediate negative feedback effect of androgen on the release of luteinizing hormone and that the formation of thin mechanism occurs within the first days of life.     

Concentration of free fatty acids in muscle following administration of corticotropin and hydrocortisone]

Experiments were conducted on rats; the gas chromatographic method was applied to the study of the free fatty acids content in the gastrocnemius 30 minutes after the intraperitoneal injection of adrenocorticotropic hormone (ACTH)--1 Unit per 100 g and hydrocortisone acetate--1 mg per 100 gm of body weight. It was shown that in the resting muscles ACTH increased the content of stearic acid, whereas hydrocortisone--of both stearic oleic acids. The changes in the content of other free fatty acids were insignificant. During the short-term activity the content of stearic acid in the regimen of single rhythmic contractions in the gastrocnemius of intact rats increased. In experiments with ACTH and hydrocortisone this elevation was much less and not significant. ACTH and hydrocortisone stimulated the stearic acid consumption by the muscles during the activity.     

Pneumadin in the ventral prostate and seminal vesicles of estradiol-treated rats: RIA and immunocytochemical studies

Pneumadin (PNM) is a decapeptide (the rat peptide: Tyr-Gly-Glu-Pro-Lys-Leu-Asp-Ala-Gly-Val-NH2) isolated from mammalian lungs. Human and rat PNM differ only by substitution of one amino acid--Tyr/Ala. PNM evokes an antidiuretic effect via a potent stimulation of arginine-vasopressin (AVP) release. By means of recently established, highly specific RIA method, high concentration of PNM had been found in the rat ventral prostate. Castration resulted in a profound drop in PNM concentration, an effect prevented by testosterone replacement. The present studies were aimed at investigating the effect of prolonged estradiol administration on PNM concentration, content and localization in the prostate and seminal vesicles of the rat. Depo estradiol (estradiolum valerianicum) administration to adult male rats resulted in a notable atrophy of ventral prostate and seminal vesicles. During the entire experiment (till day 30 after administration), PNM concentration in ventral prostate was similar to that seen in intact animals, while peptide content per gland was markedly lowered. PNM immunostaining was observed in prostate epithelium of estradiol-treated rats and its localization resembled that observed in intact animals. Nearly 40 times lower PNM concentration than in ventral prostate was found in seminal vesicles. In contrast to prostate, on days 20 and 30 of estradiol treatment PNM concentration in seminal vesicles was higher than in intact rats. However, due to profound seminal vesicle atrophy, PNM content per entire gland was notably lowered in estradiol-injected rats. By immunocytochemistry, PNM-immunoreactive substances were not found in seminal vesicles of either intact or estradiol-administered rats. High PNM concentration in the rat prostate suggests its important role in the function of the gland.     

Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver

1. Alcohol-dehydrogenase activity is first detectable in the rat foetus on about the eighteenth day of gestation, after which time it increases to about 25% of the adult activity at birth. Adult activity is reached at about 18 days after birth. The ethanol-oxidizing capacity of liver slices from rats correlates well with the increase of the enzyme activity in vitro. 2. In the guinea pig there is a steady linear increase from about 17 days before term to 5 days after birth. Adult activity is reached between the sixth and eighth postnatal day. 3. Some kinetic properties of liver alcohol dehydrogenase are very similar in newborn and adult rats. 4. Administration of ethanol to pregnant rats during the latter half of gestation had no effect on alcohol-dehydrogenase activity in the liver of the newborn offspring. Intraperitoneal injections of ethanol to newborn and young rats had no effect on the alcohol-dehydrogenase activity of the livers. 5. Intraperitoneal injections of hydrocortisone and triamcinolone to newborn and adult non-adrenalectomized rats had no significant effect on the increase of the alcohol-dehydrogenase activity as studied up to 4 days after the injection.     

Delay in the age of balano-preputial skinfold cleavage and alterations in serum profiles of testosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and gonadotropins in adult rats treated during puberty with luteinizing hormone releasing hormone

Previous work has shown that chronic treatment of intact, immature male rats with luteinizing hormone releasing hormone (LHRH) decreases sex accessory gland weights and results in retardation of the normal developmental increase in the ratio of serum testosterone (T)/5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Diol) via an apparent enhancement of testicular 5 alpha-reductase or 3 alpha-hydroxysteroid oxidoreductase activities. In the present work, androgen dependent balano-preputial skinfold cleavage was significantly delayed by approximately one week in intact, immature male rats which were treated daily for two weeks with either 1.0 micrograms, 2.5 micrograms or 5.0 micrograms of LHRH during a discrete phase of pubertal development (28-41 days of age). In intact, adult (62 day old) animals which received LHRH treatments during pubertal development, serum T concentrations and sex accessory gland weights were reduced compared to control animal values. Serum 3 alpha-Diol content in the adult rats was either unaltered or increased significantly depending on the LHRH dosage employed during sexual development. Serum luteinizing hormone concentrations were not different between control and LHRH-pretreated adult rats whereas the highest dosage of LHRH employed (5.0 micrograms) during puberty resulted in a significant elevation of adult serum follicle stimulating hormone levels. It is suggested that chronic LHRH treatment of the male rat during puberty results in a perturbation in testicular androgen biosynthetic activities and an impairment of pituitary-testicular hormone feedback mechanisms which persist at least through early adulthood.     

Neonatal exposure to vasopressin decreases vasopressin binding sites in the adult kidney

Previous experiments showed that rats injected with vasopressin (AVP) during the first seven days after birth were less sensitive as adults to the antidiuretic effects of AVP than were control rats. In the present experiment, binding sites for AVP were measured in the kidneys of similarly treated adult rats. Neonatal exposure to AVP significantly decreased the number of binding sites in the adults, but did not affect the binding affinity of the sites. It is concluded that neonatal exposure to AVP which produces a long-lasting decrease in responsiveness of the kidney to AVP is correlated with a reduction in the number of AVP binding sites in the tissue.     

Effects of prenatal ethanol exposure and sex on the arginine vasopressin response to hemorrhage in the rat

AVP synthesis, storage, and osmotically stimulated release are reduced in young adult rats exposed prenatally to ethanol (PE). Whether the reduced release of AVP to the osmotic stimulus is due to impairment of the vasopressin system or specifically to an osmoreceptor-mediated release is not known. The present experiments were done, therefore, to determine whether a hemorrhage-induced AVP response would also be diminished in PE-exposed rats. Pregnant rats were fed either a control liquid diet [no prenatal ethanol (NPE)] or a liquid diet with 35% of the calories from ethanol from days 7-21 of pregnancy. Offspring were weaned at 3 wk of life. At 11 wk of age, femoral arterial catheters were surgically placed, and blood volumes were determined at 12 wk. Three days later, two hemorrhages of 10% of the blood volume were performed with samples taken before and 10 min after the hemorrhages. After a 20% blood loss, plasma AVP was 19% higher in NPE rats than in the PE rats despite no differences in mean arterial blood pressure (MABP). Also, hypothalamic AVP mRNA and pituitary AVP content were reduced in PE rats. Furthermore, confirming an earlier report of sex differences in AVP release, the hemorrhage-induced hormone response was twofold greater in female rats than male rats, regardless of previous ethanol exposure. These studies demonstrate that the AVP response to hemorrhage is reduced in PE rats independently of differences in MABP. The data are compatible with a theory of a reduced number of hemorrhage-responsive vasopressinergic neurons capable of stimulated AVP release in PE rats.     

Hydrocortisone induces an increase of amylase content in individual zymogen granules from rat pancreas

This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses.     

Reproductive aspects in female rats exposed prenatally to hydrocortisone

We investigated the effects of hydrocortisone during the prenatal period and its later repercussion on reproductive aspects of female rats. Pregnant rats were treated (s.c.) with hydrocortisone acetate, at 1.5 mg/day on the 17th, 18th, and 19th days of pregnancy. Although the present study was not intended to identify mechanisms of toxicity, the treatment with hydrocortisone in the last period of pregnancy presented no signs of toxicity. The efficacy of the hydrocortisone in reducing the adrenal wet mass and plasma corticosterone levels immediately after delivery in both the treated mothers and in respective pups at birth may indicate impairment of the hypothalamus-pituitary-adrenal axis. In addition, the treatment with hydrocortisone did not interfere in the development of the female descendants until puberty. However, it affected the estrous cycle and fertility. Probably, the prenatal exposure to corticosteroids had altered at least partially the hypothalamus-pituitary-gonadal axis, resulting in the damages observed in adult life. These results indicate that the use of the hydrocortisone at a dose that apparently does not endanger the neonate led to undesirable effects in the adult reproductive phase, resulting in later deleterious alteration of the reproductive physiology in female rats.     

Effects of cysteamine on the hypothalamic-pituitary axis in the rat

The effects of cysteamine (CSH) on hypothalamic concentrations of neuropeptides were reviewed and correlated with available information on changes in pituitary hormone content and circulating pituitary hormone levels. In our study, we found notable changes in the morphology of lactotropes from female Long-Evans rats treated for 7 days with CSH (300 mg/(kg X day) per os). Forming granules increased in number, and crinophagy, which is the augmented incorporation of these granules into lysosomes, was evident. Storage granules were reduced in number. These changes were not suppressed by simultaneous administration of 17 beta-estradiol (50 micrograms/day s.c.) for 7 days. CSH administration failed to prevent estrogen-induced lactotrope hyperplasia. Serum prolactin levels were unaffected by CSH treatment. The morphological changes in the adenohypophysis did not resemble those observed when rats were treated with bromocriptine. The rough endoplasmic reticulum luminal density was reduced in gonadotropes from intact CSH-treated rats after 1 wk. CSH treatment suppressed the development of castration cells and significantly reduced serum luteinizing hormone levels in ovariectomized rats. The morphological effects of CSH appeared to be confined to lactotropes and gonadotropes.     

Results of the negative control chemical allyl alcohol in the 15-day intact adult male rat screening assay for endocrine activity

Development, standardization, and validation of methods to assess the potential of chemicals to disrupt hormonal homeostasis have been the focus of considerable research efforts over the past 10 years. As part of our validation effort, we evaluated the specificity of the 15-day intact adult male rat assay, using a negative control chemical, allyl alcohol, a known hepatotoxicant that was not expected to induce endocrine effects. Male rats were dosed for 15 days via oral gavage with 0, 10, 30, 40, or 50 mg/kg/day allyl alcohol. The endpoints evaluated included final body and organ weights, serum hormone concentrations, and a limited histopathology assessment. No mortality or adverse clinical signs were observed. Mean final body weight for rats in the 50-mg/kg/day dose group was decreased to 90% of control. Mean relative liver weights were increased at 40 and 50 mg/kg/day (115% and 117% of control, respectively). Serum testosterone and DHT concentrations were statistically significantly decreased at 50 mg/kg/day (72% of control). Serum prolactin concentrations were statistically significantly decreased at 40 mg/kg/day (58% of control), but not at 50 mg/kg/day. There were no effects on the other endpoints evaluated. Consistent with previous guidance for interpreting the 15-day intact adult male rat assay, histological and weight changes of target organs were given a higher weight-of-evidence than changes in serum hormone concentrations alone. Therefore, with only minimal changes in serum hormone concentrations and no effects on organ weights or microscopic alterations, the results of allyl alcohol in the 15-day intact adult male rat assay were considered negative and consistent with the predicted results.     

Effect of sex hormones on plasma T-kininogen in the rat

The influence of sex hormones on rat plasma T-kininogen concentration was examined. The level of T-kininogen in the post-pubertal female rat is about 3-times that of the male animal. Female rats castrated as adults or 15 days after birth, had low T-kininogen concentrations, near those of male rats. In contrast, castration of mature or immature male animals induced no change in T-kininogen. Treatment of castrated female or male rats with 17 alpha-ethinylestradiol significantly increased the T-kininogen level, whereas administration of testosterone or progesterone had no effect. The influence of estrogen was specific for T-kininogen, since plasma HMW kininogen concentration was the same in male and female rats and was not affected by castration or sex hormone treatment. T-kininogen concentration was not significantly changed in pregnant rat between the 12th and the 20th day of pregnancy, but increased after parturition. It was high in the newborn rat at birth and then decreased similarly over the next 3 weeks in males and females. It continued to decrease in the males, reaching the level of the adult rat, but it increased in the female from 3-4 weeks of age and reached the adult level at about 6-8 weeks. These data indicate that natural estrogens have a physiological influence on the plasma level of T-kininogen in female rats whereas testosterone had no effect on either male or castrated female rats. HMW kininogen is not physiologically dependent on sex hormones.     

Premature induction of amylase in pancreas and parotid gland of growing rats by dexamethasone.

Studies were made on changes in the contents of alpha-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal development, on the premature induction of this enzyme by hormones and on the existence of specific glucocorticoid receptors in these tissues. The amylase content in the pancreas increased from the 9th day after birth and reached the adult level on the 28th day, its content in the parotid gland increased rapidly from the 16th to the 28th day after birth and then rose more gradually to the adult level. Injection of dexamethasone into rats 6--8 days after birth induced increase in the amylase of the pancreas but not the parotid gland. However, injection of dexamethasone into weanling rats 21--23 days after birth resulted in precocious induction of amylase in both tissues. Specific glucocorticoid receptors were detectable in the parotid gland of rats from 6 days after birth but were almost undetectable in the pancreas until adolescence.     

Effect of radiation on the formation of hydrocortisone metabolites in the perfused rat liver

A study was made of the absorption of exogenous hydrocortisone and formation of its metabolites in isolated liver of intact and exposed rats in conditions of recirculating perfusion. It was shown that the absorption of the hormone by the liver of irradiated rats was greatly lowered but the content of most metabolites found in the perfused medium of irradiated liver increased as compared to the control. It is suggested that irradiation inhibits subsequent transformations of the hydrocortisone metabolism products.     

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100μg/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4, 6 or 24h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24h after surgery, and in both hypothalamic nuclei only at 6h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood.     

Effects of thyroid hormones on the receptor level in estrogen target organs

The influence of thyroid hormones on the turnover of cytoplasmic estrogen receptors in the liver, kidney and uterus of intact and ovariectomized female rats was studied under in vivo conditions. Thyroidectomy had no significant effect on the receptor level in the uterus but caused a substantial reduction of the receptor content in the liver and kidney. In livers of intact and ovariectomized animals receptor values were reduced with 70 and 80%, respectively, 30 days after thyroidectomy. Substitution with triiodothyronine (T3) restored the hepatic estrogen receptor concentration in thyroidectomized rats to the preoperative level. If rats that had been both ovariectomized and thyroidectomized were substituted with thyroid hormone for the same time period, the receptor level was increased but did not reach the level seen in animals that had been ovariectomized only. The effects of thyroid hormone substitution was found to be dose dependent and paradoxical. Thus, a high dose of 50 micrograms/day of triiodothyronine given to intact animals for nine days caused a 30% reduction in the hepatic receptor content. The same level of reduction was seen in the ovariectomized rat given a hormone dose of only 1 micrograms/day. When this type of rats was treated with the higher dose of triiodothyronine the reduction in hepatic estrogen receptors was 50%. These results are discussed in relation to existing information concerning the multihormonal regulation of estrogen receptor concentration in the rat liver.     

Basal gonadotropin hormone release rates during the period of selective follicle-stimulating hormone release in the juvenile female rat

There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three days. In contrast, anterior pituitary gland (APG) FSH and LH concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the immature female rat.