ABSTRACT
Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami of Catla catla. Gonadotropin releasing hormone activity was studied in vitro using enzymatically dispersed carp pituitary cell incubation system. Gonadotropin released into the medium was measured by carp gonadotropin-radio immuno assay. Acetic acid extracted hypothalamic material was subjected to acetone fractionation. Among the three protein pellets obtained at different time periods (AC I, AC II and AC III), AC II exhibited the gonadotropin releasing hormone activity. Gel filtration of AC II through Sephadex G-25 column showed three protein peaks (SG I, SG II SG III) and only SG II demonstrated strong gonadotropin releasing hormone activity. Elution of SG II through FPLC Mono Q column (an anion exchanger) in NaCl gradient programme showed one unadsorbed (MQ I) and three adsorbed (MQ II, MQ III and MQ IV) protein peaks. MQ III, which was eluted with 51% NaCl, exhibited gonadotropin releasing hormone activity. Surprisingly, unadsorbed fractions, MQ I, also showed gonadotropin releasing hormone activity. MQ 1 was therefore subjected to FPLC Mono S (a cation exchanger) column chromatography where a highly active gonadotropin releasing hormone enriched peak, i.e., MS III, could be eluted with 45% NaCl. These findings show that Catla catla hypothalamus has two forms of gonadotropin releasing hormones one anionic (carp gonadotropin releasing hormone I) and another cationic (carp gonadotropin releasing hormone II). These two forms of gonadotropin releasing hormones were also active in heterologous carp species, rohu (Labeo rohita), mrigal (Cirrhinus mrigala) and an exotic common carp (Cyprinus carpio). Combined activity of two forms of gonadotropin releasing hormones was significantly greater as compared to any of the single form.
ABSTRACT
Pituitaries were collected from a common carp, yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0·3% collagenase and 0·05% tsypsin. Enzymatically dispersed cells were incubated in vitro in Ca2+-free medium to observe the effect of Canna punctatus GnRH (cGnRH) and Ca2+ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 104 cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca2+ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca2+ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca2+ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca2+-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca2+-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca2+-free medium. Extent of Ca2+ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca2+-carrier protein, Addition of verapamil to this system strongly inhibited Ca2+ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca2+ in an apparently Ca2+-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca2+ and incubation of pituitary cell in Ca2+-free medium is truly not free of Ca2+.