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Biomedical subjects

M Pasmanik

Publications and source records attributed to M Pasmanik.

12 recordsLinked to original sources

Dexamethasone inhibition of cyclooxygenase expression in bovine term placenta.

Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention.

Animals↗

Bovine placental progesterone synthesis: comparison of first and second trimesters of gestation.

We have previously reported that dispersed caruncle cells from cows during the first trimester of pregnancy, in comparison to caruncle cells from cows of more than 90 days of gestation, produce little progesterone (P4) and are refractory to agents that enhance steroidogenesis. To explain this refractoriness of the first-trimester cells, we determined (1) the expression of cytochrome P450 side-chain cleavage (P450scc) and its mRNA, (2) the expression of adrenodoxin, and (3) 3 beta-hydroxysteroid dehydrogenase activity. We first determined P4 and pregnenolone (P5) production by dispersed caruncle cells from the two gestation periods using RIA. It was found that P4 synthesis by bovine maternal caruncle cells was low or undetectable in the first trimester but increased more than 10-fold in the second trimester of gestation. Addition of 25-OH-cholesterol (5 micrograms/ml) to second-trimester maternal cells increased P5 production, but no effect was observed in first-trimester cells. With [3H]P5 used as substrate, analysis of metabolites on thin-layer chromatography indicated that first-trimester maternal cells synthesized a small amount of P4 (3.02% of total radioactivity) compared to second-trimester cells (16.4%). A readily detectable amount of 17 alpha-OH-P5 was produced by the second-trimester cells (5.02%) but not by the first-trimester cells (0.6%). No other metabolites could be characterized (less than 0.5%). Cytochrome P450scc expression and its mRNA and adrenodoxin content were determined by use of Western blot or dot-blot techniques. Proteins and mRNA were detected in maternal tissues of first and second trimesters of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

Regulation of steroidogenesis in the bovine placenta.

As pregnancy progresses in the cow, the secretory activity of the corpus luteum is markedly diminished. This reduced secretion is due to a decline in the number of viable luteal cells as well as reduction in the secretory activity and responsiveness of the cells to trophic agents. The principal extra-ovarian source of progesterone (P4) by mid-gestation therefore appears to be the placenta. Uniquely this P4 biosynthesis is cyclic-nucleotide independent, but the Ca+2 dependent. It therefore appears that the Ca+2 second messenger and protein kinase C systems are responsible for regulation of sterol biosynthesis in the cow placenta. Dispersed bovine caruncle cells from the first trimester of pregnancy in comparison to caruncle cells of older than 90 days of gestation produce little P4 and are refractory to agents which enhance placental steroidogenesis. In order to explain this refractoriness of the first trimester cells, we determine (1) the expression of P450scc and its mRNA and (2) the expression of adrenodoxin. It was found that P4 synthesis by bovine maternal caruncle cells was low or undetectable in the first trimester but increased more than 10-fold in the second trimester of gestation. Addition of 25-OH-cholesterol to second trimester maternal cells increased P5 production but no effect was observed in first trimester cells. Cytochrome P450scc and its mRNA and adrenodoxin content were determined using Western blot or dot-blot techniques. Both proteins and the mRNA were detected in maternal tissue of first and second trimesters of gestation. In conclusion low P4 levels synthesized by first trimester maternal cells are not due to the absence of either cytochrome P450scc or adrenodoxin protein or production of P450scc mRNA. The data suggest that the refractoriness of the maternal caruncle cells during the first trimester is the result of post-translational regulation.

Adrenodoxin↗

Dexamethasone inhibition of prostaglandin production in human term placental cells is protein and ribonucleic acid synthesis dependent.

A key enzyme in the regulation of prostaglandin (PG) synthesis is PG synthase (PGS; cyclooxygenase), which converts arachidonic acid to PGs. Since both PGs and glucocorticoids are elevated before parturition, we studied the regulation of dexamethasone (DEX; 150 nM) on PGF2 alpha synthesis and PGS expression in human placental cells in vitro. Both first trimester and term placental cells were used. DEX reduced PGF2 alpha synthesis in human term placental cells, in contrast to first trimester cells which were unaffected by the same treatment. DEX inhibition of PGF2 alpha production by term placental cells was time and dose dependent. PGS expression was analyzed by [35S]methionine metabolic labeling and immunoprecipitation using polyclonal antibodies developed in rabbits against ram seminal vesicle PGS. DEX reduced PGS expression in term placental cells, but not in first trimester cells. In contrast to the effect of DEX on PGF2 alpha, progesterone and estradiol production by cells were unaffected at any stage of gestation examined. DEX inhibition of PGF2 alpha synthesis required de novo biosynthesis of RNA and proteins. These results suggest 1) corticosteroids play a role in the regulation of placental PG synthesis during parturition; 2) the inhibition of PG synthesis and PGS expression by glucocorticoids is RNA and protein biosynthesis dependent; and 3) induction of labor by glucocorticoids is not directly related to changes in placental progesterone or estradiol biosynthesis.

Arachidonic Acid↗

Nonmammalian vertebrate models in studies of brain-steroid interactions.

Estrogen formation in brain and pituitary mediates certain androgen actions in central targets. Goldfish (Carassius auratus) and quail (Coturnix coturnix japonica) have been advantageous for studying the role of locally formed estrogen in autoregulating aromatization and in controlling estrogen receptor occupancy, androgen receptor levels, and behavioral expression. Data from these two experimental models reveal a molecular basis for androgen-estrogen synergism in neuroendocrine tissues and for alterations in androgen sensitivity/responsiveness. These mechanisms are essential components of seasonal reproduction in the test species and may have wider relevance for cyclicity in other vertebrates, including mammals.

Androgens↗

In vivo steroid regulation of aromatase and 5 alpha-reductase in goldfish brain and pituitary.

The full expression of testosterone (T) actions in neuroendocrine tissues requires aromatization and 5 alpha-reduction to estradiol (E2) and 5 alpha-dihydrotestosterone (DHT), respectively. Recently, we documented striking changes in aromatase and 5 alpha-reductase during the annual reproductive cycle of goldfish (Carassius auratus). To investigate possible regulatory effects of sex steroids, goldfish were implanted with hormone-filled silastic capsules for 2-5 weeks. Conversion of [3H]androstenedione to estrone or 5 alpha-androstanedione by homogenates of anterior hypothalamus/preoptic area, remaining telencephalon, and whole pituitary (PIT) was used to estimate aromatase and 5 alpha-reductase, respectively. Gonadosomatic index and plasma E2, T, and DHT were monitored as an index of reproductive status and capsule effectiveness. In reproductively inactive fish in which plasma steroids and aromatase were basal (October), E2 or T increased aromatase activity in brain of both sexes but stimulated activity in PIT of females only; DHT was not effective. In a subsequent experiment initiated close to the spawning peak and prior to the seasonal decline in plasma steroids and brain aromatase (April), T increased or maintained brain aromatase in a time-dependent manner. 5 alpha-Reductase activity was unaffected by steroid treatment in both reproductively active and inactive fish. These results indicate that variations in circulating steroids are responsible, at least in part, for changes in brain aromatase during the annual reproductive cycle of goldfish and provide the first evidence for steroid control of pituitary aromatase. The steroid specificity of the induction suggests that an estrogen receptor mechanism is involved.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

Changes in brain aromatase and 5 alpha-reductase activities correlate significantly with seasonal reproductive cycles in goldfish (Carassius auratus).

Aromatization and 5 alpha-reduction are known to be required for the full expression of testosterone actions in neuroendocrine tissues. Although aromatase and 5 alpha-reductase activities in brain and pituitary can be experimentally manipulated by castration and steroid replacement, naturally occurring variations during seasonal reproductive cycles have not been examined in any species. Goldfish (Carassius auratus) were selected for study because they exhibit exceptionally high levels of aromatase in both brain and pituitary, although 5 alpha-reductase levels resemble the vertebrate norm. Four animals of each sex were tested monthly through three breeding seasons (2.5 yr). Using previously validated techniques, the enzymes were assayed by product formation from [3H]androstenedione in homogenates of anterior hypothalamus-preoptic area (AHPOA), remaining telencephalon (TEL), whole pituitary, ovary, and testis. Seasonal variations in aromatase were most dramatic in the AHPOA of female fish, exhibiting a peak in April and May that was 6-fold higher than the nadir in July. As judged by changes in the appearance and weight of the gonads, maximal aromatase coincided with the spawning season, whereas low enzyme levels corresponded to reproductive inactivity. Seasonal variations were similar but of a lesser magnitude in the TEL of females and in the AHPOA and TEL of males (2- to 3-fold, peak to nadir). Both ovarian and testicular aromatase showed cyclic changes; however, activity was much lower than that in brain at all times of the year (4.5, 1.2, and 47.0 pmol/mg protein, maximal values in ovaries, testes, and AHPOA, respectively). Pituitary aromatase varied from 5-22 pmol/mg protein, but was not consistently correlated with season. Cyclic changes in 5 alpha-reductase were distinctly different from those in aromatase, with maximal values in both brain and pituitary occurring when fish were reproductively inactive. In general, circulating sex steroids were high when aromatase was high and low when reductase was maximal; however, there was no apparent causal relationship suggested by temporal changes in a given steroid. Variations in testosterone metabolism, by regulating the quantity and quality of active hormone in close proximity to receptor sites, may be responsible for the changes in feedback sensitivity and behavioral responsiveness that are known to occur in seasonal breeders.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

A high abundance androgen receptor in goldfish brain: characteristics and seasonal changes.

Testosterone (T) exerts its actions in brain directly via androgen receptors or, after aromatization to estradiol, via estrogen receptors. Brain aromatase activity in teleost fish is 100-1000 times greater than in mammals and would be expected to significantly reduce the quantity of androgen available for receptor binding. Experiments were carried out on the goldfish Carassius auratus to determine if androgen receptors are present in teleost brain and whether their physicochemical properties reflect elevated aromatase. Cytosolic and nuclear extracts were assayed with the use of [3H]T and charcoal, Sephadex LH-20, or DNA-cellulose chromatography to separate bound and free steroids. Binding activity was saturable and had an equally high affinity for T and 5 alpha-dihydrotestosterone (Kd, approximately 2.4 X 10(-9) M). Although mibolerone was a relatively weak competitor, the putative teleost androgen 11-ketotestosterone, methyltrienolone (R1881), estradiol, progesterone, and cortisol were poor ligands. Characteristics that distinguish this receptor from a steroid-binding protein in goldfish serum are the presence of binding activity in both nuclear and cytosolic extracts, a low rate of ligand-receptor dissociation, electrophoretic mobility, sedimentation properties in low vs. high salt, and tissue distribution (forebrain greater than or equal to pituitary greater than mid-/hindbrain). DNA cellulose-adhering and nonadhering forms were detected, but these did not differ in other variables measured. Although goldfish androgen receptors resembled those of mammals in all important physicochemical characteristics, they were unusually abundant (5-68 pmol/g tissue) compared to levels in rat brain, but comparable to levels in prostate and other male sex hormone target organs. Moreover, there were seasonal variations in total receptors, with a peak at spawning (April) 4- to 5-fold higher than values in reproductively inactive fish (July/August). This temporal pattern and magnitude of change corresponded to previously reported changes in brain aromatase. Thus, both phylogenetic and physiological correlates point to a functional interdependence between androgen receptors and aromatase in the brain. These studies in goldfish indicate that brain androgen receptors have a long evolutionary history and have been highly conserved through the vertebrate series.

Animals↗

Characteristics of a testosterone-estradiol binding globulin (TEBG) in goldfish serum.

Neuroendocrine tissues in teleost fish aromatize androgen to estrogen at extraordinarily high rates. As part of a project in which we are studying the dynamics of sex steroid uptake, metabolism, and receptor binding in goldfish (Carassius auratus) brain and pituitary, we have identified and characterized a sex-steroid-binding component of serum. This protein has been designated a testosterone-estradiol-binding globulin (TEBG) since it bound testosterone (T) and estradiol-17 beta (E2) with high affinity (Kd = 1.9 and 2.1 nM, respectively) whereas other steroids were less effective ligands (5 alpha-dihydrotestosterone greater than progesterone = 11-ketotestosterone greater than estrone = estriol = diethylstilbestrol greater than cortisol). Scatchard analysis, disc gel electrophoresis and sucrose gradient centrifugation all indicate that T and E2 are bound by the same protein. The number of available serum binding sites (Bmax = 10(-7) M) greatly exceeded reported maximal levels of T and E2 in the same species and showed no obvious sex or seasonal differences. However, the steroid-TEBG interaction was unstable, exhibiting very short half-times of association (less than 15 min) and dissociation (less than 10 min). On the basis of comparison of the physicochemical characteristics of TEBG with other intracellular androgen-binding proteins in goldfish brain (androgen receptor, aromatase), we predict that the serum-binding protein would not limit but rather enhance exchange of T and E2 in central tissues.

Animals↗

Aromatase and 5 alpha-reductase in the teleost brain, spinal cord, and pituitary gland.

The distribution of aromatase and 5 alpha-reductase was investigated in the brain, spinal cord, and pituitary of adult goldfish (Carassius auratus) and toadfish (Opsanus tau) of both sexes. Tissue homogenates were incubated with [3H]androstenedione in the presence of an NADPH-generating system and, following validation of assay conditions, radiolabeled products (estradiol, estrone, 5 alpha-androstanedione), were measured as an index of enzyme activity. Neuroendocrine tissue of both species produced exceptionally large amounts of estrogen, thus confirming previously observed differences between teleosts and other vertebrates. By contrast, 5 alpha-reductase levels resembled the vertebrate norm. In general, aromatase was concentrated in the pituitary and various forebrain regions, especially the hypothalamus/preoptic area; however, estrogen yields from the medulla and anterior spinal cord of toadfish were high compared to adjacent midbrain, hindbrain, and cord regions. This same neural region in toadfish, but not in goldfish, is known to control a sex dimorphic behavior, the courtship boatwhistle. In contrast to aromatase, 5 alpha-reductase was more uniformly distributed throughout the brain, although somewhat higher activity was obtained in the pituitary. High levels of aromatase in the neuroendocrine tissues of teleosts recommend them as animal models for further studying the enzyme, its regulation, and its role in governing androgen-dependent responses in central targets.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

The role of estrogen as a parahormone in brain and pituitary.

Where aromatase and estrogen receptors are co-localized in brain and pituitary, estrogen functions as a parahormone, and estrogen levels which determine the occurrence or magnitude of a response are those in close proximity to targets. Teleost fish, a vertebrate group characterized by exceptionally high aromatase in neuroendocrine tissues, are technically advantageous animal models for studying the cellular location of aromatase, natural changes correlated with seasonal reproductive cycles, substrate-dependence of the reaction, steroid induction of enzyme activity, and possible non-genomic actions of estrogen on cultured neurons. In addition, characterization of steroid receptors reveals that the androgen receptor, like aromatase, is present in unusually high concentrations (10- to 100-fold higher than in mammalian brain). Since androgen receptors and aromatase both utilize testosterone as a ligand, their high abundance in teleost brain may be the consequence of a functional interdependence during evolution, although the primary causal factor is unknown. These studies illustrate the usefulness of unconventional species and a comparative approach for obtaining new insights into brain-steroid interactions.

Animals↗