Spatial and temporal expression of germ cell nuclear factor in murine epididymis

Aim:To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period.Methods:The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope.Results:GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis.The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis.In adults,GCNF exhibited a region-specific expression pattern,i.e.,it was expressed predominantly in the initial segment,caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis.GCNF could be found in the nuclei of the principal,apical,narrow,clear and halo cells.Conclusion:GCNF may play an important role in epididymal differentiation and development and in sperm maturation.(Asian J Andro12004 Mar,6:23-28)     

附睾相关的抗生育研究及进展

虽然至今尚没有一种直接作用于附睾并可供临床试验用的男性避孕药 ,但对附睾功能和精子在附睾成熟的研究表明 ,附睾极有可能成为男性抗生育最理想的靶器官。相关的动物试验包括直接作用于附睾精子如磺胺水杨嗪类、影响能量代谢与精子活动的氯化甘油和氯化糖苷类化合物、影响附睾环境如雷公藤单体和铜粉等。对附睾特异蛋白研究 ,可望发现免疫学途径的靶抗原 ,用于制备避孕疫苗。近年国内外对附睾特异表达基因的研究取得某些进展 ,如SC342、bin1基因的克隆和功能研究 ,有助于临床附睾炎和不育症的诊治 ,同时有望成为干扰附睾精子成熟达到避孕目的的新靶点。     

Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP1-like protein from the monkey.

A cDNA encoding an acidic epididymal glycoprotein (AEG)-like, CRISP1 (cysteine-rich secretory protein) protein from the monkey (Macaca mullata) epididymis has been cloned and sequenced. The monkey AEG (mAEG) has an open reading frame that encodes a protein containing 249 amino acids with a deduced molecular mass of 28 kDa. The mAEG protein sequence is 85% identical to human and 44% identical to mouse CRISP1, including all 16 conserved cysteine residues. mAEG also shows a significant amino acid homology with other CRISP proteins, rat AEG/DE, human TPX1/CRISP2, and guinea pig acrosomal autoantigen 1 (AA1). In addition, mAEG shows somewhat less homology to a toxin from the Mexican beaded lizard and to a human glioma pathogenesis-related protein. Northern blot analysis shows that the mRNA for mAEG is expressed in all the regions of the epididymis except the caput and was not detected in the testis, prostate, seminal vesicle, and brain. In castrated animals, mAEG gene expression in the epididymis is significantly diminished; however, testosterone enanthate replacement restored the normal level of expression, demonstrating that expression of mAEG is androgen dependent. Western blot analysis of monkey epididymal regions using mouse antirecombinant human AEG identified a 28-kDa protein only in the caudal region. Immunohistochemical analysis identified mAEG only in the principal cells of the cauda epididymal epithelium. Immunofluorescence analysis identified mAEG on the principal piece of the sperm tail and as small patches over the middle piece and head regions. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa.     

c-mos原癌基因在小鼠附睾的表达

目的研究原癌基因c-mos在小鼠附睾的表达及c-Mos蛋白在小鼠附睾上皮细胞的定位。方法采用半定量RT-PCR和间接免疫荧光的方法分别检测c-mos mRNA和蛋白在附睾不同区域的表达,并通过免疫电镜对c-Mos蛋白在附睾上皮细胞内进行精确定位。结果c-mos mRNA表达量在小鼠附睾头部最高,体部最低;仅在附睾头部管腔面检测到c-Mos蛋白表达;电镜观察见c-Mos蛋白定位在附睾上皮细胞的顶部胞质内。结论c-mos原癌基因在小鼠附睾的区域特异性表达以及c-Mos蛋白在附睾上皮细胞的定位,提示c-mos基因可能在精子成熟过程中发挥调节功能。     

小鼠Lyzls的表达特征及人LYZL6蛋白的抗菌特性研究

C类溶菌酶基因（Lyzls）属于溶菌酶基因家族,高表达于小鼠睾丸和附睾。该家族基因Lyzl4和Spaca3被报道在小鼠精卵融合和受精过程中发挥作用。然而其他两种小鼠C类溶菌酶基因Lyzll他HLyzl6的功能尚不清楚。本研究分析了小鼠C类溶菌酶基因的组织表达、表达与雄激素的关联以及重组人LYZL6蛋白（rLYZL6）在免疫中可能发挥的作用。通过RT-PCR、Westernblots、免疫组织化学和免疫荧光的方法分析了Lyz＆的表达,细菌集落形成测定实验分析了重组人LYZL6蛋白的抗菌活性。小鼠lyzls主要在睾丸和附睾中表达,受发育调控及雄激素或睾丸因子调控。免疫检测LYzL6蛋白定位于小鼠睾丸的初级精母细胞、圆形精子和成熟精子的顶体后及中段。重组人LYZL6蛋白呈现抗菌活性。我们推测Lyzls可能在精子线粒体的功能中发挥作用,LYZL6蛋白在雄性生殖道免疫中发挥作用。     

Rodent epididymal cDNAs identified by sequence homology to human and canine counterparts

Aim: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and CeS/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat. Methods:“Electronic screening“ of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis. Results: Rodent ESTs and genomic sequences homologous to HE3, HE4 and CeS/Ly6G5C were identified in the public databases and the “full-length“ rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively, mRNA expression patterns were analysed in rats, including rat HE1 and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis,Re3 and Re4 mRNAs were detected in all regions; ReS, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis.Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens. Conclusion: The novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.