Profile analysis of expressed sequence tags derived from the ovary of tilapia, Oreochromis mossambicus
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).
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These two authors contribute equally to this work.