Elsevier

Aquaculture

Volumes 350–353, 20 June 2012, Pages 1-7
Aquaculture

Effects of sex steroids on expression of myostatin in rare minnow, Gobiocypris rarus

https://doi.org/10.1016/j.aquaculture.2012.04.031Get rights and content

Abstract

Myostatin (MSTN), belonging to transforming growth factor (TGF) β superfamily, is a negative regulator of muscular development in mammals and fish. The expression of MSTN can be regulated by hormone and environmental factors. Sex steroids, androgen and estrogen can affect sex differentiation and also can affect muscle development. However, few documents are concerned about the effects of sex steroids on MSTN expression in fish. Here, we report the cloning and expressional analyses of MSTN in rare minnow (Gobiocypris rarus), an emerging model fish in China. The MSTN cDNA of rare minnow that has an open reading frame of 1128 base pairs (bp) encodes a protein of 375 amino acids belonging to fish MSTN1 with typical structures like a prodomain, a mature MSTN domain, a conservative hydrolytic site (RIRR) and 9 conservative cysteine residues in mature MSTN. MSTN was detectable in all the examined tissues by RT-PCR that indicated the ubiquitous expression of MSTN in adult tissues. MSTN was detectable from zygote till hatching showed the maternal deposition of MSTN mRNA from oocytes and continuous expression during embryogenesis. Treatment of rare minnow with 1 μg/L 17α-methyl testosterone (MT) or 17α-ethynyl estradiol (EE2) significantly impaired the body growth and increased the expression of MSTN mRNA detected by quantitative real time PCR. However, low dosage of MT (10 ng/L) had no significant effects on body growth and MSTN expression. The results suggest that the effects of exogenous sex steroids on growth can be mediated through MSTN. It is also suggested that low concentration of sex steroid (less than10 ng/L) may have little effect on MSTN expression.

Highlights

► MSTN1 named GrMSTN was isolated from rare minnow, Gobiocypris rarus. ► GrMSTN is maternal factor and expressed ubiquitously in adult tissues in rare minnow. ► 1 μg/L sex steroids significantly decrease body growth and increase MSTN expression. ► It is suggested that sex steroids can affect body growth through MSTN expression.

Introduction

Myostatin (MSTN), a member of transforming growth factor (TGF) β superfamily, negatively regulates muscular development in mammals (Grobet et al., 1997, Kambadur et al., 1997, Lee and McPherron, 2001, McPherron and Lee, 1997, McPherron et al., 1997). Fish have two MSTN genes, MSTN1 and MSTN2, involved in muscular development (Acosta et al., 2005, Amali et al., 2004, Amali et al., 2008, Biga et al., 2005, Kerr et al., 2005). Knockdown of MSTN1 induced giant phenotype and up-regulation of muscular specific genes in zebrafish (Danio rerio) (Acosta et al., 2005, Amali et al., 2004). Transgenic medaka (Oryzias latipes) with expression of dominant-negative MSTN1 exclusively in muscle increased skeleton muscle fibers at adult (Sawatari et al., 2010). Overexpression of MSTN2 in zebrafish led to muscle loss and expression of dystrophin associated protein complex, which resulted in muscle dystrophy (Amali et al., 2008).

Hormones and environmental factors can regulate the expression of MSTN in fish but the effects are different with species. Overexpression of growth hormone (GH) or administration of recombinant bovine growth hormone (rbGH) decreased MSTN2 level in coho salmon (Oncorhynchus kisutch) (Roberts et al., 2004) and rainbow trout (Oncorhynchus mykiss) (Biga et al., 2004). Overexpression of GH increased MSTN1 level in rainbow trout (Biga et al., 2004), but rbGH administration did not change MSTN1 level in coho salmon (Roberts et al., 2004). GH decreased MSTN level in giant danio (Danio aequipinnatus) while it increased MSTN level in zebrafish (Biga and Meyer, 2009). Administration of dexamethasone, a glucocorticoid resulted in loss of body weight and muscle atrophy associated with induction of MSTN expression in channel catfish (Ictalurus punctatus) (Weber et al., 2005). However, cortisol reduced MSTN level in tilapia (Oreochromis mossambicus) larvae (Rodgers et al., 2003). Fasting and refeeding did not affect MSTN mRNA levels in adult tilapia (Rodgers et al., 2003) and rainbow trout (Chauvigne et al., 2003). By contrast, larval MSTN mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting (Rodgers et al., 2003). In chronically overcrowded environment, both depression of body growth and a diminished level of MSTN mRNA in the adult zebrafish were observed (Vianello et al., 2003).

Sex steroids are important for sex differentiation and also for muscular development. Androgen can increase muscle in man and woman, and androgenic compounds have been misused in athletes to increase their muscle mass, skeletal muscle strength and performance (Miller, 2009). However, the effects of androgen on MSTN expression arouse controversy. Some people found that testosterone did not change MSTN expression in human (Homo sapiens) (Kvorning et al., 2007, Lewis et al., 2007), the others reported that testosterone increased MSTN expression (Lakshman et al., 2009). Estrogen replacement attenuated muscle growth of young ovariectomized rat (Rattus rattus) (Piccone et al., 2005). Transient (1 week) usage of estrogen decreased insulin-like growth factor-1 (IGF1) and increased MSTN expression, however long time (5 week) administration of estrogen did not apparently influence the expression of IGF1 and MSTN in rat (Tsai et al., 2007).

Some environmental endocrine disruptors can mimic the effects of sex steroid and affect growth of fish. Androgen can increase muscle mass in weakfish (Cynoscion regalis) (Connaughton and Taylor, 1995) and promote growth of tilapia (Sparks et al., 2003) and coho salmon (Larsen et al., 2004). 17alpha-ethinylestradiol (EE2) impairs growth, expression of IGF1 and IGF2 in brook trout (Salvelinus fontinalis) (Schafhauser-Smith and Benfey, 2003) and tilapia (Shved et al., 2009). However, whether sex steroids affect fish growth through MSTN has not been reported.

Rare minnow (Gobiocypris rarus), a small native cyprinid fish, is currently used in aquatic toxicology in China because of its sensitivity to environmental endocrine disruptors (Ma et al., 2007, Zha et al., 2008, Zhong et al., 2005). Here, we report the isolation of MSTN from rare minnow and the effects of androgen/estrogen on body growth and MSTN mRNA levels in rare minnow.

Section snippets

Fish

The fish of rare minnow were obtained from Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China, and were kept in our laboratory. The fish were fed twice a day with commercial diet in the environment of ambient temperature at 28 °C and light cycles of 14 hour light and 10 hour dark. The embryos were obtained by artificial fertilization with eggs and milt from normal adult fish and cultured in water at 28 °C.

The work described in this article was carried out in accordance with The

Sequence analysis of rare minnow MSTN

The sequence of rare minnow MSTN has been submitted to GenBank (ID: FJ482232) and is named as GrMSTN. The full length of GrMSTN cDNA is 2184 bp containing an open reading frame (ORF) of 1128 bp encoding 375 amino acids, the 5′ untranslated region (UTR) of 88 bp and the 3′ UTR including poly(A) tail of 968 bp. The deduced GrMSTN protein has a signal peptide (residues 1–22) predicted by the SignalP V4.0 and two conserved domains, TGF-β propeptide domain (residues 23–262) and TGF-β domain (mature MSTN

Discussion

We isolated MSTN cDNA named as GrMSTN from rare minnow. Phylogenetic analysis indicates that GrMSTN is the homologue of zebrafish MSTN1. Two MSTN genes exist in fish genome. Duplicated MSTN in fish is the result of whole genome duplication occurring in the ancestor of modern bony fish shortly after separation with tetrapod, while salmonids have two MSTN1 and two MSTN2 because of an additional event of whole genome duplication occurring in the salmonids (Kerr et al., 2005).

GrMSTN is expressed

Acknowledgments

This work was supported by the Open grant of State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences (2005FB18).

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