Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties.     

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.     

Luteal phospholipase A2 activity increases during functional and structural luteolysis in pregnant rats.

We determined cytosolic phospholipase A2 activity of the corpus luteum during luteolysis in pregnant and post-partum rats. Phospholipase A2 activity and its metabolite prostaglandin F2alpha in the corpus luteum remarkably increased just before parturition and further rose transiently during post-partum structural luteolysis. The absence of a pups' suckling stimulus delayed corpus luteum involution, being associated with an altered fluctuation in phospholipase A2 activity and depressed prostaglandin F2alpha levels. Exogenous prolactin had a reversal effect. Pharmacological and immunochemical characterization suggests multiple isoforms of phospholipase A2 in a pregnant corpus luteum. These results show the increased phospholipase A2 activity and its possible implication in luteolysis in pregnant rats.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

Hormonal control of pyruvate dehydrogenase activity in rat liver.

A detailed study of the control of liver pyruvate dehydrogenase activity by various hormones was carried out with perfused liver and isolated hepatocytes. Vasopressin produced a significant increase in the enzyme activity in fed rats, and the time course and sensitivity of the response was similar to that of glycogen phosphorylase a. The enzyme from starved animals was resistant to hormonal activation. The possible factors involved in the above effects are discussed. Angiotensin and phenylephrine also increased pyruvate dehydrogenase activity, and the magnitude of the response was of the same order as that to vasopressin by the liver enzyme. The effects of these hormones on pyruvate dehydrogenase activity were critically dependent on extracellular Ca2+, thus suggesting a role for this ion in the mechanism of action of the hormones. Insulin did not appear to have a role in the control of the enzyme activity, as shown by its lack of effect on the enzyme. Glucagon, in contrast with previous reports, produced a rapid, transient and significant increase in pyruvate dehydrogenase activity. The physiological importance of the above effects is discussed.     

Hormonal control of glycerolphosphate dehydrogenase in the rat brain

—Following hypophysectomy or adrenalectomy, glycerolphosphate dehydrogenase (GPDH) (EC 1.1.1.8) activity decreased exponentially in the cerebral hemispheres and brain stem of adult male rats. The latter region was more affected than the former. Malate dehydrogenase (EC 1.1.1.40), isocitrate dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27) and mitochondrial glycerolphosphate dehydrogenase (EC 1.1.95.5) activities remained unchanged. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats or cortisol in adrenalectomized rats restored GPDH activity. Thyroidectomy and gonadectomy had no effect on GPDH activity. Liver GPDH was not decreased by hypophysectomy or adrenalectomy. Muscle GPDH was diminished slightly by adrenalectomy and as much as brain GPDH by hypophysectomy. In young rats GPDH developmental increase in activity was inhibited by hypophysectomy. These results clearly show that brain GPDH activity is specifically regulated by cortisol (and probably closely related corticosteroids).     

Luteal 3beta-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities in the rat corpus luteum of pseudopregnancy: Effect of the deciduoma reaction

Background  

In the rat, the maintenance of gestation is dependent on progesterone production from the corpora lutea (CL), which are under the control of pituitary, decidual and placental hormones. The luteal metabolism of progesterone during gestation has been amply studied. However, the regulation of progesterone synthesis and degradation during pseudopregnancy (PSP), in which the CL are mainly under the control of pituitary prolactin (PRL), is not well known. The objectives of this investigation were: i) to study the luteal metabolism of progesterone during PSP by measuring the activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3betaHSD), involved in progesterone biosynthesis, and that of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), involved in progesterone catabolism; and ii) to determine the role of decidualization on progesterone metabolism in PSP.     

Formation and isolation of delta 5-3-ketosteroids using a purified rat liver alcohol dehydrogenase

3 beta-Hydroxy-5-androsten-17-one is converted to 5-androstene-3, 17-dione by rat liver alcohol dehydrogenase (ADH). We have reported on the purity of the enzyme which is eluted with pyrazole as a single homogeneous protein using an AMP-agarose affinity column. Rat liver ADH can oxidize hydroxyl groups not only at 3 beta-, but also at 3 alpha-, and 17 beta-positions to a lesser extent; thus it is a pure mammalian enzyme with multifunctional activity for steroids. Since it does not contain delta 5-isomerase activity, the reaction of the dehydrogenase to form the delta 5-ketosteroid intermediate can be observed at pH 7.0, 25 degrees C. Similarly, intermediary product, 5-pregnene-3,20-dione, can be isolated in the conversion of pregnenolone by ADH to progesterone. With buffer alone in a cuvette, a non-enzymatic isomerization of the delta 5-3-ketone occurs at a slow rate (t 1/2 = 6 hrs) but occurs rapidly during isolation procedures. The delta 5-3-ketosteroid intermediates were identified by their behavior on TLC plates with UV light and by their characteristic spectra in the NMR.     

Effects of unilateral orchidectomy on rat epididymal delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase

The effects of unilateral orchidectomy on the adult rat epidiymal testosterone metabolizing enzymes, delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, are investigated. Five weeks following unilateral orchidectomy, it is found that the activity of 3 alpha-hydroxysteroid dehydrogenase per organ is not altered, whereas delta 4-5 alpha-reductase activity decreased by more than 80% on the side of the orchidectomy. Neither accessory sex tissue weights, ventral prostate and seminal vesicles, nor the concentration of circulating testosterone, luteinizing hormone, follicle-stimulating hormone, or prolactin is altered by unilateral orchidectomy. These data indicate that (1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity can be maintained by circulating androgens and that (2) the major factor regulating delta 4-5 alpha-reductase activity is not a substance secreted by the testes into the peripheral circulation. It is suggested that a substance directly secreted into the epididymis by the testis regulates epididymal delta 4-5 alpha-reductase activity.     

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.     

Influence of experimental cryptorchidism on cholesterol side-chain cleavage enzyme and delta5-3beta-hydroxysteroid dehydrogenase activities in rat testes.

Testicular cholesterol side-chain cleavage enzyme (CSCCE) and delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) activities were assessed 12 hours and 2, 4, 8, 16, and 32 days after surgical induction of bilateral cryptorchidism in adult rats. Within 12 hours after surgery CSCCE activity (expressed as dpm of isocaproic acid-14C formed from cholesterol-26-14C/3 hours/testis) was significantly reduced (P less than 0.01) in cryptorchid testes to approximately 55% of sham-operated control values and remained depressed at less than 50% of control activities 2, 4, 16, and 32 days after surgery. Cryptorchid testis delta5-3beta-HSD activity (measured by a pregnenolone substrate-depletion assay and expressed as mumoles of products/30 minutes/testis) did not differ from controls (P greater than 0.05) 1/2, 2, or 4 days after translocation of testes to the abdominal cavity. By day 8 of cryptorchidism, however, delta5-3beta-HSD activity was reduced to 60% of control values (P less than 0.05) and continued to decline to approximately 30% of controls during the remainder of the experimental period. These observed alterations in enzyme activities suggest an impairment in the ability of cryptorchid rat testes to synthesize androgens and further indicate that testicular CSCCE is more acutely sensitive to the cryptorchid milieu than delta5-3beta-HSD.     

Activity of delta(5)3beta-hydroxysteroid dehydrogenase and steroid hormones content in early preimplantation horse embryos

The activity of delta (5)3 beta-hydroxysteroid dehydrogenase was examined histochemically in 6 to 10 days aged horse blastocysts. A positive reaction was noted in the blastomeres of all embryos incubated in medium with substrate. Measurable amounts of progesterone, androgens and estrogens were found in blastocysts on day 8th. The presence of enzyme and hormones suggests that steroid hormone production takes place in very early preimplantation horse embryos.     

Activity of peroxidase and delta 5-3 beta-hydroxysteroid dehydrogenase in the ovary of LH-treated cyclic female rats

Rats were treated with LH at 08:00 h on the first day of dioestrus or on Days 1 and 2 of dioestrus. Peroxidase activity increased within 3 h in females injected with LH on Day 1 and was associated with a depletion of ascorbate that lasted until the afternoon of Day 1. Values of both substances then returned to basal values by the morning of Day 2. A second LH injection on Day 2 produced effects similar to those seen after LH on Day 1. Females treated with LH did not display greater delta 5-3 beta-HSD activity than did controls on Day 1 after one LH injection, but did on Day 2 after two LH injections. Nonetheless, the changes were only modest by comparison with those of peroxidase. The peroxidase-ascorbate system appears to be involved in the mechanism responsible for the increased secretion of ovarian progesterone resulting from LH stimulation.