Thyroid hormone showed specific binding ability to mouse granulosa cells from immature mice, primed with post menopausal gonadotropin. Saturation of specific binding sites was reached by 2 nM concentration of the hormone. A Scatchard analysis of thyroid hormone binding exhibited a K_d of 42 x l0_-9M/mg nuclear DNA and a maximum binding capacity of 1 pmol/mg nuclear DNA. Competitive inhibition studies showed thyroid hormone binding to be analogue specific. Addition of 100 ng of thyroid hormone to granulosa cell incubations (1 x 10⁶ cells/well) resulted in a three-fold increase in cellular protein synthesis. Thyroid hormone resulted in a dose dependant increase in progesterone release from granulosa cell. It also stimulated the formation of pregnenolone (83%) and progesterone (81%) from radiolabeled cholesterol as compared to control. This stimulation by thyroid hormone was completely inhibited by cycloheximide. Results indicate a direct effect of thyroid hormone on granulosa cells, its binding to nuclei causing an increase in steroidogenesis through the mediation of protein(s).

Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami ofCatla catla. Gonadotropin releasing hormone activity was studiedin 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 (ACI, ACII and ACIII), 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 SGIII) and only S G 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 thatCatla 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.

Meiotic arrest of oocyte in an Indian carp,Labeo rohita Ham. has been found for the first time to be withdrawn by insulin only. Addition of insulin to oocytesin vitro caused germinal vesicle breakdown (GVBD), one of the first visual markers to determine initiation of the final maturational process. Under the influence of insulin the germinal vesicle (GV) of the oocyte migrated towards the animal pole, reached the micropyle and then dissolved (GVBD). By using different concentrations of insulin i.e., 0.063, 0.63, 6.3 and 12.6 (AM, optimum amount required was found to be 6.3 (AM. Induction of GVBD by insulin could be blocked by cycloheximide (Chx), a translation inhibitor, while actinomycin D (AcD) had no effect suggesting non-involvement of transcriptional activity in this process. Addition of the maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (DHP) stimulated (P < 0.01) GVBD of carp oocytes and its combination with insulin showed an additive effect. Gonadotropin (GtH) caused GVBD but its effect was greatly augmented by insulin. Our results demonstrate that not only can insulin alone induce GVBD in carp oocytes, but it also augments the stimulatory effect of DHP or IGF-I or GtH on GVBD. This information will be important in hormonal manipulation during induced breeding of carp.