Accepted Manuscript Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization Jian-Jun Chen, Xiao-Hua Xia, Li-Fang Wang, Yong-fang Jia, Ping Nan, Li Li, ZhongJie Chang PII:

S0093-691X(15)00002-3

DOI:

10.1016/j.theriogenology.2015.01.001

Reference:

THE 13053

To appear in:

Theriogenology

Received Date: 30 July 2013 Revised Date:

23 December 2014

Accepted Date: 1 January 2015

Please cite this article as: Chen J-J, Xia X-H, Wang L-F, Jia Y-f, Nan P, Li L, Chang Z-J, Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization, Theriogenology (2015), doi: 10.1016/ j.theriogenology.2015.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification and comparison of gonadal transcripts of testis

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and ovary of adult common carp Cyprinus carpio using

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suppression subtractive hybridization

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Jian-Jun Chen, Xiao-Hua Xia, Li-Fang Wang, Yong-fang Jia, Ping Nan, Li Li,

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Zhong-Jie Chang*

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College of Life Science, Henan Normal University, Xinxiang 453007, Henan, People’s

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Republic of China

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* Corresponding author. Tel.: +86-03733326960

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E-mail address: [email protected] (Z-J. Chang)

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ACCEPTED MANUSCRIPT ABSTRACT

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The limited number of gonad-specific and gonad-related genes that have been

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identified in fish represents a major obstacle in the study of fish gonad development

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and sex differentiation. In common carp Cyprinus carpio from China’s Yellow River

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the ovary and testis differ in volume and weight in adult fish of the same age.

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Comparing sperm, egg, and somatic cell transcripts in this carp may provide insight

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into the mechanisms of its gonad development and sex differentiation. In the present

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work, gene expression patterns in the carp ovary and testis were compared using

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suppression subtractive hybridization (SSH). Two bidirectional subtracted cDNA

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libraries were analyzed in parallel using testis or ovary as testers. Eighteen

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non-redundant clones were identified in the male library, including 15 known cDNAs.

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The expression patterns of selected genes in testis and ovary were analyzed using

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reverse transcriptase-PCR. Tektin-1, GAPDS, FGFIBP, IGFBP-5, and an unknown

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gene from the Ccmg4 clone were observed to be expressed only in testis. GSDF,

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BMI1b, Wt1a, and an unknown gene from the Ccme2 clone were expressed at higher

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levels in testis than in ovary at sexual maturity. Thirty functional ESTs were identified

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in 43 sequenced clones in the female library, including 28 known cDNAs, one

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uncharacterized cDNA (EST clone), and one novel sequence. Eight identified ESTs

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showed significant differences in expression between the testis and ovary. ZP3C and

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Psmb2 were expressed exclusively in ovary, while the expression levels of IFIPGL-1,

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Setd6, ATP-6, CDC45, AIF-1 and an unknown gene from the Ccfh2 clone were more

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strongly expressed in ovary than in testis. In addition, the expression of ZP3C, Wt1a

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and Setd6 was analyzed in male and female gonads, heart, liver, kidney, and brain.

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ACCEPTED MANUSCRIPT ZP3C was expressed only in ovary. Setd6 expression was significantly stronger in

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female tissues than in the male, except in liver, and Wt1a expression showed sexual

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dimorphism in kidney and liver. Results suggest that these genes could play key roles

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during carp growth, both in the gonad and other tissues. The results provide a resource

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for further investigation of molecular mechanisms responsible for gonad development

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and sex differentiation in Yellow River common carp.

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Keywords: suppression subtractive hybridization; differential gene expression;

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RT-PCR; common carp

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1. Introduction The common carp Cyprinus carpio is the longest-cultured and most widely

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domesticated fish species and is of significant economic important in several countries.

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It plays an essential role in China’s freshwater fisheries, which have a long history of

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carp breeding and high-yield production [1]. Male and female carp differ in growth

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rate and attained body size and, therefore, in economic value. Female carp grow

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significantly faster than males after gonad differentiation; the average weight of a

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2-year-old female carp is c. 30% greater than that of a male of similar age [1]. A

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comprehensive understanding of carp gonad development mechanisms might allow

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this to be exploited by facilitating monosex cultivation. However, despite extensive

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research into the structure and function of carp gonads at the cellular level, as well as

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the structure and development of the sperm and egg, little is known about the

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molecular mechanisms regulating gonad development and sex differentiation [2]. Few

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gonad-specific or gonad-related genes have been identified in the common carp, a

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major obstacle in the study of gonad development and sex differentiation. If we are to

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expand knowledge of the mechanisms of gonad development and sex differentiation

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in carp and other phytoplanktivorous fish, it is essential that genes that are differently

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expressed in the testis and ovary be identified.

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Suppression subtractive hybridization (SSH) using isolated mRNA is a useful

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technique for the molecular investigation of genes [3]. This method selectively

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amplifies differently-expressed target cDNA fragments and simultaneously suppresses

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non-target DNA amplification. The method is based on suppression polymerase chain

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ACCEPTED MANUSCRIPT reaction (PCR) and provides a simple and effective means of generating cDNA that is

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highly enriched for differently expressed genes of both high and low abundance

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cDNA. This method is also suitable for separating genetically related cDNA

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fragments with very small sequence differences [4].

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The goal of the present work was to identify genes that are differently expressed

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in the testis and ovary via the building of cDNA subtractive libraries for male and

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female common carp using SSH. Sequencing and BLASTx analyses of expressed

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sequence tags (EST) were used to compare the types of genes expressed and to predict

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their functions in fish gonad development and sex differentiation. This identified

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genes involved in gonad development and differentiation and allowed generation of a

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transcriptome database that expands the public EST database for this species. The

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study further analyzed expression of three candidate genes, CcWt1a, CcSetd6, and

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CcZP3C, in several male and female tissues. Results will be useful for building a

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more complete understanding of the regulatory mechanisms associated with gonad

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differentiation and reproductive processes.

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2. Materials and methods

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2.1. Fish and histology of gonads

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We used fifteen males and fifteen females of an inbred strain of common carp

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(Yellow River carp C. carpio) originally collected from the Yellow River and inbred

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by sister-brother mating for 20 generations at Henan Provincial Research Institute of

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Aquaculture. Fish of average size were selected at 22–30 weeks post-fertilization

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(spermatozoa and ova formation) and anesthetized with 400 ppm 2-phenoxyethanol,

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before killing. The sex of the individuals was confirmed by visual inspection of the

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dissected gonads. In order to understand the organizational morphology of the Yellow River carp

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gonad, the testes [gonad somatic index (GSI) 12.23%] and ovaries (GSI 18.23%) from

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mature carp were preserved in Bouin’s solution for 24 h. Fixed tissues were stored in

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70% ethanol, then dehydrated through graded ethanols, and embedded in paraffin.

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Sections were cut at 6µm thickness and stained with hematoxylin-eosin.

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2.2. Total RNA extraction and mRNA isolation

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Total RNA extraction was performed using Trizol reagent according to the

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manufacturer’s instructions (Takara, Dalian, China). Gonads from fish of the same sex

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were pooled and homogenized in 1 mL Trizol and mixed with 200 µL chloroform.

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The suspension was centrifuged at 12,000×g for 15 min. The clear upper phase was

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aspirated and placed in a clean tube. Five-hundred microliters of isopropanol was

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added, and the samples were centrifuged at 12,000×g for 10 min. The RNA pellet was

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washed with 75% ethanol and dissolved in DEPC-H2O. The concentration and purity

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of the total RNA were determined by electrophoresis gel imaging and electrophoresis

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on denaturing formaldehyde 1% agarose/ethidium bromide gel. mRNA was isolated

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using the Oligotex mRNA Spin-Column Kit (Qiagen, the Netherlands) according to

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the manufacturer’s instructions. mRNA was treated with 100% ethanol and 3 mol L−1

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NaOAc and used as the starting material to construct the SSH cDNA library.

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2.3. Construction of SSH cDNA library

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Suppression subtractive hybridization was performed using the PCR-Select

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ACCEPTED MANUSCRIPT cDNA Subtraction Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) according

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to the manufacturer’s instructions. Two SSH experiments were performed in parallel

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to construct tester-1 and tester-2 subtracted cDNA libraries. Subtracted cDNA in the

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tester-1 library was made using pooled spermatozoan mRNA as the tester and ovum

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cDNA as the driver, and cDNA in the tester-2 library was made from pooled ovum

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mRNA as the tester and cDNA from spermatozoan mRNA as the driver. After the

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synthesis of second strand cDNA, samples were digested with Rsa I to generate short,

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blunt-ended, double stranded cDNA fragments necessary for adaptor ligation.

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Subtraction was performed in two directions. RsaI-digested tester cDNA was ligated

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with either Adaptor 1 or Adaptor 2R for subtraction. The SSH products were purified

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and concentrated, and the cDNA fragments for subtracted secondary PCR products

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were inserted into the pMD 18-T vector (TaKaRa Biotechnology Co) and transformed

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into DH5α Escherichia coli cells. Colonies were grown on LB agar plates containing

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ampicillin (Kodak, New Haven, CT, USA), X-Gal (Gibco Brl., Kansas, USA), and

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isopropylthio-β-galactoside (Gibco BRL). Recombinant white clones were selected

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randomly and amplified by PCR using the M13 primer to construct the corresponding

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SSH cDNA library.

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2.4. Positive clone selection by dot blotting

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Recombinant clones were picked up and plasmids were isolated. Plasmid DNA

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was denatured at 100 °C and dotted onto duplicate Hybond-N+ nylon membranes

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(Gene Company Limited, Hong Kong, China). The secondary PCR products from

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tester-1 (spermatozoan cDNA as tester) and tester-2 (ovum cDNA as tester)

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ACCEPTED MANUSCRIPT subtraction were digested with Rsa I to remove the adaptors and labeled using the

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DIG High Prime DNA Labeling Kit and Detection Starter Kit I (Roche Applied

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Science, Mannheim, Germany). Two DIG-labeled probes were hybridized to identical

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membranes. Parallel hybridizations and signal detection were performed according to

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the manufacturer’s instructions. When the signal was stronger with the forward probes

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than with the reverse probes, the cloned inserts were compared by contrasting the

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hybridization signals of the two membranes. cDNA clones showing differential

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hybridization were isolated and sequenced.

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2.5. Sequencing and BLAST analysis

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Positive clones with significant differences in hybridization signals were selected

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for sequencing. The vector and incorrect sequences were removed using the

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chromatogram software to obtain ESTs. The ESTs were compared with the

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non-redundant

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(http://www.ncbi.nih.gov). ESTs with unknown functions were annotated using

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BLASTn after comparison with the non-redundant nucleotide sequence database and

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EST sequence database.

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2.6. Gene expression studies

sequence

database

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NCBI

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Semi-quantitative PCR using cDNA from spermatozoa and ova of Yellow River

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carp was used to determine the expression of selected up-regulated and

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down-regulated ESTs from SSH libraries and to evaluate their relative expression

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levels. Specific primers for the candidate genes were designed from EST sequences

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(Table 1). Amplification was carried out under standard cycling conditions, except for

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ACCEPTED MANUSCRIPT the annealing temperature. The expression levels of the candidate genes were

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normalized using the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH),

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which has been tested for stability of expression in tissue of Yellow River carp, as an

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internal control (Table 1) and measured as the ratio of candidate/GAPDH.

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Agarose/ethidium bromide gels were photographed and analyzed using the Gel Doc

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XR imaging system and Quantity One Software (Bio-Rad, California

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Table 1

USA).

2.7. CcWt1a, CcSetd6, and CcZP3C expression analysis by semi-quantitative and

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real-time RT-PCR

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The housekeeping gene GAPDH served as an internal reference gene, using the

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gene-specific primers GAPDH-F (forward) and GAPDH-R (reverse). CcWt1a,

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CcSetd6, and CcZP3C transcripts were detected using forward and reverse primers

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(Table 1). Semi-quantitative RT-PCR (sqRT-PCR) was performed on a volume of 25

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µl containing 1 µL of cDNA template, 2.5 µL of 10×PCR buffer (MgCl2 2.5 mM), 4

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µL of dNTP (10 mM), 1 µL of each primer (10 pmol/µL), 15.3 µL of PCR-grade water,

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and 0.2 µL (1U) of Taq polymerase (TaKaRa). PCR conditions were as follows: 30

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cycles of 95 ˚C for 3 min, 95 ˚C for 30 s, 55 ˚C for 30 s, and 72 ˚C for 30 s followed

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by 72 ˚C for 10 min. All PCR products were separated by electrophoresis on 1%

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horizontal agarose gels.

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Real-time reverse transcriptase-polymerase chain reaction was performed to

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quantify the relative expression levels of CcWt1a (from male libraries), CcSetd6 and

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CcZP3C (from female libraries) in several organ tissues, using an ABI 7500 real-time

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ACCEPTED MANUSCRIPT PCR detection system (Applied Biosystems, California

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fluorescent labeling. All samples (5 male and 5 female) were analyzed in triplicate,

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and all reactions were repeated twice, independently, to ensure reproducibility.

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GAPDH was used to normalize data among samples. The gene-specific primers for

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CcWt1a, CcSetd6, and CcZP3C are given in Table 1. Amplifications were performed

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in a reaction mixture of 50µL containing 12.5µL of 29 SYBR Premix Ex Taq (Takara),

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3.5 pM of each primer, 2µL of diluted cDNA, and 0.5 µL 50 × ROX Reference Dye II

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(Takara). Data were analyzed using 7500 system SDS Software v 1.4 (Applied

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Biosystems). Expression levels were analyzed using the 2-

Ct method [5]. The data

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from these samples were used to calculate the mean of the relative quality (RQ) value,

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2-∆∆CT [∆CT = CT (cycle threshold) of target gene minus CT of GAPDH,

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∆∆CT = ∆CT of sample minus calibrator sample] and standard error (SE). Student's

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t-test

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(http://www-01.ibm.com/software/analytics/spss).

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accepted at P < 0.05 (two-tailed test). To ensure reliability of the results, PCR

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products were detected by gel electrophoresis using 1.5% agarose gel.

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3. Results

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3.1. Histological characteristics of carp gonads

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Micrographs of the carp testis and ovary used in this study were examined to

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determine the maturation level of gonads (Fig. 1A1&2). The testes were shown to

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possess

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spermatocytes and spermatozoa). Sertoli cells were also present in the epithelium

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abundance

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precursor

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stages

(spermatogonia,

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ACCEPTED MANUSCRIPT around the tubules of the carp, which have been applied on testes in the period of

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sexual maturity. Ovaries were filled with yolk. Oocytes were readily identified by

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their large spherical nucleoli, each containing many nucleoli and a large cytoplasmic

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region bordered by a visible cell membrane. Oocytes varied in size, and each was

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surrounded by a thin layer of follicle cells (Fig. 1B1). The majority of the follicle cells

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contained ovoid nuclei and stained darkly (Fig. 1B2), which indicated the specimen

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used was in the pre-spawning period.

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3.2. Building and screening of the subtractive cDNA library.

Secondary PCR products from tester-1 and tester-2 were purified and connected

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with T vector, and transformed into an E. coli JM109 coated tablet.

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Two-hundred-eighty clones from tester-1 and 240 clones from tester-2 were selected

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and identified using nested primers by colony PCR (primer pairs). False positives

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from self-ligation and small fragments (less than 150 bp) were eliminated, and 90

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clones from tester-1 and tester-2 carrying the distinguishable insertions (insert size

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200–800 bp) were identified, selected, and dotted onto duplicate nylon membranes,

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then hybridized separately with DIG-labeled forward or reverse subtracted cDNA.

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Twenty-one clones from tester-1 and 43 from tester-2 exhibited distinct differences

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(Fig.2).

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ovarian-differential fragments and were sequenced using SP6 and T7 primers.

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Fig.2.

3.3. Functional analysis of gonad-related ESTs

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ACCEPTED MANUSCRIPT Sequence homology searches were conducted in BlastX and tBlastX entries in

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GenBank. For tester-1, 18 ESTs were identified by their functional classification in 21

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sequenced clones. These recovered genes encoded several classes of functional

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proteins, including transcription factors, DNA-binding proteins, metabolic enzymes,

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tumor-related proteins, and intracellular proteins. Twelve of the 18 (66.7%) showed

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significant similarities (≤10−3); four (22.2%) showed e-values >10−3, and two (11.1%)

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displayed no similarities or repetitive sequences (Table 2).

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For tester-2, 30 functional ESTs were identified in 43 sequenced clones.

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Twenty-seven known functional genes encoded proteins including adjustment factors,

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structural proteins, enzymes, transcription factors, and signal regulators. The

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frequencies of respiratory chain gene fragments, such as ATP synthase coupling factor

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6 (ATP-6) and NADH dehydrogenase (NDUFA1) were relatively high, while the

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frequencies of genes related to protein synthesis, such as 60S ribosomal protein and

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ribosomal protein L9, were similar. Twenty-four of the 30 (80%) showed significant

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similarities; five (16.7%) displayed e-values >10−3 and three (10%) showed no hits or

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repetitive sequences (Table 3).

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Table. 3

3.4. Expression of candidate genes

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To examine the efficacy of SSH libraries and the temporal expression patterns of

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identified genes, cDNAs corresponding to partial candidate genes from tester-1 and

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tester-2 were analyzed by semi-quantitative PCR. There was a clear difference

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ACCEPTED MANUSCRIPT between spermatozoan and ovarian samples with respect to these candidate genes.

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Nine identified ESTs from the tester-1 library exhibited significant differences in

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expression patterns, including Tektin-1, GAPDS, FGFIBP, IGFBP-5, and an unknown

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gene from the Ccmg4 clone, specifically expressed in testis (Fig. 3). Expression levels

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of GSDF, BMI1b, Wt1a, and unknown genes from the Ccme2 clone were higher in

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testis than in ovary. Eight identified ESTs from the tester-2 library also exhibited

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significant differences: ZP3C and Psmb2 were specifically expressed in ovary, and

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IFIPGL-1, Setd6, ATP-6, CDC45, AIF-1, and an unknown gene from the Ccfh2 clone

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were more highly expressed in ovary than in testis.

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3.5. mRNA expression of CcWt1a, CcSetd6 and CcZP3C

Three genes were selected and investigated in more detail because their roles in

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fish gonad development are little known, although this aspect in mammals has been

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reported like CcWt1a and CcSetd6 or was conflict in the fish gonad development

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regulation like CcZP3C (details in the discussion). The expression patterns of Ccwt1a

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gene were analyzed in adult tissues using semi-quantitative RT-PCR and fluorescent

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RT-PCR. The expression of the Ccwt1a gene varied among tissues in the male and

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female carp, with stronger expression detected in kidney, liver, and gonad of males

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and similar expression in other tested tissues in both males and females (Fig. 4). The

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CcZP3C gene was expressed exclusively in ovary in Yellow River carp (Fig. 5). The

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mRNA levels of CcSetd6 were significantly greater in tissues of females than of males

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with the exception of liver (Fig. 6).

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Fig.5.

Fig.4.

Fig.6.

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4. Discussion The mechanisms responsible for the obvious phenotypic differences between

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female and male fish are unknown. The identification and characterization of genes

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that are differentially expressed in ovary and testis will contribute to our knowledge of

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the developmental events and relevant mechanisms. Information on the relative gene

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expression patterns in ovary and testis is limited; however, available biological

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information for model fish species such as zebrafish, medaka, and salmon continues

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to increase, and the genetic databases for these species may provide a basis for

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libraries to allow the identification of corresponding genes in other fish. To gain

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insight into the molecular foundations of gonad development and growth, we

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successfully constructed female and male bi-directional subtractive cDNA libraries for

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Yellow River carp, and identified 48 EST sequences with differing functions. The

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genes with known functions coded for regulatory factors, structural proteins, enzymes,

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transcription factors, and tumor-associated and tumor suppressor proteins and were

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involved in complex biological reaction systems such as cell differentiation, ion

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transit, energy metabolism, and signal regulation. In the male SSH library, 14 of 18

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ESTs (77.8%) represented known functional genes, while the remaining four (22.2%)

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ESTs were unknown. In the female SSH library, 27 of 30 (90%) ESTs were known

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functional genes, and the other three (10%) were unknown. Similar incidences of

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61.1% and 66.7%, respectively, were identified in male and female zebrafish libraries,

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indicating that these gonad-development-related genes are conserved in fish.

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4.1. Male EST library gene function exploration Among the most abundant genes in the male library, nine selected genes were

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confirmed to exhibit differential expression in male and female adult tissues,

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including Tektin-1, GAPDS, FGFIBP, IGFBP-5, GSDF, BMI1b, Wt1a, and two

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unknown genes from Ccmg4 and Ccme2 clones. Research has revealed that these

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genes are expressed exclusively in testis in mammals and investigated fish.

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Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS), a germ cell specific isozyme,

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is tightly bound to the fibrous sheath, a cytoskeletal structure that defines the limits of

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the principal region of the sperm flagellum [6]. This localization of respiration and

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glycolysis in distinct compartments suggests that both metabolic pathways may be

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needed to provide sufficient ATP along the entire length of the flagellum to support

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activated and hyperactivated motility. There appear to be some species differences in

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spermatozoa metabolism [7], but in vitro studies have revealed that glycolysis is

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required for mouse [8], rat [9], and human spermatozoa [10] to achieve hyperactivated

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motility and penetrate the zona pellucida.

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Tektin genes are found throughout the animal kingdom and have been sequenced

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in mammals, fish, sea urchins, insects, and nematodes. They also occur in algal

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species, such as the unicellular Chlamydomonas, but not in flowering plants. They

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therefore appear to occur in all eukaryotic organisms that develop cilia or flagella [11].

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Some organisms have a single tektin; for example, zebrafish have only tektin 1, a

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testis protein. Others have several tektins: for example, sea urchins possess three in

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ACCEPTED MANUSCRIPT the spermatozoa tail, and humans have at least six, some of which are testis-specific,

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whereas others also occur in cilia and centrioles in cells of other tissues. The human

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tektin genes are found on different chromosomes. Iguchi et al. reported that the Mus

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musculus tektin-t protein, first identified in a cDNA library from haploid germ cells,

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localized specifically to the flagella at the stages from elongating spermatid to mature

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spermatozoon, suggesting that tektin-t plays an important role in flagellum formation

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and spermatozoon motility [12]. Moreover, because the gene is rarely mutated, the

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testis-specific Tektin gene can be used as a core gene to investigate the evolutionary

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distance between closely related species [13,14].

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The genes for insulin-like growth factor (IGF) and its receptor, as well as

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IGF-binding protein (IGFBP), are key regulatory genes for cell growth and

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development. The testis-immunomodulatory IGF gene is first detected in the sperm

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production tube in adult mice and in Sertoli cells in immature rats [15]. Further

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research has revealed that the IGF gene is also specifically expressed in the testis in

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other animals, and its expression patterns change as the testis matured. IGFs are

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expressed in the testis from the fetal period (100–102 days) to 25 weeks in pigs, with

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expression levels gradually increasing in a time-dependent manner [16]. In mice, IGF

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gene expression mainly occurs in Sertoli cells in the testis during the 14 days after

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birth, as well as in the testis epithelial cells during sperm production from 35 days.

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[17]. The IGF gene can stimulate DNA synthesis in spermatogonia [18], induce

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differentiation of A-type spermatogonia [19], and, importantly, induce the secretion of

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testosterone [20]. This indicates that the IGF gene and IGFBP protein are master

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ACCEPTED MANUSCRIPT regulators of vertebrate testicular development. Although this gene family has rarely

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been reported studies of in fish gonad development, evidence suggests that the

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IGFBP-5 gene may be involved in testicular development in Yellow River carp. The

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identification of this gene will provide a resource for exploring the development of

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male gonads in Yellow River carp and other fish.

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Gamete development is a complex and orderly process, involving reproductive

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cell renewal, proliferation, and finally differentiation into highly specific haploid cells.

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This process is completed by direct contact or by secretion of cytokines from

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reproductive tubes, Sertoli cells in the testes, or granulosa cells in the ovaries.

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Large-scale analysis of gene expression patterns has revealed that these cells secrete a

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new transcription factor, gonadal soma-derived factor, encoded by the GSDF gene,

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which was found to be specifically expressed in the testes of rainbow trout during

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spermatogenesis [21]. Further study found that reorganization of GSDF could

14

stimulate spermatogonia proliferation. This gene was found to be expressed in the

15

genital ridge somatic cells surrounding the primordial germ cells during

16

embryogenesis and in Sertoli cells in testis and granulosa cells in ovary at later stages

17

in medaka [22]. In the current study, we detected a gene fragment with high similarity

18

to the zebrafish GSDF gene, which was expressed in the gonad at a higher level in

19

testis than in ovary (Fig. 3). Based on previous findings, it is possible that GSDF may

20

enhance the proliferation of spermatogonial stem cells in Yellow River carp testes,

21

while its expression in ovary suggests that it may act as a potential regulator of

22

reproductive function. The fact that oocytes do not proliferate suggests that GSDF

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ACCEPTED MANUSCRIPT 1

may have a different function in the ovary from that in the testis. Multiple functions have been reported for fish activin, another member of the

3

transforming growth factor-β superfamily, including the induction of oocyte

4

maturation [23] and spermatogonia proliferation [24]. It is possible that GSDF may

5

also have multiple functions [22], and further studies are needed to clarify its roles in

6

Yellow River carp.

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The Wilms’ tumor 1 gene (Wt1) has been identified as a tumor suppressor gene

8

involved in the etiology of Wilms’ tumor. Wt1 is expressed in the urogenital ridge and

9

then becomes localized to the Sertoli cells, granulosa, and epithelial cells of the testis

10

[27] where most of the sex-determining genes are found, including SRY/Sry, MIS/Mis,

11

SF-1 / Sf-1, and DAX-1/Dax -1, which are regulated by the Wt1 gene. Two Wt1

12

homologs, Wt1a and Wt1b, have been identified in fish [26]. Both these genes consist

13

of nine exons, including sequences encoding zinc finger domains. Wt1a is expressed

14

earlier than Wt1b; the former is expressed during the mesoderm period and the latter

15

in mature nephric tubules. The Wt1 gene is also involved in regulation of the

16

aromatase gene in fish [27]. Although Wt1 plays an important role in normal testes

17

and kidney development in mammals, little information is available for teleosts with

18

the exception of Japanese medaka [28], and further exploration of its function in fish

19

is warranted. In the current study, we isolated the Ccwt1a gene from the male Yellow

20

River carp SSH library; expression showed sexual dimorphism in the gonad, kidney,

21

and liver (Fig. 4). In accordance with previous reports in mammals, the gonads and

22

metanephric kidneys are derived from a common precursor, the urogenital ridge.

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ACCEPTED MANUSCRIPT During the early stages of sexual development, the testes and ovaries cannot be

2

distinguished and are referred to as indifferent or bipotential gonads. Similarly, the

3

urogenital tracts are also similar in female and male embryos. Thickening of the

4

epithelium of the urogenital ridge marks the first stage of gonad development,

5

followed by formation of the gonadal ridge. From the gonadal ridge, the Mullerian

6

ducts are formed in the undifferentiated gonads of female and male embryos. The

7

Mullerian ducts develop into structures of the female reproductive tract, while the

8

Wolffian ducts, derived from the mesonephros, become components of the male

9

reproductive tract. Consequently, for normal embryo development to occur, one duct

10

system must develop while the other regresses. Ccwt1a may be a major gene

11

responsible for controlling kidney and male development. Further studies are needed

12

to clarify these mechanisms in carp.

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Other genes were also upregulated in the testis in male SSH libraries, including

14

the fibroblast growth factor binding protein gene (FGFBP), which, in mice, was

15

reported as expressed mainly in developmental stages of testis, decreasing with

16

maturation [29]. These proteins are highly expressed in testis, but little is known about

17

their specific function. These results provide an initial step towards more complete

18

knowledge of the molecular mechanisms regulating spermatogenesis.

19

4.2. Female SSH library gene functions

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Some gene fragments, including genes for components of the respiratory chain

21

such as ATP synthase (ATP-6) and NADH dehydrogenase (NDUFA1), were expressed

22

at higher frequencies in the female library than in the male library. Mitochondria are

19

ACCEPTED MANUSCRIPT vital for metabolic processes (citric acid cycle, electron transport, oxidative

2

phosphorylation) and are also the major site of energy production, generating more

3

than 90% of the ATP in the organism. The energy conversion of ATP is a complex and

4

orderly biological process, with each reaction requiring the activity of different

5

enzymes. It is possible that the expression levels of the mtDNA-encoded ATP

6

synthase and NADH dehydrogenase were higher in female libraries because the

7

metabolic activity of the female gonads is significantly higher than in those of the

8

male during the maturation process, and requires more ATP.

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Developing vertebrate oocytes are surrounded by an extracellular matrix

10

membrane called the zona pellucida, which is required for follicle formation,

11

fertilization, and early development. Studies on tilapia revealed that ZPC was

12

expressed exclusively in ovary [30]. ZPC in mice serves as a primary sperm receptor

13

and acrosome reaction-inducer. Knockout of the ZPC gene in mice resulted in the

14

failure of the ZP protein to accumulate around developing oocytes, and affected

15

females were infertile [31]. The vitelline envelope surrounding the egg in fish, birds,

16

and amphibians contains glycoproteins homologous to mammalian ZP proteins. These

17

glycoproteins play an essential role in the assembly of the extracellular structural coat

18

during oogenesis. They serve as a selectively permeable barrier during the early stages

19

of oocyte development, and as a protective layer for the developing embryo. They

20

also participate in sperm-egg interaction and contribute to the block of polyspermy in

21

mammals. However, in several teleost fish species, exceptions exist. For example, the

22

ZP components are transcribed in the liver in response to estrogen regulation [32,33].

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ACCEPTED MANUSCRIPT In medaka, two major proteins of the egg envelope that belong to ZPC (choriogenin L)

2

and ZPB (choriogenin H) classes are synthesized by the liver [34]. In the present study,

3

we isolated a gene with high homology to zebrafish ZP3C from the female SSH

4

library that was exclusively expressed in ovary (Fig. 5). This information will be

5

useful for studying oocyte development and the mechanisms that determine

6

sex-specific gene expression in carp. This is also the first promoter shown to drive

7

stable and efficient expression specifically in the Yellow River carp female germline,

8

and may thus have potential as a sex-specific molecular marker.

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The Setd6 gene was identified in the female library. This gene encodes a protein

10

from the SET-domain-containing protein family, an evolutionarily conserved family

11

of epigenetic regulators responsible for most histone lysine methylation. Some of

12

these genes have been revealed to be essential to embryonic development. Gene

13

expression is subject to a high degree of temporal and spatial regulation during early

14

vertebrate development, from cleavage, through blastula and gastrulation, to

15

organogenesis, and the levels and locations of histone modifications also change

16

dynamically during this process [35]. Recent genetic studies have indicated that some

17

SET-domain genes are essential for normal embryo development and survival [36],

18

but their role in fish gonad development is not known. In our study, Setd6 expression

19

was significantly higher in female tissues than in males, with the exception of in liver

20

(Fig. 6). We therefore speculated that the setd6 gene might play an important role in

21

epigenetic regulation in female development of Yellow River carp, and may be used to

22

study the biological functions of genes during early development.

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ACCEPTED MANUSCRIPT Other genes were also up-regulated or specifically expressed in the ovary in the

2

female library, such as the genes for prosome subunit beta type-2 (Psmb2),

3

interferon-inducible protein Gig1-like (IFIPGL-1), allograft inflammatory factor-1

4

(AIF-1), and cell division control protein 45 (CDC45). The molecular bases of gonad

5

development, oocyte maturation, hydration, and ovulation, as well as the mechanism

6

responsible for fertilization in carp has been unclear, but the current analysis of ESTs

7

from the SSH library has allowed the identification of genes that may contribute to

8

our understanding of the ovary’s molecular and physiological status. Some genes

9

showed differential expression not only in the reproductive tissue, but also in other

10

tissues. Further investigations into the expression and function of these genes in

11

female and male fish are needed to clarify these preliminary results. Analyses of

12

expression patterns are necessary to expand knowledge of gonad differentiation and

13

the relationship between gonad development and growth.

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Acknowledgments

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These studies were supported by the National Natural Science Foundation of

17

China (NSFC- Henan Joint Training Fund for Fostering Talents) (No. U1204329) and

18

Henan Provincial Key Scientific and Technological Project in China (No.

19

122102210168).

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ACCEPTED MANUSCRIPT

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[1] Gui JF. Genetic basis and artificial control of sexuality and reproduction in fish. BeiJing: Science Press; 2007. [2] Degani G, Baker R, Jackson K. Growth Mormons, Gonad Development, and Steroid Levefs in Female Carp. Comp Biochem Physiol 1996; 2:133-40. [3] Xu Q, Wen XP, Tao NG, Hu ZY, Yue HL, Deng XX. Extraction of high quality of RNA and construction of a suppression subtractive hybrization (SSH) library from chestnut rose (Rosa roxburghii Tratt). Biotechnol Lett. 2006; 28: 587-91. [4] Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A 1996; 93 (12): 6025-30. [5] Kenneth JL, Thomas DS. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−∆∆CT Method. Methods 2001; 25(4): 402-8. [6] Bunch DO, Welch JE, Magyar PL. Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution during mouse spermatogenesis. Biol. Reprod. 1998; 58: 834–41. [7] Mann T, Lutwak-Mann C. Male Reproductive Function and Semen (Springer, New York). 1981; pp. 198–268. [8] Urner F, Sakkas D. Glucose is not essential for the occurrence of sperm binding and zona pellucida-induced acrosome reaction in the mouse. Int. J. Androl. 1996; 19: 91–6. [9] Bone W, Jones NG, Kamp G, Yeung CH, Cooper TG. The antifertility effect of ornidazole in male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilisation in vitro. J Reprod Fert 2000; 118: 127-37. [10] Williams AC, Ford WC.The role of glucose in supporting motility and capacitation in human spermatozoa.J Androl 2001; 22(4): 680-95. [11] Setter PW, Malvey-Dorn E, Steffen W, Stephens RE, Linck RW. Tektin interactions and a model for molecular functions. Exp Cell Res 2006; 312: 2880-96. [12] Iguchi N, Tanaka H, Fujii T, Tamura K, Kaneko Y, Nojima H, et al. Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis. FEBS Lett 1999; 456: 315-21. [13] Ota A, Kusakabe T, Sugimoto Y, Takahashia M, Nakajimac Y, Kawaguchia Y, et al. Cloning and characterization of testis-specific tektin in Bombyx mori. Comp Biochem Physiol B: Biochem Mol Biol. 2002; 133: 371-82. [14] Mallarino R, Bermingham E, Willmott KR, Whinnettb A, Jiggins CD. Molecular systematics of the butterfly genus Ithomia (Lepidoptera: Ithomiinae): a composite phylogenetic hypothesis based on seven genes. Mol Phylog Evol 2005; 34: 625-44. [15] Ritzen EM. Chemical messengers between Sertoli cells and neighbouring cells. J Steroid Biochem 1983; 19: 499–504. [16] Clark AM, Samaras SE, Hammond JM, Hagen DR. Changes in the messenger ribonucleic acid for insulin-like growth factor-I and -II in the porcine testis during and between two waves of testicular development. Biol Reprod 1994; 50: 993–9. [17] Closset J, Gothot A, Sente B, Scippo ML, Igout A, Vandenbroeck M, et al. Pituitary hormones dependent expression of insulin-like growth factors I and II in the immature hypophysectomized rat testis. Mol Endocrinol 1989; 3 (7): 1125–31.

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References

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[18] Voutilainen R, Miller WL. Developmental and hormonal regulation of mRNAs for insulin-like growth factor II and steroidogenic enzymes in human fetal adrenals and gonads. DNA 1988; 7: 9–15. [19] Tajima Y, Watanabe D, Koshimizu U, Matsuzawa T, Nishimune Y. Insulin-like growth

5

factor-I and transforming growth factor-α stimulate differentiation of type A spermatogonia

6

in organ culture of adult mouse cryptorchid testes. Int J Androl. 1995; 18: 8–12. [20] Oonk RB, Grootegoed JA. Insulin-like growth factor I (IGF-I) receptors on Sertoli cells from

8

immature rats and age-dependent testicular binding of IGF-I and insulin. Mol Cell Endocrinol.

9

1988; 55: 33–43.

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[21] Mazurai D, Montfort J, Delaland C, Le GF. Transcriptional analysis of testis maturation using trout cDNA macroarrays. Gen. Comp. Endocrinol 2005; 142: 143–54. [22] Sawatari E, Shikina S, Takeuchi T, Yoshizaki G. A novel transforming growth factor-beta superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss), Dev. Biol. 2007; 301: 266–75. [23] Pang Y, Ge W, Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio). Biol. Reprod 2002; 66: 259–65. [24] Miura T, Miura C. Japanese eel: a model for analysis of spermatogenesis. Zool. Sci 2001; 18: 1055–63. [25] Rackley RR, Flenniken AM, Kuriyan NP, Kessler PM, Stoler MH, Williams BR. Expression of the Wilms' tumor suppressor gene WT1 during mouse embryogenesis. Cell Growth Differ 1993; 4: 1023–31. [26] Bollig F, Mehringer R, Perner B, Hartung C, Schäfer M, Schartl M, et al. Identification and comparative expression analysis of a second wt1 gene in zebrafish. Dev. Dyn 2006; 235: 554–61. [27] Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier AF, et al. Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 1998; 125: 4655–67.

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

[28] Lim HN, Hughes IA, Hawkins R. Clinical and molecular evidence for the role of

EP

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M AN U

SC

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

androgens and WT1 in testis descent. Mol. Cell. Endocrinol 2001; 185: 43

50.

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[29] Mullaney BP, Skinner MK. Basic fibroblast growth factor (bFGF) gene expression and

[30]

[31] [32]

[33]

protein production during pubertal development of the seminiferous tubule: follicle-stimulating hormone-induced Sertoli cell bFGF expression. Endocrinology 1992; 131: 2928–34. Chu SL, Weng CF, Hsiao CD, Hwang PP, Chen YC, Ho JM, et al. Profile analysis of expressed sequence tags derived from the ovary of tilapia, Oreochromis mossambicus. Aquaculture 2006; 251: 537−48. Wassarman PM, Jovine L, Litscher ES. Mouse zona pellucida genes and glycoproteins. Cytogenet. Genome Res. 2004; 105: 228−34. Del Giacco L, Vanoni C, Bonsignorio D, Duga S, Mosconi G, Santucci A, et al. Identification and spatial distribution of the mRNA encoding the gp49 component of the gilthead sea bream, Sparus aurata, egg envelope. Mol Reprod Dev 1998; 49: 58 69. Sugiyama H, Yasumasu S, Murata K, Iuchi I, Yamagami K. The third egg envelope subunit 24

ACCEPTED MANUSCRIPT in fish: cDNA cloning and analysis, and gene expression. Dev Growth Differ 1998; 40: 35 45. [34] Murata K, Sugiyama H, Yasumasu S, Iuchi I, Yasumasu I, Yamagami K. Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion). Proc Natl Acad Sci USA 1997; 94: 2050 2055. [35] Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 2007; 445: 214–8. [36] Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, et al. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev 2005; 19: 815–26.

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ACCEPTED MANUSCRIPT Table 1

2

Primers used in RT-PCR

3

Table 2

4

Sequence and functional analysis of ESTs in male subtracted library

5

Table 3

6

Sequence and functional analysis of ESTs in female subtracted library

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SC

7

Figure legends

9

Fig.1. Sections of carp testis and ovary after fixation in Bouin's fluid. The same

10

section was stained by hematoxylin and eosin (thickness, 6 µm). (A1 and A2)

11

Formation of a testis cavity in an adult common carp. (A1) The testis structure at low

12

magnification (×100); (A2) The testis structure at high magnification (×1000), SG:

13

spermatogonium, , SC: spermatocytes, SZ: spermatozoa, SE: sertoli cells. (B1 and B2)

14

Formation of ovarian cavity in an adult common carp. (B1) The ovary structure at low

15

magnification (×100), PO: provitellogenic oocytes, VO: vitellogenic oocytes; (B2)

16

The ovary structure at high magnification (×1000), FC: follicle cells, Y: yolks, N:

17

nucleus.

18

Fig. 2. Dot blotting of tester-1 and tester-2. (A) Dot blot using forward SSH products

19

as probe. Nine clones with arrows, including g7: IGFIBP-5; e2: Unknown gene; d3:

20

BMI1b; d2: GSDF; e7: GAPDS; g4: Unknown gene; c1: Wt1a; i1: FGFIBP; i2:

21

Tektin-1, were selected for further RT-PCR analysis; (B) Dot blotting using reverse

22

SSH products as probe. Eight clones with arrows, including c6: ZP3C; b6: Setd6; b8:

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26

ACCEPTED MANUSCRIPT Psmb2; h2: Unknown gene; c8: IFIPGL-1; b5: AIF-1; d10: ATP-6; c1: CDC45 were

2

selected for further RT-PCR analysis, Arrows indicate significantly different clones.

3

Fig. 3. The differential expression of partially homologous genes in the Yellow River

4

carp. (A) Differentially expressed genes from male SSH Library; (B) Differentially

5

expressed genes from female SSH Library.

6

Fig. 4. Expression of Ccwt1a in adult tissues of C. carpio. G: gonad; H: Heart; L:

7

liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative RT-PCR

8

and amplified using GAPDH as an internal reference; (B) Expression levels were

9

measured in tissues by fluorescent real-time RT-PCR. *P < 0.05 between experimental

M AN U

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1

group and control group. Error bars indicate standard deviation.

11

Fig. 5. Expression analysis of CcZP3C transcripts in various tissues. G: gonad; H:

12

Heart; L: liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative

13

RT-PCR and amplified using GAPDH as an internal reference; (B) Expression levels

14

were measured in tissues by fluorescent real-time RT-PCR. Error bars indicate

15

standard deviation.

16

Fig. 6. Expression analysis of CcSetd6 transcripts in various tissues. G: gonad; H:

17

Heart; L: liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative

18

RT-PCR and amplified using GAPDH as an internal reference; (B) Expression levels

19

were measured in tissues by fluorescent real-time RT-PCR. Error bars indicate

20

standard deviation.

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27

ACCEPTED MANUSCRIPT Table 1.Primers used in RT-PCR GenBank

The primers of male SSH libraries

GenBank

accession

accession

numbers

numbers

The primers of female SSH libraries

GAPDH-F: 5/GCCTCCTGCACCACCAACTG3

JZ584099

GAPDS-F: 5 TAAAGTCACCGCCATCAAC3/

RI PT

GAPDH-R: 5/CGGAAGGCCATGCCGGTCAG3/ /

ZP3C-F: 5/CCAGGAAGAGGAGGTATC3/

JZ584109

GAPDS-R: 5/GCCATCCACTGGTCTTCT3/ GSDF-F: 5/TGCGGTTCTGAGAGCACAG3/ /

GSDF-R: 5 TGCGGTCAGGAAGGCTTTC3 JZ584101

Setd6-R: 5/TCCAGATTGGCGTTGTGCTTG3/

/

Tekin-1-F: 5 TAGGAGAGACAGCAAATCAG3/

ATP-6-R: 5/CTTCGGTCAGGTTCTTCTG3/

JZ584112

IGFBP-5-F: 5/CAACAGGAGAAGCATTCGG3/ IGFBP-5-R: 5/GAAAGAGCCATGGACACG3/ BMI1b-F: 5/CGCTGTAGATTCGATGCG3/ /

IFIPGL1-R: 5/GATCTGAGTGCTGTGTCAC3/

JZ584113

Unknown-F(Ccmg4):5/ GTCTCTACAGGGAGCAACC3/

JZ584114

FGFIBP-F: 5/CCAACAAATACATCCAG3/ FGFIBP-R: 5/TGACGAGTAGTCAAGTCG3/

JZ584106

/

Wt1a-F: 5 TCGTCAGGTTCCTCTGGT3

/

TE D

CDC45-F: 5/GCAGCGTTGGTGATGTTTG3/ CDC45-R: 5/AGAGTCGGCACAATCCTG3/

Unknow(Ccfh2)-F: 5/TGAACAAAGTTATCCCTGC3/ Unknow(Ccfh2)-R: 5/CATTTTAGACGCCCTGCTT3/

Unknown-F(Ccmg2): 5/TGGTGTGTCAGTTGGTGTG3/ Unknown-R(Ccmg2): 5/GAGATGCTGTGGTATTTC3/

Fluorescent-quantitative RT-PCR

EP

Wt1a-F: 5/TCGTCAGGTTCCTCTGGT3/

Wt1a-R: 5/CACCAGAGAAGACACACAGG3/ Setd6-F: 5/ GGTATGGAGAACAAGACG 3/ Setd6-R: 5/ GAAATGGCGACAGATGC 3/

AC C

JZ584107

JZ584115

JZ584116

Wt1a-R: 5/CACCAGAGAAGACACACAGG 3/

AIF-1-F: 5/GAGGTGACAGGAGGTTG3/ AIF-1-R: 5/ CCATCTCCACTGGTCTATGG3/

Unknown-R(Ccmg4):5/TTCACTGTCAGATGTTTCGG3/ JZ584105

Psmb2-F: 5/CAAACTTCACCAGGAAAAACC3/ Psmb2-R: 5/GATGGAGAGAGTGAGGAAGGC3/

BMI1b-R: 5 CCAGATGTGGTCAGATC3/ JZ584104

IFIPGL1-F: 5/CTAACGGTCGTATGATTGC3/

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JZ584103

ATP-6-F: 5/CTTCATCGGCTCTTCCACC3/

JZ584111

Tekin-1-R: 5/TAGGCACAACTGGCGTC3/ JZ584102

Setd6-F: 5/TGACCTGTGGGACCCTGAGAA3/

JZ584110

/

SC

JZ584100

ZP3C-R: 5/GTGAGTTCTCGTCTGTTT3/

ZP3C-F: 5/CAGTAGTGCTGAATGTGAAGC3/ ZP3C-R: 5/CGAGGCAGTGTAACATCTCT3/

ACCEPTED MANUSCRIPT Table 2. Sequence and

Clone No.

functional analysis of ESTs in male subtracted library

homeotic gene

GeneBank

Species

E-value

No.

Occurence

Property of

Frequencies protein

Reverse transcriptase

XP_00293

Xenopus

0.005

_1

3173

(Silurana) tropicalis

Ccmg7

Ferritin,

heavy

NP_00100

polypeptide （Fth）

2378

insulin-like

AAM5154

growth

factor binding protein

Danio rerio

Danio rerio

9.1

significant

similarity found

Ccme2

No

significant

Ccmb4

transcript

TE D

similarity found Ccma9

9e-18

M AN U

No

2e-21

XP_00135

GA17823-RA

9326.2

novel protein

CAM1510

1

iron metabolism

5 (IGFBP-5)

Ccmb3

transcription factor

1

SC

Ccma8

1

RI PT

Ccma7

Drosophila

0.61

Binding proteins

1

Unknown

1

Unknown

1

transcription factor

Danio rerio

6e-13

1

Unknown

YP_97039

Acidovorax

0.32

1

Genes

6.1

avenae

involved

subsp.

immune

citrulli

system

Ccmb1

EP

2.1

acriflavin

resistance

AC C

protein(ACR)

Ccmd3

B Mo-MLV

in

AAC00-1

lymphoma insertion

CAQ14521

Danio rerio

2e-35

1

.1

Structural protein

region 1b（BMI1b） Ccmd2

gonadal somatic cell

NP_001108

Danio rerio

0.11

1

Regulatory

,

ACCEPTED MANUSCRIPT derived factor（gsdf） 140.1

signaling growth factor

Ccme5

structural

NP_001155

maintenance

of

Danio rerio

4e-18

1

103.1

Structural protein

1A Ccme7 Ccmi3

glyceraldehyde

NP_99825

3-phosphate

9.1

Danio rerio

dehydrogenase-s,

L35a

Ccmg4

SC

ADF97602. 1

novel protein

CAK04994

Ccmg5

EP

AC C

Ccmi2

Tektin-1

translation machinery

2

Ionic channels

us

suppressor 1a（Wt1a） 766.1

in the protein

1e-12

5265.1

NP_001117

Gene involved

Unknown

chain 3, ciliary-like

tumor

1

1

Saccogloss

Wilms'

1e-15

9e-14

XP_00273

(DNAI1)

glycolysis and

Danio rerio

Dynein intermediate

Ccmi4

Ccmc1

TE D

.1

Danio rerio

M AN U

protein

2

functions

testis-specific ribosomal

1e-20

nuclear

(GAPDS)

Ccme9

RI PT

chromosomes protein

proteins

and

transporters kowalevskii Oncorhync

3e-44

1

hus mykiss

Tumor-related proteins, tumor suppressprs

NP_00100

Danio rerio

5e-05

2

7398.1

RNA association

Ccmc8 Ccmi1

fibroblast factor

growth intracellular

binding （FGFIBP）

protein

CAM1289 8.1

Danio rerio

4e-08

1

intracellular protein

ACCEPTED MANUSCRIPT Table 3. Sequence and functional analysis of ESTs in female subtracted library Homeotic gene

GeneBank No.

Species

E-value

Occurence Frequencie s

Property of protein

Ccfc1

CDC45-related protein nicotinamide phosphoribosyl transferase-like （NAMPT） zona pellucida glycoprotein 3c （Zp3c） SET domain-containing protein 6 （Setd6）

AAI54773. 1 XP_00192 0646.2

Danio rerio

3e-31

1

Cell cycle

Danio rerio

0.001

2

cellular respiration

XP_68561 3.3

Danio rerio

3e-41

1

glycoprotei n

NP_95589 4.1

Danio rerio

2

histone lysine methylatio n protein translation Protein synthesis and stability Structural protein

Ccfb6 Ccfh10

tetraspanin-6

Ccfb8

Prosome subunit beta type-2 (Psmb2)

Ccfb9

histone protein

Ccfb10

glycoprotein M6Ba

Ccfb2

HN1 protein

EP

H1.3-like

AC C

Ccfc2

Ccfc3 Ccfc5

Ccfc9

NP_99993 9.1 NP_00100 2609.1

ankirin repeat SOCS box-containing protein 14 Atg12 protein

and

Cytosolic thiouridylase subunit 2 （CTU 2） LOC799918 protein

4e-64

Danio rerio

3e-12

1

Danio rerio

2e-11

1

Ornithorhy nchus anatinus Danio rerio

6e-10

1

8e-10

1

Enzyme

Danio rerio

4e-09

1

BAF37941. 1

Oncorhync hus mykiss

9e-07

1

Regulatory, transcriptio n factor Structural protein

AAI54047. 1 AAH89700 .1

Danio rerio

2e-12

1

Xenopus (Silurana) tropicalis Danio rerio

7e-08

1

6e-07

1

TE D

Ccfb7

SC

Ccfc6

M AN U

Ccfb3 Ccfg4

RI PT

Clone No.

XP_00151 3112.1

NP_98225 1.1 NP_991176 .1

AAI54472. 1

Enzyme, transport RNA association hypothetica l protein

ACCEPTED MANUSCRIPT

interferon-inducible protein Gig1-like(IFIPGL1)

Ccfb5

allograft inflammatory factor-1(AIF-1)

Ccfc10 Ccfd8 Ccff5 Ccfd4 Ccff2 Ccfd5 Ccfe2 Ccfd6

60S protein

Lysosome Associated Membrane Protein 2 （Lamp2） predicted protein ATP synthase-coupling factor 6 mitochondrial (ATP-6) microtubule-associate d protein, RP/EB family, member 1 （Mapre1） putative methyl-accepting chemotaxis protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like COP9 signalosome complex subunit 8 (CSN8) im:7142009

AC C

Ccff1

ribosomal protein L9

Ccff3 Ccfh5

Ccff6

Ccfg1

0.13

1

Unknown

Danio rerio

2e-09

1

BAA32796 .1

Cyprinus carpio

1e-10

1

NP_00100 1590.1

Danio rerio

9e-22

Regulatory, transcriptio n factor Regulatory, transcriptio n factor Structural protein

EFA09528. 1 EAW92929 .1 AAI39652. 1

Tribolium castaneum Homo sapiens Danio rerio

XP_00268 0878.1 NP_99847 2.1

Naegleria gruberi Danio rerio

NP_95615 8.1

3

3.3

2

1e-16

2

hypothetica l protein Structural protein RNA association

2e-04

1

7.9

2

2e-12

3

Danio rerio

3e-10

1

Structural protein

YP_00287 1741.1

Danio rerio

1e-59

1

RNA association

XP_00274 4365.1

Callithrix jacchus

3e-13

2

cellular respiration

NP_95652 3.1

Danio rerio

9e-17

1

XP_00266 2355

Danio rerio

0.008

1

signaling growth factor Enzyme

TE D

Ccfe1

TcasGA2_TC011632

EP

Ccfd7 Ccfi8 Ccfd10 Ccfi6 Ccfi9

ribosomal

Danio rerio

RI PT

Ccfc8

CAX13323 .1 XP_00266 0606.1

SC

novel protein

M AN U

Ccfc7

Structural protein cellular respiration

ACCEPTED MANUSCRIPT

gene product from transcript GJ24163-RA LOC799918 protein

Unknown

transcriptio n factor Enzyme

Drosophila virilis

1.8

2

AAI54472. 1

Danio rerio

6e-07

1

M AN U TE D EP AC C

3

XP_00205 3949.1

SC

Ccfi4

No significant similarity found

RI PT

Ccfg2 Ccfh2 Ccfi10 Ccfg3 Ccfg5

of carp testis and ovary after fixation in Bouin's fluid. The same section

EP

Fig.1. Sections

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

was stained by hematoxylin and eosin (thickness, 6 µm). (A1 and A2) Formation of a

AC C

testis cavity in an adult common carp. (A1) The testis structure at low magnification (×100);

(A2)

The

testis

structure

at

high

magnification

(×1000),

SG:

spermatogonium, , SC: spermatocytes, SZ: spermatozoa, SE: sertoli cells. (B1 and B2) Formation of ovarian cavity in an adult common carp. (B1) The ovary structure at low magnification (×100), PO: provitellogenic oocytes, VO: vitellogenic oocytes; (B2) The ovary structure at high magnification (×1000), FC: follicle cells, Y: yolks, N: nucleus.

ACCEPTED MANUSCRIPT Tester1 A

Tester2

B

EP

TE D

M AN U

A

SC

RI PT

B

Fig. 2. Dot blotting of tester-1 and tester-2. (A) Dot blot using forward SSH products

AC C

as probe. Nine clones with arrows, including g7: IGFIBP-5; e2: Unknown gene; d3: BMI1b; d2: GSDF; e7: GAPDS; g4: Unknown gene; c1: Wt1a; i1: FGFIBP; i2: Tektin-1, were selected for further RT-PCR analysis; (B) Dot blotting using reverse SSH products as probe. Eight clones with arrows, including c6: ZP3C; b6: Setd6; b8: Psmb2; h2: Unknown gene; c8: IFIPGL-1; b5: AIF-1; d10: ATP-6; c1: CDC45 were selected for further RT-PCR analysis, Arrows indicate significantly different clones.

ACCEPTED MANUSCRIPT

Ovary

Testis

Ovary

B

A

RI PT

Testis

Fig. 3. The differential expression of partially homologous genes in the Yellow River

AC C

EP

TE D

M AN U

expressed genes from female SSH Library.

SC

carp. (A) Differentially expressed genes from male SSH Library; (B) Differentially

ACCEPTED MANUSCRIPT

G

H

L

K

B 254bp 206bp 206bp

TE D

M AN U

SC

A

RI PT

GAPDH ♂ ♀

B

Fig. 4. Expression of Ccwt1a in adult tissues of C. carpio. G: gonad; H: Heart; L:

EP

liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative RT-PCR and amplified using GAPDH as an internal reference; (B) Expression levels were

AC C

measured in tissues by fluorescent real-time RT-PCR. *P < 0.05 between experimental group and control group. Error bars indicate standard deviation.

ACCEPTED MANUSCRIPT

G

H

L

K

B 254bp

GAPDH

225bp

♀

225bp

RI PT

♂

B

EP

TE D

M AN U

SC

A

Fig. 5. Expression analysis of CcZP3C transcripts in various tissues. G: gonad; H:

AC C

Heart; L: liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative RT-PCR and amplified using GAPDH as an internal reference; (B) Expression levels were measured in tissues by fluorescent real-time RT-PCR. Error bars indicate standard deviation.

ACCEPTED MANUSCRIPT G

H

L

K

B 254bp

GAPDH

202bp

♂

202bp

B

AC C

EP

TE D

M AN U

SC

A

RI PT

♀

Fig. 6. Expression analysis of CcSetd6 transcripts in various tissues. G: gonad; H: Heart; L: liver; K: kidney; B: brain. (A) Expression was analyzed by semi-quantitative RT-PCR and amplified using GAPDH as an internal reference; (B) Expression levels were measured in tissues by fluorescent real-time RT-PCR. Error bars indicate standard deviation.

ACCEPTED MANUSCRIPT Highlights

1. Two bidirectional subtracted cDNA libraries were analyzed in parallel using testis

2. We found 9 differential expressed genes in male libraries.

RI PT

or ovary testers in Yellow River carp, respectively.

3. We identified 8 significant differences ESTs between testis and ovary in female

SC

libraries.

M AN U

4. CcWt1a expression from male libraries showed sexual dimorphism in the kidney and liver.

5. Sex-specific CcZP3C may have potential as a sex-specific molecular marker. 6. CcSetd6 expression was significant higher in the female different tissues than males

AC C

EP

TE D

except for liver.