精子发生相关CG14片段的组织特异性表达及其与杂交不育的关系

目的 :确定精子发生相关 CG1 4片段的组织特异性表达及其与杂交不育的初步关系。方法 :采用 CG1 4片段的放射性同位素标记探针对心、肝、肾、脑、睾丸及附睾组织的 m RNA进行 Northern杂交。将 CG1 4片段转录为 RNA作探针 ,对成年雄性 SD大鼠、牦牛、犏牛一代 (杂交不育 )、犏牛三代 (回交生育功能恢复 )睾丸组织进行原位杂交。结果 :在睾丸组织有一条约 1 2 58bp的 m RNA特异杂交带 ;在附睾组织有一条约 1 3 51 bp的 m RNA特异杂交带 ,而在心、肝、肾及脑无杂交带。在 SD大鼠、牦牛及犏牛三代的睾丸组织冰冻切片存在明显的 CG1 4正义探针阳性杂交信号 ;在犏牛一代睾丸组织未见特异性阳性杂交信号 ;在各个组织切片中反义探针均未出现阳性杂交信号。结论 :精子发生相关 CG1 4片段在睾丸及附睾组织中特异性表达。在 SD大鼠、具有生育能力的牦牛以及生育机能恢复的犏牛三代初级精母细胞中表达 CG1 4片段的 m RNA,而在杂种不育的犏牛一代初级精母细胞中未见 CG1 4片段的 m RNA。     

5α-还原酶I型同功酶基因在人睾丸、附睾和输精管组织中的表达

我们对人５α－还原酶Ｉ型同功酶基因在正常人睾丸、附睾及输精管组织细胞内的定位表达进行了初步的研究。采用非同位素地高辛标记ｃＲＮＡ探针对人睾丸、附睾和输精管组织冰冻切片进行原位杂交。结果：人睾丸Ｓｅｒｔｏｌｉ和Ｌｅｙｄｉｇ细胞胞浆、附睾和输精管上皮的主细胞及假复层柱状上皮细胞的胞浆中均有较强的杂交信号；细胞核中未见杂交信号；睾丸生精细胞及基底膜、附睾和输精管上皮的基底膜、间质和肌层也未见杂交信号。证明人与灵长类和大鼠的５α－还原酶基因表达及其分布基本一致。本结果对深入研究５α－还原酶及其产物在人类生殖中的作用具重要意义。     

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.     

Identification of a novel testis-specific gene and its potential roles in testis development／spermatogenesis

Aim: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis.Methods: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene‘s feature. Results: A novel testis-specific gene,NYD-SPS, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved lie and Gin residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. Conclusion: NYD-SP5 is a newly found testisspecific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.     

小鼠睾丸特异性新基因TSAF76的克隆及其在睾丸组织中的表达

目的筛选与精子发生和（或）睾丸发育相关的基因。方法将不同发育阶段的小鼠睾丸组织cDNA与小鼠睾丸DNA芯片进行杂交，通过杂交信号比较，筛选出差异表达基因。利用网络信息资源对该基因进行生物学信息分析。RT-PCR方法分析该基因在小鼠不同组织及不同发育阶段睾丸组织中的表达。结果通过4日龄和18日龄小鼠睾丸芯片信号比较筛选出一个差异表达的基因，命名为TSAF76基因（GenBank登录号：AF503943），测序提示该基因全长1052bp，包含1个474bp的开放阅读框，编码157个氨基酸。对TSAF76蛋白质序列分析表明该蛋白质理论相对分子量为18．77kDa，等电点PI为9．782。TSAF76蛋白与表达于人类睾丸和成熟精子中的AAT1蛋白类似。RT-PCR分析表明TSAF76基因在处于精子发生中的睾丸组织特异性表达。结论TSAF76基因可能在小鼠精子发生过程中起作用。     

精子超活化结构蛋白——Catsper表达特性研究

目的:Catsper是精子特有的一种阳离子通道蛋白,对于精子超活化有重要的调节作用,为进一步了解其表达特性,本文主要研究其组织特异性表达及表达规律。方法:运用RT-PCR和免疫组织化学方法分析Catsper在2~8周龄昆明小鼠睾丸、附睾等8种组织中的表达情况,在精子上的精确定位以及随发育阶段变化表达量的变化。结果:Catsper特异性地表达于睾丸和附睾组织上;主要定位于精子尾部的主段;Catsper的表达量受到发育阶段的调控,该蛋白表达量随个体发育周龄的增长而呈增加的趋势,于3周龄开始检测到它的表达,随后表达量显著增加至6周龄,以后逐渐增长至一个平台期。结论:小鼠的Catsper基因特异性地表达于睾丸和附睾上,并且主要位于精子尾部的主段,其表达量与小鼠的发育阶段有密切的关系。     

Expression and regulation of aquaporins 1, 8, and 9 in the testis,efferent ducts,and epididymis of adult rats and during postnatal development

Aquaporins (AQPs) are membrane protein channels that allow the rapid passage of water through an epithelium containing tight junctions. In the present study, light microscope immunocytochemistry was utilized to localize several members of the AQP family in the testis, efferent ducts, and epididymis of normal adult animals during postnatal development and after various experimental procedures on adult animals. In the testis of normal adult animals, AQP-8 was expressed exclusively in Sertoli cells, while AQP-9 outlined Leydig cells. In the efferent ducts, AQP-1 was expressed on the microvilli, basolateral plasma membranes, and apical endosomes of the nonciliated cells and cilia of ciliated cells, while AQP-9 was present only on the microvilli of nonciliated cells. In the epididymis, AQP-9 was localized to the microvilli of the principal cells of all regions, with the most intense reaction being noted in the initial segment and cauda regions. The clear cells of the cauda region expressed only AQP-9. AQP-1 was not expressed in the testis or the epididymal epithelium, but it was expressed over the endothelial cells of the vascular channels of the efferent ducts and epididymis. After efferent duct ligation or orchidectomy, there was no change in the expression of AQP-1 or -9 over the microvilli or cilia of epithelial cells in the case of the efferent ducts, suggesting that testicular factors do not regulate their expression in this region. In contrast, AQP-9 expression in the principal cells of the initial segment, but not of other regions, and also in the clear cells of the cauda region was dramatically reduced after both treatments. As the expression was not restored to control levels by testosterone replacement, the data suggest that a luminal factor(s) derived from the testis regulates AQP-9 expression in the principal cells of the initial segment and in the clear cells of the cauda region. Postnatal studies revealed that the expression of AQP-1 and -9 in the different cell types of the efferent ducts and epididymis occurred between days 7 and 29, eliminating sperm and high androgen levels as possible regulating factors. Taken together, these data suggest cell specificity with respect to the expression of AQP-8 and -9 in the testis. In the efferent ducts and epididymis, specificity exists in cell, region, and tissue distribution with respect to the expression of AQP-1 and -9, and their expression does not appear to be regulated by androgens.     

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。     

Expression of Mammalian P1 Protamine Gene

Expression of Mammalian P1 Protamine Gene     

【原创论文】小鼠杀菌渗透增强性蛋白起源于睾丸及附睾表达并定位于精子中

杀菌渗透增强性蛋白（BPI）是具有抗革兰氏阴性菌活性的内源性杀菌蛋白。在本研究中,我们通过自行制备的多克隆抗体,检测了BPI蛋白在小鼠出生后睾丸及附睾组织中的表达,以及在附睾精子头部的亚细胞定位。实验结果表明,睾丸和附睾均独立表达BPI基因。在附睾中,自起始段至尾部,BPI蛋白的表达水平递减,并逐步特异性富集于亮细胞的胞质中。在顶体反应前的顶体基质内可见BPI蛋白,应起源于睾丸表达;顶体反应后,可见BPI蛋白分布于整个精子头部质膜表面,尤其是赤道板区域,可能有睾丸或附睾表达的两种起源。我们的研究结果提示,BPI蛋白可能参与顶体反应前后精子质膜结构的调控,并参与后续的精卵融合过程。     

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。     

Nephrocystin: gene expression and sequence conservation between human, mouse, and Caenorhabditis elegans

Juvenile nephronophthisis, an autosomal recessive cystic kidney disease, is the primary genetic cause for chronic renal failure in children. The gene (NPHP1) for nephronophthisis type 1 has recently been identified. Its gene product, nephrocystin, is a novel protein of unknown function, which contains a src-homology 3 domain. To study tissue expression and analyze amino acid sequence conservation of nephrocystin, the full-length murine Nphp1 cDNA sequence was obtained and Northern and in situ hybridization analyses were performed for extensive expression studies. The results demonstrate widespread but relatively weak NPHP1 expression in the human adult. In the adult mouse there is strong expression in testis. This expression occurs specifically in cell stages of the first meiotic division and thereafter. In situ hybridization to whole mouse embryos demonstrated widespread and uniform expression at all developmental stages. Amino acid sequence conservation studies in human, mouse, and Caenorhabditis elegans show that in nephrocystin the src-homology 3 domain is embedded in a novel context of other putative domains of protein-protein interaction, such as coiled-coil and E-rich domains. It is concluded that for multiple putative protein-protein interaction domains of nephrocystin, sequence conservation dates back at least to Caenorhabditis elegans. The previously described discrepancy between widespread tissue expression and the restriction of symptoms to the kidney has now been confirmed by an in-depth expression study.     

The role of cell adhesion molecules in ischemic epididymal injury

Objective The aim of this study was to investigate the role of adhesion molecules in epididymal injury induced by I–R in the rats. Study design A total of 20 male Sprague–Dawley rats were separated into two groups. A sham operation was performed in group 1 (control). In group 2 (I–R), following 6 h of unilateral spermatic cord torsion, 1-h detorsion of the testis was performed. Then, epididymides were removed to measure the tissue levels of malondialdehyde (MDA) and to make histological examination. Results MDA values increased in the group 2. In the group 2 rats demonstrated significant disorganization of the epithelium and loss of microvilli in the epididymal tissue. No abnormal microscopic findings of the epididymis of the rats in the control group. The tenascin expression in the interstitial area of the epididymis was intense in the group 2. ICAM-1 expression by intense brown staining was seen along the basement membrane in epididymal tissue from I to R group rats. The microvillus sites of the epithelia in I–R group were stained mildly by lectin. Conclusion The increased expression of adhesion molecules found in epididymal injury induced during postischemic reperfusion might implicate importance of inflammatory infiltration.     

实验性精索静脉曲张对青春期大鼠睾丸、附睾中CRES蛋白表达的影响

目的:研究青春期大鼠实验性左侧精索静脉曲张(ELV)对睾丸和附睾中胱蛋白酶抑制剂相关的附睾精子发生(CRES)蛋白表达的影响,探索精索静脉曲张导致不育的机制。方法:建立青春期雄性SD大鼠ELV模型,采用免疫组化及蛋白印迹方法检测CRES蛋白在ELV 2周组(n=8)、4周组(n=8)及其相应的对照组(n均=5)大鼠睾丸和附睾头、体、尾中的表达变化。结果:免疫组化显示,CRES蛋白在ELV实验组和对照组大鼠睾丸和附睾中均有表达。在睾丸中,CRES蛋白主要定位于圆形和长形精子细胞的胞质、精子的顶体以及残余体中,其表达与生精周期密切相关,Ⅰ~Ⅲ和Ⅸ~ⅩⅣ期表达最强,Ⅶ~Ⅷ期表达次之,Ⅳ~Ⅵ期表达减弱。在附睾中,CRES蛋白主要表达于从附睾起始段到尾部的上皮主细胞的胞质中,且以体、尾部表达最强,头部次之,管腔分泌物也呈阳性表达。实验组比对照组CRES蛋白表达增强。W estern印迹显示:实验组和对照组大鼠睾丸和附睾在相对分子质量(Mr)为19 000和14 000处均可见CRES蛋白条带,其中以Mr19 000处表达更为明显。实验组CRES蛋白的表达比对照组明显增强。对免疫组化和W estern印迹结果进行灰度值和积分吸光度值(IA)测定,并经统计学分析显示:ELV 2周组和4周组比其相对应的对照组表达增强且差异有显著性(P<0.05或P<0.01),而ELV各组间未见CRES蛋白表达有明显差异(P>0.05)。结论:CRES蛋白在大鼠睾丸和附睾中均有表达,睾丸中表达呈现生精周期特异性和细胞特异性,附睾中表达呈现附睾上皮区段和细胞特异性;CRES蛋白在青春期ELV大鼠睾丸和附睾中的表达比对照组明显增强。这些结果提示CRES蛋白在精子发生和精子成熟过程中可能起着重要的作用,并且可能与ELV引起的不育或生育力低下有关。