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The distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the subcellular fractions of rat adrenal gland has been determined. Based on the total activity found, 55% was in the microsomal fraction, 10% in the heavy-mitochondrial fraction, 3% in the light-mitochondrial fraction and 26% in the fraction consisting of cell-debris plus nuclei. Ninety-five percent of the total activity was recovered in the fractions. Approximately half the activity in the heavy-mitochondrial fraction could be accounted for by microsomal contamination.

The effect of follicle-stimulating hormone (FSH) and diethylstilbestrol (DES) on ovarian follicle growth was studied in hypophysectomized rats, using histologic, autoradiographic and histochemical techniques. The administration of FSH to immature hypophysectomized rats stimulated the follicle growth with thickening of the theca layer and repair of "deficiency cells". Although DES given to hypophysectomized rats also stimulated the follicle growth, the theca layer was relatively thin compared with that in FSH-treated rats. In order to detect cell division in a growing follicle, the number of labelled granulosa cells with tritiated thymidine as a proportion of the granulosa cells in a growing follicle were counted and the labelling indices of the granulosa cells were calculated. The labelling indices increased by the administration of FSH (p less than 0.05) or DES (p less than 0.05). The uptake of tritiated thymidine and leucine by the theca cells was enhanced only by FSH and was not stimulated by DES. The administration of FSH resulted in an increase of the histochemically demonstrable enzyme activity such as delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) or alkaline phosphatase in the theca cells. In contrast, the administration of DES did not stimulate the enzyme activity of the theca cells. Furthermore, FSH stimulated a release of estradiol and estriol from the ovary of hypophysectomized rat, whereas DES did not. Although ovarian follicle growth was stimulated by both FSH and DES, the effect of the two hormones on the theca cells was quite different and the secretion of estrogen was stimulated by FSH. The results suggest that FSH induced follicle growth of the ovary might be mediated by estrogen produced by the theca cells.

Graafian follicles of various sizes obtained from mares at different stages of the oestrous cycle were examined histologically and histochemically for delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and related enzymes. The 3beta-HSD activity was not found in the theca interna of any follicles but was present in the membrana granulosa of well-vascularized large follicles in the late luteal phase of the cycle and at oestrus. These findings indicate that pregnenolone cannot be converted into progesterone in the theca interna. It is suggested that this conversion occurs in the membrana granulosa and that progesterone then passes into the theca interna where it is converted into oestrogen. Participation of both granulosa and thecal cells in oestrogen production is also discussed from a morphological view-point.

Adrenal fibroblasts from adult rats acquire some adrenocortical parenchymal characteristics as a consequence of transformation in early passage with Kirsten murine sarcoma virus. To further define the effects of Kirsten murine sarcoma virus-induced transformation on the steroid enzymes of these cells, we investigated the capacity of Kirsten murine sarcoma virus-transformed and untransformed adrenocortical fibroblasts to convert progesterone to C19 and C21 steroid metabolites. Over 95% of metabolites produced were identified and quantitated, and rates of enzyme activities over 24 h were calculated. The transformed and untransformed cells exhibited 5 alpha- and 5 beta-reductase, 3 alpha-, 3 beta-, and 20 alpha-hydroxysteroid dehydrogenase; 11 beta-, 17-, and 21-hydroxylase (HY); and C17-20-lyase activities. Viral transformation resulted in several metabolites not found in untransformed cells, significantly increased 5 beta-reductase, 3 beta-hydroxysteroid dehydrogenase, and C17-20-lyase activities, and significantly decreased 5 alpha-reductase, 3 alpha- and 20 alpha-hydroxysteroid dehydrogenase, and 21-HY activities. The 11 beta-HY and 17-HY activities remained unchanged. The results support previous data suggesting that adrenocortical fibroblasts express some characteristics of adrenocortical parenchymal stem cells. In contrast to other experimental systems, viral transformation of adrenocortical fibroblasts did not cause a generalized reduction of differentiated functions. Instead, specific increases and decreases in individual enzyme activities, with persisting synthesis of fetal and adult adrenocortico-specific steroids, resulted in an altered steroid profile that may have unique effects on the biology of the malignant cells.

The hormone levels in the anterior pituitary gland and serum in Nagase analbuminemia rats (NAR), a mutant strain established from Sprague-Dawley rats with hyperlipidemia, were examined. For the anterior pituitary gland, the prolactin, TSH, GH, LH and FSH contents in male NAR were significantly lower than those of normal rats. In female NAR, prolactin, TSH and LH levels were also lower than those in normal rats, whereas FSH and GH were normal. For the serum, the concentrations of TSH, total T3, total and free T4, estradiol-17 beta and testosterone were examined. The serum testosterone concentration in NAR was lower than that of normal rats. Histochemical examination of the hydroxysteroid dehydrogenase (HSD) activity of testes was made in relation to the serum testosterone level. NAR testes, which are rather small compared with those of normal rats, have lower HSD activity. A higher level of serum TSH was seen in NAR. Total and free T4 concentrations were low in the male NAR only. Estradiol-17 beta and T3 concentrations in NAR were unchanged. Changes in serum LH and FSH levels during the estrous cycle in NAR were also studied. Their patterns of change are normal.

With a radial diffusion assay we measured the activity of six NAD- and NADP-dependent oxidoreductases: alcohol, glucose-6-phosphate, hydroxysteroid, lactate and malate dehydrogenases, and glutathione reductase. The enzyme was allowed to diffuse for 24h in an agarose gel in which the substrate was incorporated, then reacted the pyridine nucleotide coenzyme. The size of an enzyme diffusion zone could be made visible by the change of the fluorescence of the coenzyme against the background when the coenzyme was either oxidized or reduced. The procedure for each enzyme is reported. The results indicate that this new technique may be applicable to all NAD- and NADP-dependent enzymes. Because of its simplicity and potentiality for screening many samples, we think this method has applications in the clinical laboratory and in nutrition studies.

Histochemical localization of delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), 11beta-hydroxysteroid dehydrogenase (11beta-HSDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) have been studied in the kidney of white-breasted water hen, Amaurornis phoenicurus chinensis. All these enzyme activities occurred in the proximal and distal convoluted and collecting tubules, however, the intensity of these enzyme activities was more in the proximal convoluted tubules. It is suggested that these enzymes might have a role in converting certain hydroxysteroids to ketosteroids during steroid excretion.

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.

Monoclonal antibody (MAb) against 11-hydroxysteroid dehydrogenase (11-HSD) has been raised by immunization of female balb/c mice. 11-HSD from solubilized rat renal microsomal protein could be bound in a modified ELISA using antimouse IgG and MAb against 11-HSD. On Western blots of solubilized rat renal microsomes the MAb recognized a single protein band of an approximate molecular weight of 35 kD. Immunohistochemical staining of rat renal tissue with the above MAb and the APAAP staining technique displayed a heterogenous reginal and subcellular distribution: glomeruli and arterioles were practically devoid of specific staining, as were epithelial cells in inner and outer medulla. Intense immunostaining was observed in PCT and particularly in PST, appearing granular with highest density around the nuclei. Here the enzyme bound to intracellular membranes may exert an autocrine function such as signal inactivation. In contrast to cortex, staining of interstitial cells was observed in renal medulla. The latter localization is compatible with the concept of a paracrine function of 11-HSD which might prevent corticosterone from gaining access to collecting duct cells.

Corticosteroid 11 beta-dehydrogenase, the enzyme that catalyzes the oxidation of the biologically active steroid cortisol to its inactive metabolite cortisone, is present in testis. Since excess cortisol in men and other mammals and excess corticosterone in rodents cause physiological abnormalities including abnormal testicular function, it was pertinent to study the cellular distribution of 11 beta-dehydrogenase in the testis. Purified antiserum directed against homogeneous rat 11 beta-dehydrogenase was used to localize the enzyme in the developing rat testis. With immunofluorescence, the enzyme was not detectable in fetal testis or in the testis of young male rats until the 26th day of development. A few interstitial cells were stained in the testis of 26-day-old animals. In the testis of 31-day-old rats many cells in the interstitium were positive. In adult animals the entire interstitial region displayed bright fluorescence. Depleting animals of germ cells did not abolish the fluorescence. The appearance of this enzyme correlates temporally with the postnatal increase in Leydig cell number and the developmental rise in serum testosterone. We suggest that 11 beta-dehydrogenase of Leydig cells protects the testis from the deleterious effects of cortisol.

The substrate specificity of cholesterol oxidase has been further examined with respect to the size and shape of the steroid 17beta-side-chain and oxygenation of the nucleus. The relatively unreactive 17-keto-delta5-3beta-hydroxysteroids were converted to satisfactory substrates by formation of the isopentyl- or benzyloximes. The results obtained have been used to illustrate further uses of the enzyme in the gas-phase analytical characterisation and selective oxidation steroids. Oxidations were studied by kinetic experiments, gas-liquid chromatography-mass spectrometry.

The ultrastructural localization of 3 beta hydroxysteroid ferricyanide reductase, glucose-6-phosphate ferricyanide reductase and nicotinamide adenine dinucleotide and reduced form-ferricyanide reductase was investigated in some human steroidogenic tissues (corpus luteum of pregnancy, fetal adrenal gland and testis, adult testis and placenta) using ferricyanide as an electron acceptor. Copper ferrocyanide deposits were readily observed in the mitochondria, in the smooth endoplasmic reticulum profiles and in the cytoplasm. The sites of the various dehydrogenase activities could be visualized by using appropriate incubating media. The precise localization of various reactions in different electron transfer chains was determined by using different ferricyanide concentrations and intermediate electron-carriers such as menadione or exogenous nicotinamide adenine dinucleotide and reduced form-diaphorase. The use of respiratory chain inhibitors such as rotenone or antimycine A confirmed these data.

In order to localize 3beta-hydroxysteriod dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3beta-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium. A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. the addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.

Antiserum against crystallized 20beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was used for various immunodiffusion and immunoprecipitation tests to show an increase of the de novo synthesis of 20beta-hydroxysteroid dehydrogenase by Streptomyces hydrogenans after cultivation of the cells in the presence of 11beta,21-dihydroxy-4,17(20)-pregnadien-3-one. Half lives of mRNA for 20beta-hydroxysteroid dehydrogenase in induced cells and of total mRNA in non-induced cells were calculated to be 126 s and 66 s, respectively. In vivo, 20beta-hydroxysteroid dehydrogenase appears to consist of four equal subunits. The monomers, molecular weight of 27 300, show a high tendency to form dimers and tetramers in the absence of dissociating agents. The aggregation is completely reversible in the presence of increasing concentrations of sodium dodecylsulfate.

Gonadal tissue obtained at about 50-day intervals from Days 60 to 300 of gestation was examined histologically, histochemically and in the electron microscope. The marked enlargement of the gonads (ovaries or testes) reached a peak around Day 250 and was caused by hypertrophy and hypoplasia of interstitial cells. These cells had all the ultrastructural characteristics of a steroid-secreting cell but delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) was not detected at any stage of their development. There was no significant change in the ultrastructure of the interstitial cells between Days 100 and 250 except for a progressive increase in the number of dense bodies. By Day 300 the fetal gonads had begun to regress and degenerative changes were apparent in the interstitial cells. The possible mechanisms controlling the growth of the fetal gonads and their function in steroidogenesis is discussed.

Eubacterium lentum is a gram-positive, nonsporeforming, nonmotile, asaccharolytic anaerobe. In the present investigations, 3 E. lentum strains (group E) isolated from rat feces were compared with 30 E. lentum strains (groups A, B, C, and D) previously studied by Macdonald et al. (I. A. Macdonald, J. F. Jellet, D. E. Mahony, and L. V. Holdeman, Appl. Environ. Microbiol. 37:992-1000, 1979). All strains alkalized (pH 8 to 8.5) arginine-containing (2 to 15 mg/ml) culture media, and growth of the majority of the strains was stimulated by arginine. All strains converted linoleic acid into transvaccenic acid by shifting the 12,13-cis double bond of linoleic acid into an 11,12-trans(?) double bond followed by biohydrogenation of the 9,10-cis double bond. Hence, biohydrogenation of linoleic acid is a new general characteristic of E. lentum. The 33 strains were also studied for bile acid deconjugase and hydroxysteroid dehydrogenase (HSDH) activities. The 6 strains in group D were steroid inactive; the 27 strains in groups A, B, C, and E were steroid active. The steroid-active group contained bile acid deconjugase-producing strains (groups C and E, plus strain 116 in group A) and nondeconjugating strains. All nondeconjugating strains of groups A and B developed 7 alpha- and 12 alpha-HSDH activities and contained 3 alpha-HSDH-positive strains and 3 alpha-HSDH-negative strains. Deconjugating strains varied in HSDH activities.

We have proposed that 11 beta-hydroxysteroid dehydrogenase is composed of structurally independent units with 11 beta-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11 beta-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11-reductase accompanied the purification. Homogeneity of 11 beta-dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 11 beta-dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 +/- 0.06 microM for corticosterone and 17.3 +/- 2.24 microM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.

By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.

The histoenzymologic study of the adrenals in Glis glis was realized for 4 enzymatic activities (acid phosphatase, glucose 1 phosphate dehydrogenase, delta5 3beta-hydroxysteroid dehydrogenase and cholinesterase) completed by the simultaneous dosage of glucose-6-phosphate dehydrogenase. The histophotometric measures made it possible to differentiate the enzymatic activities of the different zones of the adrenal cortex and to show the persistance of an important activity of the zona fasciculata and sometimes of the zona reticulata during hibernation, independantly of the zona glomerulosa. This study confirms the activation of the adrenal cortex during hibernation with a brutal depletion at awakening and reduced activity during estivation.

Histochemical studies on the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), succinate dehydrogenase (SDH) and cytochrome oxidase (CY-O) in rabbit ovarian follicles at various times after the administration of hCG were performed to investigate the relationship between the activities of steroid biosynthesis and the respiratory system. The activities of 3 beta-HSD and SDH were studied by using the tetrazolium salt and CY-O activities by the method of Seligman with prefixation. On the granulosa cell layer prior to the administration of hCG, no activities of 3 beta-HSD and CY-O were detectable, while slight activities of SDH were observed. These three enzymes showed intense activities on the granulosa cell layer 3 hours after the administration of hCG, which were maintained till the time of ovulation. The results indicated that increased activities of steroid biosynthesis in the granulosa cell layer after LH-surge were accompanied by the accelerated TCA cycle and respiratory chain. Energy from accelerated glycolysis may be utilized for steroid biosynthesis in preovulatory granulosa cells. Increased O2 consumption due to accelerated aerobic glycolysis is in accordance with hyperactivity of perifollicular capillaries, which finally leads to rapid follicular expansion and ovulation.

Highly purified preparations of the 3alpha-hydroxysteroid:NAD- oxidoreductase (E.C.1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) which consist of two major isoenzymes, with traces of a third, have been split into two enzymatically inactive polypeptides A and B by the use of sodium dodecylsulphate, urea and guanidinium hydrochloride. Both polypeptides have a molecular weight of 25,000 +/- 2,500 as shown by thin-layer gel chromatography and ultracentrifugations. They differ, however, in charge as shown by electrophoresis on cellulose acetate strips in the presence of 8 M urea. Each of the isoenzymes, have molecular weight of about 50,000 and thus consist of two subunits. The presence of the three isoenzymes may be explained by the following combinations of the subunits, AA, AB and BB. Close to 100% of the original activity towards the three substrates, androsterone tetrahydrocortisone and desoxycholate could be restored within 24 h when the inactivated enzyme was diluted in order to remove the effect of the denaturant.

3beta-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities were localized at the ultrastructural level in amphibian interrenal (adrenocortical) cells previously fixed in a mixture of formaldehyde and glutaraldehyde. Potassium ferricyanide was used as an electron acceptor. Copper ferrocyanide deposits resulting from 3beta-HSD activity were seen in close association with the external faces of the membranes of the smooth endoplasmic reticulum. Very rare grains of precipitate appeared in mitochondrial cristae. The addition of phenazine methosulfate to the incubation medium had no effect on these localizations. The interrenal cells showed also a strong NADH-ferricyanide reductase activity. The copper ferrocyanide grains were abundant in the mitochondrial cristae and in the hyaloplasm, where they were not preferentially associated with the smooth endoplasmic reticulum.

Mouse submandibular salivary gland cells were grown in primary explant culture. After an initial period of degeneration within the explant, surviving epithelial cells proliferated rapidly and duct-like structures recolonized the explant. Autoradiographic studies showed that a peak of DNA synthesis occurred after 4 days in vitro and that proliferation was enhanced by insulin and hydrocortisone. These cells retained specialized secretory function (protease activity) for at least 2 weeks in vitro. This enzyme is a differentiated product of granular tubule cells in vivo. Between 6 and 10 days, explants attached to the substrate. An outgrowth developed, consisting largely of ultrastructurally identifiable epithelial cells which formed pseudoglandular structures in the monolayer. Epithelium survived for over 6 months in primary culture but could not be serially transferred. Secondary cultures were rapidly overgrown by mesenchymal cells.

Histochemical activity of hydroxysteroid dehydrogenases as well as alkaline and acid phosphatase was investigated in porcine granulosa cells cultured in vitro. Granulosa cells, isolated from preovulatory procine ovarian follicles, during in vitro culture showed activity of enzymes participating in steroid biosynthesis. High activity of delta53betta0H-SDH and G6P-DH as well as the activity of alkaline phosphatase, appearing in the course of culture, could be evidence of progressive luteinization of the cells. Activity of 17beta0H-SDH was lower and exhibited strong fluctuations, similarly low was 20xOH-SDH. Gonadotropic hormones caused the increase of synthesis and accumulation of intracellular lipids. They stimulated alkaline and acid phosphatase, and also the activity of the dehydrogenases. LH had the most visible effect. Estradiol stimulated the activity of acid but not alkaline phosphatase and was not influencing, even lowering the activity of dehydrogenases.

The presented paper describes the role of enzyme histochemistry in cell biological investigations. In the first chapter a general discussion has been given about enzyme histochemistry as a connecting link between biochemistry and morphology. The methods available for determination of enzymes in a particular cell or cell compartment have been reviewed. In this respect the characteristics of enzyme histochemistry have been discussed. Furthermore, attention has been paid to the possibilities and limitations of enzyme histochemistry. In chapter two a comparison has been made between histochemically judged and biochemically determined enzyme activities. Some fundamental differences between the biochemical and the histochemical approach in cell biological investigations are dealt with. To correlate histochemically and biochemically determined enzyme activities, a description has been given of the application of histochemical methods on isolated fractions and sucrose-ficoll gradients of these fractions. Several experimental results are described concerning the question whether a relation exists between histochemically and biochemically determined activities of respectively alkaline phosphatase, glucose-6-phosphatase, 5'-nucleotidase and 3ss-hydroxysteroid dehydrogenase. From these results the conclusion could be drawn that in general a good correlation exists between histochemically judged activity per volume (area X thickness) and biochemically determined activity per gram tissue. In chapter three the role of enzymes as markers of cellular particles and as parameters of metabolic pathways is described. Histochemical methods are available for most marker enzymes. Only activities of key enzymes can be regarded as parameters of metabolic pathways. The distribution in sucrose-ficoll gradients of enzymes, regarded as markers of mitochondria, lysosomes, endoplasmic reticulum and plasma membranes has been given. The changes occur ing under different experimental conditions for a number of marker enzymes in rat liver are described. Attention has been given to the contibution of enzyme histochemistry in the study of the heterogeneity of mitochondria, the dual localization of some (lysosomal) enzymes, the complexity of the microsomal fraction, the function of the Golgi apparatus and the heterogeneity and function of plasma membranes. Based on these results and on literature findings the possible role of some marker enzymes in cell metabolism has been discussed. In chapter four problems coherent with species and sex differences in enzyme activities are described. The interpretation of histochemical and biochemical results in view of these differences is discussed. Enzymes characteristic for a given cell type -3ss-hydroxysteroid dehydrogenase in steroid producing cells, ATP-ase in liver plasma membrane surrounding the bile canaliculi - do show less variations between species and sexes than enzymes not directly involved in specialized functions...

17 beta-Hydroxysteroid oxidoreductase (17-OHSD) activity in the endometrium of women with pelvic pain syndrome (PPS) and/or polycystic ovaries (PCO) was compared with that of a control group. In both groups there was a 10-fold increase in 17-OHSD activity in secretory phase tissue compared with that of the proliferative phase, measured by both oxidative and reduction pathways, and a highly significant correlation between the two directions (P less than 0.001). In normal subjects, the ratio of activity measured under oxidative conditions: reducing conditions, at all stages of the cycle except late proliferative phase, was 2.1-2.9. In the late proliferative phase the ratio was 5.5 which was significantly different from other stages of the cycle. Similar ratios were found for the PPS/PCO group (proliferative phase 2.5, secretory phase 5.6); these were also significantly different (P less than 0.01). On the basis of this study oestrogen metabolism in the endometrium of women with PPS and/or PCO appears to be no different from that of normal subjects. Measurement of enzyme activity in high speed soluble and particulate fractions of endometrial homogenate indicated the presence of two activities with different cofactor requirements. Gel filtration chromatography of the soluble fraction revealed a single peak of activity coincident with a molecular weight of 30 kDa with a strong preference for NAD + as cofactor. These preliminary findings suggest the presence of both soluble and particulate forms of 17-OHSD activity in the endometrium.