Immunoreactive dynorphin-A in bovine anterior pituitary and its in vitro release

The anterior lobe (AL) of the bovine pituitary contained and released, during in vitro culture, a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that occurring in neural tissue. Bovine AL tissue from intact females contained less ir-DYN-A than did AL tissue from castrated males. Enzymatically dispersed AL cells contained and released ir-DYN-A in vitro. Preincubation of dispersed AL cells for 18 hr, rather than 1.5 hr, increased the content and release of ir-DYN-A as well as LH. Addition of gonadotropin-releasing hormone (GnRH) to tissue slices or dispersed cells stimulated release of LH, but in contrast to published observations from rat AL, GnRH had no effect on release of ir-DYN-A. Addition of estradiol-17 beta, with or without progesterone, increased release of ir-DYN-A but not LH during 2-hr cultures. In summary, bovine AL contains and releases in vitro a large molecular weight form of ir-DYN-A. Although this ir-DYN-A was not coreleased with LH, a reproductive role was suggested by in vivo and in vitro effects of gonadal hormones on ir-DYN-A in the bovine anterior pituitary.     

Release of beta-endorphin-immunoreactivity from rat pituitary and hypothalamus in vitro: effects of isoproterenol, dopamine, corticotropin-releasing factor and arginine8-vasopressin

The release of beta-endorphin-immunoreactivity (beta E-IR) from rat pituitary anterior lobe (AL) quarters, neurointermediate lobes (NILs), and hypothalamic fragments was investigated in vitro. The beta-adrenoceptor agonist isoproterenol (ISO) and the hypothalamic neurohormone corticotropin-releasing factor (CRF) concentration-dependently stimulated the release of beta E-IR from superfused AL quarters and NILs, but not from incubated hypothalamic fragments. Dopamine (DA) inhibited the release of beta E-IR from NILs and hypothalamic tissue in a concentration-dependent manner, whereas it did not affect the release from AL quarters. Arginine8-vasopressin (AVP) stimulated the release of beta E-IR from AL quarters and hypothalamic fragments, but did not affect the release from NILs. The data indicate that the release of beta E-IR from cells in the pituitary lobes and in the hypothalamus is differentially regulated, but that common principles are involved. In particular, the results provide first direct evidence for an action of vasopressin as a stimulator of the release of POMC-derived peptides in the hypothalamus.     

Molecular forms of alpha-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe.

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of alpha-melanocyte-stimulating hormone (alpha-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of alpha-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-alpha-MSH, while monoacetyl-alpha-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-alpha-MSH was the major form of alpha-MSH. The profile of alpha-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-alpha-MSH and diacetyl-alpha-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of alpha-MSH predominate in PI tissue, while nonacetylated alpha-MSH is the major form in AL tissue. It appears, however, that acetylation of alpha-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

Molecular forms of α-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of α-melanocyte-stimulating hormone (α-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of α-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-α-MSH, while monoacetyl-α-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-α-MSH was the major form of α-MSH. The profile of α-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-α-MSH and diacetyl-α-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of α-MSH predominate in PI tissue, while nonacetylated α-MSH is the major form in AL tissue. It appears, however, that acetylation of α-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Specificity of inhibition by steroids of porcine oocyte maturation in vitro.

In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-17 beta, estradiol-17 alpha, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all at 10 microgram/ml) in defined medium that contained either BSA or dextran. Estradiol-17 beta was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in that oocytes, cultured in steroid-free medium after initial culture in estradiol-17 beta medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17 beta in both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17 beta or progesterone, nor were the oocytes saturated even when cultured in 10(-6) M estradiol-17 beta. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17 beta has shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17 beta may not act at the level of the oocyte nucleus.     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

The effect of estradiol-17 beta and actinomycin D on the release of PGF and PGE from separated cells of human endometrium

Estradiol-17 beta selectively stimulated the release of PGF from separated glandular but not stromal cells of human secretory endometrium (p less than 0.025) but had no effect on PGF release from either type of cells obtained from proliferative endometrium. PGE release was not affected by estradiol-17 beta. Actinomycin D did not antagonise the effect of estradiol-17 beta on PGF release from secretory, glandular cells. Basal release of PGF from these cells was stimulated by actinomycin D alone (100 ng/ml) (p less than 0.025) and PGE release stimulated in the presence of estradiol-17 beta. Actinomycin D had no effect on PGF or PGE release from proliferative endometrium. These findings suggest that estradiol-17 beta stimulates PGF release by a mechanism that does not affect PGE release and which is not dependent on the synthesis of new protein. The basal release of PGF and PGE by glandular cells of secretory endometrium in vitro is regulated by protein/proteins which reduce PG release.     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Effect of prostaglandins on in vitro synthesis of progesterone and oestradiol-17 beta in placenta from early human pregnancy

In vitro synthesis of progesterone and estradiol-17 beta from endogenous precursors was studied in the placenta from women in early stage of gestation (less than 7 weeks). Radioimmunoassay techniques were used to measure progesterone and estradiol-17 beta. It was shown that placental tissue from as early as six weeks of gestation can synthesize both progesterone and estradiol-17 beta in vitro. Prostaglandins F2 alpha and E2 in concentration of 100 micrograms/ml of the incubation media did not have any significant effect on the in vitro synthesis of progesterone and estradiol-17 beta in the placental tissue. It seems unlikely that the abortifacient effect of natural prostaglandins PGE2 and PGF2 alpha is due to their direct action on the synthesis of progesterone and estradiol-17 beta in the placenta.     

Estradiol-17beta activates corticosteroid synthesis and inhibits proliferation in cultured porcine adrenocortical cells

The influence of estradiol-17 beta on corticosteroids production and cell proliferation were studied in the cultured pig adrenocortical cells. Estradiol-17 beta considerably increased steroidogenesis, but inhibited cell proliferation. It is speculated, that cAMP mediated estradiol-17 beta effects in the adrenal cells.     

Effects of sex steroid hormones on differentiation of adipose precursor cells in primary culture

The effects of estradiol-17 beta and progesterone on multiplication, differentiation and lipid filling of adipose precursor cells were examined in primary cell cultures of cells prepared from adipose tissue of both male and ovariectomized female rats. Progesterone down to a concentration of 10(-7) mol/liter, alone or in the presence of estradiol-17 beta stimulated the development of glycerophosphate dehydrogenase and lipoprotein lipase activity. Estradiol-17 beta alone had no effects. These effects were essentially parallel to increases in the rate of lipid filling of the cells. Furthermore, the formation of cells with a lipid vacuole greater than 20 micron was markedly stimulated, suggesting that new fat cells were formed by the stimulation of differentiation of the adipose precursor cells. No effects of the sex steroid hormones were seen on the rate of multiplication. These results suggest a role of sex steroid hormones in the regulation of triglyceride storage capacity in adipose tissue by facilitating the differentiation of precursor cells to form new adipocytes.     

Beta-endorphin in brain limbic structures as neurochemical correlate of psychic dependence on drugs

The significance of beta-endorphin for drug dependence was explored by measuring the levels of beta-endorphin-immunoreactivity (beta E-IR) in plasma and parts of pituitary and brain of rats self-administering heroin or cocaine as compared to animals offered saline. Rats that had intravenously self-administrated heroin for 5 consecutive daily sessions of 6 h, and were decapitated immediately after the last session, showed a decreased concentration of beta E-IR in the anterior lobe (AL) of the pituitary while rats that had taken cocaine showed a decreased concentration of beta E-IR in the septum. Rats that had self-administrated heroin or cocaine and were decapitated 18 h after the last session, showed an increased concentration of beta E-IR in plasma and decreased concentrations in the AL of the pituitary and in specific areas of the brain limbic system, i.e. nucleus accumbens, septum, hippocampus and rostral striatum. The finding that self-administration of both the opiate heroin, inducing psychic and physical dependence, and the non-opiate cocaine, inducing psychic but not physical dependence, is accompanied by similar changes in beta E-IR concentrations particularly in limbic brain structures, and that these effects are present 18 h but not immediately after the last session, suggests that beta E and related peptides in limbic brain regions may represent a neurochemical correlate for psychic dependence on drugs.     

Changes in the spontaneous action potentials from the whole, isolated, intact bovine pituitary gland following an injection of various bovine pituitary intraglandular colloid fractions

Bovine pituitary intraglandular colloid (IGC) of intermediate lobe (IL) origin with accessions from the anterior lobe (AL), can modify the spontaneous action potentials (SAP) from AL, IL and posterior lobe (PL) cells. It was discovered that intraglandular colloid contains peptides ACTH, alpha-MSH, and beta-LPH when subjected to a series of radioimmunoassays. These peptides are thought, in part, to be responsible for altering the SAP.     

Estradiol-17beta triggers luteinizing hormone release in the protandrous black porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotropin-releasing hormone control.

We investigated the mechanism of estradiol-17beta (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17beta also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17beta potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2) induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2in the sex change observed in the protandrous black porgy.     

Opposing effects of androgen and estrogen on pituitary-adrenal function in nonpregnant primates

Maternal administration of androstenedione produces a sustained fall in maternal plasma adrenocorticotropic hormone (ACTH) concentrations in the pregnant nonhuman primate. We hypothesize a negative feedback influence on the maternal hypothalamo-pituitary-adrenal (HPA) axis by androgens in primates. This may reflect an important maternal adaptation during pregnancy in primates preventing premature induction of labor by maternal stress. However, androstenedione is precursor for placental estradiol-17beta synthesis, and infusion of androstenedione into pregnant primates elevates maternal plasma estradiol-17beta to term concentrations. Thus, it could be argued that 1) the effects attributed to androstenedione on the maternal HPA axis are mediated by estrogen rather than by androgen and 2) the negative influence of androgens may be on placental ACTH rather than, or in addition to, pituitary ACTH. To discriminate between androgenic and estrogenic effects of androstenedione on pituitary and/or placental ACTH function in primates we measured plasma ACTH, cortisol, and dehydroepiandrosterone sulfate (DHEAS) concentrations in nonpregnant baboons after treatment with either androstenedione or estradiol-17beta. Nine female baboons were studied between 14 and 22 days postpartum prior to estrous cycling. After 2 days of baseline, a continuous i.v. infusion of androstenedione (1.5 mg/kg per h in 10% intralipid, IL) was started at 0900 h and maintained for 9 days in 3 baboons. A similar protocol was carried out in another 3 baboons that received a continuous i.v. infusion of estradiol-17beta (10 microg/kg per h in 10% IL) instead of androstenedione. Three additional baboons received continuous i.v. IL vehicle alone and served as controls. Arterial blood samples (0.5 ml) for measurement of plasma hormones were taken during baseline and after 1, 3, 5, 7, and 9 days of infusion. Baseline plasma ACTH, DHEAS, and cortisol concentrations were similar among all groups. Plasma ACTH did not change during IL, increased following estradiol-17beta, and fell during androstenedione treatment. Accordingly, plasma cortisol and DHEAS concentrations were also unaltered by IL, and both steroids increased during estradiol-17beta treatment. In contrast, plasma cortisol and DHEAS remained unaltered from baseline during androstenedione treatment, despite the fall in plasma ACTH measured at this time. These data in the nonpregnant baboon 1) are consistent with negative feedback on pituitary ACTH by androgens and 2) demonstrate a positive influence on pituitary-adrenal function by estrogen in primates.     

Estrogen-sensitive cell lines: uptake of labeled sex steroids and growth properties

Cells of a rat pituitary cell line injected into Wistar/Furth rats produce tumors when inoculated into intact females or estradiol-17β primed males. No tumors are produced when the same number of cells are injected into spayed females, intact males or castrated males. Experiments carried out in animals and in tissue culture indicate the selective uptake of these cells for physiological amounts of estradiol-17β when compared with testosterone and when compared with an undifferentiated cell line.