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Seasonal changes in pregnenolone metabolism in toad testis
S96 P12-4
P13-2
Modification of the accumulative inactivation type K+ channels by patch excision. . * Kubo T’, Furukawa Y’ m’, ‘Dep. Biol. Sci., Fat. Sci., Hiroshima Hiroshima, Japan. ‘NIBH, Tsukuba, Japan
of Shaker-
Univ.,
Higashi-
AnAplysia K’channel, AKvl.la, and a rat K’ channel, rKvl.4, are Shaker-type K’ channels. Both channels express transient K’ currents showing a marked accumulative inactivation under on-cell patch recording. The accumulative inactivation of AKvl.la is enhanced by patch excision, whereas that of rKvl.4 is inhibited. To delineate molecular mechanisms for such modification of the inactivation, we constructed several mutant channels by site-directed mutagenesis and examined the accumulative inactivation of mutant channels in Xenopus oocytes. Because previous studies suggest that C-type inactivation is important for the accumulative inactivation, and that the structure of the external mouth of the pore affects Ctype inactivation, we made eight pore-mutants of AKvl.la (A378E, D379P, Q380T, K384Q, R406K, G409T, W411G, L4141) based on the sequence differences between AKvl.la and rKvl.4. All the mutants expressed kinetically similar transient K’ channels, but the accumulative inactivation of A378E, D379P, and R406K was not enhanced by patch excision. These results indicate that the structure of the external mouth of the pore affects the modification of the accumulative inactivation by patch excision. We are currently constructing the amino-terminal deletion mutants for these mutant channels to remove the effects of N-type inactivation, and making chimeras between AKvl.la and rKvl.4 to examine the contribution of other domains.
Cloning hormone brain.
and expression of corticotropin-releasing and urotensin I precursors in the goldfish
Bemier NJ, Lin X and Peter RE Department of Biological Sciences, University Edmonton, Alberta, Canada T6G 2E9.
of Alberta,
Corticotropin-releasing hormone (CRH; 966 bp) and urotensin I (UI; 769 bp) precursor cDNAs were cloned and sequenced from a goldfish brain cDNA library. The CRH and UI cDNAs encode a 163 and 146 amino-acid precursor, respectively, and consist of a signal peptide sequence, a cryptic region, and a 41 amino-acid mature peptide at the carboxyl terminal. While the deduced 41 amino-acid sequences of the mature CRH and UI peptides show a high degree of homology (76-100%) with their respective homologues in other vertebrates, a comparison of their sequence exhibits a 54% sequence homology. Although the mRNA encoding CRH and UI are not expressed in the pituitary, they ate found in the telencephalon-preoptic, hypothalamic, optic tectum, and posterior brain regions. In addition, the mRNA encoding CRH is expressed in the olfactory bulbs. Elevation of plasma cortisol with sillastic implants reduces CRH mRNA expression in the telencephalonpreoptic region. Overall, the goldfish CRH and UI precursors are structurally homologous to other vertebrate corticotropinreleasing precursors and the expression of the goldfish CRH gene appears to be negatively influenced by cortisol.
P13-1
P13-3
Induction of apoptosis by salmon GnRH and chicken GnRH-II in the Goldfish ovarian follicles. Andreu CV and Habibi HR. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
Seasonal changes in pregnenolone metabolism in toad testis L.F. Canosa, A.G. Pozzi and N.R. Ceballos. Dpto. Ciencias Biologicas, FCEyN, USA, PRHOM-CONICET, Buenos Aires, Argentina.
Gonadotropin-releasing hormone (GnRH) is a decapeptide with a significant degree of functional diversity. There is evidence for the presence of variant GnRH and GnRH receptors in both reproductive and non-reproductive tissues in a number of vertebrates investigated including goldfish. Previous studies in mammals have demonstrated that GnRH increases apoptosis which is a form of cell death believed to be implied in different physiological events including atresia of ovarian follicles. No information is available on the role of GnRH in the onset of apoptosis in the fish ovary. In this study, we tested the hypothesis that GnRH play a regulatory role in follicular atresia in the goldfish ovary. Follicular apoptosis was studied here by means of an ELISA for oligonucleosome which indicates spontaneous DNA breakdown. Preovulatory goldfish ovarian follicles (0.9 mm) were cultured for 24h in the presence of 2% fetal bovine serum (FBS) which was found to minimize the onset of apoptosis. Treatments with 100 nM of either salmon GnRH (sGnRH) or chicken GnRH-II (cGnRH) increased oligonucleosome content compared to controls indicating follicular apoptosis in goldfish. In the presence of gonadotropin (GtH) (500 ng/ml), however, the DNA fragmentation was reduced to 50%. The results demonstrate that treatment of fully mature goldfish follicles with GnRH alone causes apoptosis which is reduced in the presence of GtH. Supported by NSERC grants.
Testicular androaen biosvnthesis could proceed through the A5 pathway charact&sed by the presence of 3j3-old-ene mtermediates or follows an alternative pathway called A4 which involve 3-oxo-4-ene steroids. The rermlation of this choice could be the result of steroidogenic enzym’e properties. Thus, if 17-hydroxyla se-Cr7.aa lyase shows a greater a@inity for pregnenolone (Ps) than for progesterone (P4), the A5 pathway would predominate. A similar assumption about 3P-hydroxysteroid dehydrogenaseisomerase (3PHSD/I) could be made. In Bufo arenarum, previous results from our laboratory suggested seasonal changes in steroid metabolism. In order to study these changes, testis fiagment were incubated with 3H-P~ and-the products-were separated and character&d bv TLC. HPLC and recrvstallisation. The results showed that in nor&productive per&d, PS is manly converted in DHEA, androstenedione, Sandrosten-3 $, 17@diol, 17a-hydroxy-P5, testosterone and Sa-DHT. On the other hand, in the reproductrve period, testis produced a higher yield of P4 and its reduced derivatives like See-pregnanedione, Sa-pregnan3a,20j3diol, and fia-pregnan3ao1, 2Oone, while the A’ metabolites became undetectable, and Crs steroids tend to diminish. The enzymatic activities responsible for this changes could be the Ct7.20 lyase, 38HSD/I and, in a minor fashion, Sa-reductase. However, only the relative contribution to total steroid metabolism of C17_20 lyase drastically falls. In vitro studies show that gonadotropins have no direct influence on the S@HSD/I or ~CZreductase activities. These results rise the possibility that gonadotropins could regulate the changes in biosynthetic pathways by controlling the Cr7.20lyase activity.
P13-2
Modification of the accumulative inactivation type K+ channels by patch excision. . * Kubo T’, Furukawa Y’ m’, ‘Dep. Biol. Sci., Fat. Sci., Hiroshima Hiroshima, Japan. ‘NIBH, Tsukuba, Japan
of Shaker-
Univ.,
Higashi-
AnAplysia K’channel, AKvl.la, and a rat K’ channel, rKvl.4, are Shaker-type K’ channels. Both channels express transient K’ currents showing a marked accumulative inactivation under on-cell patch recording. The accumulative inactivation of AKvl.la is enhanced by patch excision, whereas that of rKvl.4 is inhibited. To delineate molecular mechanisms for such modification of the inactivation, we constructed several mutant channels by site-directed mutagenesis and examined the accumulative inactivation of mutant channels in Xenopus oocytes. Because previous studies suggest that C-type inactivation is important for the accumulative inactivation, and that the structure of the external mouth of the pore affects Ctype inactivation, we made eight pore-mutants of AKvl.la (A378E, D379P, Q380T, K384Q, R406K, G409T, W411G, L4141) based on the sequence differences between AKvl.la and rKvl.4. All the mutants expressed kinetically similar transient K’ channels, but the accumulative inactivation of A378E, D379P, and R406K was not enhanced by patch excision. These results indicate that the structure of the external mouth of the pore affects the modification of the accumulative inactivation by patch excision. We are currently constructing the amino-terminal deletion mutants for these mutant channels to remove the effects of N-type inactivation, and making chimeras between AKvl.la and rKvl.4 to examine the contribution of other domains.
Cloning hormone brain.
and expression of corticotropin-releasing and urotensin I precursors in the goldfish
Bemier NJ, Lin X and Peter RE Department of Biological Sciences, University Edmonton, Alberta, Canada T6G 2E9.
of Alberta,
Corticotropin-releasing hormone (CRH; 966 bp) and urotensin I (UI; 769 bp) precursor cDNAs were cloned and sequenced from a goldfish brain cDNA library. The CRH and UI cDNAs encode a 163 and 146 amino-acid precursor, respectively, and consist of a signal peptide sequence, a cryptic region, and a 41 amino-acid mature peptide at the carboxyl terminal. While the deduced 41 amino-acid sequences of the mature CRH and UI peptides show a high degree of homology (76-100%) with their respective homologues in other vertebrates, a comparison of their sequence exhibits a 54% sequence homology. Although the mRNA encoding CRH and UI are not expressed in the pituitary, they ate found in the telencephalon-preoptic, hypothalamic, optic tectum, and posterior brain regions. In addition, the mRNA encoding CRH is expressed in the olfactory bulbs. Elevation of plasma cortisol with sillastic implants reduces CRH mRNA expression in the telencephalonpreoptic region. Overall, the goldfish CRH and UI precursors are structurally homologous to other vertebrate corticotropinreleasing precursors and the expression of the goldfish CRH gene appears to be negatively influenced by cortisol.
P13-1
P13-3
Induction of apoptosis by salmon GnRH and chicken GnRH-II in the Goldfish ovarian follicles. Andreu CV and Habibi HR. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
Seasonal changes in pregnenolone metabolism in toad testis L.F. Canosa, A.G. Pozzi and N.R. Ceballos. Dpto. Ciencias Biologicas, FCEyN, USA, PRHOM-CONICET, Buenos Aires, Argentina.
Gonadotropin-releasing hormone (GnRH) is a decapeptide with a significant degree of functional diversity. There is evidence for the presence of variant GnRH and GnRH receptors in both reproductive and non-reproductive tissues in a number of vertebrates investigated including goldfish. Previous studies in mammals have demonstrated that GnRH increases apoptosis which is a form of cell death believed to be implied in different physiological events including atresia of ovarian follicles. No information is available on the role of GnRH in the onset of apoptosis in the fish ovary. In this study, we tested the hypothesis that GnRH play a regulatory role in follicular atresia in the goldfish ovary. Follicular apoptosis was studied here by means of an ELISA for oligonucleosome which indicates spontaneous DNA breakdown. Preovulatory goldfish ovarian follicles (0.9 mm) were cultured for 24h in the presence of 2% fetal bovine serum (FBS) which was found to minimize the onset of apoptosis. Treatments with 100 nM of either salmon GnRH (sGnRH) or chicken GnRH-II (cGnRH) increased oligonucleosome content compared to controls indicating follicular apoptosis in goldfish. In the presence of gonadotropin (GtH) (500 ng/ml), however, the DNA fragmentation was reduced to 50%. The results demonstrate that treatment of fully mature goldfish follicles with GnRH alone causes apoptosis which is reduced in the presence of GtH. Supported by NSERC grants.
Testicular androaen biosvnthesis could proceed through the A5 pathway charact&sed by the presence of 3j3-old-ene mtermediates or follows an alternative pathway called A4 which involve 3-oxo-4-ene steroids. The rermlation of this choice could be the result of steroidogenic enzym’e properties. Thus, if 17-hydroxyla se-Cr7.aa lyase shows a greater a@inity for pregnenolone (Ps) than for progesterone (P4), the A5 pathway would predominate. A similar assumption about 3P-hydroxysteroid dehydrogenaseisomerase (3PHSD/I) could be made. In Bufo arenarum, previous results from our laboratory suggested seasonal changes in steroid metabolism. In order to study these changes, testis fiagment were incubated with 3H-P~ and-the products-were separated and character&d bv TLC. HPLC and recrvstallisation. The results showed that in nor&productive per&d, PS is manly converted in DHEA, androstenedione, Sandrosten-3 $, 17@diol, 17a-hydroxy-P5, testosterone and Sa-DHT. On the other hand, in the reproductrve period, testis produced a higher yield of P4 and its reduced derivatives like See-pregnanedione, Sa-pregnan3a,20j3diol, and fia-pregnan3ao1, 2Oone, while the A’ metabolites became undetectable, and Crs steroids tend to diminish. The enzymatic activities responsible for this changes could be the Ct7.20 lyase, 38HSD/I and, in a minor fashion, Sa-reductase. However, only the relative contribution to total steroid metabolism of C17_20 lyase drastically falls. In vitro studies show that gonadotropins have no direct influence on the S@HSD/I or ~CZreductase activities. These results rise the possibility that gonadotropins could regulate the changes in biosynthetic pathways by controlling the Cr7.20lyase activity.