Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis)

Accepted Manuscript Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis)

Zhongdian Dong, Ning Zhang, Yang Liu, Wenteng Xu, Zhongkai Cui, Changwei Shao, Songlin Chen PII: DOI: Reference:

S0378-1119(18)31028-X doi:10.1016/j.gene.2018.10.003 GENE 43259

To appear in:

Gene

Received date: Revised date: Accepted date:

22 May 2018 23 September 2018 1 October 2018

Please cite this article as: Zhongdian Dong, Ning Zhang, Yang Liu, Wenteng Xu, Zhongkai Cui, Changwei Shao, Songlin Chen , Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis). Gene (2018), doi:10.1016/ j.gene.2018.10.003

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ACCEPTED MANUSCRIPT Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis) Zhongdian Dong 1,2,#, Ning Zhang 1,2,#, Yang Liu2,3, Wenteng Xu 2,3, Zhongkai Cui 2,3, Changwei Shao2,3, Songlin Chen 2,3,*

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1 Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong higher Education Institutes, Fisheries College, Guangdong Ocean University, Zhanjiang, China 2 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Yellow Sea Fisheries Research Institute, CAFS, Qingdao 266071, China 3, Key Lab for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao 266071, China # These authors contributed equally to this work *Corresponding author. E-mail address: [email protected] (S. Chen)

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Abstract: Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein that regulates the interaction between somatic cells and germ cells during gonad

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developmental process in mammals. In this study, the zglp1 of Chinese tongue sole, Cynoglossus semilaevis (cysezglp1), was cloned and characterized for the first time in fish. Cysezglp1 had an open reading frame with five exons and was located to

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chromosome 9. The open reading frame of cysezglp1 consisted of 1692 nucleotides

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and encoded a 583 amino acid polypeptide. The predicted protein contained two zinc finger structures (Znf1 and Znf2), one of which was highly homologous to the

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GATA-type zinc finger domain. Multiple sequence alignment showed that Znf1 was conserved across different species while Znf2 was more divergent. Through

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quantitative Real-time PCR (qRT-PCR), we found that cysezglp1 was predominantly expressed in gonads, and the expression level of the ovary was significantly higher than that of the testis. We compared expression level in different embryonic stages and found that cysezglp1 mRNAs were mainly expressed in the fertilized egg to the cleavage stage, subsequently declining in the blastula stage. Cysezglp1 expression was not detected from the gastrulation stage onward. In the ovary, cysezglp1 expression was detected at 120 days after hatching and expression gradually increased with the maturation of the ovary. In situ hybridization showed that the cysezglp1 was mainly expressed in oocytes. Taken together, our results suggest that cysezglp1 may play an

ACCEPTED MANUSCRIPT important role in the process of oogenesis in Chinese tongue sole.

Keywords: Cynoglossus semilaevis; zglp1; qRT-PCR; Oogenesis 1. Introduction Chinese tongue sole (Cynoglossus semilaevis) is an important economic marine fish species in China. It employs a ZW/ZZ sex chromosome system, and has obvious

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sexual dimorphism where females grow 2-4 times faster than males. With the establishment of the Chinese tongue sole genome, this species has emerged as a new

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model to study sex determination and differentiation (Chen et al., 2014). In some

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environmental conditions, such as high temperature, the female can undergo sex reversal to become a pseudo-male that have effectively the same physiology as males

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(Chen et al., 2014; Shao et al., 2014). In addition, there is a very high probability that offspring of pseudo-male fish will become pseudo-male fish (Shao et al., 2014).

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Breeding all-female stock increases productivity in tongue sole culture (Chen et al., 2007), and elucidating the mechanism of Chinese tongue sole sex determination and

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differentiation is crucial for controlling the sex of the stock. To this end, there has

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been a focus on functional analysis of Chinese tongue sole reproduction related genes to better understand the molecular mechanisms that regulate sex differentiation in this species (Deng et al., 2009; Dong et al., 2011; Zhang et al., 2014; Dong et al., 2016; Li

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et al., 2016; Xu et al., 2016; Cui et al., 2017). Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein

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containing two zinc finger structures at the C-terminus, one highly homologous to the GATA-type zinc finger domain and the other with less conserved across different species. Therefore, ZGLP1 does not generally bind to the GATA motif on DNA (Li et al., 2007; Strauss et al., 2011). ZGLP1 plays an important role in regulating the interaction between somatic and germ cells during gonad development, and it is important for maintaining normal development and reproductive ability (Li et al., 2007). ZGLP1 is not necessary for pregranulosa cell function but is required for germ cell survival (Li et al., 2007; Strauss et al., 2011). In mice, ZGLP1 is expressed at high level in the somatic cells of the developing gonads, including Leydig cells in the

ACCEPTED MANUSCRIPT testes and granulosa cells in the ovaries. Expression of ZGLP1 in ovarian somatic cells is required for normal fertility in female mice, as its deficiency leads to the absence of oocytes (Li et al., 2007; Strauss et al., 2011). Thus far, studies on the zglp1 have been performed on mice, while there is no report on the function of zglp1 in fish (Li et al., 2007; Strauss et al., 2011). In this study, we cloned and characterized the

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zglp1 in the Chinese tongue sole (cysezglp1) for the first time. qRT-PCR and in situ hybridization analysis revealed that the cysezglp1 is mainly expressed in ovary and

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may play an important role in oogenesis of the Chinese tongue sole.

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2. Materials and methods 2.1. Fish

All experimental Chinese tongue soles and embryos were obtained from the

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Haiyang 863 High-tech Experimental Base (Haiyang, Shandong Province, China). 18-month old fish were anesthetized with MS-222, and samples of brain, gill, heart,

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intestine, kidney, liver, muscle, ovaries, spleen and testes were collected from three male and female. In addition, the testes of three pseudo-male fish were collected using

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the same method. The gonads of the fish at 52, 80, 120, 150, 210, and 365 days after

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hatching (d) were collected. One side of the gonad was collected for RNA extraction, while the other was fixed in 4% PFA solution for histological examination. Every 20-30 embryos at various developmental stages (zygote, cleavage, blastula, gastrula,

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somite, heart-beating, crustal phase, brain differentiation, embryo twist and hatching stages) were collected as one sample.

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2.2. Total RNA isolation and cDNA synthesis Total RNA from the above collected samples was extracted according to the manufacturer protocol for TRIzol Regent (Invitrogen, USA). The total RNA treated with the RNase-free DNase I (Takara, Dalian, China) to remove genomic DNA. Reverse transcription and cDNA synthesis was carried out using PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. 2.3. Cloning and sequencing According to the sequence of cysezglp1 on NCBI (Gene ID: 103384171), we designed several pairs of specific primers to amplify cysezglp1 sequence and detect its

ACCEPTED MANUSCRIPT expression pattern (Table S1). Primer pairs zglp1-f1/zglp1-r1 and zglp1-f2/zglp1-r2 were used to amplify the coding sequence of cysezglp1 and ovarian cDNA was used as template. The PCR conditions were as follows: 95 °C for 2 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s; and then 72 °C for 10 min. The PCR product was purified using DNA Purification Kit (Tiangen, Beijing, China) and was cloned

Genewiz company (Genewiz, Suzhou, China) for sequening.

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2.4. qRT-PCR and statistical analysis

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into pMD-T18 vector (Transgen, Beijing, China). The positive clone was sent to

qRT-PCR analysis was conducted to analyze the expression pattern of cysezglp1 in

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different tissues, and different stages in embryonic and gonadal development. We carried out stability screening of some housekeeping genes in our experimental

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samples and found that the rpl7 was most stable (Table S2, S3 and Fig. S1, S2, S3). Therefore, we used rpl7 as the internal reference gene (rpl7-f/rpl7-r, Efficiency = 96%,

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Genebank: XM_008307613.1) (table S1). Primers, zglp1-rtf/zglp1-rtr (Efficiency = 94%) were used for analyzing of cysezglp1 expression. The reaction was carried out at

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a final volume of 15.0 µL, containing 7.5 µL 2 x SYBR Premix Ex Taq II (Tli

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RNaseH Plus), 0.6 µL of each forward and reverse primer, 0.3 µL ROX Reference DyeⅡ, 1.5 µL cDNA and 4.5 µL ddH2O. PCR amplification was performed in triplicate using the 7500 Fast Real-Time PCR System (Applied Biosystems, USA),

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and amplification reaction with ddH2O was used as negative control. The PCR program was as follows: 30 s at 95 °C for predenaturation, followed by 40 cycles of

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95 °C for 5 s and 60 °C for 30 s. Dissociation curve analysis was performed after the end of amplification to determine target specificity. rpl7 was amplified in parallel with the cysezglp1, and the relative gene expression was calculated using the 2-ΔΔCt method (Livak and Schmittgen, 2001). Statistical analyses were performed using SPSS 19.0 and analysis of variance at a significance level of 0.05 was carried out to determine the expression level of cysezglp1. 2.5. In situ hybridization In situ hybridization of gonads at one-year-old was conducted as described by Xu et al (Xu et al., 2016). To synthesize RNA probes, a pair of primers, zglp1-ishf and

ACCEPTED MANUSCRIPT zglp1-ishr (Table S1) were designed and synthesized. A 349 bp cDNA fragment of cysezglp1 was amplified and cloned into the pBluescript II SK (+) plasmid. The recombinant plasmid was linearized with Xho I and EcoR V. The linearized plasmid was used as template for antisense and sense probes synthesis with T7 or T3 RNA polymerase. DIG RNA Labeling Mix (Roche Diagnostics GmbH, Mannheim,

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Germany) was used to mark the RNA probes. At least three samples were processed in In situ hybridization.

3.1. Cloning and sequence analysis of cysezglp1

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3. Results

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The genomic sequences of the cysezglp1 was obtained by PCR and sequenced to verify the sequence identity. The cysezglp1 is located on chromosome 9 with genomic

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sequence of 3501 nucleotides (nt) and has five exons. The ORF region is 1692 nt, encoding a protein with 563 amino acid residues (Fig. 1). The predicted molecular

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weight is 62.56 kDa and the theoretical isoelectric point is 6.50. The predicted protein does not contain a signal peptide, indicating it was an endogenous protein (Petersen,

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Brunak et al. 2011). Predict Protein (https://www.predictprotein.org) analysis

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predicted that the localization of the protein was nuclear (Goldberg et al., 2012). The predicted protein contains two zinc fingers (Znf1 and Znf2) near the C-terminus (Fig.

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1, underlined sequence).

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Fig. 1 cysezglp1 genome sequence analysis of Chinese tongue sole. The exon regions are indicated in uppercase and intron regions are in lowercase. The initiation codon and stop codon is indicated in boxes. The double underline and single underline indicate zinc finger domains.

3.2. Alignment, phylogenetic and synteny analysis

ACCEPTED MANUSCRIPT We compared the zinc finger domains of Cysezglp1 and GATA transcription factors in Chinese tongue sole, and found that the Znf1 (Cys-X2-Cys-X17-Cys-X2-Cys, 493-517 aa) is high homologous to the GATA-type zinc finger domain, and the Znf2 (Cys-X2-Cys-X13-Cys-X2-Cys, 528-548 aa) lacks four amino acids that is commonly found in the GATA-type zinc finger domain (Fig. 2). When protein sequences were

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compared using BLASTp, we found that Cysezglp1 was not well-conserved compared to other species, including fish Zglp1s. However, the region where the two

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zinc fingers located was highly consistent among different species. For example, the Cysezglp1 zinc finger domain shared 96% identity to Zglp1s from Oreochromis

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niloticus and Larimichthys croceaetc (Fig. 2). By aligning the Znf1 domain of different species, we found that it contained 18 completely conserved amino acid

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residues (18/25) and three highly conserved amino acid residues (3/25). All differential amino acids were concentrated within the middle two cysteine residues

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(Cys-X17-Cys), and the differences were mainly located at the 5, 11, 14, and 19 aa positions of Znf1. Except for Xenopus tropicalis, all Zglp1s that were included in the contained

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zinc

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analysis

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Cys-X2-Cys-X13-Cys-X2-Cys motif, and Znf2 differ greatly among species (15/21). A neighbor-joining phylogenetic tree was constructed with selected Zglp1s from fish and other species (Fig. 3a). Zglp1s were clustered into two groups, one group

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containing teleost, amphibians and reptiles, and another group with mammalian and invertebrate sequences. Furthermore, synteny analysis revealed the organization of the

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genomic region surrounding zglp1 in teleost, amphibians and mammals. As shown in Fig. 3b, the gene distribution patterns around zglp1 were almost consistent among teleost, and that were relatively consistent between amphibian and mammals. Zglp1 and its downstream Fdx1l showed conserved synteny in teleost and mammals. In Chinese tongue sole, the GTPase IMAP family member 8-like is upstream of zglp1, whereas in other teleost, it is GTPase IMAP family member 6.

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Fig. 2 Alignment of zinc finger domains in Zglp1s and GATA families. Asterisks indicate the amino acid sequence is the same. Gaps are shown by dashes. Shading and arrows indicate conserved cysteine residues. Transparent boxes indicate zinc finger domains. The GenBank accession number of Zglp1s and species used in the sequence alignment are shown in Fig. 3a.

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Fig. 3 Phylogenetic and synteny analysis of Zglp1s from different species. (a) Phylogenetic tree of Zglp1 proteins of species was constructed by MEGA 6.0, and numbers of each node indicate the percentage of bootstrapping in 1000 replications. (b) Synteny maps compared genes flanking Zglp1 in teleost, amphibians and mammalian using the Ensembl Genome Brower (http://asia.ensembl.org) and National Center for Biotechology Information (https://www.ncbi.nlm.nih.gov). Gene symbols were described according to Ensembl and NCBI database. The bar lengths are not proportional to the distances between genes. Dotted lines represent the omitted genes on the chromosome. The direction of the arrows indicated the gene orientation.

3.3. Expression of cysezglp1 mRNA in different tissues We conducted qRT-PCR analyses to test for expression of cysezglp1 in different tissues of the Chinese tongue sole (Fig. 4a). cysezglp1 showed significant expression

ACCEPTED MANUSCRIPT level difference in different tissues of sexually mature Chinese tongue sole. Cysezglp1 was mainly expressed in gonads and was significantly enriched in ovaries than testes. 3.4. Expression of cyseglp1 mRNA in the gonad of female, male and pseudo-male Gonads from18-month old female, male and pseudo-male were used to further detect cysezglp1 mRNAs expression pattern. We found that cysezglp1 mRNA levels

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were significantly higher in ovaries than in testes of male and pseudo-male (Fig. 4b).

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3.5. Expression of cysezglp1 mRNA in embryos

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In embryos, we found that cysezglp1 mRNA level was high at the cleavage stage (1-cell to 32-cells) and its expression was reduced to a relatively low level in the

blastocyst stage until hatching (Fig. 4c).

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subsequent blastocyst stage. We did not detect expression of cysezglp1 after the

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3.6. Expression of cyseglp1 mRNA in the developmental stages of ovaries and testes

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During individual development, cysezglp1 mRNAs expression levels in the

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ovaries were higher than that in testes. In ovaries, cysezglp1 mRNAs were almost undetectable between 52 d to 80 d, and low levels of cysezglp1 mRNAs were detected at 120 d stage. At the one-year stage, there was a significant increase in cysezglp1

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expression (Fig. 4d). In testes, cysezglp1 mRNAs were detected at 120 d and

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expression lasted until the one-year old stage (Fig. 4d).

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Fig. 4 cysezglp1 mRNA expression pattern in Chinese tongue sole. (a) Expression pattern of cysezglp1 in different tissues. Data are expressed as mean ± S.D. of three different tissues. (b) Expression pattern of cysezglp1 in gonads of female, male, and pseudo-male fish. Data are expressed as mean ± S.D. of four different gonads. (c) Expression pattern of cysezglp1 in different stages of embryonic development. Data are expressed as mean ± S.D. of two different embryo pools at each developmental stage. (d) Expression pattern of cysezglp1 in the developmental stages of ovaries and testes. Data are expressed as mean ± S.D. of three different samples at each developmental stage. Different letters indicate significant difference of cysezglp1 in Chinese tongue sole (p < 0.05).

3.7 Cellular localization of cysezglp1 in the gonad

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We examined the cellular localization of cysezglp1 in one-year old ovaries and testes using in situ hybridization (Fig. 5). Sense probes were used as negative control

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and no specific signals were observed (Fig. 5a, 5c). The intensive signals were observed in ovaries and cysezglp1 mRNA was mainly localized in oocytes (Fig. 5b), and the weak signals were detected in sperm of testis (Fig. 5d), consistent with cysezglp1 expression in gonads at one-year old stage (Fig. 4d). When samples from other individuals were tested, the same results were obtained.

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Fig. 5 In situ hybridization of cysezglp1 mRNA in gonads of Chinese tongue sole. Ovary of one year old female fish with sense RNA probe (a), and antisense RNA probe (b). Testis of one - year old male fish with sense RNA probe (c), and antisense RNA probe (d). SL: seminiferous lobules, SP: spermatid, OC: oocyte, NU: nuclear, Scale bars, 100 μm.

4. Discussion

In this study, we cloned and characterized cysezglp1 for the first time in fish

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(Chinese tongue sole). Besides the zinc finger, an extended region rich in basic amino acids (arginine, lysine and histidine) near the C-terminal zinc finger is required for

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GATA factor DNA binding (Merika and Orkin, 1993; Blobel et al., 1995; Li et al., 2007). There were only a few basic amino acids adjacent to Zfn1 in Cysezglp1 (Fig. 1), which indicated that the Cysezglp1 may not bind to the classic the GATA factor binding motif (5′-WGATAR-3′). This result is consistent with studies of Zglp1 in mice (Li et al., 2007; Strauss et al., 2011). Zinc finger domains of cysezglp1 and GATA family in Chinese tongue sole have high level homology (Fig. 2) , which indicating that zglp1 and GATA family gene may have been derived from a common ancestry gene. The relative positions of the four cysteines in Znf1 are consistent across invertebrates and vertebrates (Fig. 2),

ACCEPTED MANUSCRIPT indicating its conservative role of this domain among eukaryotes. Except for the four conserved cysteines, almost all amino acid residues were different across species (Fig. 2), suggesting that Znf2 is highly polymorphic and the C-terminus of the protein may be more susceptible to environmental influences. Both phylogenetic and synteny analysis demonstrated that zglp1 had high protein sequence divergence and was

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distributed in different regions of the genome during evolution. Quantitative results showed that mRNAs of cysezglp1 were expressed in the gonad of female, male and

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pseudo-male fish, and the expression level in the ovary was significantly higher than that in the testis (Fig 4a, 4b). In mature mice, ZGLP1 is mainly expressed in ovarian

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granulosa cells and Leydig cells, regulating the interaction between somatic cells and germ cells during gonad development (Li et al., 2007; Strauss et al., 2011).

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Here, we conducted in situ hybridization analysis and found that cysezglp1 is mainly expressed in the oocytes, suggesting that the function of Zglp1 on the ovary

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may not be conserved between fish and mammals. Zglp1 is likely to play an important role in the oogenesis and maturation of fish oocytes. Ma et al. (2006) found that the

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tongue sole ovary began to reach adequate ovarian cavity at 100 d, which is one of

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evidences of plaice gonad differentiation, and the presumptive ovary developed primary oocytes at 120 d (Ma et al., 2006). Wu et al. (2010) also found that the ovarian cavity began to form the ovarian plate at 100 d in Chinese tongue sole, and

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the cavity continued to increase in size to form a closed ovarian cavity at 120 d to 190 d (Wu et al., 2010). The expression pattern of cysezglp1 that we found in our study

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indicate that cysezglp1 does not play an important role in the ovarian differentiation of Chinese tongue sole but may be involved in oogenesis and the maturation of the oocyte. We detected very low expression of cysezglp1 in testis, which may indicate that cysezglp1 does not play a key role in testis differentiation and spermatogenesis. During embryonic development, cysezglp1 mRNAs were expressed at higher levels in fertilized eggs and cleavage stage, possibly due to maternal effects resulting from high expression of the oocyte (De Sousa et al., 1998; Bullock and Ish-Horowicz, 2001). The expression level of cysezglp1 at the blastocyst stage was significantly decreased until hatching, which may indicate that it is not involved in the

ACCEPTED MANUSCRIPT differentiation of embryonic cells and the formation of early tissues.

5 Conclusion We cloned and characterized the full ORF of zglp1 in Chinese tongue sole. Experimental data shows cysezglp1 mRNA is mainly expressed in germ cells in the ovary, which may indicate that the gene is involved in oogenesis and maturation.

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However, whether and how cysezglp1 regulates oogenesis and oocyte maturation

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remains unclear and further studies are needed to investigate this.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation

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of China (31730099), Special Scientific Research Funds for Central Non-profit Institutes, Yellow Sea Fisheries Research Institute (20603022017003), AoShan

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Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology (No.2017ASTCP-OS15), Taishan Scholar Climbing Project of Shandong, Start-up Fund from GDOU.

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ACCEPTED MANUSCRIPT Abbreviations list

ORF: Open reading frame d: days after hatching PFA: Paraformaldehyde ZGLP1: Zinc finger GATA like protein-1

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nt: nucleotide

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aa: amino acid PCR: Polymerase Chain Reaction

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s: second min: minute

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Zinc finger GATA like protein-1 (zglp1) was cloned and characterized for the first time in fish. RT-PCR and ISH demonstrated that cysezglp1 is mainly expressed in oocytes. Cysezglp1 may play an important role in oogenesis in the Chinese tongue sole.