The Pig Lhx8 Gene: cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

Agricultural Sciences in China

December 2009

2009, 8(12): 1503-1510

The Pig Lhx8 Gene: cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos FANG Wei, LI Gui-qiang, LI Mei-li, WANG Wei and XU Yin-xue College of Animal Science & Technology, Nanjing Agricultural University, Nanjing 210095, P.R.China

Abstract Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends (RACE) gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage. Key words: pig, Lhx8, molecular cloning, mRNA expression, qRT-PCR, preimplantation embryos

INTRODUCTION LIM-homeobox gene family is one of several subfamilies of homeobox genes. LIM-homeobox genes encode a family of transcription regulators that share common structure features. They all contain two tandemly repeated cysteine-rich double-zinc finger motifs called LIM domains, in addition to a homeodomain (Hobert and Westphal 2000). The regulation function of LIMhomeobox genes is based on their characteristic LIM domains by the unique potential to cornbinatorial interact with others. As transcription regulators, functions of the LIM-homeobox genes have been analyzed in various invertebrate and vertebrate organisms, including caenorhabditis elegans, drosophila, xenopus, and mouse. These extensive studies have revealed that the LIMReceived 4 March, 2009

homeobox genes play essential roles in patterning and differentiation of diverse cell types during embryonic development. Lhx8 is a member of the LIM-homeobox transcription factor family which encodes a LIM homeodomain transcriptional regulator preferentially expressed in germ cells and is a critical regulator of mammalian oogenesis. Human Lhx8 gene was localized in chromosome 1p31.1 while that of mice was in chromosome 3H3-H4 (Kitanaka et al. 1998). Lhx8 transcripts localize to oocytes of germ cell clusters and primordial, primary and antral follicles in the mouse ovary (Pangas et al. 2006). The physiological function of Lhx8 in early folliculogenesis has not been described, but Lhx8 deficiency accelerates postnatal oocyte loss in the ovary and causes infertility in female. In mice, Lhx8-deficient ovaries fail to maintain the primordial follicles and the transition

Accepted 17 March, 2009

FANG Wei, MSc, E-mail: [email protected]; Correspondence XU Yin-xue, Professor, Tel: +86-25-84395278, Fax: +86-25-84395314, E-mail: [email protected] © 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60365-X

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from primordial to growing follicles does not occur. Lhx8-deficient ovaries misexpress oocyte-specific genes such as Gdf9, Pou5f1 and Nobox (Choi et al. 2008). Sohlh1, Nobox, Lhx8, Figla, and the genes that they regulate are all important candidate genes for nonsyndromic ovarian failure. Sohlh1, a germ cellspecific basic helix-loop-helix transcription factor, is upstream of Lhx8, and which in return regulate Nobox and Figla. In recent study, mutations in the coding region of Lhx8 are not the reason of Caucasian women with premature ovarian failure (Qin et al. 2008). Some studies also demonstrated the LIM-homeobox gene Lhx8 was required for many cholinergic neurons in the mouse forebrain (Zhao et al. 2003). Early research demonstrated that Lhx8 was selectively expressed in the medial ganglionic eminence (Matsumoto et al. 1996). Recently studies in mice have demonstrated that L3/Lhx8-null mice presented significantly less basal forebrain cholinergic neurons, and was further supported with a study showing that an L3/Lhx8-knockdown cell line specifically lacked the cholinergic phenotype (Mori et al. 2004; Manabe et al. 2005). The Lhx8 gene had been identified in mice and humans, but there was little studies in pigs. In the present study, we cloned the full-length cDNA of porcine Lhx8 gene and used the basic bioinformatic method to analysis the protein. The pig Lhx8 mRNA level in tissues was analyzed by RT-PCR while its level in preimplantation embryos was analyzed by real-time quantitative PCR.

MATERIALS AND METHODS Tissue preparation and oocytes treatment The tissue samples were obtained from slaughtered pigs and the fresh samples were put into liquid nitrogen immediately, and then stored at -70°C before use. Cumulus-oocyte complex (COCs) were aspirated from antral follicles (3-5 mm in diameter) using an 18gauge needle fixed to a 10-mL disposable syringe. COCs with intact, compact cumulus vestments were selected as suitable for in vitro maturation (IVM). After a brief wash in PBS, pooled COCs were transferred to tissue culture medium-199 (TCM-199; Gibco, USA) supplemented with 10% (v/v) porcine follicular fluid, 10 IU

FANG Wei et al.

mL-1 PMSG, 10 IU mL-1 hCG, 75 mg L-1 penicillin G, and 50 mg L-1 streptomycin sulfate. The COCs were cultured for 44 h at 38°C in 5% CO2 in air. The COCs were treated with 0.1% hyaluronidase to separate the oocytes and cumulus cells. Maturation of oocytes was checked under the microscope by the presence of polar bodies in denuded oocytes (DOs). Denuded oocytes were then washed three times in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS). Matured oocytes were selected using an inverted microscope (Zeiss, Axiovert S100, Germany) based on the presence of the metaphase-II plate and the first polar body. Selected oocytes were preactivated with 25 —mol L-1 of calcium ionophore (CaI, C-7522, Sigma, Germany) for 20 min and then incubated for 4 h with 2 mmol L-1 6-dimethylaminopurine (6-DMAP, D-2629, Sigma, Germany). Reconstructed and activated embryos were cultured in NCSU23 medium supplemented with 0.4% BSA for 48168 h essentially (Uhm et al. 2007). All cultures were done at 38.5°C under 5% CO2 in air with high humidity (> 95%).

RNA extraction and first-strand synthesis RNA was isolated from each samples using Trizol (Invitrogen, California, US). RNA quality was analyzed by agarose gel electrophoresis. 2 mg of RNA was reverse transcribed in a 20-—L reaction mixture at 42°C for 60 min and then at 70°C for 15 min with M-MLV reverse transcriptase (TaKaRa, Dalian, China) using d(T)18. The product was stored at -20°C until use.

Isolation of full-length cDNA of Lhx8 by rapid amplification of cDNA ends Basically, BLAST searches against the public databases using full-length cDNA sequences of these human genes as references were performed to retrieve all porcine orthologous sequences (Wang et al. 2008). BLAST searches retrieved 3 porcine expressed sequence tags (ESTs) (GenBank accession no. BG733086, BG733085, DQ499450) for Lhx8 gene. ESTs were assembled into contigs and carried out by DNAStar SeqMan program (DNAStar, Madison, US). Therefore, primers designed for amplification complete coding sequence were: for-

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The Pig Lhx8 Gene: cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

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ward (GSP1) 5´-ATG TCA GAG GAG TGC GGG CG3´/reverse (GSP2) 5´-GGG GTA ACA AGG GCT GGA GT-3´; and forward (GSP3) 5´-AAT GGC TTA TTC TGC CTA CG-3´/reverse (GSP4) 5´-ATT CAT CAA CCA CAA ACA CA-3´. In order to obtain a full-length gene, 5´- and 3´-RACE were used. For this purpose, total RNA was extracted and reverse transcribed follwing the manufacturer’s instruction of GeneRacerTM Kit (Invitrogen, California, US), which were then used for the 3´/5´-RACE PCR with GeneRacerTM 5´ primer (5´CGA CTG GAG CAC GAG GAC ACT GA-3´), GeneRacerTM 5´ nested primer (5´-GGA CAC TGA CAT GGA CTG AAG GAG TA-3´), GeneRacerTM 3´ primer (5´-GCT GTC AAC GAT ACG CTA CGT AAC G-3´), and GeneRacerTM 3´ nested primer (5´-CGC TAC GTA ACG GCA TGA CAG TG-3´). According to the GeneRacerTM Kit, special primers designed for amplification the 5´ UTR and 3´ UTR were: 5´ primer, 5´-GGG CAC CTT CAA CAC TTA TTC CA-3´, 5´ first nested primer, 5´-GAC CCA GTC GGT AGA ATG GAT GTG3´, 5´ second nested primer, 5´-TCC CTC GGC TCA CCA GTC CCT CTT-3´; 3´ primer, 5´-GGA CTC CAG CCC TTG TTA CCC CAT T-3´, 3´ first nested primer, 5´-TCA GGG ATA GAA ATG ATA AAG GAT A-3´, and 3´ second nested primer, 5´-TTT GCT GCC CAG GTA TGT ATC TCT A-3´. Polymerase chain reaction (PCR) was performed using Taq polymerase (TaKaRa, Dalian, China) according to the manufacturer’s instructions. First-strand cDNA was used as a template for PCR reactions. The PCR products were separated on 1% agarose gels, gel-purified and subcloned using pMD19-T vector (TaKaRa, Dalian, China). A positive clone was sequenced on an ABI3730 sequencer (Invitrogen, Shanghai, China).

of the new protein sequence were analyzed via ScanProsite (http://www.expasy.ch/tools/ scanprosite/), while signal peptide was analyzed by SignalP 3.0 (http://genome.cbs.dtu.dk/services/ SignalP) and the PSORT II program (http://psort.ims. u-tokyo.ac.jp/form2.html), respectively.

Bioinformatic analysis

RESULTS

The full-length cDNA sequence was used to search homologous sequences via BLASTX in NCBI (Liu et al. 2009). Translation program in ExPASy was performed to identify the open reading frame (ORF). Multiple alignments of amino acid sequences were performed between porcine and other animals using ClustalW/X. The phylogenetic relationship tree was then constructed by the neighbor-joining (NJ) method. The physico-chemical parameters and typical motifs

Cloning of full-length cDNA of Lhx8 gene

Real-time PCR analysis of pig Lhx8 gene in tissues and preimplantation embryos The expression level of Lhx8 was analyzed by real-time quantitative PCR (continuous fluorescence detector, MJ Research. Inc., BIO-RAD, South San Francisco, US). Quantitative system was carried out as follows: 20 —L volume with 200 nmol L-1 primers and 2 —L cDNA using 10 —L SYBR Premix Ex TaqTM (TaKaRa, Dalian, China). PCR amplification was performed consisting of 45 cycles of denaturizing for 10 s at 95°C, annealing for 10 s at 56.5°C, and extension for 10 s at 72°C, after the initial denaturizing step (95°C for 3 min). The primers were designed as follows: forward 5´-AAT GGC TTA TTC TGC CTA CG-3´/reverse 5´-TAT TGG CAG TTG TGT CAT TG-3´. The relative amount of mRNA was calculated with GAPDH mRNA as the invariant control, which were amplified using a pair of primers, forward 5´-AGA GCA GAG CGA GGA TGG A-3´/reverse 5´-CGG CAG AGG AAA GAG AAC ATT AG-3´. The amplified products were assessed by 8% polyacrylamide gel electrophoresis. Experiments were performed in triplicate for each sample. One-way ANOVA test method (SPSS Inc., Chicago, IL) was used to compare differernce expression level among these samples.

The retrieved 3 porcine ESTs were assembled 2 contigs, and on the basis of them, we amplified ORF by using GSP1, GSP2, GSP3, and GSP4. In order to isolate full length of Lhx8 gene, 3´ and 5´ RACE-PCR was carried out using the corresponding GSPs. About 260-bp fragments at 5´ UTR were amplified by touchdown and nested PCR, while about 600-bp fragments

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at 3´ UTR were amplified. This gene whose full cDNA length was 1 681 bp, contains a 885-bp ORF (Fig.1-A, B) flanked by a 244-bp 5´ UTR (Fig.1-C) and a 550-bp 3´ UTR, and encodes a 295 amino acid polypeptide (GenBank accession no. FJ587986). A potential polydenylation signal (AATAAA) was found at 3´ end. Sequence analysis of Lhx8 showed that it was completely same as corresponding sequence of the contig, implying the precision and accuracy of the sequence.

Physico-chemical parameters of Lhx8 protein The theoretical molecular of pig Lhx8 was 33.147 kDa and isoelectric point was 8.85, predicted by the Compute pl/Mw program (Gasteiger et al. 2003). The signal peptide prediction performed by SignalP 3.0 (Bendtsen et al. 2004) on the basis of a combination of several artificial neural networks and hidden Markov models revealed that this polypeptide contained no signal peptide in its amino acids, and was a nonsecretory protein. Hydropathy analysis (Christine et al. 2008) using the Protscale program showed that the Lhx8 protein contained no typical hydrophobic regions, but was a soluble protein. Using a hidden Markov model algorithm, transmembrane region predictions made by the TMHMM program (Moller et al. 2001) indicated that the protein was not a potential membrane protein, but a soluble protein, in accordance with hydropathy

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analysis. For subcellular localization analysis, the amino acid sequence was submitted to the PSORT II program, using the k-nearest neighbor classification algorithm (Nakai and Horton 1999), indicating that the protein resides in the nucleolus with a probability of 95.7%.

The pig Lhx8 amino acid sequences and comparative characterization Homeobox genes encode proteins that bind DNA and function as transcription factors to control development and differentiation of tissues. Like other members of the LIM-homeobox gene familiy, Lhx8 contains two tandemly repeated LIM domains, which are characterized by cysteine-rich, double-zinc finger motifs and a diatinct homeodomain (Fig.2). Sequence analysis by BLASTX program in NCBI database revealed that the putative amino acid sequence of pig Lhx8 shared high identities with known LIM-homeobox genes, such as Lhx6 and Lhx7. To analyze phylogenetic relationship between pig Lhx8 and other orthologous LIM-homeobox genes from other animal species, a neighbor-joining phylogenetic tree was constructed based on amino acid sequences of pig Lhx8 and other representative animals, which showed that pig Lhx8 was most related to human Lhx8 as they were clustered into the same clade, while it was most distant to Caenorhabditis elegans Lhx4 (Fig.3).

Fig. 1 Analysis of PCR products on 1% agarose gels. A, B, results of PCR products from Lhx8 ORF. A-1, PCR product by GSP1 and GSP2; B-1, nested PCR product by GSP3 and GSP4. C, results of PCR products from 5´ UTR. C-1, the second nested PCR product of 5´ RACE; C-2, the first nested PCR product of 5´ RACE. M, DL 2000 DNA marker (Bioer, Hangzhou, China).

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The Pig Lhx8 Gene: cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

LIM

Query seq.

LIM

HOX

Specific DNA base contacts DNA binding site

Specific hits Superfamilies

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Homeodomain LIM superfamily

LIM superfamily

Homeodomain superfamily

Fig. 2 Schematic representation of the porcine Lhx8 protein, showing the positions and types of conserve domains. A, the result of searching domains in porcine Lhx8 protein using Smart v4.0. B, the result of searching domains in porcine Lhx8 protein using CDD v2.11.

Fig. 3 Phylogenetic tree for several proteins of the LIM superfamily. Neighbor-joining tree was constructed by MEGA software. Numbers in the branch are the neighbor-joining bootstrap values. The length of branch indicates evolutionary distance with its scale being 0.05.

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The pig Lhx8 expression in tissues and preimplantation embryos To define the stage dependent differences of Lhx8 expression level in various tissues and preimplantation embryos, pig Lhx8 mRNA in these tissues and different developmental stages of preimplantation embryos was analyzed by RT-PCR. The continuously expressed gene, GAPDH, was used, and served as an endogenous reference for determination of targeted mRNA profiles. Multitissue RT-PCR with RNA derived from adult tissues showed that Lhx8 was expressed in brain, skeletal muscle, ganod, and immunity tissues. Other tissues, such as liver, heart, lung, spleen, kidney, and lymph were not detected (Fig.4). In preimplantation embryos, Lhx8 mRNA was detected in all of granulocytes, oocytes, 4-cell embryos, 8-cell embryos, morula, and blastula. Its expression profiling revealed that its mRNA levels were firstly increased from GV stage oocytes to

FANG Wei et al.

4-cell stage embryos and the highest in 4-cell stage embryos, and then gradually decreased until the blastocyst stage (Fig.5).

DISCUSSION Sequence assay In the current work, we presented data indicating that a LIM-heomobox gene (Lhx8) had been successfully cloned from pig, which showed a high degree of identity with Lhx8 from other mammals at the amino acid level. Relationship analysis based on the phylogenetic tree constructed on the basis of putative amino acid sequences demonstrated that pig Lhx8 was most closely related to that from human Lhx8 in the surveyed animal species. It also revealed that Lhx8 from mammalian species first clustered with each other, but distinct from caenorhabditis elegans Lhx4, suggesting clear separa-

Fig. 4 Result of Lhx8 gene expression by multiple-tissue RT-PCR. M, pBR322 DNA/Msp I (Tiangen, Beijing, China).

Fig. 5 Quantification of Lhx8 mRNA in porcine granulocytes, oocytes and early embryos using real-time RT-PCR. Maturation and development stages were studied. GV, germinal vesicle oocytes; MII, metaphase II oocytes; 4-cell, 4-cell embryos; 8-cell, 8-cell embryos.

tion between these two groups. These distances and the dendogram revealed the evolutionary relationship of various species, and in addition validated the correctness of the current classification of these subfamily proteins. LIM-homeobox genes encode a family of transcription factors that are highly conserved throughout evolution (Dawid et al. 1998). Multiple alignments of amino acid sequences between pig Lhx8 and others showed that the former was also well conserved. It is characterized by two tandemly repeated cysteine-rich double zinc finger motifs called LIM domains located aminoterminally of the homeodomain. Whereas the homeodo-

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The Pig Lhx8 Gene: cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

main in the protein is used for DNA binding, the LIM domains are thought to be involved in protein-protein interactions required for assembly of multi-protein complexes that regulate transcription of downstream target genes. Two factors that bind directly to the LIM domains, Ldb1 (also termed NL1, CLIM-2) and Ldb2 (NL2, CLIM-1), have recently been identified (Agulnick et al. 1996; Bach et al. 1997; Jurata et al. 1996).

Involvement of Lhx8 in tissues and early embryos Sequence alignments of Lhx8 gene indicated that it was evolutionally conserved in mammals, suggesting that it possessed the similar biological functions. Lhx8 was initially implicated in the development of cholinergic neurons and telencephalon, as well as palate (Zhao et al. 1999), however it was not until recently that Lhx8 preferential expression within oocytes of the gonad was discovered (Pangas et al. 2006). Lhx8 therefore represents the first member of the LIM homeodomain family to be expressed in germ cells. Indeed, our present study found that the Lhx8 gene is heavily involved in gonad tissues in pigs, which is in consistence with findings observed in mice. In the loss-of-function mice model, Lhx8-/- females lose oocytes within 7 d after birth (Choi et al. 2008). It is not something surprising to see that Lhx8 are heavily involved in tesitis. The kit tyrosine-kinase receptor and its ligand, KL, are essential for the maintenance of primordial germ cells (PGCs) in both sexes. However, kit is known to play important roles also during post-natal stages of spermatogenesis (Zhao and Xi 2007). KL up-regulates in differentiating spermatogonia the expression of early meiotic genes, for instance Lhx8. Our present study showed Lhx8 expression level was heavily in gonad especially in ovary and oocyte. Moreover, Lhx8 mRNA had been detected in immunity tissues which also demonstrated it was correlated with immunologic mechanism. There are numerous genes affected by Lhx8, such as Nlrp5, Zp1, Zp2, and Zp3. Although transcription of Nlrp5 and Zp genes commences in small oocytes, knockouts show that their function is beyond early folliculogenesis. Nlrp5 is essential for embryonic development past 2-cell and 4-cell stages in mice (Tong et al. 2000). Zp genes are essential components of the

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zona pellucida that surrounds the oocyte (Bleil and Wassarman 1980). Many genes down-regulated in Lhx8 deficient mice are functionally uncharacterized, and include several that are preferentially expressed in germ cells such as BQ175373, 9230115E21Rik, and Pramef12 (Su et al. 2002). Some of these genes may as well be critical components of oocyte differentiation and survival. In the present study, we found that there was little expression in porcine granulocytes, but heavily expression in germinal vesicle oocytes and metaphase II oocytes. It demonstrated that Lhx8 was essential for maintain oocytes differentiation and survival and it was important to maintain Zp genes effectiveness in this process. We also found that the Lhx8 mRNA level was significant increase from MII to 4-cell stage and its expression level was the hightest in 4-cell stage. Therefore, we infered that Lhx8 was essential for Nlrp5.

Acknowledgements This study was supported by the High Technology Research and Development Program of China (2006AA10Z136).

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