Ethics statement
All experiments were carried out in compliance with the Guide for Care and Use of Laboratory Animals after obtaining institutional ethical clearance from the Institutional Ethics Committee, North-Eastern Hill University, Shillong, India.
Experimental Animals And Their Maintenance
All experiments were conducted on the pineal organ of sexually mature male Clarias gariepinus, which is readily available throughout the year in Shillong. For all experiments, Clarias gariepinus (length: 23–27 cm; body weight: 95–100 g) were procured from local fish suppliers, and acclimatized for 15 days in a fish room under 12 h light: 12 h dark cycle (lights ‘on’ at 06:00 h, lights ‘off’ at 18:00 h) and water temperature 25 ± 2oC at Shillong (Latitude 25o30' N, Longitude 91o52' E; Altitude 1450 ASL). During acclimatization, the fish were fed with commercial dry fish pellets and minced earthworms ad libitum. Water was changed every day to avoid infections.
Pineal organ culture and in vitro treatments
In order to collect the pineal organ, the acclimatized fish was anesthetized with MS-222 (Sigma-Aldrich, USA), decapitated, the pineal window was exposed, the pineal organ was rapidly removed, washed and placed in the culture medium in a multi-wells culture plate (Nunclon™ Delta Surface, Thermo Scientific, USA). All dissections were carried out between 10:30 h – 11:30 h. Pineal organs were collected and pooled into three samples of three pineals each (9 in total), and were used for estimating the relative expression of aanat2 gene. Sterile Dulbecco’s Modified Eagle Medium (DMEM; Gibco Thermo Fisher Scientific, USA) supplemented with Bovine Serum Albumin (BSA, 1mg/ml), Ascorbic acid (0.1 mg/ml), Penicillin-Streptomycin (10 µl/ml), HEPES salt (4.77 mg/ml, Sigma, USA), and calcium carbonate (0.125 mg/ml, HIMEDIA, India) were used for the pineal culture. Pineals were pre-incubated at 25 ± 2°C for 1 h in an atmosphere of 85% O2, 5% CO2 and 95% relative humidity in a gas (O2-CO2) incubator (Heraeus: Cytoperm, Germany).
In vitro treatment of the pineal organ with cAMP, cGMP and selective inhibitors:
In order to study the role of cAMP-dependent protein kinase (PKA) in aanat2 gene regulation, 36 pineal organs were divided into 12 samples of 3 pineal organ each, then 12 samples were divided into 4 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), H-89 (10− 6 M; selective inhibitor of db-cAMP-dependent protein kinase), H-89 (10− 6 M) + db-cAMP (10− 6 M) with a suitable control group.
In order to study the role of cGMP-dependent protein kinase (PKG) in aanat2 gene regulation, 36 pineal organs were divided into 12 samples of 3 pineal organs each, then 12 samples were divided into 4 treatment groups of 3 samples each, and treated with db-cGMP (10− 6 M), KT5823 (10− 6 M; selective inhibitor of cGMP-dependent protein kinase), KT5823 (10− 6 M) + cGMP (10− 6 M) with a suitable control group.
In order to study the role of calcium-dependent protein kinase (PKC) in aanat2 gene regulation, 72 pineal organs were divided into 24 samples of 3 pineal organs each, then 24 samples were divided into 8 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), db-cGMP(10− 6 M), db-cAMP (10− 6 M) + db-cGMP (10− 6 M), chelerythrine chloride (Che, 10− 6 M; specific inhibitor of calcium-dependent protein kinase), Che (10− 6 M) + db-cAMP (10− 6 M), Che (10− 6 M) + db-cGMP (10− 6 M), and Che (10− 6 M) + db-cAMP (10− 6 M) + db-cGMP (10− 6 M) with a suitable control group.
In order to study the role of MeK in aanat2 gene regulation, 72 pineal organs were divided into 24 samples of 3 pineal organs each, then 24 samples were divided into 8 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), db-cGMP (10− 6 M), db-cAMP (10− 6 M) + db-cGMP (10− 6 M), U0126 ethanolate ( UE, 10− 6 M; specific inhibitor of MeK), UE (10− 6 M) + db-cAMP (10− 6 M), UE (10− 6 M) + db-cGMP (10− 6 M), and UE (10− 6 M) + db-cAMP (10− 6 M) + db-cGMP (10− 6 M) with a suitable control group.
In order to study the role of p38 MAP kinase (p38 MAPK) in aanat2 gene regulation, 72 pineal organs were divided into 24 samples of 3 pineal organs each, then 24 samples were divided into 8 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), db-cGMP(10− 6 M), db-cAMP (10− 6 M) + db-cGMP (10− 6 M), SB 202190 monohydrochloride hydrate (SB, 10− 6 M; specific inhibitor of p38 MAP kinase), SB (10− 6 M) + db-cAMP (10− 6 M), SB (10− 6 M) + db-cGMP (10− 6 M), and SB (10− 6 M) + db-cAMP (10− 6 M) + db-cGMP (10− 6 M) with a suitable control group.
In order to study the role of serine/threonine phosphatase type 1 and 2A in aanat2 gene regulation, 72 pineal organs were divided into 24 samples of 3 pineal organs each, then 24 samples were divided into 8 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), db-cGMP (10− 6 M), db-cAMP (10− 6 M) + db-cGMP (10− 6 M), calyculin A (CA, 10− 6 M; specific inhibitor of serine/threonine phosphatase type 1 and type 2A), CA (10− 6 M) + db-cAMP (10− 6 M), CA (10− 6 M) + db-cGMP (10− 6 M), and CA (10− 6 M) + db-cAMP (10− 6 M) + db-cGMP (10− 6 M) with a suitable control group.
In order to study the role of serine/threonine phosphatase type 2B in aanat2 gene regulation, 72 pineal organs were divided into 24 samples of 3 pineal organs each, then 24 samples were divided into 8 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), db-cGMP(10− 6 M), db-cAMP (10− 6 M) + db-cGMP (10− 6 M), cypermethrin (Cyp, 10− 6 M; specific inhibitor of serine/threonine phosphatase type 2B), Cyp (10− 6 M) + db-cAMP (10− 6 M), Cyp (10− 6 M) + db-cGMP (10− 6 M), and Cyp (10− 6 M) + db-cAMP (10− 6 M) + db-cGMP (10− 6 M) with a suitable control group.
In order to study the role of CREB and CBP-CREB interaction in aanat2 gene regulation, 54 pineal organs were divided into 18 samples of 3 pineal organs each, and then 18 samples were divided into 6 treatment groups of 3 samples each, and treated with db-cAMP (10− 6 M), CREB inhibitor (CI, 10− 6 M), CBP-CREB interaction inhibitor (CCII, 10− 6 M), CI (10− 6 M) + db-cAMP (10− 6 M), and CC11 (10− 6 M) + db-cAMP (10− 6 M) with a suitable control group.
In all experiments, after pre-incubation the culture medium was replaced with sterile culture medium containing H-89 dihydrochloride hydrate (selective inhibitor of cAMP-dependent protein kinase, 10− 6 M, Sigma, USA), KT5823 (specific inhibitor of cGMP-dependent protein kinase, 10− 6 M, Sigma, USA), chelerythrine chloride (specific inhibitor of calcium-dependent protein kinase, 10− 6 M, Sigma, USA), U0126 ethanolate (MeK inhibitor, 10− 6 M, Sigma, USA), SB 190202 monohydrochloride hydrate (selective inhibitor of p38 MAP kinase, 10− 6 M) (Sigma, USA), calyculin A (specific inhibitor of serine/threonine phosphatase type 1 and 2A, 10− 6 M, Sigma, USA), cypermethrin (specific inhibitor of serine/threonine phosphatase and 2B, 10− 6 M, Sigma- Aldrich, USA), CREB inhibitor (10− 6 M, 666-15-Calbiochem®; Merck, Germany), and CBP-CREB interaction inhibitor (10− 6 M, CAS 92-78-4-Calbiochem, Calbiochem®; Merck, Germany) for 30 min. The timing of pre-treatment was kept in such a way that each group treated only with H-89, KT5823, chelerythrine chloride, cypermethrin, CREB inhibitor CBP-CREB interaction inhibitor, U0126 ethanolate, SB 190202, dibutyryl-cAMP (db-cAMP, an analogue of cAMP, 10− 6 M) (Enzo, USA), dibutyryl-cGMP (db-cGMP, an analogue of cGMP, 10− 6 M) (Enzo, USA), and db-cAMP (10− 6 M) + db-cGMP (10− 6 M) for 30 min and treatment of pre-treated pineals with db-cAMP (10− 6 M), db-cGMP (10− 6 M) and db-cAMP (10− 6 M) + db-cGMP (10− 6 M), respectively for 30 min were kept the same. Pineal organs incubated in DMEM supplemented with BSA (1mg/ml), CaCO3 (0.125 mg/ml), Ascorbic acid (0.1 mg/ml), Penicillin-Streptomycin (10 µl/ml) and HEPES salt (4.77 mg/ml) without any drugs were treated as control. After treatment with desired drugs for 30 min, the pineal organs were removed and collected in numbered micro-centrifuge tubes containing TRI reagent (500 µL) and processed for isolating total RNA.
Total RNA extraction, cDNA synthesis, and analysis by Quantitative Real-Time PCR
Total RNA was extracted from the pineal organs using TRI reagent (Sigma, USA) by following the manufacturer’s protocol. Then quantity and quality of extracted RNA were checked by using QIAxpert (QIAGEN, Germany). In order to maintain good quality of the samples, RNA samples with a ratio of sample absorbance at 260 and 280 nm between 1.8 and 2.1 were used for all experiments. Synthesis of cDNA was performed using 300 ng of total RNA using Verso cDNA Synthesis Kit (Thermo Scientific, USA) in a total reaction volume of 20 µL containing 4 µL of 5X cDNA synthesis buffer (1X final concentration), 2 µL of dNTP mix (500 µM each), 1 µL of anchored oligo-dt (500 ng/µL), 1 µl of RT Enhancer, 1 µl of Verso Enzyme mix, template RNA and nuclease free water was added to make the volume of reaction mixture 20 µl and then incubated at 42°C for 60 min and then at 95°C for 2 min. qPCR was performed in triplicate reactions along with non-template control and negative control in each plate using a 7500 Fast Real-Time PCR system (Applied Biosystems, USA). The reactions were carried out in a total volume of 10 µl containing power SYBR® Green Master Mix, gene-specific primers, RNase-free water, and cDNA. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was used as the reference gene. Following primers were used:- aanat2 gene-specific primers: CGNAT-F; 5'-ACT GGA CGA GGT GCT GAA CT-3', CGNAT-R; 5'-CGT GGA TGT GGA CAG TAG GA-3', and gapdh gene-specific primers: CGGAP-F; 5'-ACC GGA GTC TTC CTC AGC A-3', CGGAP-R; 5'-TCA TGT TGG AGG GGT CGT A-3' (Singh et al. 2017). Real-Time RT-PCR was performed using the following protocol: 8 minutes at 95oC to activate the enzyme, then 40 cycles of denaturation at 95oC for 15 seconds and annealing at 60oC for 1 minute. Relative expressions of aanat2 mRNA of all samples were calculated using the 2−ΔΔCT (threshold cycle) method (Livak and Schmittgen, 2001).
Statistical analysis
All data were presented as the mean of three independent experimental results ± standard error of the mean. The data for aanat2 gene expression was analysed using One-way ANOVA followed by Tukey’s Multiple Comparison test. A p < 0.05 was considered as significant for checking the levels of significance between control and treated groups or between treated groups. All the data were analysed by using GraphPad Prism V4.03.