ERp29蛋白在BALB／c小鼠附睾头部和尾部精子中的鉴定和定位

目的 对在BALB/c小鼠附睾精子中内质网蛋白29(endoplasmic reticulum protein 29,ERp29)进行鉴定以及定位.方法 应用免疫印迹方法鉴定BALB/c小鼠附睾头、尾部精子ERp29蛋白,同时利用激光共聚焦显微镜和间接免疫荧光定位的方法研究ERp29在附睾头、尾部精子上的定位.结果 证实ERp29蛋白存在于BALB/c小鼠附睾头部和尾部精子中,且其在尾部精子中含量较头部明显增高.ERp29蛋白主要定位于BALB/c小鼠附睾头部精子的头部前端,而在附睾尾部精子则主要定位于头部和尾部主段.结论 通过对ERp29蛋白在BALB/c小鼠附睾头部和尾部精子上的鉴定和定位,可以帮助进一步研究该蛋白对小鼠精子的作用.     

【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。     

OCTN2在1例正常生育男性附睾中的表达报告

目的：研究人类附罩有机阳离子转运子2COCTN2）的mRNA表达特征及蛋白表达特征,为进一步探讨附睾肉碱转运机制提供理论依据。方法：采用RT-PCR方法检测人类附睾组织头部、体部及尾部OCTN2基因的表达;并用Westernblot方法检测该基因在附睾中的蛋白表达,计算其在蛋白水平的相对表达量。结果：OC-TN2mRNA在人附睾头、体、尾组织中均有OCTN2表达,表现为附睾头部表达较弱,而附睾体、尾部分表达丰富;OCTN2蛋白在人附睾头部表达较弱,表达量为（0．71±0．09）,附睾体、尾部分表达较丰富,表达量分别为（0．95±0．22）与（0．99±0．15）。结论：两种方法均证实有机阳离子转运子2在人类附睾中有表达,且呈现附睾头部表达较弱,附睾体、尾部分表达卡富的特,止,为进一步研究附睾肉碱转运机制奠定基础。     

环氧合酶-2在成年雄性大鼠睾丸和附睾中的表达

目的:探讨环氧合酶 2在雄性大鼠睾丸和附睾组织中的表达及其意义。　方法:应用免疫组化SP法检测 40只雄性SD大鼠睾丸和附睾组织中环氧合酶 2的表达及其定位情况。　结果:环氧合酶 2在雄性大鼠的睾丸和 附睾组织中有较强的表达。在附睾头部主要表达于腔上皮细胞的细胞核内,部分细胞质内也可见表达,睾丸精原 细胞胞质和细胞核内均可见棕黄色颗粒。　结论:免疫组化法可较为敏感地检测环氧合酶 2在雄性大鼠睾丸和 附睾组织的表达。     

Ccdc70基因在小鼠睾丸精子发生过程中的表达特征

目的:探讨Ccdc70(Coiled-coil domain containing 70)基因在小鼠睾丸中的表达特征并分析其在生精过程中的潜在功能。方法:经表达谱芯片筛选出小鼠睾丸特异性基因Ccdc70,通过RT-PCR、real-time PCR、Western印迹及免疫组化检测Ccdc70基因在成年小鼠睾丸中表达特征,并对该蛋白做相关的生物信息学分析。结果:RT-PCR、real-time PCR和Western印迹结果表明Ccdc70基因在小鼠睾丸中高表达,在附睾中低表达;免疫组化结果表明Ccdc70蛋白在睾丸中主要表达于小鼠精母细胞和圆形精子胞质,在附睾中主要表达在附睾管上皮细胞。生物信息学分析显示该蛋白存在一个CCDC结构域,并在哺乳动物进化过程中高度保守。结论:Ccdc70为小鼠睾丸高表达基因,且主要表达于精母细胞、圆形精子及附睾管上皮细胞,提示其可能参与调控小鼠精子发生过程及附睾精子的进一步成熟。     

血管内皮生长因子和其受体Flt-1在青春期大鼠睾丸、附睾及附睾内精子上的表达研究

目的:通过对血管内皮生长因子(VEGF)及其受体Flt-1在大鼠睾丸、附睾及附睾内精子上表达的研究,探讨其在雄性生殖系统中的作用。方法:免疫组化SP法和免疫荧光法检测20只青春期SD大鼠睾丸、附睾及精子上VEGF和Flt-1蛋白的表达情况。结果:VEGF和Flt-1在大鼠睾丸和附睾组织及精子上均有特征性表达。睾丸内VEGF蛋白表达于生精细胞、精子细胞发育中的顶体、Sertoli和Leydig细胞胞质内;Flt-1只见于精子细胞发育中的顶体及Leydig细胞胞质中。附睾中VEGF表达于各段上皮主细胞胞质内,而Flt-1表达于头、尾段上皮主细胞胞质内,体部免疫染色阴性;两者在附睾上皮亮细胞、晕细胞和基细胞中均为阴性表达。免疫荧光染色显示,VEGF和Flt-1共同定位于附睾内精子头部的顶体,尾部的颈、中和主段。结论:VEGF和Flt-1蛋白在大鼠睾丸、附睾及精子中的特异性表达提示,他们可由不同生精上皮细胞、间质细胞和附睾主细胞产生,可能以自分泌或旁分泌的形式单独或共同作用于睾丸和附睾的生殖细胞或Leydig细胞,直接或间接地影响精子的发生、发育和成熟过程,并与精子的活动和受精能力有关。     

附睾分泌蛋白2β1在青春期雄性大鼠睾丸和附睾中的表达

目的:探讨附睾分泌蛋白2(HE2/EP2)的一种异构体———HE2β1在青春期雄性大鼠睾丸和附睾中的表达及其意义。方法:应用免疫组化SP法检测15只青春期SD大鼠睾丸和附睾组织中HE2β1的定位及其表达情况。结果:HE2β1在青春期大鼠睾丸和附睾组织中均有表达。在附睾中,HE2β1主要表达于附睾管上皮主细胞胞质内,而在亮细胞、晕细胞及基细胞内未见阳性表达;其表达水平在附睾头部远段较弱,在体部近、中段及尾部较强,而在附睾始段未见阳性表达。在睾丸生精小管中,部分精原细胞核及支持细胞核均可见明显的棕褐色的阳性颗粒,其他生精细胞以及间质细胞均为阴性。结论:HE2β1在青春期雄性大鼠的睾丸和附睾上皮中均有表达,其定位及表达水平具有区域特异性和细胞特异性,提示其在大鼠精子发生、成熟及附睾上皮天然抗感染机制中发挥重要的作用。     

鼠附睾中生殖细胞核因子的表达部位和时间

[Zhou ZY, Xu C, Guo QS, et al. Asian J Androl 2004;6: 23-28]目的:研究产后小鼠和大鼠附睾中生殖细胞核因子(GCNF)表达的部位及时间。方法:产后不同时间的附睾切片染色后用间接免疫荧光技术及CarlZeiss共聚焦显微统计数码摄影检测GCNF。结果:小鼠附睾首先在第12d测得GCNF,而大鼠则为第14d。大小鼠附睾均在第35d呈GCNF的最高表达。在成年鼠GCNF呈区域特异性表达,即主要表达在大鼠附睾的初始段,头部及近侧体部,小鼠附睾则在近侧体部较丰富,GCNF可在主、尖、狭、清及晕细胞核中发现。结论:GCNF可在附睾分化及发育和精子成…     

水通道蛋白1和3在小鼠前列腺组织的表达及意义

目的:揭示水通道蛋白(AQP)在小鼠前列腺组织的表达及意义,为深入研究前列腺正常及某些病理状态下AQPs表达调控及生理功能奠定基础。方法:取小鼠前列腺组织,采用RT-PCR方法检测AQP 0~4mRNA在前列腺组织的表达,同时应用Western印迹和免疫组化研究AQP1及AQP3在前列腺组织的定位表达。结果:RT-PCR显示前列腺组织中有AQP1、3 mRNA表达,而未见AQP0、2、4 mRNA的表达。Western印迹结果显示在正常前列腺组织中表达相对分子质量为28 000的AQP1蛋白,以及相对分子质量分别为35 000和27 000的糖基化和非糖基化AQP3蛋白。免疫荧光和免疫组化结果表明小鼠前列腺近管腔处分泌细胞的胞内囊泡及细胞膜均可见较强的AQP1蛋白表达;而在前列腺基质上皮细胞有AQP3的表达。结论:AQP1和AQP3基因在前列腺分泌上皮细胞表达,提示AQP1和AQP3可能通过促进前列腺上皮细胞对水的渗透,在前列腺液分泌过程中发挥重要的生理作用。     

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.     

Postnatal changes in the expression of claudin-11 in the testes and excurrent ducts of the domestic rabbit (Oryctolagus cuniculus domesticus)

We examined the expression of claudin-11 (CLDN11) in the testes and male reproductive tracts of rabbits. The rabbit CLDN11 cDNA sequences were nearly identical with human, mouse, and bovine CLDN11. The levels of CLDN11 mRNA and protein (22 kDa) were markedly increased in the testis during adult development. On postnatal day (PND) 10, CLDN11 was colocalized with ZO-1 at the lateral contacts between adjacent Sertoli cells and was perpendicular to the basal lamina. In adult testis on PND 180, CLDN11 was codistributed with ZO1, and the pattern of immunoreactivity consisted of wavy linear tracts parallel to the basal lamina, which was different according to the spermatogenic stage. These results suggest that CLDN11 participates in inter-Sertoli cell tight junctions (TJs) at the blood-testis barrier in adult rabbits. CLDN11 was also found in the basal regions of Sertoli cells adjacent to the basal lamina in adult testis, suggesting that CLDN11 also participates in the adhesion between Sertoli cells and the basal lamina. CLDN11 mRNA and protein expressions were decreased in the adult epididymis compared with those in immature animals. In adults, CLDN11 mRNA levels were relatively high in the efferent duct, followed by those in the vas deferens, proximal corpus, and distal cauda, although low levels were observed in the initial segment and caput. On PND 10, CLDN11 immunoreactivity was identified at the apicolateral contacts between adjacent epithelial cells in the epididymis and vas deferens. In adults, CLDN11 was found in the nonciliated cells in the efferent duct and at the lateral contacts in the epithelial cells in the epididymal segments. In the caput, CLDN11 was found at the apicolateral contacts between adjacent epithelial cells, but expression was weak to negligible in the corpus of the vas deferens. CLDN11 may play an important role in TJs and cell adhesion in immature rabbit excurrent duct epithelia. In adult rabbits, CLDN11 in efferent duct epithelium and epididymal epithelium may contribute to the specific environment for sperm maturation.     

Secretion of glycosidases in human epididymal cell cultures

The dynamics of glycosidase secretion was evaluated in human epididymal cell culture. Epithelial cells from caput, corpus, and cauda epididymis were isolated from tissue obtained from patients undergoing therapeutic orchidectomy due to prostatic carcinoma. The activities of alpha-glucosidase, N-acetylglucosaminidase, beta-glucuronidase, and alpha-mannosidase were analyzed in conditioned culture media. Glycosidase activity was significantly higher in corpus and/or cauda than in caput epididymis. There was a time-dependent increase in enzyme activities that was maximal between 10 and 14 days of culture in all epididymal regions. Epididymal glycosidases are secreted by cultured epithelial cell from human epididymis with an increase toward the distal regions of this organ, which may be related to the dynamics of sperm maturation. Cultures from different epididymal regions may represent a valuable tool to study of human epididymal function.     

有机阳离子转运子2在人类附睾中的表达及其意义

目的:研究人类附睾有机阳离子转运子2(OCTN2)mRNA的表达,探讨附睾肉碱转运机制,为探索男性避孕节育新技术提供理论依据。方法:应用RT-PCR方法检测人类附睾头、体、尾组织中OCTN2 mRNA的表达。结果:人类附睾头、体、尾组织中都存在OCTN2 mRNA表达。结论:人类附睾可能依赖OCTN2转运肉碱进入附睾管,为精子提供能量,促进精子成熟。对人类附睾OCTN2的进一步研究,将成为男性节育研究中新的分子靶标。