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Biomedical subjects

H J Karavolas

Publications and source records attributed to H J Karavolas.

At least 37 records · Page 2Linked to original sources
PubMed1981

Ovarian regulation of hypothalamic and pituitary progestin-metabolizing enzyme activities.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities (NADH-linked) were measured in hypothalamic and pituitary tissues from rats during various stages of the estrous cycle and in ovariectomized rats treated with 17 beta-estradiol or vehicle. Enzyme assays were performed using 3H-labeled steroid substrates and reverse isotopic dilution analyses. Pituitary 5 alpha-reductase activity fluctuated over the estrous cycle, with peak levels observed on proestrus, estrus, and metestrus and lowest levels on diestrous 1 and 2 of 5-day cycling rats. Pituitary NADH-linked 3 alpha-HSOR activity also fluctuated over the estrous cycle, paralleling 5 alpha-reductase activity. The pituitary NADPH-linked 3 alpha-HSOR activity and three hypothalamic enzyme activities, the 5 alpha-reductase, NADH-linked, and NADPH-linked 3 alpha-HSOR activities, did not change over the estrous cycle. After ovariectomy (10 days), pituitary 5 alpha-reductase activity was elevated 10- to 12-fold relative to mean levels in intact rats, while the pituitary NADH-linked 3 alpha-HSOR activity was elevated 4- to 5-fold. Treatment for 3 days with estradiol benzoate (10 micrograms/day) significantly reduced both pituitary 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities to about half the levels observed in ovariectomized rats. The pituitary NADPH-linked 3 alpha-HSOR activity was unaffected by ovariectomy. After ovariectomy, a small decrease was observed in the hypothalamic 5 alpha-reductase; no differences were observed in the NADH-linked or NADPH-linked 3 alpha-HSOR activities of the hypothalamus compared to mean levels in cyclic rats. The results suggest a role of the ovary in the regulation of the pituitary and hypothalamic progesterone 5 alpha-reductase and pituitary NADH-linked 5 alpha-dihydroprogesterone 3 alpha-HSOR activities.

Animals↗
PubMed1981

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the rat during pregnancy.

Metabolites of [3H]progesterone were quantitated from incubations of hypothalamus, pituitary, and uterus of rats during different stages of pregnancy. The hypothalamus, anterior pituitary, and a section of uterus from five rats on Days 1, 8, 15, and 21 of pregnancy were incubated individually with [3H]progesterone and analyzed for metabolite formation by reverse isotopic dilution analysis. The radioactive metabolites present were 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one, 20 alpha-hydroxy-4-pregnen-3-one, 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol. The major metabolite formed by the hypothalamus and pituitary was 5 alpha-DHP. In the pituitary samples, formation of 5 alpha-DHP was decreased on Days 15 and 21 of pregnancy compared to Day 1, and formation of 20 alpha-hydroxy-5 alpha-pregnan-3-one was decreased on Day 21 compared to Day 1. In the uterine samples, 3 alpha-hydroxy-5 alpha-pregnan-20-one was the major metabolite formed at all stages of pregnancy. The formation of all metabolic products of progesterone by the uterus was increased on Day 21 compared to Days 1, 8, and 15 of pregnancy. No changes in the formation of progesterone metabolites were observed in the hypothalamic samples during pregnancy. It is concluded that there are different profiles in the in vitro metabolism of [3H]progesterone by the hypothalamus, pituitary, and uterus of the rat during the course of pregnancy.

Animals↗
PubMed1981

Effect on ovulation of 20 alpha-hydroxy-4-pregnen-3-one and its 5 alpha-reduced metabolites in immature rats treated with pregnant mare serum gonadotrophin.

The ability of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-dihydroprogesterone, 20 alpha-DHP) and its 5 alpha-reduced metabolites, 20 alpha-hydroxy-5 alpha-pregnan-3-one and 5 alpha-pregnane-3 alpha,20 alpha-diol, to facilitate ovulation was examined in immature female rats which had been treated on day 22 of age with a non-ovulatory dose (12 i.u.) of pregnant mare serum gonadotrophin. An increased incidence of ovulation occurred in rats treated on the morning of day 24 with 20 alpha-DHP. However, a dose of 20 alpha-DHP three to four times that of progesterone was required to induce ovulation in all animals. In contrast, neither 20 alpha-hydroxy-5 alpha-pregnan-3-one (at doses ranging from 0.5 to 5.0 mg) nor 5 alpha-pregnane-3 alpha,20 alpha-diol (tested at 3.0 or 4.0 mg) facilitated ovulation. Therefore both 20 alpha-DHP and progesterone, the two major progestins of the rat oestrous cycle, have the ability to facilitate ovulation. It is concluded that the ability of 20 alpha-DHP to facilitate ovulation does not appear to be by way of conversion to its 5 alpha-reduced metabolites.

20-alpha-Dihydroprogesterone↗
PubMed1980

The effects of neurotransmitters and other cellular modulators and factors on hypothalamic and anterior pituitary delta 4-steroid (progesterone) 5 alpha-reductase activity.

A number of diverse biological compounds involved in the regulation of the hypothalamo-hypophyseal-ovarian axis have been examined for effects on the conversion of 3H-progesterone to 3H-5 alpha-dihydro-progesterone and 3H-3 alpha-hydroxy-5 alpha-pregnan-20-one by female rat hypothalamus and/or anterior pituitary. Broken cell preparations were incubated with 3H-progesterone and NADPH, and product 5 alpha-reduced progestins were quantitated by reverse isotopic dilution analysis. Progesterone 5 alpha-reductase activity was reduced up to 50% in the presence of 10(-2) to 10(-3) M serotonin in both preparations. At 10(-3) M, various indoles including n-acetylserotonin, melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-hydroxyindole acetic acid decreased by 10 to 30% 5 alpha-reduced product formation. At 10(-2) M, carbamylcholine and norepinephrine were without effect, while 10(-2) M dopamine reduced by 20% the 5 alpha-reduction of progesterone only in pituitary homogenates. The LHRH protease inhibitor bacitracin (2 X 10(-3) M) decreased by 10 to 40% progesterone 5 alpha-reductase activity in both tissues. By itself, LHRH did not affect the 5 alpha-reduction of progesterone nor did it potentiate the bacitracin effect. In the presence of 1 mM ATP, 100 micronM cAMP and 100 micronM cGMP increased 5 alpha-reduced product formation in the hypothalamus by 19 and 14%. The gonadotropins LH and FSH and the prostaglandins E1, E2, F1 alpha, and F2 alpha were without effect. Thus, these results and others indicate that a number of cellular components and other factors can affect the in vitro 5 alpha-reduction of progesterone in broken cell preparations.

20-alpha-Dihydroprogesterone↗
PubMed1979

Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus.

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [3H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [3H]-progesterone, [3H]-5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone) and [3H]-17 beta-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol. These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol.

20-alpha-Dihydroprogesterone↗
PubMed1978

The effect of progesterone analogues, naturally occurring steroids, and contraceptive progestins on hypothalamic and anterior pituitary delta4-steroid (progesterone) 5alpha-reductase.

The effects of a number of steroids on the conversion of progesterone to 5alpha-dihydroprogesterone by hypothalamic and pituitary progesterone 5alpha-reductase have been investigated. Using enzyme preparations from female rats and 3H-progesterone as substrate, 5alpha-reduced products (5alpha-dihydroprogesterone and 3alpha-hydroxy-5alpha-pregnan-20-one) were analyzed by reverse isotopic dilution analysis. The amount of total 5alpha-reduced products formed was compared in the presence and absence of the test steroid. Derivatives lacking the delta4 and/or the 3-keto moiety were without effect. Corticosterone had no effect. 16beta-Methylprogesterone inhibited progesterone 5alpha-reduction in both tissues by at least 65%, while the 2alpha-, 6alpha-, and 7alpha-methylated derivatives had lesser effects. 3-Oxo-4-pregnene-20beta-carboxaldehyde and 21-fluoroprogesterone were potent inhibitors. 17-Hydroxyprogesterone was a competitive inhibitor (substrate) with Ki's of 0.27 micrometer (pituitary) and 0.29 micrometer (hypothalamus). Medroxyprogesterone exerted little inhibitory effect. Of the 19-nor-steroids examined, only norethindrone appreciably inhibited the 5alpha-reduction. These results suggest that some natural delta4-3-ketosteroids can modify enzymatic activity. Also, inhibitory analogues may be useful for studies on the role of this 5alpha-reduction of progesterone.

3-Hydroxysteroid Dehydrogenases↗
PubMed1977

Progesterone metabolism in the pineal, brain stem, thalamus and corpus callosum of the female rat.

Specific brain regions, namely, thalamus, tectum, tegmentum, cerebellum, medulla and pineal, from five proestrous rats were incubated for 30 min with [3H]progesterone. After reverse isotopic dilution analysis, the following metabolites were identified in all incubations by purification to constant specific activity, derivative formation and/or gas liquid chromatography trapping: [3H]5alpha-pregnane-3, 20-dione (10-20% of the starting substrate except pineal -- 0.7%), [3H]3alpha-hydroxy-5alpha-pregnan-20-one (1.6-3.8% except for pineal -- 0.5%) and [3H]20alpha-hydroxy-4-pregnen-3-one (0.05-0.11%). Preliminary results from the corpus collosum incubation indicated the presence of the same metabolites. Although some apparent constant specific activities were obtained for 20alpha-hydroxy-5alpha-pregnan-3-one and 5beta-pregnane-3, 20-dione, the low levels of 3H associated with these steroids did not permit a definitive identification. The results indicate the presence of at least delta1-steroid 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities with progesterone as substrate in the brain regions examined.

Animals↗
PubMed1977

Effect of progesterone and its 5 alpha-reduced metabolites on gonadotropin levels in estrogen-primed ovariectomized rats.

In an effort to determine whether the metabolic conversion of progestrone may be important in the feedback effects of this steroid, serum LH and FSH levels were measured after administration of progesterone, 5 alpha-dihydroprogesterone or 3 alpha-hydroxy-5 alpha-pregnan-20-one to estrogen-primed ovariectomized rats. A single injection of 2 or 4 mg progesterone, 4 mg 5 alpha-dihydroprogesterone, or 4 mg 3 alpha-hydroxy-5 alpha-pregnan-20-one 72 h (Day 3) after estrogen pretreatment induced a highly significant increase in serum LH and FSH 6 h later (1800 h). Although serum gonadotropin levels had begun to decrease 12 h after administration of the progestins, they were still significantly higher than control values and did not return to baseline levels until noon on Day 4. When either progesterone or 3 alpha-hydroxy-5 alpha-pregnan-20-one was administered at noon on Days 3 and 4, there was a significant reduction in LH levels 6 h after the second injection. In contrast, serum LH levels were slightly elevated 3 to 6 h (1500 to 1800 h) after the second injection of 5 alpha-dihydroprogesterone and did not decrease until 2100 h. There was no effect on FSH concentrations after a second injection of any of the progestins. Loss of uterine luminal fluid was observed within 24 h after a single injection of progesterone. Neither of the 5 alpha-reduced metabolites had an effect on uterine ballooning until after the second injection, and, even then, nonfluid-filled uteri were observed in only 20 to 30% of the animals. The results suggest that the conversion of progesterone to 5 alpha-dihydroprogesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one by neuroendocrine tissues may be necessary for the positive and negative feedback effects of progesterone on gonadotropin secretion. Thus, the diverse effects of progesterone may be due to progesterone per se (e.g., in the uterus) and/or its metabolites (e.g., in the hypothalamus and pituitary).

5-alpha-Dihydroprogesterone↗
PubMed1976

Quantitative changes in the metabolism of 20alpha-hydroxy-4-pregnen-3-one by rat hypothalamus and pituitary during proestrus.

The in vitro conversion of 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP) by medial basal hypothalamus and anterior pituitary was investigated throughout the day of proestrus in the 4-day cyclic rat. Reverse isotopic dilution analysis was utilized to quantitate the substrate remaining and three metabolic products: 20alpha-hydroxy-5alpha-pregnan-3-one, 5alpha-pregnane-3alpha,20alpha-diol and progesterone. Serum levels of 20alpha-DHP, progesterone, LH and FSH were measured by radioimmunoassay. Conversion of 20alpha-DHP to its 5alpha-reduced metabolites (20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha,20alpha-diol) by the pituitary was constant throughout proestrus except for a significant decrease at 1600 h, near the end of the critical period. Although 5alpha-reduction of 20alpha-DHP by the hypothalamus fluctuated, it was relatively high at 1600 h and was lowest at 1400 h. Small amounts of progesterone (less than2%) were formed but there was not variation with time. The decrease in pituitary enzymic activity coincided with the time when serum levels of LH, FSH and progesterone were increasing but not with later times when the elevated serum levels were maintained. Thus, there may be endogenous regulation of 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase activity in rat pituitary and perhaps hypothalamus on the afternoon of proestrus. The regulation and subsequent effects of quantitative changes in 5alpha-reduction of 20alpha-DHP by pituitary and hypothalamus remain to be elucidated.

20-alpha-Dihydroprogesterone↗
PubMed1976

Uptake of (3H)progesterone and (3H)5alpha-dihydroprogesterone by rat tissues in vivo and analysis of accumulated radioactivity: accumulation of 5alpha-dihydroprogesterone by pituitary and hypothalamic tissues.

These in vivo studies explored the possibility that the metabolism of progesterone to 5alpha-dihydroprogesterone (5alpha-DHP) and 3alpha-hydroxy-5alpha-pregnan-20-one in hypothalamus and pituitary may influence gonadotropin release. [3H]progesterone or [3H]5alpha-DHP was injected iv into ovariectomized or ovariectomized-adrenalectomized rats for 10 0r 30 min. 3H content was determined for plasma, anterior pituitary, medial basal hypothalamus (MBH), cerebral cortex, muscle and uterus. Isotopic dilution analyses of the accumulated 3H were made for progesterone, 5alpha-DHP, 3alpha-hydroxy-5alpha-pregnan-20-one, 20alpha-dihydroprogesterone, 20alpha-hydroxy-5alpha-pregnan-3-one, and 5alpha-pregnane-3alpha, 20alpha-diol on samples from the 10 min groups. With progesterone injections, most of the tissue 3H was distributed among progesterone, 5alpha-DHP, and 3alpha-hydroxy-5alpha-pregnan-20-one. Progesterone was the predominant 3H-steroid in uterus, MBH, cerebral cortex and muscle. [3H]5alpha-DHP was the other major 3H-steroid in MBH and the predominant one in pituitary. In terms of tissue/plasma concentration comparisons, no tissue concentration of [3H]progesterone was greater than that in plasma except for MBH in the ovariectomized-adrenalectomized group. In contrast, [3H]5alpha-DHP levels in pituitary, MBH and cerebral cortex were many fold greater than those in plasma and muscle. MBH and pituitary levels were significantly greater than that in cerebral cortex. With 5alpha-DHP injections, most tissue 3H was associated with 5alpha-DHP and/or 3alpha-hydroxy-5alpha-pregnan-20-one. No [3H]progesterone was detected. [3H]5alpha-DHP predominated in pituitary and MBH, while 3alpha-hydroxy-5alpha-pregnan-20-one predominated in the others. In terms of tissue and plasma concentration comparisons, MBH, pituitary and cerebral cortical concentrations of [3H]5alpha-DHP were markedly higher than plasma, muscle, and uterine levels. Pituitary and MBH concentrations were also greater than that in cerebral cortex. Tissue levels of [3H]3alpha-hydroxy-5alpha-pregnan-20-one were not significantly greater than that in plasma. Thus, 10 min after injection of either [3H]progesterone or [3H]5alpha-DHP, high and significant amounts of 5alpha-DHP are accumulated in pituitary and hypothalamus, but not in uterus, which suggests that its presence may be functionally important in governing progesterone-sensitive processes in these feedback tissues.

20-alpha-Dihydroprogesterone↗
PubMed1975

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.

20-alpha-Dihydroprogesterone↗
PubMed1975

Properties and subcellular distribution of delta4-steroid (progesterone) 5alpha-reductase in rat anterior pituitary.

The properties and subcellular distribution of anterior pituitary delta4-steroid (progesterone) 5alpha-reductase, which stimulates the conversion of progesterone to 5alpha-pregnane-3,20-dione, have been investigated utilizing 3H-substrate and a reverse isotopic dilution assay system. The enzymic activity was stimulated by NADPH but not NADH and exhibited a Km of 2.7+/-0.9 times 10(-7) M for progesterone. The substrate specificity of the enzyme for other delta4-3-ketosteroids and the effect of estradiol-17beta were also studied. 20alpha-hydroxy-4-pregnen-3-one was more reactive than progesterone, while testosterone was less reactive. Estradiol-17beta in vitro had an inhibitory effect on the 5alpha-reduction of progesterone. Studies on the subcellular distribution of the 5alpha-reductase activity indicate that the bulk of the activity was widely distributed amongst particulates sedimenting at 1,000, 15,000 and 100,000xg; with the 15,000xg pellet containing the most enzymic activity. The 100,000xg supernatant possessed only a small fraction of the total activity. After further fractionation of the 1,000xg pellet, the activity was distributed equally between the purified nuclear and cell debris-membranes fractions.

Animals↗