K.P. Joy

Development, characterization, conservation and storage of fish cell lines: a review

Cell lines provide an important biological tool for carrying out investigations into physiology, virology, toxicology, carcinogenesis and transgenics. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain, skin. One hundred and twenty-four new fish cell lines from different fish species ranging from grouper to eel have been reported since the last review by Fryer and Lannan (J Tissue Culture Methods 16: 87–94, 1994). Among the cell lines listed, more than 60% were established from species from Asia, which contributes more than 80% of total fish production. This includes 59 cell lines from 19 freshwater, 54 from 22 marine and 11 from 3 brackish water fishes. Presently, about 283 cell lines have been established from finfish around the world. In addition to the listing and a scientific update on new cell lines, the importance of authentication, applications, cross-contamination and implications of overpassaged cell lines has also been discussed in this comprehensive review. The authors feel that the review will serve an updated database for beginners and established researchers in the field of fish cell line research and development.

Induction of maturation and ovulation by pimozide-LHRH analogue treatment and resulting high quality egg production in the Asian catfish, Clarias batrachus (L.)

Abstract In Clarias batrachus (250–310 g), collected from nature in the early spawning season, simultaneous injections of pimozide (a dopamine antagonist) and the luteinizing hormone-releasing hormone analogue (LHRHa), des-Gly10 [D-Ala6]-LHRH ethylamide, caused a high rate of ovulation (85.7%), while injections of either pimozide or LHRHa alone or the vehicle medium did not. The ovulated fish produced an average of 21.15±2.6 g eggs with a very high hatching rate (91.88±1.51%) and a high yield of normal fry (85.5±3.1%). The results suggest that the combination can be used as a simple and efficient method for inducing ovulation in the catfish.

The effect of luteinizing hormone-releasing hormone analogue (LHRHa) in combination with different drugs with anti-dopamine and anti-serotonin properties on gonadotropin release and ovulation in the African catfish, Clarias gariepinus

Under hatchery conditions the reproduction of the African catfish depends on artificial induction of egg maturation and ovulation. In this study the effect of a number of potential psychotropic drugs with variable anti-dopamine and/or anti-serotonin properties in combination with LHRHa on gonadotropin release and ovulation was investigated. Drugs with a potent anti-dopaminergic character caused a preovulatory gonadotropin surge, which seems to be independent of their anti-serotonergic properties. One drug, however, did not follow this rule. Drugs exhibiting low interaction with dopamine and high interaction with serotonin receptors had no effect on the LHRHa-induced gonadotropin release.

Toxic effects of three mercurial compounds on survival, and histology of the kidney of the catfish Clarias batrachus (L.).

In Clarias batrachus methylmercuric chloride (CH3HgCl) was 1.18 and 10.05 times more toxic than mercuric chloride (HgCl2) and emisan 6 (methoxyethyl mercury chloride), respectively, for 96 hr. A similar trend was observed at 24, 48, and 72 hr. Exposure of the catfish to sublethal concentrations of these mercurials for 14 and 28 days caused progressive changes in the kidney. The diameter of the proximal convoluted tubules was increased, the epithelial cells were hypertrophied, and the lumen was reduced and filled with secretory material and sloughed-off cells. In 14-day emisan 6-treated fish, some glomeruli were also degenerated. Exposure of the catfish to toxicologically safe concentrations of the mercurials for 90 days caused vacuolation, atrophy, and extensive damage of the tubules in HgCl2-and CH3HgCl-treated fish, and hypertrophy and vacuolation of epithelial cells of some tubules in the emisan 6-treated fish. Regeneration was noticed in the 90-day HgCl2-exposed group. After 180 days of exposure, however, histology of the kidney appeared normal in all the groups.

Immunocytochemical localization, HPLC characterization, and seasonal dynamics of vasotocin in the brain, blood plasma and gonads of the catfish Heteropneustes fossilis

Immunocytochemical distribution and dynamics of vasotocin (VT) were studied in the air-breathing catfish Heteropneustes fossilis in relation to the reproductive cycle. Vasotocin was localized in the brain and ovary by streptavidin-biotin immunocytochemistry. The immunoreactivity was found throughout the hypothalamo-hypophysial neurosecretory system consisting of the magnocellular and parvocellular neurons of the nucleus preopticus, neurosecretory axonal tract and neurohypophysis (NH). The VT neurons showed seasonal changes; they were numerically less in resting phase but increased during the recrudescent phase. The neurons were hypertrophied and degranulated in pre-spawning phase and heavily degranulated and vacuolated in spawning phase. In the NH, the density of VT fibers increased up to the pre-spawning phase and decreased thereafter. In the ovary, VT immunoreactivity was noticed in the follicular layer and varied with the growth of the follicles. Vasotocin was characterized and quantified by a high performance liquid chromatography with UV detection method in the brain, plasma and ovary. Brain and plasma VT concentrations were also assayed with an EIA method, which was more sensitive than the HPLC method with values about 2-fold higher. Vasotocin levels showed significant seasonal and sexual differences with higher concentrations in females in the recrudescent (preparative, pre-spawning and spawning) phase. Brain VT recorded the highest concentration in the preparative phase (both sexes) while plasma (both sexes) and ovarian VT in the spawning phase. The ovarian concentration of VT was 15- and 25-fold higher in the pre-spawning and spawning phases (when expressed per mg protein), respectively, than plasma but lower than brain levels. In testis, VT concentration was relatively low and apparently did not show any significant seasonal variation. The seasonal activity patterns and gonadal distribution of VT indicate a reproductive function of the peptide.

Relative in vitro seasonal effects of vasotocin and isotocin on ovarian steroid hormone levels in the catfish Heteropneustes fossilis

In the present investigation, catfish (Heteropneustes fossilis) ovarian tissues were incubated in vitro with vasotocin (VT) or isotocin (IT) to demonstrate their effects on estradiol-17beta (E(2)), progesterone (P(4)), 17alpha-hydroxy-4-pregnene-3, 20-dione (17-P) and 17alpha, 20beta-hydroxy-4-pregnen-3-one (17, 20beta-DP). Parallel incubations with human chorionic gonadotropin (hCG) alone or in combination with VT were used for a comparison. In pre-vitellogenic phase (preparatory phase, GSI-0.48+/-0.03%), both VT and hCG stimulated E(2) significantly, VT in a biphasic manner and hCG in a dose-dependent manner. In pre-spawning (post-vitellogenic, GSI-9.05+/-0.11%) and spawning (post-vitellogenic, GSI-8.01+/-0.12%) phases, both hormones decreased E(2) levels in a dose- and duration-dependent manner; the VT effect being biphasic in the spawning phase. The co-incubation with VT+hCG stimulated E(2) in the preparatory phase but inhibited it in the pre-spawning and spawning phases. The incubations with VT or hCG increased P(4) levels in a dose- and duration-dependent manner, the magnitude of the effect was higher in the pre-spawning and spawning phases. The co-incubation with VT+hCG stimulated P(4) without any additive effect. The P(4) derivatives (17-P and 17, 20beta-DP) showed similar changes except 17-P in the spawning phase which decreased at 16h of the incubation. The incubations with IT produced similar but low responses. In conclusion, like hCG, VT has differential effects on ovarian steroidogenesis and may be involved directly or indirectly in ovarian functions, as a paracrine/autocrine factor or a neurohormone.

Seasonal and Daily Variations in Hypothalamic Monoamine Levels and Monoamine Oxidase Activity in the Teleost Channa Punctatus (Bloch)

In Channa punctatus, a significant daily variation in hypothalamic 5-HT level and monoamine oxidase (MAO) activity was noticed in preparatory phase (February), but not in prespawning (May) or postspawning (November) phases. Hypothalamic dopamine (DA) and noradrenaline (NA), on the other hand, showed marked daily variation in their levels during all the three seasons with peak values in the photophase. The overall activity of MAO (mean +/- SEM on 24-hr period) increased from November to May through February, whereas the 5-HT content which was high in November decreased during February and May. The NA and DA levels were low in November and February and high in May. The catecholamine (CA) content and MAO activity increased with increasing photoperiod and temperature which is indicative of an enhanced CA metabolism.

Seasonal effects of administration of melatonin and 5-methoxytryptophol on ovarian activity in the catfish Heteropneustes fossilis (Bloch)

k >y KP, Agha AK. Seasonal effects of administration of melatonin and 5‐methoxytryptophol on ovarian activity in the catfish Heteropneustes,ti,ssilis (Bloch). J Pineal Res 1991:10:65‐70.

Pineal-gonadal relationship in the teleost Channa punctatus (Bloch): Evidence for possible involvement of hypothalamic serotonergic system

Abstract: In Channa punctatus, pinealectomy results in an accelerated growth (stimulatory effect) of the ovary in preparatory phase (March), but had no significant effect in prespawning (May‐June) or postspawning (September) phases. Administration of melatonin (25 (μg/fish or 0.4 mg/kg BW at 2‐day intervals for 30 days) inhibits the stimulatory effect of a long photoperiod and high temperature (16L:8D; 22 ± 1°C) on the ovary in the early preparatory phase (February). In the late preparatory phase (April), the administration of both melatonin (0.5 mg/kg BW at 2‐day intervals) and parachlorophenylalanine (pCPA, a serotonin synthesis blocker; 100 mg/kg BW at 3‐day intervals) for 30 days inhibits ovarian activity in comparison to that of the saline‐treated control group. In the dose‐response study, ≥0.5 mg/kg BW of melatonin induced a significant increase in hypothalamic 5‐HT content and ≥10 mg/kg BW of pCPA decreased it significantly. In the time‐course study, melatonin (2.0 mg/kg BW) elevates the 5‐HT content significantly after an hr of the injection and maintained it up to 48 hrs. The administration of pCPA (200 mg/kg BW) had significantly inhibited the 5‐HT content which was sustained for 72 hr. In another study, a single injection of melatonin (0.5 mg/kg BW) increased the 5‐HT content significantly. A single injection of pCPA (100 mg/kg BW) decreased significantly both the content and activity of 5‐HT. It is inferred that hypothalamic 5‐HT may play a central role in photosexual mechanisms and mediate long photoperiodic effects on neuroendocrinereproductive axis.

Brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: annual and circadian variations, and sex and regional differences in enzyme activity and some kinetic properties.

Dynamics of tyrosine hydroxylase (TH) was studied in various brain regions and pituitary in relation to annual/seasonal and circadian variations in either sex of the catfish Heteropneustes fossilis. alpha-Methylparatyrosine inhibited TH activity significantly in a time-(in vivo study) and concentration-(in vitro study) dependent manner. The inhibition was higher in resting phase than preparatory phase and in females than males (time-course study). The apparent kinetic constant of inhibition (K(i)) varied both seasonally and regionally. The values were higher in resting phase and in the forebrain regions (telencephalon and hypothalamus) than medulla oblongata. No significant sex difference was noticed in the K(i) values in the same season. TH activity showed significant annual/seasonal variations with telencephalon and hypothalamus showing higher activity than medulla oblongata. The females showed significantly higher enzyme activity than males with a distinct activity peak in June (prespawning phase). Such an activity peak was apparently absent in males. TH activity showed significant circadian/diurnal variations, the highest activity was noticed at 12 h and the lowest at 24 h. The apparent Michaelis-Menten constant (K(m)) values (hypothalamic TH) for substrate (L-tyrosine) and cofactor (DMPH(4)) showed seasonal variations with the highest values in the resting phase, decreasing through preparatory and prespawning phases, to the lowest values in spawning phase. V(max) was the lowest in the resting phase and highest in the spawning phase. TH activity was low in the pituitary and could be detected in pooled samples from March (preparatory phase) to July (spawning phase). Activity showed significant variations, which could be correlated with the gonadosomatic index. The results show that TH activity could be positively correlated with the annual reproductive cycle. The enzyme activity could be also correlated with seasonal, sex, and regional variations in the apparent K(m) and V(max) values suggesting apparent differences in the affinity of the enzyme towards substrate and cofactor.