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Molecular cloning, spatial and temporal expression analysis of CatSper genes in the Chinese Meishan pigs.

Song C, Gao B, Wu H, Xie Y, Wang X, Li B, Chen G, Mao J - Reprod. Biol. Endocrinol. (2011)

Bottom Line: Among the four CatSpers, CatSper2, 3, and 4 were more conserved across species, compared with CatSper1.CatSper3 and CatSper4 mRNAs were present in mature sperm cells.Substantial upregulation of CatSper genes was initiated at Day 60 and maintained this marked production until sexual maturity.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China. chengyilab@hotmail.com

ABSTRACT

Background: Sperm ion channel proteins (CatSpers) are essential for sperm hyperactivated motility, and then penetration through the zona pellucida. The CatSper class of proteins have well been characterized in the mouse and human. However, such data for pigs are not available. In the present study, we cloned the porcine CatSper 1-4 genes, analysed their spatial expression in various organs and temporal expression in the testes from birth until sexual maturity in Meishan boars.

Methods: Rapid amplification of cDNA ends (RACE) was performed to clone the full length cDNAs of porcine CatSper genes and bioinformatics analysis of inferred CatSper proteins was also determined. Various organs were collected from 150 day-old pigs to characterize the spatial expression of CatSper genes by qualitative reverse transcriptase polymerase chain reaction (RT-PCR), and testes from birth to 150 day-old boars were sampled to detect the temporal expression of CatSper genes by quantitative real-time RT-PCR.

Results: The mRNA sequences of CatSper1 (2452 bp), CatSper2 (2038 bp), CatSper3 (1408 bp), and CatSper4 (1799 bp), including full length of cDNAs, 5' and 3' flanks, were obtained. The bioinformatics analysis indicated that coding regions spanning the ion transport domains were conserved for different species analyzed. Among the four CatSpers, CatSper2, 3, and 4 were more conserved across species, compared with CatSper1. In addition, six conservative trans-membrane domains, a pore forming motif, and a coiled-coil motif were also identified. The spatial analysis from different organs showed that CatSper1 was detected in both testes and hypothalamus, CatSper2 was restricted in testes only, CatSper4 was expressed in testes and rete testes; whereas CatSper3 was more ubiquitously. CatSper3 and CatSper4 transcripts were also detected in ejaculated sperm. At Days 1 and 30 of age, CatSper mRNAs exhibited only sparse expression in the testes. However, these transcripts highly expressed at Day 60 and onward till sexual maturity (Day 150 of age).

Conclusions: The spatial and temporal expression profiles of CatSper genes were reported herein for the first time in pigs. CatSper1, CatSper2 and CatSper4 were primarily expressed in testes, while CatSper3 transcript was prevalent in a variety of organs. CatSper3 and CatSper4 mRNAs were present in mature sperm cells. Substantial upregulation of CatSper genes was initiated at Day 60 and maintained this marked production until sexual maturity.

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Fragments of CatSper1, CatSper2, and CatSper4 cDNAs. A. approximately 650 bp of CatSper1; B. 450 bp of CatSper2; C. 900 bp of CatSper4, amplified by using primers designed according to the available CatSper nucleotide sequence of pig and the conserved sequences of CatSpers among animals. Lane 1 is the PCR products from testis cDNAs, lane 2 is the negative control, lane M is the DL2000 DNA marker.
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Figure 1: Fragments of CatSper1, CatSper2, and CatSper4 cDNAs. A. approximately 650 bp of CatSper1; B. 450 bp of CatSper2; C. 900 bp of CatSper4, amplified by using primers designed according to the available CatSper nucleotide sequence of pig and the conserved sequences of CatSpers among animals. Lane 1 is the PCR products from testis cDNAs, lane 2 is the negative control, lane M is the DL2000 DNA marker.

Mentions: The available porcine CatSpers cDNA sequences from the NCBI database [25] included 125 bp for CatSper1 [GenBank:AM947645], 137 bp for CatSper2 [GenBank:AM947646], 169 bp for CatSper3 [GenBank:AM947647] and 174 bp for CatSper4 [GenBank:AM947648]. To facilitate the 5'RACE, three sets of primers correspond to CatSper1, CatSper2 and CatSper4 cDNAs were designed, according to the available CatSper nucleotide sequences of pig and the conserved sequences of CatSpers among other mammals. Three PCR products of CatSper1, CatSper2 and CatSper4 cDNAs with expected size of 650 bp, 450 bp, and 900 bp were obtained (Figure 1). Then, the purified cDNA products were inserted into TA clone vectors and sequenced. The sequencing results were confirmed by analyzing for similarity with known CatSper sequences of other species using the BLASTN program. The mRNA sequences, including the 5' and 3' un-translated regions and full-length cDNAs of porcine CatSper1, CatSper2, CatSper3 and CatSper4 genes, were successfully obtained by RACE method. These sequences have been deposited into GenBank with accession number of HQ654519, HQ654520, HQ654521, and HQ654522. The characteristics of porcine CatSper mRNA sequences were predicted by DNAStar and are summarized in Table 3. The lengths of cloned mRNA sequences of CatSper1, CatSper2, CatSper3 and CatSper4 genes were 2452, 2038, 1408, and 1799 bp, respectively. They contain 2169, 1599, 1203, and 1350 bp open reading frames (ORFs), respectively, flanked by 5' and 3' terminal un-translated regions (Table 3).


Molecular cloning, spatial and temporal expression analysis of CatSper genes in the Chinese Meishan pigs.

Song C, Gao B, Wu H, Xie Y, Wang X, Li B, Chen G, Mao J - Reprod. Biol. Endocrinol. (2011)

Fragments of CatSper1, CatSper2, and CatSper4 cDNAs. A. approximately 650 bp of CatSper1; B. 450 bp of CatSper2; C. 900 bp of CatSper4, amplified by using primers designed according to the available CatSper nucleotide sequence of pig and the conserved sequences of CatSpers among animals. Lane 1 is the PCR products from testis cDNAs, lane 2 is the negative control, lane M is the DL2000 DNA marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198926&req=5

Figure 1: Fragments of CatSper1, CatSper2, and CatSper4 cDNAs. A. approximately 650 bp of CatSper1; B. 450 bp of CatSper2; C. 900 bp of CatSper4, amplified by using primers designed according to the available CatSper nucleotide sequence of pig and the conserved sequences of CatSpers among animals. Lane 1 is the PCR products from testis cDNAs, lane 2 is the negative control, lane M is the DL2000 DNA marker.
Mentions: The available porcine CatSpers cDNA sequences from the NCBI database [25] included 125 bp for CatSper1 [GenBank:AM947645], 137 bp for CatSper2 [GenBank:AM947646], 169 bp for CatSper3 [GenBank:AM947647] and 174 bp for CatSper4 [GenBank:AM947648]. To facilitate the 5'RACE, three sets of primers correspond to CatSper1, CatSper2 and CatSper4 cDNAs were designed, according to the available CatSper nucleotide sequences of pig and the conserved sequences of CatSpers among other mammals. Three PCR products of CatSper1, CatSper2 and CatSper4 cDNAs with expected size of 650 bp, 450 bp, and 900 bp were obtained (Figure 1). Then, the purified cDNA products were inserted into TA clone vectors and sequenced. The sequencing results were confirmed by analyzing for similarity with known CatSper sequences of other species using the BLASTN program. The mRNA sequences, including the 5' and 3' un-translated regions and full-length cDNAs of porcine CatSper1, CatSper2, CatSper3 and CatSper4 genes, were successfully obtained by RACE method. These sequences have been deposited into GenBank with accession number of HQ654519, HQ654520, HQ654521, and HQ654522. The characteristics of porcine CatSper mRNA sequences were predicted by DNAStar and are summarized in Table 3. The lengths of cloned mRNA sequences of CatSper1, CatSper2, CatSper3 and CatSper4 genes were 2452, 2038, 1408, and 1799 bp, respectively. They contain 2169, 1599, 1203, and 1350 bp open reading frames (ORFs), respectively, flanked by 5' and 3' terminal un-translated regions (Table 3).

Bottom Line: Among the four CatSpers, CatSper2, 3, and 4 were more conserved across species, compared with CatSper1.CatSper3 and CatSper4 mRNAs were present in mature sperm cells.Substantial upregulation of CatSper genes was initiated at Day 60 and maintained this marked production until sexual maturity.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China. chengyilab@hotmail.com

ABSTRACT

Background: Sperm ion channel proteins (CatSpers) are essential for sperm hyperactivated motility, and then penetration through the zona pellucida. The CatSper class of proteins have well been characterized in the mouse and human. However, such data for pigs are not available. In the present study, we cloned the porcine CatSper 1-4 genes, analysed their spatial expression in various organs and temporal expression in the testes from birth until sexual maturity in Meishan boars.

Methods: Rapid amplification of cDNA ends (RACE) was performed to clone the full length cDNAs of porcine CatSper genes and bioinformatics analysis of inferred CatSper proteins was also determined. Various organs were collected from 150 day-old pigs to characterize the spatial expression of CatSper genes by qualitative reverse transcriptase polymerase chain reaction (RT-PCR), and testes from birth to 150 day-old boars were sampled to detect the temporal expression of CatSper genes by quantitative real-time RT-PCR.

Results: The mRNA sequences of CatSper1 (2452 bp), CatSper2 (2038 bp), CatSper3 (1408 bp), and CatSper4 (1799 bp), including full length of cDNAs, 5' and 3' flanks, were obtained. The bioinformatics analysis indicated that coding regions spanning the ion transport domains were conserved for different species analyzed. Among the four CatSpers, CatSper2, 3, and 4 were more conserved across species, compared with CatSper1. In addition, six conservative trans-membrane domains, a pore forming motif, and a coiled-coil motif were also identified. The spatial analysis from different organs showed that CatSper1 was detected in both testes and hypothalamus, CatSper2 was restricted in testes only, CatSper4 was expressed in testes and rete testes; whereas CatSper3 was more ubiquitously. CatSper3 and CatSper4 transcripts were also detected in ejaculated sperm. At Days 1 and 30 of age, CatSper mRNAs exhibited only sparse expression in the testes. However, these transcripts highly expressed at Day 60 and onward till sexual maturity (Day 150 of age).

Conclusions: The spatial and temporal expression profiles of CatSper genes were reported herein for the first time in pigs. CatSper1, CatSper2 and CatSper4 were primarily expressed in testes, while CatSper3 transcript was prevalent in a variety of organs. CatSper3 and CatSper4 mRNAs were present in mature sperm cells. Substantial upregulation of CatSper genes was initiated at Day 60 and maintained this marked production until sexual maturity.

Show MeSH
Related in: MedlinePlus