Cloning and sequencing of Tert gene in gilthead seabream, Sparus aurata, and European seabass, Dicentrarchus labrax: Expression patterns in germ and somatic cells

Highlights

• Two Tert transcripts from seabream and one from seabass were characterized.

• The different coding transcripts of seabream may contribute to regulation of telomerase activity.

• Tert gene is expressed ubiquitously in most organ systems tissues, particularly in gonads.

• The high expression levels of Tert in testis and ovary suggested an important role in gametogenesis.

• Tert expression was not evidenced in mature gametes.

Abstract

Tert gene encodes a catalytic subunit of the enzyme telomerase, which protects telomeres from abnormal folds and their degradation. In mammals, the activity of telomerase in tissues of adults is limited to stem cells with high potential for proliferation, finding expression in the cells of the germline, tumors and neoplastic cells, although the Tert gene seems to be ubiquitous in fish. To gain insight on Tert implication for fish gonad cell differentiation and gametogenesis progress, we cloned the Tert cDNA of two reared marine fish species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax), and their quantitative and qualitative Tert mRNA expression were analyzed. Two Tert transcripts encoding proteins which differ at their functional C-terminal end were isolated from gilthead seabream, whereas only one Tert transcript was identified from European seabass. The qPCR assays showed that Tert genes are expressed ubiquitously in both fish species, and the highest expression levels were found in gonads and particularly in differentiating or maturating germ cells, which could suggest an important role of Tert genes in gametogenesis and cell-tissue development of fish species.

Introduction

Telomeres have an important role in the maintenance of structural stability of eukaryotic chromosomes, protecting them from nuclease degradation and fusion events. The telomeric complexes are found at the ends of chromosomes and they are composed of tandem repeated sequences of non-coding DNA and associated specific proteins (Blasco, 2002, de Lange, 2002, Grabowski et al., 2005). During the process of DNA replication and cell division, telomeres are shortened as a result of the inability of DNA polymerases to replicate chromosomal linear ends (Levy et al., 1992). The attrition of the telomeres during successive cell division cycles leads to cellular senescence in which the cells stop dividing, meaning a limited proliferative capacity of cells (Olovnikov, 1973). Nevertheless, cells can counteract the loss of telomeric repeats by the presence of the telomerase which adds de novo hexameric repeats to the ends of linear chromosomes (Blackburn and Collins, 2011). Many authors reported that the canonical telomerase activity prevents telomere erosion, allowing chromosome stability and cellular multiplication or proliferation (reviewed by Liu et al., 2004a).

It has been proposed that cellular senescence can be triggered by the loss of telomerase expression, together with other factors like DNA damage, considering that telomere shortening limits cell proliferation and growth (Campisi et al., 2001, Shay and Wright, 2005). Telomerase is a ribonucleoprotein complex composed of a telomerase reverse transcriptase (TERT), a telomerase RNA component (Terc) used as template for the transcriptase reaction, and multiple telomerase associated proteins (Blasco, 2005). The maintenance of telomeric length is very important for the proper meiotic progress of germ cells during mammalian gametogenesis (Liu et al., 2004b), because telomeric shortening has been related to apoptosis, generation of aneuploid gametes, and reproductive aging (Reig-Viader et al., 2014). High levels of Tert expression can trigger an uncontrolled cell proliferation, which plays an important role in cancer, considering that most tumors in humans seem to depend on telomerase reactivation through direct regulation by the cancer genes or through transformation of progenitor cell population expressing telomerase (Artandi and DePinho, 2010, Blasco, 2005). However, over the last few years several studies have demonstrated that TERT protein also exhibits other different non-telomeric activities, such as cell cycle regulation and cell proliferation, modulation of cell signaling pattern and gene expression, as well as protection again oxidative damage and apoptosis (for reviews see, Ale-Agha et al., 2014, Bollmann, 2008, Chiodi and Mondello, 2012).

Telomerase has been characterized and/or detected in a wide variety of eukaryotes, from unicellular life forms to multicellular organisms, including vertebrates (reviewed by Hrdličková et al., 2012, Sýkorová and Fajkus, 2009). In humans, most somatic cells lack the telomerase activity, which is only present in early embryogenesis, in proliferative cells of renewal tissues, hematopoietic stem cells and germ cells, as well as in tumor and cancer cells (Huang et al., 2014). The activity of telomerase is tightly regulated principally at the level of Tert transcription, since human Tert is only detected in cells with high proliferative capacity whereas Terc appears to be ubiquitous, being expressed in most cell types, although there is evidence that expression of Terc might also be regulated among tissues (Cairney and Keith, 2008, Wright et al., 1996). Nevertheless, telomerase activity and its expression in somatic cells varies greatly among organisms as in different species such as birds, fish and inbred strains of laboratory mice, telomerase is expressed in many somatic cells, in contrast to preferential expression in proliferating human cells, such as stem cells and tumor or cancer cells (Haussmann et al., 2007, McChesney et al., 2005, Prowse and Greider, 1995).

In contrast to mammals, some organisms such as several fish species grow throughout their life with little senescence (Patnaik et al., 1994). Therefore, all cells from these “immortal” fish would require a high proliferation capacity, and thereby high telomerase activities should be active in all fish cells (Klapper et al., 1998). High telomerase activity is conserved in somatic tissues of fish species at all ages and it has been associated with their continuous growth throughout life and their ability to regenerate injured tissues, which demands a high proliferative potential (Elmore et al., 2008, Hartmann et al., 2009, Hatakeyama et al., 2008, Klapper et al., 1998, Pfennig et al., 2008, Rao et al., 2011). In fish species, the telomerase activity and Tert expression in both germ and somatic cell types have been related to reproductive aspects and differences in growth patterns. Interestingly, Tert was found to be highly expressed in regenerative tissues (brain, gills, gut, kidney…). Moreover, increases in Tert expression can be induced by environmental stressors, such as hypoxia, food availability or exposure to carcinogens (Kong et al., 2008, Peterson et al., 2015, Yu et al., 2006).

The global aquaculture production of marine fish species has been increasing for the last few years (FAO, 2005a, FAO, 2005b). The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are two important commercial marine fish species for the aquaculture and fishing of Mediterranean countries (Arechavala-Lopez et al., 2012). Both teleostean species have different reproductive strategies and show some sexual differential growth pattern (Franch et al., 2006, Piferrer and Guiguen, 2008). For synchronous and reliable maturation and spawning of fish species in aquaculture systems it is necessary to understand and to control reproduction (Devlin and Nagahama, 2002). Although the basic mechanisms of reproduction of both teleost species have been extensively studied (Chaves-Pozo et al., 2004, Zanuy et al., 2001), the application of genetic and cellular markers will allow for advancement in the understanding of the mechanisms that can be involved in the genetic and molecular regulation of the reproductive process in fish. As it is largely known, there are numerous genes (Cyp19a, Amh, vasa, dmrt) involved in sex determination/differentiation and in gametogenesis in fish species (Martínez et al., 2014, Piferrer and Guiguen, 2008). Currently, other genes and dependent-enzymatic activities (i.e. Tert, telomerase) are being analyzed for their involvement in cell proliferation and anti-apoptotic processes, which are also directly implicated in the progress of oogenesis and spermatogenesis, somatic growth, regenerative processes, and protection stress mechanisms (Klapper et al., 1998, López de Abechuco et al., 2014).

In this paper, for the first time, a phylogenetic, molecular and cellular approach to the Tert gene is reported for two marine fish species, European seabass and gilthead seabass, which show different reproductive strategies and some differential sex-specific growth-patterns. For this purpose, we have isolated and cloned their Tert mRNA and cDNA, and have characterized the quantitative and qualitative gene expression patterns in different organ systems and tissues (gonads, brain, hypophysis, heart, kidney, spleen among other somatic tissues) from both male and female adult specimens.