Molecular Characterization and Expression of Cytochrome P450 Aromatase in Atlantic Croaker Brain: Regulation by Antioxidant Status and Nitric Oxide Synthase During Hypoxia Stress.

Md Saydur Rahman,Peter Thomas

doi:10.3389/fphys.2021.720200

Md Saydur Rahman, Peter Thomas

Open Access

https://doi.org/10.3389/fphys.2021.720200

Copy DOI

Abstract

We have previously shown that nitric oxide synthase (NOS, an enzyme) is significantly increased during hypoxic stress in Atlantic croaker brains and modulated by an antioxidant (AOX). However, the influence of NOS and AOX on cytochrome P450 aromatase (AROM, CYP19a1, an enzyme) activity on vertebrate brains during hypoxic stress is largely unknown. In this study, we characterized brain AROM (bAROM, CYP19a1b) cDNA in croaker and examined the interactive effects of hypoxia and a NOS-inhibitor or AOX on AROM activity. The amino acid sequence of croaker bAROM cDNA is highly homologous (76–80%) to other marine teleost bAROM cDNAs. Both real-time PCR and Northern blot analyses showed that bAROM transcript (size: ∼2.8 kb) is highly expressed in the preoptic-anterior hypothalamus (POAH). Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 4 weeks) caused significant decreases in hypothalamic AROM activity, bAROM mRNA and protein expressions. Hypothalamic AROM activity and mRNA levels were also decreased by pharmacological treatment with N-ethylmaleimide (NEM, an alkylating drug that modifies sulfhydryl groups) of fish exposed to normoxic (DO: ∼6.5 mg/L) conditions. On the other hand, treatments with Nω-nitro-L-arginine methyl ester (NAME, a competitive NOS-inhibitor) or vitamin-E (Vit-E, a powerful AOX) prevented the downregulation of hypothalamic AROM activity and mRNA levels in hypoxic fish. Moreover, NAME and Vit-E treatments also restored gonadal growth in hypoxic fish. Double-labeled immunohistochemistry results showed that AROM and NOS proteins are co-expressed with NADPH oxidase (generates superoxide anion) in the POAH. Collectively, these results suggest that the hypoxia-induced downregulation of AROM activity in teleost brains is influenced by neuronal NOS activity and AOX status. The present study provides, to the best of our knowledge, the first evidence of restoration of AROM levels in vertebrate brains by a competitive NOS-inhibitor and potent AOX during hypoxic stress.

Highlights

The teleost brain is a major target organ for investigating the molecular, cellular, physiological, neuroendocrine, and behavioral responses of fishes to environmental stressors (Zohar et al, 2009; Diotel et al, 2011)
In marked contrast to findings in the majority of other teleost species, higher cytochrome P450 aromatase (AROM) activities were detected in the olfactory bulb (OB), TEL, hypothalamus and pituitary of male than in female seabass and pituitary brain aromatase (bAROM) mRNA expression was greater in male South American catfish than in females (Gonzalez and Piferrer, 2003; de Assis et al, 2018). These results show that the bAROM gene is highly expressed in brain tissues in fish, ubiquitously found in the entire hypothalamic region, and show sex differences in expression, these sex differences are observed in different brain regions in the various teleost species examined to date (Garcia-Segura et al, 2003; Piferrer and Blázquez, 2005; Roselli, 2007). bAROM is presumed to be localized in radial glial cells in croaker hypothalami, this was not confirmed in the present study, because it has been shown to be localized in glial cells in the brains of all the teleost species examined to date (Forlano et al, 2001; Page et al, 2010; Diotel et al, 2011; Xing et al, 2014; Chaube et al, 2015)
The present results show that hypoxia exposure drastically decreases in bAROM mRNA and protein expressions and AROM activity in hypothalamic tissues, which is accompanied by decreases in GSI of both and female croaker, in agreement with our field results from hypoxic sites in the northern Gulf of Mexico (Thomas and Rahman, 2012)

Summary

Introduction

The teleost brain is a major target organ for investigating the molecular, cellular, physiological, neuroendocrine, and behavioral responses of fishes to environmental stressors (Zohar et al, 2009; Diotel et al, 2011). Most tetrapods have a single AROM (CYP19a1) transcript in the brain, gonads, and other tissues, while teleost fishes have two distinct AROM transcripts: (i) an ovarian-type AROM (oAROM, CYP19a1a) transcript expressed mainly in the ovary, and (ii) a brain-type AROM (bAROM, CYP19a1b) transcript expressed mainly in the brain (Tchoudakova and Callard, 1998; Gonzalez and Piferrer, 2002, 2003; Piferrer and Blázquez, 2005; Diotel et al, 2011). The cellular expression of AROM in the Abbreviations: AROM, cytochrome P450 aromatase; bAROM, brain-type aromatase (Cyp19a1b); oAROM, ovarian-type aromatase (Cyp19a1a); NOS, nitric oxide synthase; ROS, reactive oxygen species; RNS, reactive nitrogen species; NEM, N-ethylmaleimide; AOX, antioxidant; NAME, Nω-nitro-L-arginine methyl ester; RT-PCR, reverse-transcription polymerase chain reaction; RACE, rapid amplification of cDNA ends; GSP, gene specific primer; UTR, untranslated region; ORF, open reading frame; DO, dissolved oxygen; POAH, preoptic-anterior hypothalamus; PIT, pituitary; OB, olfactory bulb; MT, mid-brain tegmentum; TEL, telencephalon

Objectives

Methods

Results

Discussion

Conclusion