First Report of Tomato yellow spot virus Infecting Leonurus sibiricus in Brazil.

In Brazil, serious epidemics of begomovirus diseases have been successively reported since the mid-90s, among them those caused by Tomato yellow spot virus (ToYSV) (1). In July 2009 and October 2010, high incidences (40 to 60%) of plants of the weed Leonurus sibiricus (Lamiaceae) exhibiting symptoms of yellow leaf mosaic were found near soybean (Glycine max) crops within the municipalities of Marechal Cândido Rondon and Tapejara, in the states of Paraná and Rio Grande do Sul. Leaves from 21 symptomatic and seven asymptomatic L. sibiricus plants were collected from both localities and tested for the presence of begomovirus. Total DNA was extracted from each sample using Dneasy Plant Mini Kit (Qiagen) and submitted to PCR using begomovirus universal oligonucleotides PAL1v1978/PAR1c496 (3). One fragment of approximately 1,300 bp comprising the 5'-region of the replication-associated protein (Rep) gene, the entire intergenic region (IR), and the 5'-region of the coat protein (CP) gene was amplified from all symptomatic, but not from asymptomatic samples. Amplified fragments corresponding to all isolates were directly sequenced and nucleotide sequence comparisons indicated 98 to 99% nucleotide identity among themselves, and 93 to 94% identity with the corresponding nucleotide sequences for the DNA-A of the begomovirus ToYSV (GenBank Accession No. DQ336350). To confirm these results, the full genome of ToYSV Mc-7 isolated from Marechal Cândido Rondon was cloned and completely sequenced by primer walking (Macrogen Seoul, Korea). The DNA-A of ToYSV Mc-7 (JX513952) was 2,592 nt long and shared 92 and 91% identity with isolates of ToYSV from Argentina (FJ538207) and Brazil (DQ336350), respectively. The DNA-B of ToYSV Mc-7 (JX513952) was 2,568 nt long and shared 91% identity with DNA-B of a Brazilian isolate of ToYSV (DQ336351). The ToYSV Mc-7 isolate is a new strain named Tomato yellow spot virus (Brazil:Marechal Candido Rondon 7:Leonurus:2009) [ToYSV-(BR:MCR7:Le:09)]. To demonstrate pathogenicity, virus-free adults of Bemisia tabaci biotype B were confined on symptomatic L. sibiricus plants for a 48-h acquisition period. The whiteflies were then transferred to healthy L. sibiricus, bean (Phaseolus vulgaris), soybean, and tomato (Solanum lycopersicum) plants. L. sibiricus plants showed the original symptoms on the leaves (five symptomatic plants, seven inoculated plants), whereas bean (3/7), soybean (4/10), and tomato plants (5/10) exhibited mild yellow leaf mosaic. The infection in these symptomatic plants was confirmed by PCR with oligonucleotides PAL1v1978/PAR1c496 (3) and subsequent direct nucleotide sequencing of the 5'-region of the CP gene, which confirmed the identity of the transmitted virus as ToYSV. ToYSV was first reported infecting tomato plants in Minas Gerais state, Brazil (1). Recently, ToYSV was found infecting bean and soybean plants in northwestern Argentina (2). Because L. sibiricus is a weed widely distributed throughout Brazil, and the ToYSV vector B. tabaci is also common, this weed may become a potential source of inoculun of ToYSV to bean, soybean, and tomato crops. To our knowledge, this is the first report of L. sibiricus as a natural host of ToYSV. References: (1) R. F. Calegario et al. Pesq. Agropec. Bras. 42:1335, 2007. (2) P. E. Rodríguez-Pardina et al. Ann. Appl. Biol. 158:69, 2011. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

A Sida sp. Is a New Host for "Candidatus Phytoplasma brasiliense" in Brazil.

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated "Candidatus Phytoplasma brasiliense" (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). "Ca. Phytoplasma brasiliense" has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.

Properties of microsomal delta4-3-ketosteroid 5alpha-reductase in immature rat ovary. Inhibition by estradiol-17beta.

The subcellular distribution of immature rat ovarian 5alpha-reductase has been studied by utilizing 20alpha-hydroxypregn-4-en-3-one as substrate. The main enzyme activity was found to be associated with the microsomal fraction, with lower activities in the 1000 X g and 10 000 X g fractions. The enzyme activity associated with the microsomes exhibited an apparent Km of 3.0 +/- 1.1 micrometer for 20alpha-hydroxypregn-4-en-3-one and 36.3 +/- 6.7 micrometer for testosterone as substrate. Progesterone and testosterone competitively inhibited the 5alpha-reduction of 20alpha-hydroxypregn-4-en-3-one; estradiol-17beta was found to be a noncompetitive inhibitor of this reaction. A concentration of estradiol-17beta as low as 1 micrometer when added to 25 micrometer concentration of 20alpha-hydroxypregn-4-en-3-one exerted a significant inhibition of 5alpha-reductase activity.

Regulation of ovarian 17 beta-hydroxysteroid dehydrogenase activity by gonadotropin.

Serum testosterone levels are elevated prior to the lutropin surge, and decline abruptly following the release of endogenous lutropin. To investigate this phenomenon, the activity of 17 beta-hydroxysteroid dehydrogenase, the enzyme directly related to testosterone production from androstenedione, was measured. This was done in immature rats in which follicular maturation and ovulation were induced by pregnant mare serum gonadotropin administration. It appears that the effect of the gonadotropin on the enzyme activity is sharply divided into two phases that match with the follicular and the luteal phases. One day following gonadotropin administration, there was already a 7.67-fold increase in the original activity which further increased 48 h following hormone administration. At the peak of the lutropin surge, when follicular development is at its maximum, a 18.44-fold increase was measured. The activity fell abruptly 10 h following ovulation, at a time when fresh corpora lutea are already present in the ovary. It seems that the elevation of serum testosterone followed by its abrupt decline, is directly related to the increased and decreased ovarian 17 beta-hydroxy-steroid dehydrogenase activity. The possible importance of the observed changes to the mechanism of the onset of puberty are discussed.

Metabolic pathways for androstanediol formation in immature rat testis microsomes.

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone resulted in a large preference of 17 alpha-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [3H]progesterone together with [14C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low 14C/3H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17 alpha-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.

Periovulatory changes in steroid C17,20-lyase activity in ovaries of immature rats treated with pregnant mare serum gonadotropin.

It has been established that the effect of LH on ovarian steroid formation results initially in a rise of overall steroid production, which 2-4 h later is followed by a decrease in the formation of C19-steroids. To determine the reason for this decrease, the course of 17 alpha-hydroxyprogesterone (17 alpha-OHP) metabolism in immature rat ovaries (10,000 X g supernatant) stimulated to ovulate with pregnant mare serum gonadotropin (PMSG) was investigated. Employing optimal conditions the C17,20-lyase was estimated from the formation of androstenedione from 17 alpha-OHP as substrate. The Michaelis-Menten constant Km was 61 +/- 4 microM and V varied according to the time after PMSG administration. The initial activity at 0 h was 340 pmol min-1 mg-1 protein. It decreased 48 h after PMSG to 160 pmol, followed by a slight rise at 54 h and followed by a 60% fall at 57 h, at the height of the LH-surge. The activity declined to undetectable levels at 60 and 64 h (8 h before ovulation) and remained undetectable after ovulation at 72 and 96 h after PMSG. The enzymes 5 alpha-reductase and 20 alpha-hydroxysteroid dehydrogenase were estimated from the formed 3 alpha, 17 alpha-dihydroxy-5 alpha-pregnan-20-one and 17 alpha, 20 alpha-dihydroxy-4-pregn-en-3-one, respectively. In the unstimulated ovary the 5 alpha-reductase was 405 pmol; it decreased to 110 pmol 48 h after PMSG and continued to decrease. The 20 alpha-hydroxysteroid dehydrogenase activity which was undetectable in untreated rats, rose to 330 pmol 60 h after PMSG, 8 h before ovulation. The changes in enzyme activities when tracer amounts of [3H] 17 alpha-OHP were used agreed with the pattern of kinetic studies. From these incubations eight metabolites were isolated, one of them, 5 alpha-pregnane-3 alpha,17 alpha, 20 alpha-trioi, has not been previously identified. It is concluded that the decrease in C17,20-lyase activity limit the formation of C19-steroids in preovulatory follicles shortly before ovulation.

Effect of human chorionic gonadotropin and prolactin on 20 alpha-hydroxysteroid dehydrogenase activity in granulosa cells of immature rat ovary.

The effects of FSH, hCG, and PRL on the activity of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) of separate ovarian components of hypophysectomized, diethylstilbestrol-treated rats were studied. Enzyme activity was found to reside mainly in granulosa cells. FSH induced an increase in enzyme activity. A preparation of FSH was purified by adsorbing its LH contamination on rat corpora lutea membranes and by further neutralizing LH traces with an antiserum to the beta-subunit of LH. This purified FSH retained the ability to induce a 6-fold increase in specific enzyme activity in granulosa cells of hypophysectomized, diethylstilbestrol-treated rats. Administration of hCG to rats treated with purified FSH, further enhanced 20 alpha-SDH activity in granulosa cells up to 11.5-fold above control. PRL, which is known to inhibit 20 alpha-SDH activity in regressing rat corpora lutea, suppressed the FSH-induced increase in enzyme activity in the granulosa cells.

Identification of two 5alpha-reduced pregnanes as major metabolites of progesterone in immature rat ovaries (1000 x g supernatant) in vitro.

Incubation of the 1000 x g supernatant obtained from 23-day-old rat ovarian homogenate with labeled progesterone resulted in the production of 3 major metabolites; 5alpha-androstane-3alpha,17beta-diol, and two 5alpha-reduced pregnanes that were identified as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one. The 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one has not been hitherto isolated from mammalian ovaries. The steroids were identified by their mobilities on thin-layer and gas-liquid chromatography, by mass spectroscopy, derivative formation and by recrystallization to constant specific activity. In another experiment, incubation of the 1000 x g supernatant from 23-day-old rat ovaries with 3alpha-hydroxy-5alpha-pregnan-20-one as substrate resulted in the production of 5alpha-androstane-3alpha,17beta-diol. It is suggested that 5alpha-androstane-3alpha,17beta-diol is produced in immature rat ovaries by a pathway in which the identified 5alpha-reduced pregnanes serve as intermediates.

The steroid C-17,20-lyase complex in isolated Graafian follicles: effects of human chorionic gonadotropin.

Androstenedione synthesis was studied in isolated rat preovulatory follicles and compared with that of rat testicular tissue using [14C]progesterone together with 17 alpha-hydroxy-[3H]progesterone as substrates in the presence of NADH or NADPH as cofactors. The amount of androstenedione formed was measured by addition of carrier, reisolation, and crystallization to constant specific activity. The labeling patterns of androstenedione and 17 alpha-hydroxyprogesterone (17-OHP) confirmed that both tissues preferentially catalyzed the synthesis of androstenedione from progesterone rather than from 17-OHP. It appears, therefore, that free 17-OHP was not an obligatory intermediate in this reaction. When hCG (5 IU) was administered sc and the follicles were isolated 3 h later, androstenedione synthesis was inhibited whether NADH or NADPH was added as cofactors. By contrast, 17-hydroxylase activity was inhibited only with NADH as cofactor. Hence, the gonadotropin, with NADH as cofactor, specifically reduced progesterone incorporation into androstenedione without affecting incorporation of 17-OHP. Thus, hCG appears to affect androstenedione production from progesterone at two different sites of the lyase complex.

Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge.

It has been shown that 3 h after the preovulatory gonadotropin surge, an abrupt decrease occurs in follicular C-17,20-lyase (lyase) activity which causes a decrease in C19-steroid production. To determine the reason for the reduced lyase activity, we used rats that were induced to ovulate by means of PMSG administration. In these rats, a 54% decrease in lyase activity occurred at the peak of the LH surge. When the gonadotropin surge and ovulation were blocked by pentobarbitone the decrease was prevented. Administration of LH to the pentobarbitone-blocked rats reduced lyase activity to well below the level reached after the endogenous gonadotropin surge. In cycling proestrous rats as well, human CG (hCG) decreased the lyase activity, as measured in isolated follicles 3 h after hCG administration. Out of three potential inhibitory steroids for lyase activity; progesterone, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnen-20-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, only the last compound inhibited competitively ovarian lyase activity. The inhibition constant (Ki) value was 29 microM. In order to explore which of the two activities of the lyase complex is regulated by the gonadotropin, a double label double substrate experiment was conducted using [14C]progesterone with [3H]17 alpha-hydroxyprogesterone (17 alpha-OHP). With this assay procedure, we could determine in the same experiment the site of stimulation, the preferred substrate, and the amount of conversion. The conversion of progesterone to 17 alpha-OHP, as well as the conversion to androstenedione were significantly inhibited throughout the reaction by the gonadotropin. Thus, the changes in ovarian lyase after hCG mimic those of 17 alpha-hydroxylase. The labeling pattern of androstenedione showed that the ovarian lyase complex catalyzed the conversion preferentially from progesterone. Whereas the 3H/14C ratio in androstenedione varied between 0.29 to 0.76, the ratios in the 17 alpha-OHP were from 5 to 22. Thus, the exogenous 17 alpha-OHP did not equilibrate with the product formed from progesterone. The effect of the hCG was to decrease the preference of progesterone over 17 alpha-OHP as substrate. It is concluded that: the LH of the surge inhibits both the 17 alpha-hydroxylase and lyase activities. The ovarian lyase complex shows a preference for progesterone as a substrate rather than 17 alpha-OHP. 17 alpha-OHP is not an obligatory intermediate in androstenedione production in ovarian tissue. hCG affects the ovarian lyase complex by shifting the relative preference of substrates towards 17 alpha-OHP.

Steroid production in testicular tissue of the European eel.

Testicular tissue of normal and hCG-stimulated European eels was incubated in vitro with tritiated progesterone or androstenedione as substrates. The following compounds were isolated and identified: 5 beta-androstane-3,17-dione; 17 beta-hydroxy-5 beta-androstan-3-one; androst-4-ene-3,11,17-trione (adrenosterone); 11 beta-hydroxyandrost-4-ene-3,17-dione; 11 beta-hydroxytestosterone; 3 alpha,11 beta-dihydroxy-5 beta-androstan-17-one, and an additional steroid for which the oxidation product was identified as 5 beta-androstene-3,11,17-trione. Four of these steroids have not been hitherto identified in gonadal tissue of any vertebrate. The pattern of steroid production in this tissue is unique for its 5 beta-reduction, for the appearance of adrenosterone as a major metabolite, and for the lack of production of 11-ketotestosterone, which is a regular metabolite of gonadal tissue of teleosts. Thus, it appears that steroid metabolism in the eel testis deviates considerably from the known pattern of steroid production in gonads of other vertebrates.

Decrease in blood and ovarian 3 alpha- and 3 beta-androstanediol levels in rats induced to ovulate with pregnant mare serum gonadotropin.

Several hours before the first ovulation progesterone metabolism in the rat ovary, in vitro, is shifted from the production of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) as the major metabolite toward the production of 4-ene-3-oxosteroids. In the present paper, changes in levels of 3 alpha-diol and its 3 beta-epimer as well as testosterone in blood and ovaries around the time of the first ovulation have been studied in immature PMSG-treated rats. Forty-eight hours after PMSG, a considerable increase in blood and ovarian testosterone concentration was observed, whereas the concentrations of both androstanediols in blood decreased sharply. At 52 h, the level of ovarian 3 beta-diol was only one third of the control level and continued to fall. The decrease in ovarian 3 alpha-diol was less pronounced, but reached about half, or less, of the control value. In PMSG-treated rats in which the LH surge was blocked by pentobarbitone, the decrease in blood diols was delayed but not prevented. It is concluded that the decrease in production of the androstanediols preceding the first ovulation observed previously in isolated ovaries, also occurs in the intact rat. The decrease in androstanediols occurs very shortly before an ovulation induced with an injection of PMSG and is dependent on the occurrence of an LH surge. Since it is assumed that the androstanediols have a prepubertal role in inhibiting uterine and ovarian growth and in preventing cyclic LH release, it is essential that their concentration decrease several hours before the first ovulation.

Effect of androstanediol sulfates on luteinizing hormone release in ovariectomized rats.

Androstanediols are the major products of the immature rat ovary and are present in peripheral circulation mainly as sulfate conjugates. In this paper we identified 5 alpha-androstane-3 beta, 17 beta-diol-3-monosulfate (3 beta-A-MS) as one of the forms found in blood and subsequently synthesized and administered it to ovariectomized rats at a dose of 100 microgram/100 g BW . day from 21-45 days of age. This dose effectively inhibits postcastrational LH elevation. Other androstanediols examined, like 5 alpha-A-3 alpha, 17 beta-diol-disulfate, 5 alpha-A-3 beta, 17 beta-diol-disulfate, and the free 5 alpha-A-3 beta, 17 beta-diol do not exert such an effect on LH release. The MCR of 3 beta-A-MS was 441 +/- 64 ml/h, independent of the infusion rate between 0.15-15.0 microgram/h, and its production rate was calculated to be 37 microgram/day at the age of 30 days. The quantitative relations of the steroid level in serum to its capacity to inhibit LH release was studied using Silastic capsules. A steady concentration of 1.1 ng 3 beta-A-MS/ml serum inhibits postcastrational LH release in the immature female rat. Since a similar or higher concentration of the steroid is present in the intact rat, it is assumed that 3 beta-A-MS controls pituitary LH release in the intact immature female rat. (Endocrinology 108: 500, 1981)

Placental secretion of androgens in the rat.

In contrast to the human placenta, which does not secrete androgens, the rat placenta synthesizes significant amounts of these steroids. The purpose of this study was to determine why the rat placenta does not secrete androgens before day 12 of pregnancy, to ascertain whether the rat placenta secretes more androstenedione than testosterone, to compare the capacity of luteal and placental tissue to secrete androgen, and to determine whether the rat placental produces androstenedione via the delta 4- or delta 5-steroidogenic pathway. To determine whether the inability of the rat placenta to produce androstenedione before midpregnancy was due to the absence of active 17 alpha-hydroxylase and 17,20-lyase enzymes and also to investigate the ontogeny of both placental production of androstenedione and enzyme activities, placentas were isolated from rats between days 8-21 of pregnancy and either incubated or used to determine the activities of 17 alpha-hydroxylase and 17,20-lyase. Before day 11, enzyme activity was not detectable. From day 11, both enzyme activities and placental secretion of androstenedione steadily increased to peak values by day 18 and declined just before parturition. To investigate the principal aromatizable androgen secreted both in vivo and in vitro approaches were used. Levels of androstenedione and testosterone found in the uterine vein as well as those produced by placental tissue were determined. Rat placentas secreted markedly more androstenedione than testosterone, both in vivo and in vitro. When placental and luteal secretion of androstenedione and testosterone were compared, it was found that luteal tissue had a higher capacity for androgen synthesis than did the placenta. Yet, because of its greater mass, each placenta secreted 15 times more androstenedione and 4.5 times more testosterone than each corpus luteum. To determine the preferential usage of progesterone or pregnenolone as substrate by the placenta, [14C] progesterone and [3H]pregnenolone were added in equimolar concentrations. The resulting 14C to 3H ratio of the androgen produced indicates that the preferred substrate is progesterone. In summary, results of this investigation describe, for the first time, the development of 17 alpha-hydroxylase and 17,20-lyase activities in the rat placenta and demonstrate that the placenta does not produce androgen before day 11 due to the absence of active enzymes. The results further demonstrate that the rat placenta secretes significantly more androstenedione that testosterone both in vivo and in vitro, produces more androgen than the corpus luteum because of its greater mass, and forms its androgen primarily via the delta 4-st

The nature of the defect in familial male pseudohermaphroditism in Arabs of Gaza.

Studies in six Arab individuals from Gaza with familial male pseudohermaphroditism (MPH) due to 17-ketoreductase deficiency revealed several metabolic aberrations associated with the disorder. Plasma LH, FSH, testosterone, and androstenedione concentrations were low in the two prepubertal patients. After hCG administration plasma androstenedione increased markedly. The four postpubertal MPH patients had very high plasma gonadotropin and androstenedione concentrations, the latter increasing further after hCG administration. Plasma testosterone concentrations in all six patients were moderately low or normal for age and increased little after hCG administration. Spermatic venous testosterone concentrations, measured in three adults, were within the normal range in two and low in one, while androstenedione concentrations were markedly elevated (15- to 32-fold) in all three patients. Kinetic analyses of progesterone and androstenedione metabolism were performed in testicular tissue of these patients and compared to the results in two control subjects. While testicular tissue from the two prepubertal patients metabolized progesterone only to androstenedione, and that to a limited extent, the tissue from the four postpubertal patients metabolized progesterone to 16 alpha- and 16 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testosterone and metabolized androstenedione to testosterone. The Michaelis constants of these reactions were similar in the tissue from the MPH and the control subjects. The production of 16 alpha- plus 16 beta-hydroxyprogesterone was 5.4- to 10.3-fold greater, and 17-hydroxylase activity was 5.8- to 8.1-fold lower in the testes of the postpubertal MPH patients compared to values in the control subjects. The preference of androstenedione production through the delta 4- or delta 5-pathways was examined in the testes of two adult MPH patients using an equimolar concentration of [14C]progesterone and [3H]pregnenolone as substrates. While the flow of substrates in the control testes was equal or slightly greater through the delta 4-pathway, the delta 5-pathway predominated in the testes of the MPH patients. A large amount of dehydroepiandrosterone accumulated when NAD, the cofactor for 3 beta-hydroxysteroid dehydrogenase-isomerase, was omitted, supporting the contention that androstenedione was produced in the testes of the MPH patients mainly through the delta 5-pathway. Additional support for this suggestion was the finding that the 3H/14C ratio in androstenedione and testosterone produced from both substrates was 8 times higher in the testes from MPH patients than in those from the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of pancreatic amylase release in vitro: effects of ions, cyclic AMP, and colchicine.

1. The time course and concentration-response relationship of amylase release from pieces of guinea-pig pancreas in vitro in response to bethanechol and pancreozymin was determined.2. Removal of Ca(++) from the medium had no effect on basal amylase release but abolished the stimulating effect on release of bethanechol.3. Elevation of the concentration of Mg(++) in the medium increased basal amylase release and reduced the response to bethanechol.4. Elevation of the concentration of K(+) in the medium increased amylase release; this effect was blocked by a concentration of atropine which blocked also the response to bethanechol.5. Cyclic AMP, dibutyryl cyclic AMP and theophylline failed to stimulate amylase release. Pancreatic cyclic AMP concentrations were found not to be increased by bethanechol, pancreozymin or an elevated concentration of K(+) in the medium.6. Colchicine had no effect on basal amylase release or the response to bethanechol or pancreozymin.7. It is concluded that the coupling of stimulus to secretion involves ionic control but that neither cyclic AMP production nor microtubular mechanisms play a major role in controlling exocytosis in the pancreatic acinar cell. These findings are discussed in relation to the stimulus-secretion coupling processes in other cells.

Androstanediol sulphates in peripheral blood of immature rats and some of their biological effects.

The conjugation of 5alpha-androstane-3alpha,17beta-diol (3alpha-A) and its 3beta epimer (3beta-A) was determined in the peripheral blood of immature female rats. About two thirds of these steroids were present in blood as sulphates and one third as glucuronides; no free steroids were detected. Administration of 3beta-A sulphate (25mug/100 g body weight/day) and of 3alpha-A sulphate (50 mug/100 g/day) from day 21 of life until the day of vaginal opening, advanced the day of the first ovulation. Administration of the 3beta-A sulphate did not induce precocious vaginal opening whereas the free alcohol was active in this respect. Implantation of 3beta-A sulphate, but not of the 3alpha epimer, into the basal medial hypothalamus resulted in the dealth of all animals within 24 h.

Difference in luteal and placental P450(17) alpha: substrate preference and hormonal regulation in the rat.

The purpose of this study was to assess the substrate specificity of P450(17) alpha in both the corpus luteum and placenta of pregnant rats, and to analyse the site at which LH/human chorionic gonadotrophin (hCG) regulates the activities of this enzyme. To distinguish the substrate preference, placentas and corpora lutea were obtained from rats on day 15 of pregnancy. Tissues were homogenized and the 10,000 g supernatants incubated in the presence of equimolar concentrations of [14C]progesterone and [3H]17 alpha-hydroxyprogesterone as substrate with either NADH or NADPH as cofactors for 2, 8, 16 and 24 min. The labelling pattern of both 17 alpha-hydroxyprogesterone and testosterone indicated that the corpus luteum produced testosterone preferentially from progesterone, whereas the placenta principally used 17 alpha-hydroxyprogesterone and synthesized six times as much testosterone from 17 alpha-hydroxyprogesterone than from progesterone. Addition of either NADPH or NADH as cofactors had no effect on substrate preference. The products of the two enzymatic activities were identified by recrystallization to constant 14C/3H ratios. The ratio of 14C/3H in testosterone produced by the corpus luteum was 16-fold higher than in that produced by the placenta. To explore which of the two activities of P450(17) alpha is regulated by the gonadotrophin, rats were treated with either 1.5 IU hCG or vehicle between days 13 and 15 of pregnancy. Hydroxylase and lyase activities were determined on day 15 after incubation for 2, 8, 16 or 24 min in the presence of either NADH or NADPH. Administration of hCG significantly inhibited NADH-dependent 17 alpha-hydroxylase in the placenta at each time-point studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Pre-ovulatory changes in steroidogenesis in ovaries from immature rats treated with pregnant mare serum gonadotrophin.

The metabolism of progesterone in the 1000 g supernatant fraction of homogenates of ovaries from PMSG-treated immature rats was determined. As early as 52 h after a single injection of 50 i.u. PMSG, still before the LH surge, 5alpha-pregnane-3alpha, 20alpha-diol was identified as the main metabolite, together with small quantities of 3alpha-hydroxy-kalpha-pregnan-20-one and 5alpha-androstane-3alpha, 17beta-diol. Similar incubations of untreated rat ovaries at the same age did not produce 5alpha-pregnane-3alpha, 20alpha-diol. The quantities of 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-androstane-3alpha, 1mbeta-diol were reduced in PMSG-treated rat ovaries as compared with control ovaries. When progesterone metabolism was examined 64 h after PMSG administration, 5-7 h after the peak of LH surge but still before ovulation, 75% of the substrate was converted to 5alpha-pregnane-3alpha, 20alpha-diol, while 3alpha-hydroxy-5alpha-pregnan-20-one as well as 5alpha-androstane-3alpha, 17beta-diol could not be detected.

Changes in steroid concentration in the ovaries of immature rats treated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin.

The concentrations of testosterone, progesterone and 20alpha-hydroxypregn-4-en-3-one (20alpha-OHP) were measured in the ovaries of immature rats in which ovulation was induced by treatment with pregnant mare serum gonadotrophin (PMSG) and, 48 h later, with human chorionic gonadotrophin (HCG). The concentration of testosterone in the tissue increased significantly 48 h after treatment with PMSG, reached a peak 4 h after the administration of HCG and declined to the basal level 4 h later. Increases in the levels of progesterone and 20alpha-OHP were observed 4 h after the administration of HCG. Whereas the level of 20alpha-OHP continued to rise during the subsequent 30 h, progesterone levels declined near the presumed time of ovulation (12 h after administration of HCG). It is concluded that 20alpha-hydroxysteroid dehydrogenase activity is present in the immature rat ovary before ovulation and that an increase in the production of testosterone in the ovaries of rats treated with PMSG and HCG precedes increased production of progesterone and 20alpha-OHP in these ovaries.