Abstract [Objectives] This study was conducted to construct forward and reserve libraries of suppression subtractive hybridization （SSH） in the testis of male tilapia under the stress of methomyl by using SSH technology.
　　[Methods] Using male tilapia as the test animal， the forward and reserve libraries of SSH in the testis of tilapia under the stress of methomyl were constructed by using the SSH technology.
　　[Results] 45 expressed sequence tags （ESTs） were obtained， and 25 expressed sequence tags were successfully noted， including 13 forward libraries and 12 reverse libraries. The genes with confirmed functions were classified into five types. Genes related to catalytic activity and cell characteristics were up regulated， while genes related to structural molecules activity and biological process were down regulated. The expression amount of integrin β1 was up regulated， while serine/threonine protein kinase pim 3， Ca 2+  ATPase， Na+ K+ ATPase and ribosomal protein L22 were down regulated.
　　[Conclusions] The research results could lay a foundation for revealing the molecular mechanism of methomyls reproductive toxicity to tilapia.
　　Key words Methomyl; Tilapia; Suppression subtractive hybridization; cDNA library; Testis
　　 Methomyl is a broad spectrum carbamate type pesticide. It has strong contact and stomach toxicities and has no uptake transmission function. It is one of the most widely used pesticides. In 1997， the World Wildlife Fund listed methomyl as an estrogen type endocrine disruptor[1]. Its biological toxicity to the environment is to destroy the predation relationship between animals， thereby threatening the balance of ecosystems[2]. Due to the high solubility of methomyl in water and its high dosage， coupled with unreasonable discharge in industrial and agricultural production， it has been detected in some water bodies and foods[3]. At present， methomyl has received extensive attention due to its high detection rate in environmental waters and its endocrine disrupting effect[4-6].
　　Suppression subtractive hybridization （SSH） is a subtractive technique with the ability to select genes for amplification[7]. Sequences with different abundances can finally agree on their relative content by using this technique， and then， according to the principle that intra chain annealing is prior to the inter chain annealing， the primers cannot be paired with the non target sequence fragments， which means that the non target sequence fragments are suppressed， and the gene of interest is amplified. Compared with other differential gene acquisition methods， this technology has the characteristics of high sensitivity， low false positive and strong specificity， and is thus used to screen and clone differentially expressed genes. With male tilapia as the experimental animal， and the forward and reverse SSH libraries for testis of tilapia in the threat of estrogen pesticide methomyl was constructed by suppression subtractive hybridization， and differentially expressed genes in the tilapia testis tissue under the stress of the estrogen pesticide methomyl were identified， with an attempt to lay a foundation for revealing the molecular mechanism of the reproductive toxicity of methomyl on tilapia. 　　Materials and Methods
　　Materials
　　Male tilapia （Yixing Breeding Base of Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences） with an average body mass of （112.24±9.48） g and an average body length of （17.06±0.91） cm was selected as the test animal. 30 male tilapia individuals were randomly placed in an aquarium with a volume of 300 L （containing 200 L of water） and domesticated for 4 weeks. During domestication， feed was supplied once a day at 08：00， and the amount of feed was calculated at 2% of the tilapia body mass. The ratio of dark to light was 12 h∶12 h. An air compressor was used for aeration.
　　Test water
　　The test water was dechlorinated tap water aerated for 7 d. The water had a dissolved oxygen content of 6.3-7.0 mg/L with a temperature at （25.0±0.5） ℃ and a pH value of （7.0± 0.5）.  The test water satisfied the fishery water quality standard （GB11607 89）.
　　Main reagents and instruments
　　Main reagents
　　Methomyl with a mass fraction greater than 97%， purchased from Shanghai Focus Biotechnology Co.， Ltd.; Oligotex mRNA kit and Maxi Plasmid kit， purchased from Qiagen; 1 kb DNA Ladder and UltraPure Agarose， purchased from Zrbiorise; Taq DNA polymerase and 10 mmol/L dNTP， purchased from TaKaRa; TRIzol Reagent extraction kit，  purchased from Invitrogen; RT Enzyme， purchased from ABI; PCR Select cDNA Subtraction kit， purchased from Clonetech.
　　Main instruments
　　Desktop high speed freezing centrifuge （Eppendorf， Centrifuge 5810R）; electroporator （BTX， ECM630）; PCR instrument （Bio rad， MyCycler）; electrophoresis apparatus （tanon， HE 120）; gel imager （Tanon， 2500）.
　　Experimental design
　　According to the result of the previous acute test （96 h LC50 of methomyl： 940 μg/L）， the residual amount of methomyl in natural water （0-97 μg/L）[8-10] and the maximum limit of methomyl stipulated by US water quality standards （200 μg/L）[11]， the experiment adopted following five concentration gradients： 0， 0.2， 2.0， 20.0 and 200.0 μg/L. Each concentration gradient was set with three parallels， and 200 L of the methomyl solutions at above concentrations were prepared in 300 L aquaria， respectively. Into each of the parallels， 20 tails of healthy male tilapia were randomly input.
　　The experiment was carried out by semi static water contact exposure method. 50% of the water in the aquaria of each concentration group was replaced every day， and the drug was added to the original concentration. The experiment period was 48 d， in which the first 30 d was the methomyl exposure test phase， and the last 18 d was the clear water recovery test phase （no methomyl）. 　　The actual methomyl concentrations of the various test group were measured by the method of Zhang et al.[12] and Zhang  et al. [13]. The 0， 0.2， 2.0， 20.0 and 200.0 μg/L groups had the concentration of 0， 0.23， 2.12， 21.50 and 182.00 μg/L， respectively， at the time of initial exposure， and after 24 h， the concentration was 0， 0.21， 1.92， 18.52 and 179.00 μg/L， respectively. The concentration of methomyl in each concentration group after 24 h was similar to that at the time of exposure.
　　Sample collection
　　Sample collection was performed on the 30 μg/L methomyl treatment group and the control group on the 30 th  day of exposure. Feeding was stopped 24 h before sampling. During the experiment， tilapia was active and healthy. At the time of sampling， tilapia was anesthetized with 250 mg/L MS 222， followed by the measurement of the body length and body weight and collection of the testis after quickly killing. The collected samples were placed in a RNAlater preservation solution， and finally in a -80 ℃ ultra low temperature freezer for later use.
　　Total RNA extraction and mRNA separation
　　The samples stored in the ultra low temperature freezer were taken out and ground into a powder. The total RNA of the tilapia testis tissue was extracted using TRIzol Reagent extraction kit. The specific operation was carried out according to the instructions of TRIzol Reagent extraction kit. The total RNA was precipitated with ethanol and dissolved in DEPC treated water to determine its quality and concentration. The sample mRNA was isolated and purified by the Oligotex mRNA kit， and the specific procedure was carried out according to the instruction of the kit.
　　SSH library construction
　　The SSH library was constructed using PCR Select cDNA Subtraction kit. The purified mRNA was reverse transcribed into cDNA. The reverse transcriptase was added to the synthesis system of the first strand of cDNA， and the DNA polymerase I of E. coli was used to carry out the synthesis of the second strand. The SSH forward and reverse libraries were constructed by using the double stranded cDNA of the testis tissue of the methomyl treatment group and the double stranded cDNA of the control group as the driving or experimental side， respectively. The double stranded cDNA of the experimental side was divided into two parts， which were ligated with Adaptor 1 and Adaptor 2R， respectively. After successful ligation， the cDNA of the experimental side was hybridized twice with the cDNA of the driving side， and the hybridization product was subjected to specific PCR twice. The PCR product was subjected to T/A cloning， during which 50 μl of the library liquid was taken and plated on a plate with corresponding resistance， and incubated at 37 ℃ overnight， and positive clones were selected by colony PCR， obtaining the strains， which were preserved. Specifically， positive clones were identified by PCR with M13 F and M13 R as primers. The PCR reaction procedures were as follows： 94 ℃ for 4 min，  30 cycles of 94 ℃ for 30 s and 56 ℃ for 30 s， and 72 ℃ for 5 min. The reaction system was  20 μl ， and the PCR product was stored at 4 ℃. Sixty four positive clones were randomly selected for sequencing. 　　Sequence alignment
　　After removing the EST efficient sequence vector and the linker sequence， similarity alignment was performed with the GenBank database to analyze the function of sequences.
　　Results and Analysis
　　Total RNA extraction
　　The total RNA of the tilapia testis tissues from the methomyl treatment group and control group was extracted and detected by 2% agarose gel electrophoresis using TRIzol Reagent kit. It can be seen from Fig. 1 that both the clear bands of 18S and 28S were over 1 000 bp， and there was no genomic contamination， that is， the extracted RNA was of good quality.
　　Synthesis and enzymatic digestion of double stranded cDNA
　　The tilapia double stranded cDNA was detected by electrophoresis （Fig. 2）， and the double stranded cDNA bands before and after Rsa I digestion were compared. It was found that the fragments after Rsa I digestion were significantly smaller， the bands were moved down， and the enzyme digestion effect was good， satisfying the experimental requirements for subsequent construction of the libraries.
　　
　　Discussion
　　In the forward libraries of SSH in the tilapia testis tissue under the stress of the estrogen pesticide methomyl， the expression levels of low density lipoprotein receptor and 17 α hydroxylase/17，20 lyase were up regulated. The low density lipoprotein receptor （LDLR） is a cell surface glycoprotein involved in the lipoprotein metabolism of steroid producing tissues such as ovaries and testes. The LDLR pathway takes in cholesterol for steroid hormone synthesis， cell proliferation， etc. α hydroxylase/17，20 lyase is a cytochrome P450c17 enzyme that controls the synthesis of steroid hormones in the cholesterol synthesis pathway[14]. For fish， the P450c17 enzyme is expressed more in the testis and less in the ovary[15]. Fish steroid hormone testosterone and 11 ketotestosterone are derivatives of cholesterol in the body， which play an important regulatory role in spermatogenesis by affecting spermatogenic cells and supporting cell activity[16]. Methomyl has an endocrine disrupting effect of environmental estrogen[1]， and may have an effect on the secretion of steroid hormones in tilapia. Meng[17] found that the testosterone and 11 ketotestosterone levels were significantly reduced in tilapia under the stress of a high concentration of methomyl. Under the threat of the environmental estrogen methomyl， the expression amounts of low density lipoprotein receptor protein and 17α hydroxylase/17， 20 lyase in the testis tissue was up regulated， which was possibly due to that the lipoprotein metabolism or steroid hormone secretion in the tilapia testis tissue was interfered by methomyl， which caused the bodys feedback mechanism， which further up regulated the expression of low density lipoprotein receptor and 17α hydroxylase/17，20 lyase. 　　The adherens junction is mainly to connect two adjacent cells together by adhesion molecules， while the spermatogenic cells are connected to the supporting cells through adherens junction. When the number of adhesion molecules changes or the relationship with the cytoplasmic linker protein changes， the adhesion between cells will change[18]. The expression level of integrin β1 was up regulated in the forward libraries of SSH in this study. Integrin α6 and integrin β1 are two important adhesion molecules in the spermatogenic epithelium. They can form heterodimers on the surface of the supporting cells[19]， interact with the laminin on the surface layer of sperm cells， and participate in the formation of top ES of the spermatogenic epithelium[20]. Therefore， the up regulation of the integrin β1 expression in the testis tissue might be due to the feedback mechanism caused by the effect of environmental estrogen methomyl on the adhesion of tilapia testis， which makes the expression of integrin β1 up regulated to maintain the stability of the internal structure of testis.
　　In the reverse libraries of SSH in the testis tissue of tilapia， the serine/threonine kinase pim 3 expression was down regulated. The serine/threonine kinase pim 3 has the biological action of phosphorylating the apoptosis protein Bad and transmitting cell survival signal. The pro apoptotic protein Bax of the mitochondrial outer membrane forms a homodimer under the action of the apoptotic protein Bad， which increases mitochondrial membrane permeability， releases cytochrome Cytc and activates caspase， which induces apoptosis[21]. The active form of Bad is non phosphorylated， and it is inactivated after phosphorylation， which inhibits apoptosis[22-23]. Thus， the down regulation of serine/threonine kinase pim 3 may limit the phosphorylation of the apoptosis protein Bad. In the reverse libraries of SSH in this study， the expression of plasma membrane Ca 2+  ATPase and Na+ K+ ATPase was down regulated. Under normal conditions， the ion concentration in plasma membrane is in a dynamic equilibrium state， which can maintain cell structure and normal physiological functions. Ca 2+  AT Pase and Na+ K+ ATPase have an important regulatory effect on the homeostasis of Ca 2+ ， Na+， and K+ ions， and they have specific ion transport functions in active transport to maintain the ion balance on the cell membrane of tissues or organs. Under the stress of methomyl， the expression of Ca 2+  ATPase and Na+ K+ ATPase was down regulated， which may affect the regulation of ion concentration on the cell membrane of tilapia testis， leading to the imbalance of ion concentration on the cell membrane and affecting the normal physiological function of testis cells. 　　In the reverse libraries of SSH in the tissue of tilapia testis， the expression of the ribosomal protein L22 was down regulated. Li[24] found that knocking out L22 in Drosophila melanogaster embryo and larval S2 cells had an effect on cell cycle. Sun et al.[25] found that cyclin D1 decreased significantly when L22 expression was silenced， and the corresponding cell proliferation was significantly inhibited. This indicates that the ribosomal protein L22 is closely related to the cell proliferation cycle. Therefore， the down regulation of the expression of the ribosomal protein L22 induced by the methomyl stress may have an effect on the cell cycle of the testis tissue. In addition， the differentially expressed gene of phospholipase A2 was also down regulated. Phospholipase A2 mainly hydrolyzes phospholipids and is involved in phospholipid metabolism. The down regulation of phospholipase A2 may affect phospholipid metabolism in the testis tissue.
　　Conclusions
　　The forward and reserve libraries of SSH in the testis of tilapia under the stress of methomyl were constructed by using the SSH technology， and eight gene sequences with confirmed functions were obtained. The identification and analysis of some important differentially expressed genes showed that under the stress of environmental estrogen， the lipoprotein metabolism or steroid hormone secretion of tilapia testis tissues was disturbed， causing the bodys defense mechanism， which induced the up regulation of the expression levels of proteins related to low density lipoprotein receptor and 17α hydroxylase/17， 20 lyase; the expression of integrin β1 was up regulated to maintain the stability of the internal structure of the testis; and the expression of the serine/threonine kinase pim 3 was down regulated， limiting the phosphorylation of the apoptosis protein Bad. The expression of Ca 2+  ATPase， Na+ K+ ATPase and ribosomal protein L22 was down regulated， and the ion balance on cell membrane and cell proliferation of testis cells may be affected.
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