Cloning and characterization of estrogen hydroxylase (cyp1a1 and cyp1b1) genes in the stinging catfish Heteropneustes fossilis and induction of mRNA expression during final oocyte maturation.

Estrogen hydroxylases (EHs) are cytochrome P450 Family 1 (Cyp1, Clan 2) proteins involved in estrogen hydroxylations at 2-, 4- or 16- carbon positions to form catecholestrogens. EHs are encoded by CYP1A1, CYP1A2 and CYP1B1 in mammals. In the catfish Heteropneustes fossilis, cyp1a1 and cyp1b1 cDNAs were cloned and characterized from liver and ovary. The cyp1a1 cDNA is 2071 bp long and codes for a 518 amino acids (aa) long protein. The cloned cyp1b1 cDNA is 1927 bp long and codes for a 509 residue protein. The deduced proteins clustered distinctly into teleost Cyp1a1 and Cyp1b1 clades, distinct from the tetrapod clusters and featured common function domains and homology with other teleost proteins. In the qPCR assay, the transcripts were the most abundant in the liver, followed by brain and ovary, and moderate in gill, kidney and muscle. Evidence was presented to show the involvement of the genes in reproduction. Expression of brain and ovarian transcripts showed significant seasonal variations with the highest abundance in the spawning phase. In situ hybridization showed the transcripts in the follicular layer (theca and granulosa) of the ovarian follicles. Periovulatory changes in the expression cyp1a1 and cyp1b1 were obtained during final oocyte maturation (FOM) and ovulation induced by human chorionic gonadotropin (hCG), both in vivo and in vitro, and by 2-hydroxyestradiol-17ß (catecholestrogen) in vitro. In the brain, the transcript levels increased with time but in the ovary, the increase was maximal at 16 h and decreased at 24 h. The periovulatory activation of the cyp1 genes was reported in this study and discussed on the basis of complex regulation of FOM and ovulation.

Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17ß on mRNA expression.

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish.

Use of a radioimmunoassay which detects C21 steroids with a 5beta-reduced, 3alpha-hydroxylated configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

Two radioimmunoassays (RIAs) have been developed which detect C21 (pregnane) steroids with a 5beta-reduced, 3alpha-hydroxyl (5beta, 3alpha) configuration. One RIA only detects 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one and 3alpha, 17-dihydroxy-5beta-pregnan-20-one, whilst the other detects a range of 5beta,3alpha steroids, including 5beta-pregnane-3alpha,17,20 beta-triol, a major metabolite of 17, 20beta-dihydroxy-4-pregnen-3-one, the putative oocyte maturation-inducing steroid in plaice Pleuronectes platessa. The RIAs, in conjunction with reverse-phase high-performance liquid chromatography (HPLC), have identified and quantified the steroids in plasma and urine of reproductively mature females. Total levels of 5beta,3alpha metabolites which can be extracted with diethyl ether (i.e., free steroids) are relatively low (<10 ng/ml). However, total levels of 5beta,3alpha metabolites released by solvolysis (i.e. , sulphated steroids) are very high (up to 1000 ng/ml in plasma and 20 microg/ml in urine). On HPLC, these metabolites have been identified (in order of their abundance in plasma) as: 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one, 5beta-pregnane-3alpha,17, 20beta-triol, 5beta-pregnane-3alpha,17,20alpha-triol, 3alpha,11beta, 17,21-tetrahydroxy-5beta-pregnane-20-one, and 3alpha, 17-dihydroxy-5beta-pregnan-20-one. Levels of the first three steroids are significantly elevated in female plaice undergoing natural or gonadotrophin-induced final oocyte maturation.

Use of a radioimmunoassay which detects C21 steroids with a 17, 20beta-dihydroxyl configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

A radioimmunoassay (RIA) has been developed to detect a range of C21 (pregnane) steroids with a 17,20beta-dihydroxyl (17,20beta) configuration. In conjunction with reverse-phase high-performance liquid chromatography (HPLC), it identifies and quantifies the metabolites of 17,20beta-dihydroxy-4-pregnen-3-one, the putative "maturation-inducing steroid" in female plaice Pleuronectes platessa. Total levels of 17,20beta metabolites which can be extracted from plasma or urine with diethyl ether (i.e., free steroids) are very low (<3 ng/ml). However, total levels of 17,20beta metabolites which can be released by solvolysis (i.e., sulphated steroids) are very high (up to 1 microg/ml in plasma and 10 microg/ml in urine). On HPLC, these sulphated metabolites have been identified (in order of abundance in plasma) as: 5beta-pregnane-3alpha,17,20beta-triol, 5beta-pregnane-3beta,17,20beta-triol, 17, 20beta-dihydroxy-4-pregnen-3-one, and 17, 20beta-dihydroxy-5beta-pregnan-3-one. These steroids are absent from plasmas of fish which have not yet begun final oocyte maturation. The results support the hypothesis that 17, 20beta-dihydroxy-4-pregnen-3-one is the maturation-inducing steroid in plaice but that it is rapidly metabolised to render it inactive. The results also show that the '17,20beta'-RIA, in combination with an overnight acid solvolysis procedure, is a useful procedure for monitoring the effects of exogenous factors (such as gonadotrophin injections) on final oocyte maturation in female plaice.

In vitro effect of mammalian pituitary hormones on germinal vesicle breakdown in oocytes of major carps, Labeo rohita, Cirrhinus mrigala, Catla catla and Cyprinus carpio.

Among all the mammalian pituitary hormones, luteinizing hormone (LH) was the most potent in vitro inducer of oocyte maturation in L. rohita, C. mrigala, C. catla and C. carpio. It induced significant germinal vesicle breakdown (GVBD) at concentrations of 10, 1, 0.1 and 0.01 micrograms/ml. At the highest concentration used, LH induced 77.9 +/- 5.9, 73.8 +/- 4.6, 50.3 +/- 2.8 and 40.8 +/- 1.4% GVBD in oocytes of L. rohita, C. mrigala, C. catla and C. carpio, respectively. Among other hormones, follicle stimulating hormone (FSH) induced only a marginally significant GVBD (13.2 +/- 0.8%) in the oocytes of C. carpio, but not in other three species. Thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) had no effect on GVBD.

In vitro effects of cyanoketone and epostane on LH-induced germinal vesicle breakdown in oocytes of Indian major carps.

The effect of cyanoketone (inhibitor of delta 5-3 beta-hydroxysteroid dehydrogenase) and epostane (inhibitor of 3 beta-hydroxysteroid dehydrogenase) on LH-induced in vitro germinal vesicle breakdown (GVBD) in the oocytes of Indian major carps, Labeo rohita, Cirrhinus mrigala, and Catla catla, was investigated using five concentrations for each inhibitor. Both of these inhibitors could significantly inhibit the LH-induced GVBD at all of their concentrations in L. rohita and C. catla. However, in C. mrigala, when the oocytes were incubated with cyanoketone or epostane along with LH, the rate of GVBD was not induced significantly at their lowest concentration but four other higher concentrations could inhibit the LH-induced GVBD. A probable mechanism of inhibition of the oocyte maturation by these inhibitors is discussed in the light of available literature.

Relativein vitro effectiveness of estradiol-17ß, androgens, corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown in oocytes of Indian major carps,Labeo rohita, Cirrhinus mrigala andCatla catla.

The relative effectiveness of estradiol-17ß, androgens, corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown (GVBD) was investigatedin vitro using folliculated oocytes of three carps,Labeo rohita, Cirrhinus mrigala, andCatla catla. In all three species progesterone and 17α-hydroxyprogesterone could induce GVBD but relatively 17α,20ß-dihydroxyprogesterone was consistently found to be the most potent maturation-inducing steroid. Both estradiol-17ß and testosterone were ineffective in inducing GVBD. Androsterone and dehydroepiandrosterone were found to be effective inC. catla at all the concentrations used. Deoxycorticosterone (DOC), hydrocortisone (HC) and cortisone were effective inducer of GVBD inC. catla whereas inL. rohita andC. mrigala only cortisone was found to be effective. All 5ß-reduced pregnenes were effective in inducing GVBD inL. rohita but inC. catla, only 5ß-pregnane-17α-01-3,20-dione and 5ß-pregnane-3α,17α,20ß-triol and inC. mrigala, 5ß-pregnane-3α-ol-20- one could induce oocyte maturation.

Effect of malathion and endosulfan on brain acetylcholinesterase and ovarian steroidogenesis of Channa punctatus (Bloch).

The effect of sublethal concentrations of malathion (organophosphorus insecticide) and endosulfan (organochlorine insecticide) was investigated in Channa punctatus. Brain acetylcholinesterase (AchE) and ovarian delta 5,3 beta-hydroxysteroid dehydrogenase (delta 5,3 beta-HSD) and glucose-6-phosphate dehydrogenase (G-6-PD) activities were studied. Apart from the loss of stage II and III oocytes, the absence of delta 5,3 beta-HSD and G-6-PD activity indicating the inhibition of steroidogenesis was seen in the malathion- and endosulfan-treated fish ovaries. Malathion demonstrated a dose-dependent inhibition of brain AchE activity, whereas endosulfan caused no significant reduction of AchE activity.

Relative toxicity of technical material and commercial formulation of malathion and endosulfan to a freshwater fish, Channa punctatus (Bloch).

Relative toxicity of technical and commercial formulations of malathion and endosulfan were evaluated by determining LC50 values and their 95% confidence interval end points for 24, 48, 72, and 96 hr exposure to Channa punctatus using the "trimmed Spearman-Karber method." The commercial formulations of malathion and endosulfan were 1.176 and 1.88 times more toxic than their technical materials, respectively. Ninety-six-hour LC50 values (0% trimming) of technical and commercial formulations of malathion and endosulfan (95% confidence interval in parentheses) were 4.51 (4.11-4.96) and 3.89 (3.46-4.38) mg/liter, and 5.78 (4.49-7.44) and 3.07 (2.43-3.84) micrograms/liter, respectively. Fishes showed characteristic changes in behavior when exposed to various concentrations of these insecticides.