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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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CatSper1 (A), CatSper2 (B), CatSper3 (C), and CatSper4 (D) channel coiled-coil predictions. X-axis, amino acid residue numbering of query sequence. Y-axis, probability score for a sequence adopting a coiled-coil configuration calculated for a scanning window of 14, 21 or 28 amino acid residues.
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Figure 3: CatSper1 (A), CatSper2 (B), CatSper3 (C), and CatSper4 (D) channel coiled-coil predictions. X-axis, amino acid residue numbering of query sequence. Y-axis, probability score for a sequence adopting a coiled-coil configuration calculated for a scanning window of 14, 21 or 28 amino acid residues.

Mentions: Based on the gene sequences, the amino acid sequences of the four porcine CatSpers were deduced. The amino acid identity between pig and human, mouse, rat, cattle, and dog (access number refer to Table 4) was then analyzed by using CLUSTALW multiple sequence alignment program. The alignment data showed that the regions across the ion transport domains were predictably highly conserved (Figure 2). In contrast low sequence identity in the N and C-terminals, and other regions was observed. Six transmembrane domains (S1-S6) in the protein alignment were predicted by tmap program. A short, conserved hydrophobic stretch representing the pore-forming region is present in a longer loop region between the fifth and sixth transmembrane domains (Figure 2). The sequences in the pore loop represented a similar conserved motif (T×D×W) (Figure 2), which is characterized in voltage-gated calcium channels [26]. Overall, the amino acid sequences of these motifs also appear to be highly conserved across species (Figure 2). A coiled-coil domain at the C terminus of each member of the porcine CatSper family was predicted by COILS program and shown in Figure 3. This domain is well characterized as potential protein-protein interaction motif with various complexes [27].


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)

CatSper1 (A), CatSper2 (B), CatSper3 (C), and CatSper4 (D) channel coiled-coil predictions. X-axis, amino acid residue numbering of query sequence. Y-axis, probability score for a sequence adopting a coiled-coil configuration calculated for a scanning window of 14, 21 or 28 amino acid residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: CatSper1 (A), CatSper2 (B), CatSper3 (C), and CatSper4 (D) channel coiled-coil predictions. X-axis, amino acid residue numbering of query sequence. Y-axis, probability score for a sequence adopting a coiled-coil configuration calculated for a scanning window of 14, 21 or 28 amino acid residues.
Mentions: Based on the gene sequences, the amino acid sequences of the four porcine CatSpers were deduced. The amino acid identity between pig and human, mouse, rat, cattle, and dog (access number refer to Table 4) was then analyzed by using CLUSTALW multiple sequence alignment program. The alignment data showed that the regions across the ion transport domains were predictably highly conserved (Figure 2). In contrast low sequence identity in the N and C-terminals, and other regions was observed. Six transmembrane domains (S1-S6) in the protein alignment were predicted by tmap program. A short, conserved hydrophobic stretch representing the pore-forming region is present in a longer loop region between the fifth and sixth transmembrane domains (Figure 2). The sequences in the pore loop represented a similar conserved motif (T×D×W) (Figure 2), which is characterized in voltage-gated calcium channels [26]. Overall, the amino acid sequences of these motifs also appear to be highly conserved across species (Figure 2). A coiled-coil domain at the C terminus of each member of the porcine CatSper family was predicted by COILS program and shown in Figure 3. This domain is well characterized as potential protein-protein interaction motif with various complexes [27].

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