In the catfish Heteropneustes fossilis and Clarias batrachus, ovarian oestrogen-2-hydroxylase (OE-2-H) activity increased significantly at 8 h after the injection of an ovulatory dose (0.15 microg/g body weight) of a mammalian GnRH analogue ([d -Ala(6)-Pro(9)]-LHRH ethylamide) and was restored to the 0 h (control) level after egg-stripping at 16 h. On the other hand, ovarian oestradiol-17beta (OE2) level and catechol-O-methyltransferase (COMT) activity decreased significantly at 8 h. While the OE2 level was restored to the 0 h level, COMT activity increased significantly at 16 h. Changes in ovarian OE2 level and enzymes indicate higher synthesis of 2-hydroxylated catecholoestrogens and their degradation during the periovulatory period. Under in vitro conditions, the synthetic catecholoestrogens (CEs, 2- and 4-hydroxylated oestradiol17beta and oestrone (OE1)) induced germinal vesicle break down (GVBD) in a dose- (0.01-10 microg/ml) and duration- (1-36 h) dependent manner, the mean values of the responses being in the order 2-OH OE2>4-OH OE2> 2-OH OE1>4-OH OE1. The CE-induced GVBD response (8 h induction) was not blocked by prior and subsequent incubations with steroid synthesis inhibitors (cyanoketone, epostane and aminoglutethimide) up to 36 h, suggesting that de novo steroidogenesis is not essential for the response. The percentage of GVBD response to 2-h induction by CEs was significantly inhibited by actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor), indicating the involvement of both RNA and protein synthesis. The CE-induced 8-h stimulation of GVBD was mildly blocked by propranolol, the beta-adrenergic inhibitor, suggesting the response was partly mediated through a beta-adrenergic receptor mechanism. Incubations with phentolamine, an alpha-adrenergic inhibitor, did not interfere with the CE-induced GVBD response. The results demonstrate CE-related enzymatic changes in teleost (catfish) ovaries and maturation-inducing substance activity of CEs.
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R Chaube and KP Joy
In Heteropneustes fossilis, ovariectomy inhibited in vivo brain (hypothalamus-pituitary, telencephalon and medulla oblongata) tyrosine hydroxylase (TH) activity with significant effects in weeks 2, 3, 4 and 5 of the gonadal resting phase and in weeks 3, 4 and 5 of the prespawning phase (P<0.05, Tukey's test). Oestradiol-17beta (OE(2)) replacement in 3-week ovariectomised fish produced biphasic responses in both seasons; the low dosages of 0.05 and 0.5 micro g/g body weight (BW) elevated TH activity, whereas the high dosages of 1.0 and 2.0 micro g/g BW decreased it. The magnitude of the inhibition was higher in the resting phase than in the prespawning phase. The inhibitory effect of ovariectomy may be produced by elevating the apparent K(m) values (decreased affinity) of the enzyme for both L-tyrosine (substrate) and dimethyltetrahydropteridine (cofactor) and consequently decreasing the V(max). Significant changes (P<0.05) in both these parameters were noticed but showed minor differences with regard to the length of ovariectomy, season or brain regions. The biphasic effects of OE(2) replacement on TH activity seemed to be produced by differential effects on apparent K(m) and V(max). The stimulatory effect of the low dosages of OE(2) coincides with a decrease in the apparent K(m) values (increased affinity) for both substrate and cofactor and an increase in the V(max) of the enzyme. The inhibitory effect of the high dosages of OE(2) correlated with an increase in the apparent K(m) values (decreased affinity) for both substrate and cofactor, and a decrease in the V(max) compared with the lower dosage groups. The results strongly suggested that OE(2) can modulate brain catecholaminergic activity at the level of tyrosine hydroxylation which, in turn, may alter gonadotrophin secretion. OE(2) may elicit biphasic effects by differentially altering the enzyme affinity towards the substrate and cofactor.
R Chaube and KP Joy
In the female catfish Heteropneustes fossilis, administration of thyroxine (T(4))(,) 1 micro g/g body weight, i.p., in both gonadal resting and preparatory phases for 7, 14 and 21 days caused hyperthyroidism, as evidenced from a duration-dependent significant increase in serum triiodothyronine (T(3)), and of tyrosine hydroxylase (TH) activity in telencephalon, hypothalamus-pituitary and medulla oblongata (Newman-Keuls' test; P<0.05). Hypothyroidism induced by adding 0.03% thiourea to aquarium water holding the catfish for 7, 14 and 21 days decreased serum T(3) levels in a duration-dependent manner (Newman-Keuls' test; P<0.05) and inhibited TH activity in the brain regions. T(4) replacement in 21day thiourea-treated fish restored and even elevated significantly serum T(3) levels as well as brain TH activity in a duration-dependent manner. In general, the changes in enzyme activity were higher in the forebrain regions than medulla oblongata and in the resting phase than preparatory phase. Kinetic studies by Lineweaver-Burk plots showed that the stimulatory effect following T(4) administration and T(4) replacement on TH activity was due to increased affinity of the enzyme for its cofactor (6,7-dimethyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine), as evident from a significant decrease in apparent Michaelis-Menten constant (K(m)) and an increase in apparent velocity maximum (V(max)). The TH inhibition due to the thiourea treatment can be related to decreased affinity of the enzyme for its cofactor, as evident from a significant increase in apparent K(m) value and a significant decrease in V(max). These data clearly show that circulating levels of T(4)/T(3) modulate brain TH activity by altering the kinetic properties of the enzyme, which, in turn, influence catecholaminergic activity and dependent functions.
KP Joy, B Senthilkumaran, and CC Sudhakumari
In Heteropneustes fossilis, administration of a single dose (0.15 micrograms/g body weight, i.p.) of [D-Ala6,Gly10]-gonadotrophin-releasing hormone analogue (GnRHa) induced ovulation (in 35 of 35 fish) when mild-stripped at 16 h. Plasma gonadotrophin II (GTH II) levels showed a highly significant increase at 2, 4, 8, 12 and 16 h with the peak at 8 h. Plasma cortisol, progesterone and testosterone showed significant elevations at 2, 4, 8 and 12 h with peaks at 8 h (cortisol and testosterone) and 4 h (progesterone). The levels declined to control values at 16 and 48 h except that of testosterone which decreased even further. In contrast, plasma levels of oestradiol-17 beta decreased significantly at 2, 4, 8 and 12 h, with the lowest value at 8 h, but increased at 16 and 18 h. The contents of hypothalamic and pituitary serotonin and noradrenaline increased at 8 h, coinciding with the peak GTH II rise, and decreased at 16 h. In contrast, dopamine content declined at 8 h in both the hypothalamus and pituitary, but increased at 16 h only in the hypothalamus. The hypothalamic adrenaline level decreased at 8 h but increased significantly at 16 h. Hypothalamic levels of monoamine oxidase, catechol O-methyltransferase and dopamine beta-hydroxylase were elevated significantly at 8 h; the dopamine beta-hydroxylase activity decreased at 16 h. Phenylethanolamine N-methyltransferase activity was elevated only at 16 h, coinciding with the rise in adrenaline content. It is inferred that the preovulatory decrease in dopamine content concomitant with rises in serotonin and noradrenaline levels, triggered by the low titre of oestradiol, might have potentiated the GnRHa/GnRH (endogenous)-induced release of GTH II for a prolonged period.