Endocrine regulation of cytochrome P-450 in the rat brain and pituitary gland

In an effort to understand the physiological functions of cytochrome P-450 in the central nervous system and pituitary gland, we evaluated changes in the level of the enzyme as a function of the endocrine status of rats and the ability of these tissues to synthesize or degrade steroids. The P-450 content of microsomes prepared from the hypothalamic preoptic area (HPOA), the olfactory lobes and the cerebrum was 0.040 +/- 0.009 and in the pituitary gland 2.2 +/- 0.6 (S.D.) nmol/g tissue. The P-450 content of the HPOA and olfactory lobes, but not of the rest of the cerebrum, was influenced by the endocrine status of rats. In microsomes it increased five- to tenfold over control levels during late pregnancy in the olfactory lobes and during lactation in the HPOA, and in both brain regions treatment of rats with 5 alpha-dihydrotestosterone (DHT) caused an eight- to tenfold increase in the P-450 content. Androstenedione was not a good substrate for brain P-450. The level of androstenedione 19-hydroxylase in the olfactory lobe microsomal fraction was 0.50 +/- 0.06 nmol 19-hydroxyandrostenedione formed/g tissue per h. This activity was tenfold lower in other brain areas and was not detectable in the pituitary gland. The rate of aromatization of androstenedione to oestradiol in the HPOA and olfactory lobe of lactating rats was 0.46 +/- 0.14 and 0.38 +/- 0.05 pmol/oestradiol formed/g tissue per h respectively. 5 alpha-Androstane-3 beta,17 beta-diol (A-5 alpha-3 beta,17 beta-diol) was a much better substrate for P-450 throughout the brain and pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the feedback regulation of gonadotropin concentrations in male rats. (II). Partial characterization of 5 alpha-reductase in the medial basal hypothalamus and anterior pituitary gland

To evaluate the testosterone (T) 5 alpha-reductase activity in medial basal hypothalamus (MBH) and anterior pituitary gland of the adult male rat, whole homogenate of MBH or anterior pituitary glands was incubated with 14C-T in the presence of coenzymes under various experimental conditions. Major metabolites were 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha (beta), 17 beta-diol(3 alpha(beta)-diol) and delta 4 androstenedione. The activity of 5 alpha-reductase was expressed as the sum of the amount of DHT and 3 alpha(beta)-diol formed after incubation. The metabolites converted from T was separated and identified by thin layer chromatography and confirmed by recrystallization. The anterior pituitary gland possessed about four times higher 5 alpha-reductase activity compared with that in MBH. NADPH was essential for 5 alpha-reductase activity. They existed in microsomal fraction. They had same optimum temperature and almost same optimum pH. The Michaelis constant of the 5 alpha-reductase for T in MBH and anterior pituitary gland was 3.1 X 10(-7) M and 5.6 X 10(-7) M, respectively. These results suggest that 5 alpha-reduced metabolites of T, especially DHT have some physiologically significant roles in not only MBH but also anterior pituitary gland. Furthermore, it is noteworthy that 5 alpha-reductase in the anterior pituitary showed higher activity than that in MBH.     

Metabolism of testosterone in the anterior pituitary gland and the central nervous system of the European starling (Sturnus vulgaris)

The metabolism of testosterone was studied in vitro in anterior pituitary, hypothalamic and hyperstriatal tissues taken from male European starlings in the autumn. In all the tissues studied, testosterone was converted into 5alpha-androstan-17beta-ol-3-one (5alpha-DHT), 5beta-androstan-17beta-ol-3-one (5beta-DHT), 5beta-androstane-3alpha,17beta-diol (5beta-THT), 5beta-androstane-3,-17-dione and androst-4-ene-3,17-dione. The 5alpha-DHT was produced in significantly greater amounts by the pituitary gland than by the hypothalamus and hyperstriatum. The amount of 5alpha-DHT produced, however, was very low in comparison with the amounts of 5beta-reduced metabolites. The amount of 5beta-reductase was also higher in the pituitary gland than in the two nervous tissues. The ratios between the production of 5beta-DHT, 5beta-THT and 5beta-androstane-3,17-dione were, however, different in the three tissues: 5beta-DHT was produced in the greatest quantities by the hyperstriatum, while the production of 5beta-THT, 5beta-androstane-3,17-dione and androst-4-ene-3,17-dione was greatest in pituitary tissue. The role of 5alpha- and 5beta-reduced metabolites in the pituitary gland and in the brain of birds is unknown, but some possibilities arising from the present results are discussed.     

Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland

Testosterone metabolism in the brain and pituitary and cloacal glands of male and female Japanese quail was studied in vitro during sexual maturation (from 1 day to 5 weeks after hatching). The production of 5 alpha-dihydrotestosterone in the hyperstriatum and cloacal gland and that of androstenedione in the cloacal gland of males was highest at 1 day after hatching, which could be related to the peak of plasma androgens previously demonstrated in neonatal quail. 5 beta-Reductase activity was very high in the brain, but not the pituitary or cloacal glands of young chicks and decreased markedly, especially in the hypothalamus, during sexual maturation. As 5 beta-reduced metabolites of testosterone are inactive androgens, it is suggested that the decrease of 5 beta-reductase activity with age corresponds to a potentiation of the effects of testosterone at the level of the brain.     

Photoperiodic control of testosterone metabolism, plasma gonadotrophins, cloacal gland growth, and reproductive behavior in the Japanese quail.

The changes in plasma gonadotrophins and testosterone, in cloacal gland area, and in reproductive behavior were observed in male Japanese quails after transfer to long days or testosterone implantation and were correlated with the testosterone metabolism in the hyperstriatum, hypothalamus, pituitary, and cloacal gland. Long days stimulate the growth of the cloacal gland and at the same time enhance its production of androstenedione from testosterone. This increased 17β-hydroxysteroid dehydrogenase activity is correlated at the individual level with cloacal gland area but not with plasma testosterone. Similarly the changes observed in some reproductive activities (aggressive behavior and struts) are correlated with individual differences in the brain metabolism of testosterone (hyperstriatal or hypothalamic production of androstenedione), but not with plasma testosterone. The plasma luteinizing hormone is also related to testosterone metabolism rather than to the circulating level of the hormone (negative correlation with the production of 5α-dihydrotestosterone in the pituitary). The testosterone metabolism in target organs thus appears of critical importance in the control of reproduction and seems largely responsible for the occurrence of individual differences. After exposure to 12 long days the mean testosterone metabolism in the pituitary is also strongly affected (increased 5β-reduction) though not in the same way as after 2 months of photostimulation. It is suggested that these metabolic changes at the pituitary level may play some role in the induction of the increased responsiveness to luteinizing hormone-releasing hormone which can be observed in quails after exposure to 7 long days.     

Identification of human luteinizing hormone, follicle-stimulating hormone, luteinizing hormone beta-subunit and gonadotrophin alpha-subunit in foetal and adult pituitary glands.

Specific radioimmunoassays were used to assess the content of LH, FSH, the gonadotrophin alpha-subunit and the LH beta-subunit in four adult, 19 normal foetal pituitary glands (9-5--32 weeks of gestation) and a pituitary extract from an anencephalic foetus (36 weeks). The hormones and subunits were further identified by column chromatography on Sephadex G-100. All pituitary glands contained free alpha-subunit and intact LH but the alpha-subunit:LH ratio was significantly higher in the early foetal pituitaries (9-5--16 weeks) than in the four adult pituitaries. Only small or undetectable amounts of LH beta-subunit and 'undetectable' FSH were found in these early foetal pituitaries (9-5--11-5 weeks). The concentration of intact hormones or subunits in the pituitaries showed no significant sex difference in any of the groups. In contrast to these results, only alpha-subunit was detectable in the pituitary of the anencephalic foetus. For 14 early foetuses (age of gestation 10--16 weeks) the serum levels of LH-HCG, FSH, and alpha-subunit in the circulation were significantly higher than in 26 foetuses at term (37--41 weeks). On the basis of these results a theory for the development of the gonadotrophin secretion from the foetal pituitary gland is outlined.     

Releasing factor and hormonal changes in the hypothalamic-pituitary-gonadotrophin and -adrenocorticotrophin systems before and after birth and puberty in male, female and androgenized female rats.

The hypothalamic content of LH releasing factor (RF), pituitary ACTH and pituitary and plasma LH and FSH were measured by radioimmunoassay from foetal Day 15 to postnatal Day 65. Bioassayable corticotrophin releasing activity was also measured during the postnatal period. Hypothalamic LH-RF was detectable as early as foetal Day 15, increasing gradually until postnatal Day 2 and then steeply between Days 5 and 16. The levels of LH-RF were similar in both male and normal female rats until Day 41, after which the increase which had been occurring from Day 16 continued in the male but not the female. In female rats treated with testosterone propionate neonatally ('androgenized females') the hypothalamic content of LH-RF at Day 9 was significantly less than that in the male or normal female, levels reaching those found in the latter two groups by Days 16-22. The lower level of LH-RF in the androgenized female was associated with pituitary gonadotrophin and plasma FSH levels which were lower than in the normal female until Day 30. In the normal female, vaginal opening was associated with a marked drop in hypothalamic LH-RF content and in pituitary LH and FSH, but in the androgenized female, vaginal opening occurred while hypothalamic LH-RF and pituitary LH levels were still rising. The peaks in pituitary FSH and LH and in plasma LH seen on Days 22, 30 and 41, respectively, in the normal female were each delayed by 8-9 days in the androgenized female. In all three types of animal there was a significant drop in plasma FSH between Days 50 and 65 which was associated with a significant increase in pituitary FSH in the male and a significant decrease in pituitary FSH in the androgenized female rats. The day 17 foetal pituitary gland also contained ACTH, and again levels of this hormone rose steeply between Days 5 and 9. In contrast to the gonadotrophins, there was a marked divergence between the pituitary content and concentration of ACTH: content rose while concentration remained relatively steady after Day 9. There was no major difference in pituitary ACTH levels between the three types of animal throughout the study; however, around Days 16 and 50, corticotrophin releasing activity was higher in males and androgenized females compared with that in normal females.     

Effects of in vivo corticosterone treatment on the in vitro metabolism of testosterone in the comb and brain of the young male chicken

Male chickens kept in constant light were injected from their second to their 12th day of life daily either with 2.5 mg of corticosterone or with a control solution. In both groups of birds, the in vivo metabolism of labeled testosterone by the comb and by brain tissues (hyperstriatum, hypothalamus, pituitary gland) was then studied.The experimental treatment blocked the growth of the comb observed in control birds. In this tissue, it also reduced the production from testosterone of 5α-dihydrotestosterone and of 5α-androstane-3α,17β-diol, but it increased the production of 5β-androstane-3α,17β-diol. These modifications did not appear in any brain tissue; they were not accompanied by changes either of LH or of testosterone circulating levels or of the testes' weights.     

Leu-Phe cleaving endopeptidase activity, gamma-endorphin, and beta-endorphin in the rat pituitary gland and brain. Effect of adrenalectomy and corticosterone substitution

The influence of adrenalectomy and corticosterone substitution was investigated on Leu-Phe cleaving endopeptidase activity and on the levels of gamma-endorphin and beta-endorphin in the pituitary gland and the brain. The enzyme activity was quantitated by a specific radiometric assay based on the cleavage of the Leu17-Phe18 bond in a NH2- and COOH-terminally protected synthetic substrate which was analogous to beta-endorphin-(15-19). This activity may mimick the formation of gamma-endorphin. beta-Endorphin and gamma-endorphin were measured by specific radioimmunoassays. After 14 days of adrenalectomy enzyme activity had increased in anterior (15%) and neurointermediate lobes of the pituitary gland (30%), hypothalamus (25%), and liver (15%). This increase was prevented when the adrenalectomized animals were subjected to chronic corticosterone substitution by subcutaneous implantation of a pellet of 100 mg. Extirpation of only the adrenal medulla did not affect the Leu-Phe cleaving activity. Enzyme activity in the septum, hippocampus, and cerebellum had not changed after adrenalectomy. Determination of immunoreactive levels of gamma- and beta-endorphins showed that in the anterior pituitary gland gamma- and beta-endorphins had increased by 275 and 300%, respectively, 14 days after adrenalectomy. No significant changes were observed in endorphin levels of the intermediate lobe of the pituitary gland, hypothalamus, hippocampus, and septum. The results indicate that Leu-Phe cleaving endopeptidase activity in sensitive to glucocorticoids in tissues containing proopiomelanocortin-producing cells, i.e., anterior and neurointermediate pituitary gland and the hypothalamus. In the anterior pituitary gland it is correlated with the levels of gamma- and beta-endorphins.     

Pituitary Leptin Gene Expression Is Reduced by Neonatal Androgenization of Female Rats

We have previously reported evidence of leptin gene expression (ob mRNA) in adult rat brain and pituitary gland. We have also shown that ob mRNA levels in female rat brain and pituitary are regulated in an age- and tissue-dependent fashion. In view of the known sexual dimorphism in adipose tissue leptin expression, we have extended our original work to include an assessment of ob mRNA levels in brain, pituitary and fat of developing male and female rats. In addition we determined the effects of neonatal androgenization of female rat pups with testosterone propionate. Leptin (ob) mRNA expression was evaluated using semi-quantitative RT-PCR analysis. Leptin mRNA levels were developmentally regulated in the pituitary and cortex of male rats, paralleling the changes previously observed in female rats. In the pituitary, leptin expression was significantly higher during the early postnatal period and dropped abruptly by postnatal day (PD) 22. In the cortex, leptin expression was lowest at PD 4 and rose significantly by PD 14. In addition gender differences, most notably in the pituitary, were also observed. In pituitary gland, ob mRNA was significantly higher in female rats than in males at PD 14 (+60%; p < 0.05) but there were no sex differences at PD 4 and PD 22. Testosterone treatment of neonatal female rats profoundly reduced ob mRNA at PD 14 (3.5-fold; p < 0.01) and PD 22 (3-fold; p = 0.05). In subcutaneous adipose tissue and hypothalamus we observed no sex difference in ob mRNA levels nor an effect of testosterone. We conclude that leptin gene expression in rat pituitary gland is sexually dimorphic and sensitive to neonatal manipulation of sex steroid levels.     

Sexual differences in the Japanese quail: behavior, morphology, and intracellular metabolism of testosterone

Three experiments were carried out to study whether differences in the intracellular metabolism of testosterone (T) can explain sexually differential responses to T in Japanese quail. In the first experiment, a series of dose-response curves in which length of Silastic testosterone implants was related to effects on several behavioral and physiological variables was established. In Experiment 2, adult males and females were assigned to six experimental groups: intact males and females (I-males and I-females), castrated males and females implanted subcutaneously with 40-mm Silastic implants of T (T-males and T-females), and castrated males and females without hormone treatment (CX-males and CX-females). No CX-bird (male or female) and no I-female exhibited male sexual behavior. However, I-males and T-males regularly copulated during the behavioral tests. No crowing was ever heard in CX-animals and I-females. T-females crowed less than T-males and their crowing sounded weaker than those of males. The cloacal glands of T-females were less developed than those of males. Radioimmunoassay of T and 5 alpha-DHT showed that T-males and T-females have similar plasma levels of androgens. No striking differences were observed in the way testosterone is metabolized by the pituitary gland and central nervous tissues of males and females. By contrast, the production of 5 alpha-dihydrotestosterone (5 alpha-DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha, 3 alpha-diol) was higher in the cloacal glands of males than in those of females. These sex differences were not detected between T-males and T-females. In experiment 3, the cloacal gland of males produced more 5 alpha-reduced metabolites than those of females. The pituitary gland of females also produced more 5 beta-androstane-3 alpha, 17 beta-diol (5 beta, 3 alpha-diol). In syringeal muscles, the production of 5 beta-dihydrotestosterone (5 beta-DHT) and 5 beta, 3 alpha-diol was higher in females compared to males.     

Androgen aromatization and 5 alpha-reduction in ferret brain during perinatal development: effects of sex and testosterone manipulation

Ferrets of both sexes were killed 8 or 5 days before expected parturition as well as 7, 15, 30, or 51 days after birth, and the activities of aromatase (using 19-[3H]hydroxyandrostenedione as substrate) and of 5 alpha-reductase (using [3H] testosterone as substrate) were assayed in whole homogenates of preoptic area plus anterior hypothalamus (POA), mediobasal hypothalamus (MBH), temporal lobe (TL), and cerebral cortex. Aromatase and testosterone 5 alpha-reductase activities were also measured in these regions in adult gonadectomized male and female ferrets. Compared with adults of both sexes in which aromatase activity was low in all brain regions studied, fetal ferrets had high levels of aromatase activity in POA plus MBH and in TL. At these prenatal ages, aromatase activity in POA plus MBH was significantly higher in males than in females. Aromatase activity in POA, MBH, and TL remained high in both sexes on postnatal days 7, 15, and 30, before declining by postnatal day 51. Cortical aromatase activity was uniformly low across all perinatal ages. The existence of a sex difference in aromatase activity in fetal POA plus MBH cannot be explained by a concurrent sex difference in circulating testosterone. Administration of testosterone to pregnant female ferrets over days 30-41 of gestation caused 150- to 350-fold increases in maternal plasma concentrations of testosterone and 2- to 5-fold increases in fetal plasma testosterone. However, aromatase activity was not affected in the POA and MBH of fetuses or mothers, although activity was significantly increased in the TL of mothers given testosterone. Furthermore, castration of neonatal or adult breeding males decreased plasma androgen levels by factors of 8 and 480, respectively, but resulted in only modest reductions in POA, MBH, and TL aromatase activity (a significant reduction occurred only in the adult male TL). Relatively high levels of testosterone 5 alpha-reductase activity were found in all brain regions across all perinatal ages, as well as in gonadectomized adult ferrets; there was no sex differences at any postnatal age studied. Prenatally, males had higher levels of 5 alpha-reductase activity than females only on day -8 in the POA plus MBH. The results show that estrogen and 5 alpha-reduced androgens can be synthesized in the brains of ferrets of both sexes during the perinatal period of sexual differentiation. A functional role for this neural metabolism of androgen remains to be demonstrated in this carnivorous species.     

The nuclear accumulation of [3H]testosterone and [3H]estradiol in the brain of the female primate: evidence for the aromatization hypothesis

To identify the metabolites of estradiol (E2) and testosterone (T) in nuclei obtained from the female primate brain and, hence, to investigate the mechanism of their actions on behavior, 9 ovariectomized adult rhesus monkeys were studied. Two of these females were injected with 5.5 mCi [3H]T, and 30 min later, samples of 14 brain areas, pituitary gland, and peripheral tissues were removed and homogenized. Purified cell nuclei and a crude cytosol fraction were prepared, extracted with ether, and fractionated by HPLC to identify steroid metabolites. In nuclei from the hypothalamus, preoptic area, and amygdala, [3H]E2 formed locally was the major form of radioactivity. In nuclei from the clitoris, [3H]dihydrotestosterone was the major form of radioactivity, and in nuclei in all other brain samples and in the pituitary gland and uterus, [3H]T predominated. Two females (controls) were pretreated for 5 days with oil sc, injected with 1 mCi [3H]E2, and killed 60 min later. In these females, elevated nuclear concentrations of [3H]E2 were found in the hypothalamus, preoptic area, amygdala, pituitary gland, and uterus. Similar results were obtained in 2 females that were pretreated for 5 days with 2 mg/day dihydrotestosterone propionate, sc, and then injected with 1 mCi [3H]E2. In 3 females that were pretreated for 5 days with 2 mg/day T propionate, sc, and then injected with 1 mCi [3H]E2, levels of [3H]E2 were reduced by 100% (P less than 0.01) in nuclei from preoptic area and amygdala compared with control values and by 78% (P less than 0.05) in nuclei from the hypothalamus. There were no comparable reductions in steroid levels in cerebral cortex, pituitary gland, or uterus. This is the first direct evidence in the brain of a female primate that the actions of T and E2 involve the same receptor systems.     

Demonstration of a pituitary gonadotrophin hormone activity in the male foetal mouse.

The testosterone production by 18-days-old foetal mouse testis was measured in an organ culture system, by RIA in the culture medium. This production was time-dependent, and could be stimulated by ovine LH and age-matched foetal pituitary. The gonadotrophin activity derived from foetal pituitary appeared to be released into the culture medium as a limited reserve. These data clearly show that a biologically active gonadotrophin material is present in the pituitary of the 18-days-old mouse foetus.     

Specific, high-affinity binding of 17beta-estradiol in cytosols from several brain regions and pituitary of intact and castrated adult male rats.

Specific 17beta-estradiol binding capacities of cytosols from several brain regions and pituitary were determined in intact and castrated adult male rats. The binding capacity of the pituitary was approximately 10 times higher than that of any of the 5 brain region studied. Of these brain regions, the highest 17beta-estradiol binding capacities were present in the anterior hypothalamus followed by progressively lower capacities in the posterior hypothalamus, amygdala, midbrain, and cerebral cortex. The specific 17beta-estradiol binding capacity of cytosol from the anterior hypothalamus was significantly higher in castrated males than in intact rats. No such difference was found in any of the other tissues studied. Using sucrose gradient ultracentrifugation, an 8S sedimentation coefficient was found for the specific estradiol binding macromolecules present in cytosols from pituitary as well as anterior and posterior hypothalamus of castrated rats. The affinity for estradiol of cytosols from anterior and posterior hypothalamus was very high, with the mean association constants being 2.9 and 2.4 X 10(10) M-1, respectively. In competition experiments the 17beta-estradiol binding molecules present in cytosols from pituitary and anterior hypothalamus showed a higher affinity for 17beta-estradiol than for either estrone or estriol. In both tissues these 17beta-estradiol binding molecules showed a moderate affinity for the anti-estrogens MER-25 and cis-clomiphene citrate as well as for the androgen 3beta-androstranediol, but almost no affinity for 3alpha-androstanediol, 5alpha-dihydrotestosterone, testosterone, or corticosterone. These findings suggest that a true cytoplasmic receptor for estradiol exists in the male rat brain and pituitary which may play an important role in regulating reproductive function.     

Effect of insulin-like growth factors on human foetal, adult normal and tumour pituitary function in tissue culture

To determine the direct effects of insulin-like growth factors (IGFs) on hormone release by the human pituitary gland, human foetal, adult normal and tumour pituitary tissues were maintained in culture for 2 to 4 weeks and tested with acute (3 h) exposures to different preparations of IGF peptides. Adult normal pituitaries and adenomas were tested with a semipurified preparation of IGFs, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. Human foetal pituitaries were tested with the semipurified IGFs as well as more purified preparations of IGF-I and IGF-II. Culture media were assayed for hGH, hPrl, hACTH and hLH using specific radioimmunoassays. Both foetal (n = 16 (No. of pituitaries), 33 (No. of observations] and normal adult (n = 3, 16) human pituitaries cultures responded to the semipurified IGFs (2-25 ngEq/ml for foetal and 2-4 ngEq/ml for adult pituitaries) with a significant decrease in hGH release compared to basal (P less than 0.01) whereas the GH-secreting pituitary tumours showed no effect when tested with from 2 to 25 ngEq/ml (n = 8, 129, NS). The effect of IGFs on human foetal somatotrope activity was dose-related for both the semipurified IGFs (2-25 ngEq/ml, n = 16, 33) and IGF-I or IGF-II (10-100 ng/ml; n = 3, 18).(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain testosterone metabolism in thyroidectomized and thyroxine-treated chickens

The metabolism of testosterone to reduced derivatives was studied in the pituitary gland, the hypothalamus, and the hyperstriatum dorsale of thyroidectomized, sham-operated, and thyroxine (T4)-injected immature cockerels. The levels of plasma thyroid hormones were markedly reduced (P less than 0.001) in thyroidectomized cockerels whereas thyroidectomized or sham-operated birds injected daily with 100 micrograms/kg thyroxine had significantly elevated (P less than 0.001) levels in comparison with sham-operated control birds. Each tissue was found to produce significant amounts of 5 beta-androstane-17 beta-ol-3-one (5 beta-DHT), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-3 alpha-diol), and androstenedione. Irrespective of thyroid state 5 beta-DHT and 5 beta-3 alpha-diol were produced to the greatest extent by the hyperstriatum dorsale whereas androstenedione was maximally produced in the pituitary gland. In comparison with the hyperstriatum dorsale and the hypothalamus only small quantities of 5 beta-DHT were produced in the pituitary gland. In the hyperstriatum dorsale of thyroidectomized birds both 5 beta-DHT (P less than 0.05) and 5 beta-3 alpha-diol (P less than 0.1) were formed to a greater extent than in sham-operated birds. This effect was reversed by administration of T4 to the operated birds which reduced the levels to those measured in the sham-operated controls. Similarly, injection of T4 into sham-operated birds decreased (P less than 0.05) the production of 5 beta-DHT in the hypothalamus while in the pituitary gland injection of T4 into thyroidectomized birds reduced the production of androstenedione (P less than 0.05). It was concluded that in the cockerel thyroid hormone is likely to play a role in the metabolism of testosterone. The physiological significance of 5 beta-reductase activity in the neuroendocrine tissues is discussed.     

Hormonal, drug, and dietary factors affecting peptidyl glycine alpha-amidating monooxygenase activity in various tissues of the adult male rat

The factors controlling levels of peptidyl glycine alpha-amidating monooxygenase (PAM) activity in its major tissue sources in the adult male rat were investigated by carrying out a variety of endocrine, pharmacological, and dietary manipulations. Levels of PAM activity and alpha MSH immunoactivity in the neurointermediate lobe of the pituitary gland rose and fell in parallel in rats treated with the dopamine antagonist haloperidol or the dopamine agonist bromocriptine, respectively. PAM activity in the anterior pituitary lobe was increased after adrenalectomy or castration and decreased after thyroidectomy or treatment with haloperidol. PAM activity in the submandibular gland was increased after treatment with the alpha-adrenergic antagonist phenoxybenzamine and decreased after treatment with the alpha-adrenergic agonist phenylephrine. Serum levels of PAM activity were unaltered after hypophysectomy, adrenalectomy, sialectomy, or castration, but rose after thyroidectomy and declined after treatment with the ganglionic blocker chlorisondamine or phenoxybenzamine. Chronic dietary copper deficiency in rats resulted in increased PAM activity in homogenates of anterior pituitary lobe and submandibular gland assayed under optimized conditions; chronic dietary ascorbate deficiency in guinea pigs did not produce consistent changes in PAM activity in the tissues examined.     

Demonstration of desacetyl alpha-melanocyte-stimulating hormone in fetal and adult human anterior pituitary corticotrophs

Immunocytochemistry, radioimmunoassay and high-performance liquid chromatography (HPLC) techniques have been used in combination to investigate the presence of immunoreactive (ir)-alpha-MSH in the normal human pituitary gland, and to investigate the possible origin of these cells from the fetal pars intermedia. Two separate antisera to alpha-MSH were employed in immunocytochemistry to distinguish between authentic alpha-MSH and the desacetyl form. Only desacetyl alpha-MSH was detected in the pituitary gland of fetal and adult man, in both the pars (zona) intermedia and the pars anterior. In the fetus, a large proportion of the ACTH-containing cells of the anterior lobe also contained ir-alpha-MSH, while ir-alpha-MSH containing cells were more sparse in adults. Radioimmunoassay of acid extracts of adult pituitary tissue showed alpha-MSH levels representing less than 0.05% of the ACTH content of the gland. HPLC analysis of these extracts confirmed that only the desacetyl form was present. These results suggest that alpha-MSH peptides are synthesized by anterior lobe cells of the human pituitary gland, which are not derived from the fetal pars intermedia. Possible regulatory mechanisms affecting cells which contain ir-alpha-MSH are discussed, and by comparison with the intermediate lobe of other species it is concluded that there is little evidence for a true intermediate lobe in the human pituitary gland.     

Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry.

32P-labeled oligonucleotides derived from the coding region of rat dopamine D2 receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body. In these areas the distribution was comparable to that of the dopamine D2 receptor binding sites as visualized by autoradiography using [3H]SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D2 receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D2 receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptors and their alteration following selective lesions or drug treatments.