在中国梅山猪中 CatSper 基因的分子克隆、时空表达分析。
Molecular cloning, spatial and temporal expression analysis of CatSper genes in the Chinese Meishan pigs.
机构信息
College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China.
出版信息
Reprod Biol Endocrinol. 2011 Oct 4;9:132. doi: 10.1186/1477-7827-9-132.
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.
背景
精子离子通道蛋白（CatSper）对于精子超激活运动至关重要，然后穿透透明带。CatSper 类蛋白在小鼠和人类中得到了很好的描述。然而，猪的数据尚不可用。本研究克隆了猪 CatSper1-4 基因，分析了它们在各种器官中的空间表达以及在性成熟前 Meishan 公猪睾丸中的时间表达。
方法
采用快速扩增 cDNA 末端（RACE）技术克隆猪 CatSper 基因全长 cDNA，并对推测的 CatSper 蛋白进行生物信息学分析。从 150 天大的猪身上采集各种器官，通过定性逆转录聚合酶链反应（RT-PCR）鉴定 CatSper 基因的空间表达，从出生到 150 天大的公猪睾丸中采集样品，通过定量实时 RT-PCR 检测 CatSper 基因的时间表达。
结果
获得了 CatSper1（2452bp）、CatSper2（2038bp）、CatSper3（1408bp）和 CatSper4（1799bp）的 mRNA 序列，包括 cDNA 的全长、5'和 3'侧翼。生物信息学分析表明，不同物种分析的离子转运结构域编码区具有保守性。在这四种 CatSpers 中，CatSper2、3 和 4 在物种间的保守性更高，而 CatSper1 则较低。此外，还鉴定了六个保守的跨膜结构域、一个孔形成基序和一个卷曲螺旋基序。来自不同器官的空间分析表明，CatSper1 在睾丸和下丘脑均有检测到，CatSper2 仅局限于睾丸，CatSper4 则在睾丸和输精管中表达；而 CatSper3 则更为普遍。CatSper3 和 CatSper4 的转录本也在射出的精子中检测到。在 1 天和 30 天龄时，睾丸中仅稀疏表达 CatSper mRNA。然而，这些转录本在 60 天龄时高度表达，并持续到性成熟（150 天龄）。
结论
本文首次在猪中报道了 CatSper 基因的时空表达谱。CatSper1、CatSper2 和 CatSper4 主要在睾丸中表达，而 CatSper3 转录本在多种器官中广泛存在。CatSper3 和 CatSper4 的 mRNA 存在于成熟的精子细胞中。CatSper 基因在 60 天龄时开始大量上调，并保持这种显著的产生直到性成熟。
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