Diurnal variation in plasma concentrations of testosterone, 5α-dihydrotestosterone,and corticosteroids in the Australian brush-tailed possum, Trichosurus vulpecula (Kerr)

Plasma concentrations of testosterone, 5α-dihydrotestosterone, and total adrenal corticosteroids were measured in intact and castrated males and female possums over a 24-h period. In males, testosterone levels varied diurnally, being significantly higher in the morning than in the evening, with an overall mean concentration of 3.30 ± 0.43 (SEM) ng/ml. Levels of 5α-dihydrotestosterone showed a similar variation but of lower magnitude and the overall mean plasma concentration was 0.63 ± 0.10 ng/ml. Total adrenal corticosteroid concentrations in males also appeared to cycle, but inversely to the androgens with a peak in the early evening, and the overall mean concentration was 0.61 ± 0.08 μg/100 ml. Testosterone concentrations averaged 0.46 ± 0.02 and 0.35 ± 0.01 ng/ml in castrated males and females, respectively, and the corresponding values for 5α-dihydrotestosterone were 0.20 ± 0.03 and 0.21 ± 0.03 ng/ml. These measurements indicate that plasma testosterone levels of the marsupial possum fall within the range reported for eutherian species.     

Oestrogen receptors in the genital tract of the Australian marsupial Trichosurus vulpecula

Macromolecules with high binding affinity for oestrogens (K_a = 10⁹–10¹⁰M^?1 at 0°) are detected in cytosols of vaginal and uterine endothelium of the marsupial brush-tailed possum Trichosurus vulpecula. There are two distinct classes, of high (8 S) and low (4 S) molecular weight, the proportions of which vary in relation to the reproductive state or hormone treatment. In the endometrium, the 8-S form predominates during all phases of the breeding cycle; but there is a relative increase in the 4-S form, up to 40% of the total, during lactation. In the vaginal endothelium, an 8-S form is predominant in cytosols from follicular and lactating animals, with little or no 4-S binding, but the reverse occurs in luteal or pregnant animals. Only the 4-S form is found in cytosols of endometrium or vaginal endothelium of 16-day castrate females. After treatment of castrates with oestradiol, testosterone, or progesterone, however, only the 8-S form is detected in the endometrium. In the vaginal endothelium, the high-affinity binding remains in the 4-S region in castrates treated with oestradiol or testosterone, but is in the 8-S form after treatment with progesterone. The concentration of high-affinity binding sites per milligram of protein is significantly increased in both endometrial and vaginal endothelial cytosols during the follicular phase of the cycle and in castrates treated with oestradiol. This increase is associated with a marked increase in size of the two organs. Treatment with progesterone or testosterone has no effect on the concentration of binding sites in castrates. These findings are discussed in relation to the unique method of reproduction in marsupials.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. IV. The effect of exposure to placental steroids

The effect upon steroidogenesis of adding various steroids produced by the placenta was studied in short term cultures of human fetal adrenal cells. The addition of high concentrations (10(3) ng/ml) of estrone or estriol inhibited the production of cortisol, but only the former elicited a parallel increase in dehydroepiandrosterone (DHA) production. Estradiol was effective in inhibiting delta-4-3-ketosteroid production at concentrations of 10-100 ng/ml, levels which approach those found in the fetal circulation, while DHA production was increased at concentrations of 1 microgram/ml. The addition of progesterone (4 microgram/ml) to the medium caused increased production of cortisol and corticosterone, but had no effect on DHA production. Pregnenolone (4 microgram/ml) increased the basal production of DHA and slightly impaired both basal and ACTH-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect of estrogen is not influenced by other peptide hormones such as hCG, human prl, beta-lipotropin, corticotropin-like intermediate lobe peptide, or beta-endorphin. A revised model of the fetoplacental steroidogenic unit is presented which may explain both normal and fetal hyperplasia and postnatal involution of the adrenal cortex and the variations from this pattern seen in apituitary children.     

Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.     

Sex differences in the regulation of oxytocin receptors by ovarian steroids in the ventromedial hypothalamus of the rat.

The facilitation of sexual receptivity by oxytocin (OT) in female rats is related to the regulation of oxytocin receptors (OTR) by ovarian steroids in the ventromedial nuclei (VMN) of the hypothalamus. In a previous study, we have shown that estradiol benzoate (EB) causes a twofold increase in OTR binding in the VMN. Progesterone (P) then modulates levels of the estrogen-induced OTR and increases the area occupied by the receptors by acting on the neuronal membrane. In the present study, we compared the effects of EB and P on OTR binding between males and females. In both sexes, EB increased the density of OTR and the area covered by the receptors at the level of the medial and caudal VMN. In estrogen-primed females, P further increased OTR levels in the medial VMN and the area covered by OTR at the level of the caudal VMN. By contrast, P did not modulate OTR binding in estrogen-primed males. Thus, the behavioral insensitivity of male rats to ovarian hormones, in particular to P, may be related to sex differences affecting the modulation of OTR binding.     

Evidence of the in vitro formation of cortisol by the adrenal gland of embryonic and young chickens (Gallus domesticus)

The aim of this study is to elucidate the ontogenetic aspect of corticosteroidogenesis in the chicken. The adrenal gland of embryonic and very young chicks contains an enzyme system which converts progesterone to 17α-hydroxyprogesterone. 4-¹⁴C-Labeled cholesterol, pregnenolone, progesterone, and 17α-hydroxyprogesterone were incubated with the homogenates of adrenal gland from 17- and 21-day-old chick embryos and chickens. The metabolic products were identified by their mobilities on a thin-layer chromatogram and recrystallization to constant ³H:¹⁴C ratio after adding the corresponding ³H-labeled steroid. Cholesterol was metabolized to pregnenolone in the tissue homogenates from chick embryos and chickens at all ages. Pregnenolone was metabolized to progesterone, 11-deoxycorticosterone, and other minor metabolites, but not to 17α-hydroxyprogesterone. The major products from progesterone were 11-deoxycorticosterone and 17α-hydroxyprogesterone. The yield of 17α-hydroxyprogesterone decreased with advancing age and became zero at 7 days posthatching. 11-Deoxycortisol and cortisol were produced from progesterone by the homogenates from 17- and 21-day-old embryos and 3-day-old chicks, but neither was produced by those from 7-day-old chicks or those from 150-day-old hens. Radioactive 17α-hydroxyprogesterone was converted to 11-deoxycortisol and cortisol in large amounts and to cortisone in small amounts. Androstenedione and testosterone were detected in the adrenal homogenate from 17 days of incubation to 7 days posthatching, but not in the tissue from 14 days posthatching. The activity of 17α-hydroxylase was high at 17 days of incubation, decreasing with advancing age, and disappeared between 7 and 14 days posthatching. These results represent definite evidence of cortisol and testosterone formation in vitro by embryonic and very young chick adrenals.     

Development of the adrenal stress response in the Florida scrub-jay (Aphelocoma coerulescens)

Nestlings of altricial species undergo a period of substantial growth and development in the nest after hatching. The hypothalamic-pituitary-adrenal axis regulates the release of stress hormones such as corticosterone, which in adults is critical in allowing an animal to respond to a stressor. However, activation of this axis in young birds may be detrimental to growth and possibly survival. The developmental hypothesis predicts that altricial nestlings should display a dampened corticosterone response to stress as a means of protection against the potentially harmful effects of elevated corticosterone. We examined this hypothesis in Florida scrub-jays, a cooperatively breeding species with altricial young. Blood samples were collected from nestlings, nutritionally independent young, and yearlings for measurement of corticosterone levels. Baseline corticosterone levels did not differ between age-classes; however, stress-induced corticosterone levels were highest in yearlings, intermediate in independent young, and lowest in nestlings. The nestling stress response was also of a shorter duration than the response in independent young and yearlings. This variation in stress responsiveness across ages may be an adaptive mechanism to protect the developing bird from the negative effects of corticosterone on growth and cognitive development.     

The establishment of the assay-system of blood adrenal steroids in the method of gaschromatograph/mass spectrometry (GC/MS)

In this study, we established a method for the quantitative measurement of native adrenal steroids with GC-MS equipped with capillary column (cross-linked methyl silicone 25 m X 0.2 mm I.D., 0.11 m thin film). 1 ml of serum sample containing 5 alpha-cholestane as internal standard (IS) was elicited by organic solvent using extrelunt column. These samples were derived by n-butylboronic acid, o-methylhydroxylamine and trimethyl-silylating agents, then were finally applied to GC-MS. The intensities of molecular ions were used for the measurement of the serum concentration of steroids. The molecular ion peaks of steroids were obtained at m/z460 (17 alpha-hydroxyprogesterone; 17OHP), m/z548 (corticosterone; B), m/z470 (11-deoxycortisol; S), m/z417 (pregnenolone; PL), m/z372 (progesterone; PT), m/z558 (cortisol; F), m/z389 (dehydroepiandrosterone; DHEA), m/z371 (estrone; E1), m/z416 (estradiol; E2), m/z504 (estriol; E3), m/z389 (testosterone; T), m/z344 (androstenedione; A) and m/z372 (IS). The curve of calibration for each steroid showed good linearity. The sensitivities of the GC/MS method were less than 5pg/one shot of each sample. The coefficients of variations of accuracies and precisions in this GC/MS method were less than 15% of each steroid. The samples from normal subjects after metyrapone and ACTH loading tests, and the patients of congenital adrenal hyperplasia showed a good correlationship between the data of GC/MS and the data of RIA after sephadex LH-20 column-chromatography. These results implied the usefulness of our system in clinical application. Moreover, this assay takes only 3 hrs. Thus it saves much time in comparison with the time-consuming radioimmunoassay system.     

Sex and regional differences in estradiol content in the prefrontal cortex, amygdala and hippocampus of adult male and female rats

In general, the behavioral and neural effects of estradiol administration to males and females differ. While much attention has been paid to the potential structural, cellular and sub-cellular mechanisms that may underlie such differences, as of yet there has been no examination of whether the differences observed may be related to differential uptake or storage of estradiol within the brain itself. We administered estradiol benzoate to gonadectomized male and female rats, and compared the concentration of estradiol in serum and brain tissue found in these rats to those of gonadectomized, oil-treated rats and intact rats of both sexes. Long-term gonadectomy (3 weeks) reduced estradiol concentration in the male and female hippocampus, but not in the male or female amygdala or in the female prefrontal cortex. Furthermore, exogenous treatment with estradiol increased estradiol content to levels above intact animals in the amygdala, prefrontal cortex and the male hippocampus. Levels of estradiol were undetectable in the prefrontal cortex of intact males, but were detectable in all other brain regions of intact rats. Here we demonstrate (1) that serum concentrations of estradiol are not necessarily reflective of brain tissue concentrations, (2) that within the brain, there are regional differences in the effects of gonadectomy and estradiol administration, and (3) that there is less evidence for local production of estradiol in males than females, particularly in the prefrontal cortex and perhaps the hippocampus. Thus there are regional differences in estradiol concentration in the prefrontal cortex, amygdala and hippocampus that are influenced by sex and hormone status.     

Prolactin acts on the hypothalamic-pituitary axis to modulate follicle-stimulating hormone gene expression in the female brushtail possum (Trichosurus vulpecula)

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic-pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 μg) resulted in an 18-fold stimulation (P < 0.05) of progesterone (P₄) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n = 10) or saline (n = 8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P < 0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P < 0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P₄ concentrations were not different (P = 0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.     

In vitro production of steroids by the adrenal and testicular tissues of the cobra (Naja naja)

The capacity of cobra adrenals and testes to produce steroids in vitro from endogenous precursors was investigated by thin-layer and gas chromatographic analysis. The following adrenal steroids were separated and tentatively identified: corticosterone, aldosterone, 18-hydroxy-corticosterone (as the etiolactone by infrared spectroscopy), cortisone, testosterone, dehydro-epi-androsterone and estrone. The pattern of testicular steroids obtained from the same group of animals suggested the presence of dehydro-epi-androsterone, androstenedione, 17α-hydroxypregnenolone and estrone.These results are discussed in relation to the known biosynthetic mechanisms operative in these tissues.     

Effect of urophysial homogenates on plasma ion levels in Gillichthys mirabilis (Teleostei: Gobiidae)

In seawater-acclimated Gillichthys mirabilis, 2 and 4 hr after receiving an intraperitoneal injection of Gillichthys urophysial homogenate (two urophyses per fish), plasma sodium, magnesium, and chloride levels were significantly greater than those observed following a saline injection. When sodium and magnesium levels were measured in injected urophysectomized fish, similar increases were noted. No differences were observed in plasma potassium or calcium or in hematocrit between fish receiving saline and those receiving the urophysial homogenate. No differences were observed between urophysectomized and sham-operated fish in plasma sodium, potassium, calcium, and magnesium or in hematocrit 1, 3, 7, or 14 days postoperatively. Transection of the spinal cord at the level of the seventh or eighth preterminal vertebra had no effect on plasma sodium, potassium, calcium, or magnesium compared with sham-operated fish 14 days postoperatively. These findings demonstrate an effect of urophysial factor(s) on osmotic and ionic regulatory mechanisms in a euryhaline marine teleost.     

Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis

Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex. The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx). From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain. Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm. The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function. The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported. When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism. Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.