碳酸酐酶CAIV在母鸡贮精腺调控精子贮存机制的研究

Birds have evolved blind-ended sperm storage tubules (SSTs) that can maintain sperm viability for long periods. In hens, SSTs are located in the utero-vaginal junction (UVJ) and in the infundibulum, although the primary storage site for sperm is the SSTs in the UVJ. The spermatozoa are transported to the infundibulum, which is the site of fertilization and also serves as a secondary sperm storage site. It is normal in chicken production that the fertility will decline if fresh semen is stored at room temperature for more than 0.5 h. In contrast, female chicken with 41℃ body temperature can maintain viable and fertile sperm in the oviduct for 3 to 4 weeks. Sperm storage in chicken SSTs is related to carbonic anhydrase (CA). Carbonic anhydrases are the products of a gene family that encodes 16 different functionally active proteins. It is not known which carbonic anhandrase isoform is involved in regulating sperm storage in hens. We found that the mRNA expression level of one of the carbonic anhydrase isoforms (CAIV) in UVJ and infundibulum was significantly higher than that in other parts of hen’s reproductive tract, and the difference is thousands times. Furthermore, the mRNA expression level of CAIV in UVJ (the primary sperm –storage site) was significantly higher than that in infundibulum (the secondary sperm-storage site). This project will study the CAIV mRNA and protein in the hen’s reproductive tract before and after sexual maturation using in situ hybridization and immunohistochemistry, silence the CAIV mRNA expression in cultured cells of sperm storage tubules using siRNA, express the chicken CAIV in Pichia Pastoris, and study the effect of CAIV on SST microenviroment and its relationship with sperm storage using increasement and reduction of CAIV method, aims to ①find the cell which express the CAIV, the CAIV position in the cell and the expression character in the hen’s reproductive tract; ②find the mechanism of CAIV regulating chicken sperm storage, hoping to find clues on sperm storage mechanisms in chicken. The results will facilitate developing new methods for storing liquid semen.

禽类精子主要在母禽子宫阴道交接部的贮精腺长期贮存，在伞部短暂贮存后受精。生产中鸡精子在室温半小时后受精力降低，而在母鸡子宫阴道交接部贮精腺却可存活3-4周，碳酸酐酶(CA)参与该贮存过程。碳酸酐酶有16种亚型，目前不清楚哪一类亚型参与精子贮存。我们发现CAIV在母鸡子宫阴道交接部和伞部的mRNA表达量显著高于其它部位，是其它部位的数千倍，且贮精腺主要部位子宫阴道交接部的CAIV表达量显著高于伞部。故本项目从蛋白和mRNA水平检测母鸡输卵管各部CAIV的表达，用RNA干扰沉默CAIV在贮精腺培养细胞内的表达，并在毕赤酵母中表达该基因，研究增加和减少CAIV对母鸡贮精腺微环境的影响及其与精子贮存的关系，旨在：①确定参与母鸡生殖道精子贮存的碳酸酐酶亚型、细胞类型及CAIV的表达规律；②揭示CAIV参与母鸡贮精腺精子贮存的机制，为探明禽类贮精腺的贮精原理奠定基础，为开发液态精液体外保存提供依据。

CAIV可能参与禽类精子在母禽生殖道贮精腺的长期贮存，因此我们分析鸡CAIV基因及其编码蛋白的序列，定量该基因在母鸡生殖道的表达，酵母表达鸡CAIV蛋白，研究其对精液环境和精子活力的影响，用基因干扰手段研究其对鸡成纤维细胞（DF-1细胞）培养液的pH值和L-乳酸浓度的影响，并通过基因免疫方法研究母鸡内源性CAIV降低后对活体基因表达、受精率、蛋黄膜精子穿孔数、血清L-乳酸含量的影响。结果表明鸡CAIV蛋白的序列保守，存在多个抗原表位。鸡CAIV基因存在6个SNP，其中4个为非同义突变，母鸡含贮精腺的子宫阴道交接部CAIV的mRNA表达量最高。酵母表达的鸡CAIV降低贮存精液的pH值和精子活力。干扰降低DF-1细胞CAIV基因的mRNA表达，MCT4的mRNA表达量也显著降低，培养液的pH值没有发生变化，但培养液的左旋乳酸含量显著下降。降低母鸡CAIV的表达，子宫阴道交接部和伞部CAIV基因的mRNA水平显著降低，受精率、蛋黄膜精子穿孔数和血清L-乳酸含量也显著降低。CAIV可能与MCT4共同作用，为母鸡生殖道长期维持精子活力提供适宜微环境。研究结果探明CAIV在母鸡生殖道的表达规律，证实CAIV参与母鸡生殖道的精子贮存，为探明禽类贮精腺的贮精原理奠定基础，为开发液态精液体外保存提供依据。