Steroid 5α-Reductase in Adult Rat Brain After Neonatal Dihydrotestosterone Administration

Testosterone (T) is known to play an important masculinizing role in the developing brain of rat, including the regulation of 5α-reductase (5α-R) isozymes. However, the effects of dihydrotesterone (DHT), a more potent androgen than T, have not been elucidated. In this study, DHT was administered from day 5 through day 20 of postnatal life (period of postnatal sexual differentiation of the central nervous system) at doses of: 12 mg/kg/d on days 5, 6, 7, 8, 19, and 20; 15 mg/kg/d on days 9, 10, 11, 12, 16, 17, and 18; and 18 mg/kg/d on days 13, 14, and 15. In adulthood, quantitative RT-PCR was used to measure mRNA levels of 5α-R1 and 5α-R2 isozymes in the prefrontal cortex (PFC) of male and female rats with varied androgenic status. Under our study conditions, neonatal DHT administration influenced on adult PFC 5α-R isozymes levels and their regulation pattern by androgens, and this pattern was the inverse of that reported in adult neonatally T-treated rats.     

Regulation of the genes for insulin-like growth factor (IGF) I and II and their receptors by steroids and gonadotropins in the ovary

Insulin-like growth factors (IGFs) I and II are two single-chain polypeptide hormones that are structurally related to each other and to proinsulin. Among the large number of growth factors involved in ovarian physiology, IGF-I and IGF-II are considered to be important progression factors for ovarian follicular development. To explore the ovarian expression of IGF-I, IGF-II and their receptor genes, a solution hybridization/RNase protection assay, was used. IGF-I mRNA was seen in the granulosa cells, and IGF-II mRNA in the theca-interstitial compartment. To study the hormonal regulation of the IGF-I and IGF-II gene, immature (21-day-old) hypohysectomized rats were treated with FSH (10 μg/day),GH (150 μg/day) and diethylstilbestrol (DES subcutaneous implant/5 days). Estrogen differentially regulated ovarian IGF-I and IGF-II gene expression. In concert with GH, estrogen up-regulated ovarian IGF-I mRNA, but significantly decreased hepatic IGF-I gene expression. Both IGF receptors (type I and type II) as well as the insulin receptor gene, were expressed in both ovarian cells. The expression of the type IIGF receptor gene (but not the type II IGF gene) was up-regulated by FSH and estrogen in vivo. In conclusion, these studies may serve to better understand the auto paracrine role of IGF, and their receptors in the pathophysiology of follicle recruitment, oocyte maturation and potentially embryo development.     

Decidualization in rats given 5alpha-dihydrotestosterone or its propionate neonatally.

In Experiment 1, female rats were given a single subcutaneous injection of 1.25 mg 5alpha-dihydrotestosterone (DHT) or its propionate (DHTP) on day 5 of postnatal life. All of them showed regular estrous cycles as adults like untreated control animals. At about 60 days of age, the rats were ovariectomized and given 7 daily injections of 2 mg progesterone (P) plus 0.2 mug estradiol-17beta (ED). Uterine trauma applied on the 4th day of P-ED injections resulted in well developed deciduomata in all animals by the day after the last injection. This made a sharp contrast to the failure of female rats receiving testosterone propionate (TP) neonatally to give a positive response under similar experimental conditions (Takewaki and Ohta, 1974). The mean weight of traumatized horns was significantly larger in DHTP-treated rats (but not in DHT-treated rats) than in controls. In Experiment 2, rats were ovariectomized on day 4 and given a dose of 1.25 mg DHT or DHTP on day 5. Controls were ovariectomized on day 4 but not given any steroid on the next day. A series of 7 daily injections of 2 mg P plus 0.2 mug ED was started at about 60 days of age, after the animals had received 3 daily injections of 0.2 mug ED or 30 daily injections of 0.1 mug ED. Incidence of deciduomata following uterine traumatization was markedly lowered only in animals treated with DHTP neonatally and given 0.1 mug ED for 30 days as adults, no significant differences being found in both incidence and size of deciduomata among the other groups. It was suggested that the effects of neonatal steroid administration on uterine responsiveness in adulthood are specific to the steroid. The previous conclusion that persistent estrus in androgen-sterilized rats plays a part in the reduction of uterine responsiveness was confirmed. An exposure of rats to estrogen for a prolonged postpuberal period was without effect, unless the animals had received enough androgen neonatally.     

Masculinization of growth hormone (GH) secretory pattern by dihydrotestosterone is associated with augmentation of hypothalamic somatostatin and GH-releasing hormone mRNA levels in ovariectomized adult rats.

The role of androgen in the sexual dimorphism in hypothalamic growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SS) gene expression was examined in rats. In the first study, the SS and GHRH mRNA levels were measured in both male and female rats at 4, 6, 8, and 10 weeks of age. A significant sex-related difference in the SS and GHRH mRNA levels was observed after 8 weeks of age, when sexual maturation is fully attained. Male rats had higher SS and GHRH mRNA levels than the female rats. In the second study, adult ovariectomized rats received daily injection of dihydrotestosterone (DHT), nonaromatizable testosterone, at a dose of 2 mg/rat for 21 days. The DHT treatment masculinized the GH secretory pattern, which was indistinguishable from that of intact male rats, and simultaneously augmented the SS and GHRH mRNA levels. The DHT treatment of ovariectomized rats after hypophysectomy significantly raised the level of SS mRNA, but not that of GHRH mRNA compared to the control animals. These findings suggest that the activation of the SS gene expression through androgen receptor plays an important role in the maintenance of sexual dimorphism in GH secretion in rats.     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.     

Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.

This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.     

Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats

The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves

Glucocorticoids and colostrum feeding influence postnatal maturation of the somatotropic axis. We have tested the hypothesis that dexamethasone (Dexa) affects the somatotropic axis in neonatal calves dependent on colostrum intake. Calves were fed either with colostrum or with a milk-based formula (n = 14/group), and, in each feeding group, one-half of the calves were treated with Dexa (30 micro g. kg body wt-1. day-1). Pre- and postprandial blood samples were taken on days 1, 2, 4, and 5, and liver samples were taken on day 5 of life. Dexa increased insulin-like growth factor (IGF)-I, but decreased growth hormone (GH) and IGF-binding protein (IGFBP)-1 and -2 plasma concentrations and increased GH receptor (GHR) mRNA levels in liver. Dexa increased IGF-I mRNA levels only in formula-fed calves and increased hepatic GHR binding capacity, but only in colostrum-fed calves. Colostrum feeding decreased IGFBP-1 and -2 plasma concentrations and hepatic IGFBP-2 and -3 mRNA levels. In conclusion, Dexa and colostrum feeding promoted maturation of the somatotropic axis. Dexa effects partly depended on whether colostrum was fed or not.     

Developmental and steroid hormonal regulation of insulin-like growth factor II expression

We have investigated the influence of steroid hormones on insulin-like growth factor II (IGF-II) expression. Hepatic IGF-II mRNA decreased gradually during postnatal development, reaching adult levels at 3 weeks of age. Treatment of 1-day-old rats for 4 days with 10 micrograms/day of the glucocorticoid dexamethasone (DEX) reduced IGF-II mRNA levels 10-fold in liver and inhibited body weight gain. Estradiol and testosterone did not affect IGF-II expression. A dose-response relationship between IGF-II mRNA levels and the different amounts of DEX injected was seen. IGF-II levels remained low after withdrawal of DEX, indicating an irreversible effect. Albumin expression was increased in newborn rat livers after DEX treatment. Our results suggest that glucocorticoids play an important role in the regulation of IGF-II expression. The mechanism for glucocorticoid-induced reduction of IGF-II mRNA is still unclear; however, our findings indicate that DEX inhibits IGF-II by causing premature differentiation of the liver.     

Androgen regulation of aldehyde dehydrogenase 1A3 (ALDH1A3) in the androgen-responsive human prostate cancer cell line LNCaP

Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in human prostate cancer epithelial (LNCaP) cells. In the present study, we attempted to identify if any of the three ALDH1A/RA synthesis enzymes are androgen responsive and how this may affect retinoid-mediated effects in LNCaP cells. We demonstrated that exposure of LNCaP cells to the androgen dihydrotestosterone (DHT) results in a 4-fold increase in ALDH1A3 mRNA levels compared with the untreated control. The mRNA for two other ALDH1A family members, ALDH1A1 and ALDH1A2, were not detected and not induced by DHT in LNCaP cells. Inhibition of androgen receptor (AR) with both the antiandrogen bicalutamide and small interfering RNA for AR support that ALDH1A3 regulation by DHT is mediated by AR. Furthermore, specific inhibition of the extracellular signal-regulated kinase and Src family of kinases with PD98059 and PP1 supports that AR's regulation of ALDH1A3 occurs by the typical AR nuclear-translocation cascade. Consistent with an increase in ALDH1A3 mRNA, DHT-treated LNCaP cells showed an 8-fold increase in retinaldehyde-dependent NAD(+) reduction compared with control. Lastly, treatment of LNCaP with all-trans retinal (RAL) in the presence of DHT resulted in significant up-regulation of the RA-inducible, RA-metabolizing enzyme CYP26A1 mRNA compared with RAL treatment alone. Taken together, these data suggest that (i) the RA biosynthesis enzyme ALDH1A3 is androgen responsive and (ii) DHT up-regulation of ALDH1A3 can increase the oxidation of retinal to RA and indirectly affect RA bioactivity and metabolism.     

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1–4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation. J. Cell. Biochem. 71:449–458, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Dihydrotestosterone activates male mating behavior in castrated King—Holtzman rats

Having previously found that King-Holtzman rats respond behaviorally to dihydrotestosterone (DHT), this strain was used to compare the effectiveness of DHT and dihydrotestosterone propionate (DHTP) in maintaining and reinstating copulatory behavior. The 5α-reduced androgens were capable of stimulating mating behavior in these castrated male rats. DHT and DHTP were equally effective in maintaining ejaculatory behavior, whereas DHT was slightly more potent behaviorally than DHTP in restoring mating responses. It was found that as little as 200 μg hormone/day restored ejaculatory behavior in 78% of the DHT-treated and 50% of the DHTP-treated rats. In both the maintenance and restoration paradigms, the mating performance of the DHT(P) treated males declined over time. The present data suggest that the conversion of androgen to estrogen may not be critical for the activation of male mating behavior.     

Age- and sex-related changes in type-1 iodothyronine deiodinase messenger ribonucleic acid in rat liver and kidney.

To evaluate the age- and sex-related changes in Type 1 iodothyronine deiodinase gene expression in the liver and kidneys, we measured 5'-deiodinating activity and deiodinase mRNA in developing rats. The activity in the liver increased after birth, and that in neonates was approximately half that in adults. In contrast, the activity in neonatal kidneys remained very low. The relative importance of activity in male kidneys compared to the liver increased from the ages of 1 to 20 days. The male adult rat liver showed a higher level of activity than the female liver. Deiodinase mRNA in the male liver gradually increased from 1 to 20 days, in correlation with the activity. In kidneys, deiodinase mRNA was low before day 20, and there was no significant sex difference in all age groups. In orchiectomized male rats, the activity and mRNA in the liver was similar to the low levels found in females; however, the levels in the kidneys were not significantly different than those of normal males. These data suggest that the age- and sex-related changes in iodothyronine deiodinase gene expression are regulated mainly at the pretranslational level, and that the relative importance of kidneys to liver in iodothyronine deiodinase increases from birth to age 20 days due to the difference in the gene expression.     

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-beta1 gene expression in the rat, Syrian hamster and guinea pig

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-beta1 (TGF-beta1) gene expression. Male Sprague-Dawley rats, Syrian hamsters and Dunkin-Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during study days -1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-beta1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-beta1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-beta1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.