Elsevier

Aquaculture

Volume 251, Issues 2–4, 28 February 2006, Pages 537-548
Aquaculture

Profile analysis of expressed sequence tags derived from the ovary of tilapia, Oreochromis mossambicus

https://doi.org/10.1016/j.aquaculture.2005.05.040Get rights and content

Abstract

Precise regulation of gene expression is required for a proper transition through the different phases of the ovarian cycle. To study ovarian gene expression in teleosts, we constructed an adult ovary cDNA library of tilapia, Oreochromis mossambicus, and analyzed the gene expression profile using an expressed sequence tag-based strategy. Of 768 random clones, 530 clones, with insert lengths of > 100 base pairs (bp), were assembled into 474 tentative unique genes (TUGs), including 34 contigs and 440 singlets. Among these, 230 TUGs were highly homologous to known genes in public databases, while others exhibited a low sequence homology or no significant matches to known sequences; these are called novel genes here. The abundance of each identified gene was assayed to evaluate its relative importance in ovarian functions. In addition, we analyzed the expression patterns for 6 development-and reproduction-related genes, including cofilin, fibulin-4, glucosamin-6-phosphate deaminase 2 (GNPDA), MCM-6, stathmin, and zona pellucida C5 (ZPC5), as well as 9 novel genes. Results showed that GNPDA, MCM6, ZPC5, and 7 of the novel genes, including O34, O141, O148, O149, O243, O258, and O319, were predominantly expressed in the ovary and/or testis. In addition, the expression patterns of gonad-specific novel genes in tilapia larvae were differentially affected by treatment with 17 β-estradiol and 17 α-methyltestosterone during the sex-sensitive period.

Introduction

The ovary is a female reproductive organ with dual functions: the production of ova and the timely secretion of hormones for regulating growth, development, and structural maintenance of animal life. Precise regulation of gene expression is required for the proper transition through different phases of the ovarian life cycle (Richards, 1994). Principally through the study of known genes, we have learned the basics of genes critical for the sequence of the hormone-dependent ovarian cycle. A primordial follicle is initiated in order to assemble extracellular coat proteins, such as zona pellucida 2 and zona pellucida 3, around the oocyte (Millar et al., 1991). An oocyte in the early growth stage expresses specific signaling factors, such as c-kit, a tyrosine kinase receptor (Packer et al., 1994). However, the factor which triggers the growth of primordial oocytes remains unclear. After initiation of early follicular growth, preovulatory follicular growth is mainly regulated by luteinizing hormone (LH) and follicular-stimulating hormone (FSH). LH alone or in cooperation with FSH elicits the growth, atresia, development, ovulation, and ultimately the formation and breakdown of the corpus luteum. The combination of LH and FSH induces highly synchronized and exquisitely timed gene expression for precise control of the ovarian cycle (Richards, 1994). Although, a few LH/FSH downstream effectors, such as adenylyl cyclase (Richards and Rolfes, 1980), are known, the regulatory mechanisms of the ovarian cycle are still far from being fully understood. It is very likely that other undiscovered genes may be involved in the control of ovarian function. In this regard, the expressed sequence tag (EST) strategy is remarkably suitable for the large-scale screening for novel genes and gene expression profile analysis.

EST cloning has been widely applied in a variety of organisms since its first use in the human genomic project (Adams et al., 1991). In addition to gene identification, one advantage of EST analysis is its ability to measure the relative abundance of expressed transcripts, which allows the evaluation of the relative importance of expressed genes. EST libraries of various organs and tissue from teleosts (Davey et al., 2001, Douglas et al., 1999, Gong et al., 1997, Hirono and Aoki, 1997, Inoue et al., 1997, Ju et al., 2000, Karsi et al., 2002, Kocabas et al., 2002, Martin et al., 2002, Zeng and Gong, 2002) have been constructed, including those for tilapia (Hamilton et al., 2000, Shiue et al., 2004). Of particular interest to us, an ovary library was produced for zebrafish (Zeng and Gong, 2002). The ovary cDNA library has yielded a substantial amount of novel genes. In addition, several genes were shown to be specifically expressed in the ovary that may serve as useful molecular markers for the analysis of oogenesis and development.

Oreochromis mossambicus and its close relative, O. niloticus, are important fish species for both research and aquaculture. Tilapia are the second most important food fish, next to carp worldwide. They are also excellent laboratory animals because (1) they have a short generation time of 6∼7 months, (2) the embryos and fry are easily obtained and reared in laboratory, and (3) transgenic animals can be produced relatively easily (Maclean et al., 2002). Despite their importance to fisheries, studies of their genome are still very preliminary and far behind other fish species like zebrafish or medaka. In addition, the feasibility of controlling the sexual differentiation by various methodologies, including genetics, changes in the environment, and drug treatments, make tilapia favorable models for the functional study of gonads. Therefore, in this study, we sequenced and analyzed 768 EST clones derived from an ovary cDNA library of adult O. mossambicus and identified 474 tentative unique genes. In addition, we also analyzed 6 development- and reproduction-related genes and 9 novel genes for their gene expression patterns. Furthermore, the expression patterns of 6 gonad-specific novel genes under the influences of 17 α-methyltestosterone and 17 β-estradiol were also analyzed during the sex differentiation-sensitive period from days 24 to 33 after fertilization. The establishment of the gene profile and its analysis of this tilapia ovary cDNA library are thus proving to be a fruitful gene identification source, which will benefit research of ovarian functions and sex differentiation of tilapia as well as other economically important cultured fishes.

Section snippets

Construction of a cDNA library

Adults of O. mossambicus were obtained from the Mariculture Research Center, Fisheries Research Institute, Tainan, Taiwan. PolyA+ RNA was extracted using the NucleoTrap® mRNA Kit (BD Biosciences Clontech) from the ovaries of 3 females. One-half microgram of polyA+ RNA was utilized for the construction of the library using a Creator™ SMART™ cDNA Library Construction Kit (BD Biosciences Clontech) according to the manufacturer's instructions. The titer of the primary cDNA libraries was about 1.5 × 10

Summary of EST clones in the tilapia ovary cDNA library

In total, 768 random clones from the tilapia, O. mossambicus, ovary cDNA library were partially sequenced from the 5′ end, and the insert sizes were determined by PCR, among which, 665 clones were successfully determined. Most clones (643) contained an insert of between 0.1 and 2.5 kb. The most-abundant clone insert sizes ranged 0.9∼1.5 kb (364, 55.6%) and 0.1∼0.3 kb (92, 14%) (Fig. 1). After vector sequence trimming, 69% (530) of the EST sequences were found to exceed 100 bp in length. These

Discussion

In this study, 768 random clones were sequenced and 474 tentative unique genes (TUGs) were identified, including 230 known genes. Among the known genes, only 6 of them have previously been reported in O. mossambicus. We were unable to annotate 244 (51.5%, 244/474) unique sequences due to the low sequence homology or no significant match (44 clones) compared to existing known sequences in public databases. The high percentage of un-annotated sequences implies that the genetic diversity of

Acknowledgements

We would like to thank Dr Y.C. Yang for help in analyzing data. We are also in debt to Dr. W.L. Liao for preparing the tilapia feed, Dr. S.W. Lou for supplying 17 α−methyltestosterone, and Dr. J.R. Hseu for providing the tilapia. This work was supported by the National Science Council of the R.O.C. (NSC91-2317-B-002-022 and NSC92-2317-B-002-024).

References (37)

  • S.A. Martin et al.

    EST-based identification of genes expressed in the liver of adult Atlantic salmon (Salmo salar)

    Biochem. Biophys. Res. Commun.

    (2002)
  • A.I. Packer et al.

    The ligand of the c-kit receptor promotes oocyte growth

    Dev. Biol.

    (1994)
  • J.S. Richards et al.

    Hormonal regulation of cyclic AMP binding to specific receptor proteins in rat ovarian follicles. Characterization by photoaffinity labeling

    J. Biol. Chem.

    (1980)
  • Y.L. Shiue et al.

    EST-based identification of genes expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus

    Biochem. Biophys. Res. Commun.

    (2004)
  • J.C. Sible et al.

    Developmental regulation of MCM replication factors in Xenopus laevis

    Curr. Biol.

    (1998)
  • A. Sobel et al.

    Intracellular substrates for extracellular signaling. Characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin)

    J. Biol. Chem.

    (1989)
  • M.D. Adams et al.

    Complementary DNA sequencing: expressed sequence tags and human genome project

    Science

    (1991)
  • P. Amireault et al.

    Cloning, sequencing, and expression analysis of mouse glucosamine-6-phosphate deaminase (GNPDA/oscillin)

    Mol. Reprod. Dev.

    (2000)
  • Cited by (24)

    • Transcriptome analysis of the pearl oyster (Pinctada fucata) hemocytes in response to Vibrio alginolyticus infection

      2016, Gene
      Citation Excerpt :

      For annotation based on sequence similarity, only 29,615 unigenes (40.01%) were annotated in at least one database in this paper, and the same problem also existed in other groups of marine organism transcriptome (Meyer et al., 2009; Zhao et al., 2011). The poor annotation efficiency could be due to the limited genome researches on aquaculture species and the insufficient sequences in the public databases for phylogenetically closely related species to date (Chu et al., 2006; Dong and Xiang, 2007; Zhao et al., 2011). On the other hand, the low expression level and short length of unigenes may be another reason for rarely matched to the known genes (Novaes et al., 2008).

    • Effects of acclimation salinity on the expression of selenoproteins in the tilapia, Oreochromis mossambicus

      2014, Journal of Trace Elements in Medicine and Biology
      Citation Excerpt :

      The tilapia mRNA sequences for twelve selenoprotein and selenoprotein synthesis factor genes were identified by using templates from known protein sequences of zebrafish; two genes were identified from the protein sequence and cDNA library of Nile tilapia; one was identified from the protein sequence of rainbow trout. The mRNA sequence of SelW was employed as previously published [30]. Accession numbers for each cDNA sequence found after conducting tBLASTn are provided in Table 1.

    • Identification of reproduction-related genes and SSR-markers through expressed sequence tags analysis of a monsoon breeding carp rohu, labeo rohita (hamilton)

      2013, Gene
      Citation Excerpt :

      Genomic resources (i.e. ESTs and type 1 markers) in commercially important fish species are useful for many purposes such as stock identification, stock enhancement, genome mapping, marker assisted breeding, genetic management, and preservation of genetic diversity (Tassanakajon et al., 1997) as well as functional genomics (Gui and Zhu, 2012; Liu, 2007). Genomic resources are currently available for other commercially important fishes, including rainbow trout (Oncorhynchus mykiss) (Gohin et al., 2010; Von-Schalburg et al., 2005), coho salmon (Oncorhynchus kisutch) (Luckenbach et al., 2008), tilapia (Oreochromis mossambicus) (Chu et al., 2006), Atlantic halibut (Hippoglossus hippoglossus) (Mommens et al., 2010), senegalese sole (Solea senegalensis) (Cerdà et al., 2008a,b), Atlantic salmon (Salmo salar) (Leong et al., 2010) and cod (Gadus morhua) (Goetz et al., 2006). The relationship between the transcriptome and physiological indicators of reproduction have been studied for some commercially important fish species e.g. rainbow trout; Hook et al., 2011, coho salmon; Luckenbach et al., 2008 etc. but no similar information is available about reproduction for L. rohita.

    • Constructing and random sequencing analysis of normalized cDNA library of testis tissue from oriental river prawn (Macrobrachium nipponense)

      2012, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics
      Citation Excerpt :

      They are important in reproduction because iron metabolism plays an important role in both male and female fertility (Beninger et al., 2003; Huang et al., 2003). Ferritins have been reported to be present in both ovary and testis in Oreochromis mossambicus and played an important role in fertility of both sexes (Chu et al., 2006). They reportedly play a similar role in L. vannamei and M. rosenbergii (Zhang et al., 2006; Zhou et al., 2008).

    View all citing articles on Scopus
    1

    These two authors contribute equally to this work.

    View full text