Progesterone metabolism in vitro by rabbit testes at different stages of development.

Testicular homogenates from white rabbits of 0.4, 1, 3, 4, 5, 8, 18, and 24 months of age were incubated with [3H]progesterone and NADPH. At 12 days of age, major C21-17-OH-and C19- steroids formed from progesterone were 17alpha-hydroxyprogesterone and testosterone. However, at 4-24 months of age, 17alpha-hydroxyprogesterone, 3beta,17alpha-dihydroxy-5alpha-pregnan-20-one, and 5alpha-reduced C19-steroids such as 17beta-hydroxy-5alpha-androstan-3-one, 3beta-hydroxy-5alpha-androstan-17-one, and 5alpha-androstane-3beta,17beta-diol were the major products. The formation of significant quantities of 5alpha-reduced C19-steroids, which had been demonstrated previously only in prepubertal testes of rats and mice, was present in prepubertal as well as adult testes of rabbits. These findings clearly indicate that the metabolic patterns of progesterone in the adult rabbit differ from those in the adult rat and mouse. The major C19-steroids formed by testes are testosterone in the adult rat and mouse, but 5alpha-reduced C19-steroids in the adult rabbit.     

Detection of unconjugated and conjugated steroids in the ovary, eggs, and haemolymph of the decapod crustacean Nephrops norvegicus

Gas chromatography/mass spectrometry with selected ion monitoring has been employed to examine extracts from the ovary, eggs, and haemolymph of the marine prawn, Nephrops norvegicus, to demonstrate the presence of steroids. Both free and conjugated steroids were isolated by solvent partitioning and chromatography (lipophilic Sephadex, reversed-phase Sep Pak, and normal phase medium-pressure liquid chromatography) and steroidal conjugates were cleaved enzymatically. Steroids were determined as their methyloxime derivatives, trimethylsilyl (TMS) ethers or methyloxime-TMS ethers. All assignments were based on the detection of characteristic ions and cochromatography with the authentic steroid derivatives. 5 alpha-Dihydrotestosterone, testosterone, pregnenolone, and 20 alpha-hydroxypregn-4-en-3-one were detected in unconjugated form in the ovary. The eggs and haemolymph were found to contain unconjugated 17 beta-estradiol. Conjugated 5 alpha-dihydrotestosterone was detected in both the ovary and haemolymph, but no conjugated steroids were found in the eggs.     

N-Acetyltransferase, hydroxyindole-O-methyltransferase and melatonin in the optic lobes of the giant tiger shrimp Penaeus monodon

Abstract: The activities of the enzymes N-acetyltransferase (NAT) and hydroxyin-dole-O-methyltransferase (HIOMT) and the hormone melatonin were studied in the optic lobe of the subadult giant tiger shrimp Penaeus monodon . Compared with the level in other species, a relatively high level of NAT activity that was temperature- and pH-dependent were observed. The NAT enzyme had a relatively high maximum velocity (Vmax, 100 pmol/hr/μg protein) and low Michaelis constant (Km, 22 μM), when tryptamine is used as substrate. In contrast to the high level of NAT activity, HIOMT activity and melatonin levels were low in the optic lobe of the giant tiger shrimp. Sex differences in the levels of NAT activity and melatonin, which are observed in a freshwater species Macrobrachium rosenber-gii , were not noticeable in the saltwater species P. monodon , at least not when they were in their subadult stage.     

Progesterone metabolism in adipose cells

The aim of the present study was to investigate pathways of progesterone metabolism in human adipose cells. Adipose tissue samples from the omental (OM) and subcutaneous (SC) fat compartments were surgically obtained in women. In isolated mature adipocytes, progesterone was converted to 20alpha-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo-keto reductases 1C1, 2 and 3 (20alpha-HSD, 3alpha-HSD type 3 and 17beta-HSD type 5, respectively). In cultured preadipocytes, progesterone was converted to several metabolites identified using bidimensional thin layer chromatography, with or without the dual inhibitor of 5alpha-reductase type 1 and 2 (17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan-3-one (4-MA)). Major metabolites identified in OM and SC preadipocytes which were incubated for 24h with (14)C-labelled progesterone were 20alpha-hydroxyprogesterone, 5alpha-pregnane-3alpha/beta-ol-20-one, 5alpha- and 5beta-pregnanedione, 5alpha- and 5beta-pregnane-20alpha-ol-3-one, 5alpha-pregnane-3alpha/beta-ol-20-one and 5beta-pregnane-3alpha/beta-20alpha-diol. Induction of preadipocyte differentiation increased expression levels of AKR1C1 and modified the pattern of progesterone metabolism substantially, leaving 20alpha-hydroxyprogesterone as the main metabolite generated. On the other hand, progesterone itself showed no consistent effect on adipocyte differentiation. In conclusion, preadipocytes and lipid-storing, mature adipocytes efficiently generate progesterone metabolites in women, which is consistent with rather modest effects progesterone on abdominal fat cell differentiation.     

Separation and biological effects of CNS factors affecting water balance in the decapod crustacean Thalamita crenata

Two active factors have been separated from the CNS of the crab that produce opposite affects on rates of water influx in crustaceans. The two factors are differentially soluble in water and acetone, and the water-soluble factor was partly purified by column chromatography and found to be a low molecular weight compound.When injected into crabs and crayfish held in hypotonic medium, the water-soluble fraction caused a decrease in ³H₂O influx, while the acetone-soluble fraction increased the influx rate. Neither of these factors affected sodium influx rates. It is suggested that these active factors play a role in influencing crustacean osmoregulation, and their neuroendocrine significance is discussed.     

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the aged rat

Progesterone metabolism was examined in tissues of rats in three stages of reproductive senescence (constant estrus, repeated pseudopregnancies, and anestrus) and in young rats. Metabolites were quantitated by reverse isotopic dilution analysis after incubation of the hypothalamus, pituitary, and uterus with [3H]progesterone. The metabolism of progesterone to 5 alpha-dihydroprogesterone and to 20 alpha-hydroxy-5 alpha-pregnan-3-one and the formation of total 5 alpha-reduced products was significantly reduced (by half) in pituitaries of constant estrous rats compared to rats in all other stages. The formation of 3 alpha-hydroxy-5 alpha-pregnan-20-one and total 3 alpha-reduced products was about 2-fold higher in pituitaries and hypothalami of pseudopregnant and anestrous rats than in constant estrous and young rats, but these differences were statistically significant only in the pituitary samples. In the uterus, progesterone metabolism to 20 alpha-dihydroprogesterone was significantly increased in anestrous rats compared to that in constant estrous and pseudopregnant rats. The results indicate that progesterone metabolism by target tissues, particularly the pituitary, is altered during reproductive senescence. They suggest the possibility that changes in the tissue metabolism of progesterone may be one means by which the effectiveness of progesterone is decreased during aging.     

The presence and distribution of gonadotropin-releasing hormone-liked factor in the central nervous system of the black tiger shrimp, Penaeus monodon

The distribution and presence of gonadotropin-releasing hormone (GnRH) in the central nervous system (CNS) of Penaeus monodon were examined by immunocytochemistry, high performance liquid chromatography (HPLC), and radioimmunoassay (RIA). We demonstrated the existence of octopus (oct)GnRH-liked immunoreactivity (ir-octGnRH) and lamprey (l)GnRH-III-liked immunoreactivity (ir-lGnRH-III) in cell bodies of medium-sized neurons of the anterior part (protocerebrum) of the supraesophageal ganglion (brain). In addition, only the ir-octGnRH was detected in the nerve fibers located in the brain and segmental ganglia (subesophageal, thoracic, and abdominal ganglia). Moreover, some branches of these fibers also innervated the neurons in the middle (deutrocerebrum), posterior (tritocerebrum) brain and segmental ganglia. There was no ir-lGnRH-I and ir-salmon (s)GnRH detected in the shrimp CNS. The results from HPLC and RIA showed ir-GnRH in the CNS using anti-lGnRH-III, but not with anti-mammalian (m)GnRH. The data from immunocytochemistry, HPLC and RIA suggest that ir-GnRH in shrimp may be more similar to octGnRH and lGnRH-III than the other forms. These findings support the hypothesis that GnRH-liked factor(s) may be an ancient peptide that also exists in this decapod crustacean.     

Hemostasis of Tiger Prawn Penaeus monodon Affected by Vibrio harveyi,Extracellular Products,and a Toxic Cysteine Protease

ABSTRACT: The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied. The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease or PBS (control) was observed for 2 h at 25 C. Of these, only the control group was clottable while all the other groups were unclottable. A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (Rα coagulogen) to neutralize the clotting ability of normal prawn hemolymph. The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels. The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using Rα coagulogen. In addition, the apparent disappearance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using Rα coagulogen or by reducing the time for pre-incubation from 2 h to 30 min. Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph. These complex events may significantly contribute to the pathogenicity of V. harveyi in the prawn.     

Hemostasis of tiger prawn Penaeus monodon affected by Vibrio harveyi, extracellular products, and a toxic cysteine protease

The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied. The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease of PBS (control) was observed for 2 h at 25 C. Of these, only the control group was clottable while all the other groups were unclottable. A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (R alpha coagulogen) to neutralize the clotting ability of normal prawn hemolymph. The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels. The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using R alpha coagulogen. In addition, the apparent disapperance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using R alpha coagulogen or by reducing the time for pre-incubation from 2 h to 30 min. Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph. These complex events may significantly contribute to the pathogenicity of V. harveyi in the prawn.     

Progesterone metabolism in normal human endometrium during the menstrual cycle and in endometrial carcinoma.

Different subcellular fractions (purity checked by electron microscopy and respective marker enzymes) were incubated with 0.1 muCi 14C-progesterone (10 muM) in 0.15 M phosphate buffer at pH 7.4 and 37 C under air for varying periods of time in the presence of NAD(P)H (500 muM). By the preparation of chromic acid oxidation products and acetates, thin-layer chromatography, and crystallisation to constant specific activity, the following metabolites were identified: 20alpha-hydroxypregn-4-en-3-one, 20alpha-hydroxy-5alpha-pregnan-3-one, 20alpha-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3,20-dione, and 5beta-pregnane-3,20-dione, indicating the presence of a 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and 5alpha- and 5beta-reductases. Most of the 20alpha-HSD activity was located in mitochondria (associated mainly with outer membranes) and microsomes. Purified nuclei and cytosol contained 1/6 to 1/18 of the activity of mitochondria and microsomes, respectively. SUBFRACTIONS OF ENDOMETRIAL CELLS ONLY CONTAINED EITHER 5ALPHA- OR 5BETA-REDUCTASE ACTIVITY. 5alpha-reductase activity was mainly associated with microsomes, 5beta-reductase activity was found only in the cytosol. While in normal endometrium specific enzyme activities in subcellular fractions depended on the phase of the cycle, in endometrial carcinoma it depended on the degree of tumour differentiation. The highest values of 5alpha-reductase activity were found in the early proliferative phase. 20alpha-HSD activity was highest in the middle of the secretory phase. The specific activity of the 5alpha-reductase increased with decreasing differentiation of the tumour while the specific activity of the 20alpha-HSD decreased. Kinetic parameters (Km-values, coenzyme requirements and maximum velocities) were determined. The Km-value for progesterone of the 20alpha-HSD in proliferative endometrium was significantly higher than in secretory endometrium, while the Km-values of the 5alpha- and 5beta-reductases were considerably lower during the proliferative than secretory phase.