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。     

Animal models for epididymoepididymostomy. Development of an alternative microsurgical procedure for vasoepididymostomy.

The present success rate for high level microsurgical vasoepididymostomy in patients with obstructive azoospermia is generally poor in comparison with more distal vasoepididymal bypasses, suggesting that the development of a high level bypass method which preserves the distal maturation and storage functions of the epididymis might increase the fertility success rate. To achieve this we have developed a segmental bypass of the distal caput-proximal corpus regions of the epididymis (epididymoepididymostomy) using the rat and rabbit as animal models. In this procedure the epididymides of 11 adult male Sprague-Dawley rats were exposed through a scrotal incision and a convolution of the duct in the proximal corpus region was attached to an opened convolution in the mid-caput epididymides using a standard microsurgical technique. Each male was rested for at least 3 months after the microsurgical bypass operation to allow the preoperative sperm contents of the caudal storage region to be replaced by post-bypass spermatozoa. Six of the 11 rats were proven fertile after surgery by siring litters from mating trials up to 11 months later. The patency of anastomoses was confirmed in 3 animals by laparotomy and recovery of large numbers of sperm with normal motility from the cauda epididymidis distal to the anastomosis site and also from the vas. Similar unilateral surgery in 2 adult male rabbits resulted in normal post-operative semen profiles in 1 and an in vivo fertilisation rate of 100% from 1 mating trial 8 months after surgery. The successful development of a reliable animal model for epididymoepididymostomy provides an experimental tool for studying the function of the epididymis. This technique may also have clinical application as an alternative to high level vasoepididymostomy in selected patients with obstructive azoospermia.     

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。     

Expression of Cdv-iR gene in mouse epididymis as revealed by in situ hybridization

We have previously studied mouse Cdv (carnitine deficiency-associated gene expressed in ventricle)-1 related gene Cdv-1IR and its human counterpart CDV-1R, and revealed that mouse Cdv-1R was predominantly expressed in testis by multiple tissue northern analysis. To further localize the Cdv-1R mRNA in mouse testis and epididymis tissue, in situ hybridization study was reported in this article. In the adult mice, the Cdv-1R expression was intensively found in the epithelial cells of the caput and corpus epididymis, whereas it was moderately detected in the initial segment, and weakly in the cauda epididymis. In the seminiferous tubles of the testis, no obvious hybridization signals were observed above the background level. This Cdv-1R region-specific expression pattern in the epididimis suggests Cdv-1R may play an important role in sperm maturation. Moreover, considering the Cdv-1R has a similar expression distribution in epididymis to the OCTN2, it would appear that Cdv-1R might be involved in the carnitine pathway in the epididimis.     

Conjugation of maturation-related wheat-germ-lectin-binding proteins to caput epididymal sperm in co-cultures with corpus epididymal epithelial cells of BALB/c mouse

In BALB/c mice, two maturation-related wheat-germ-binding glycoproteins (GP-49 and GP-83) are synthesized and secreted by corpus and cauda epididymis. A co-culture technique was used to investigate these glycoproteins in principal cells of corpus epididymis and the conjugation of these molecules on caput sperm. The principal cells were recovered from corpus epididymides of 4-week-old mice and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. After culturing for 3-4 days, most cells revealed epithelial cell-specific keratins in immunofluorescent localization with monoclonal antibody. By electron microscopy, a prominent nucleolus with well-extended euchromatin was revealed in the nucleus and the cytoplasm contained multivesicular bodies, and a well-developed Golgi apparatus with endoplasmic reticulum. By SDS-PAGE, GP-83 and GP-49 were revealed in the cell extracts and cell culture supernatants after incubation with 35S-methionine. Radiolabeled binding sites were also found on the surface of caput sperm co-cultured with the principal cells for 4 h in the presence of 35S-methionine. WGA-binding glycoproteins may be synthesized and secreted by the principal cells of corpus epididymis and conjugated to caput sperm during the epididymal transit.     

Seminal carnitine content in obstructive azoospermia. Correlation with the anatomic level of obstruction

Free carnitine in human semen originates predominantly in the epididymis. The role of carnitine in the evaluation of different forms of obstructive azoospermia was studied in 42 patients. In 14 of the men, a bilateral vasectomy had been performed. In the remaining 28 patients, the occlusion was located within the epididymis. In postvasectomy cases and where the occlusion was located in the cauda epididymidis, carnitine concentrations were low, with mean values of 115.57 mumol/l and 121.28 mumol/l, respectively. When the occlusion was located in the corpus epididymidis, the mean value increased to 194.72 mumol/l. In patients having obstruction of the caput epididymidis or of the rete testis, the mean value of free carnitine was 416.0 mumol/l. After vasovasostomy, a return of free carnitine concentration to the normal range was observed in 10 of 12 cases. The results indicate that there is a significant correlation in patients with obstructive azoospermia between the concentration of free carnitine and the anatomic site of the obstruction. These findings may lead to important conclusions concerning therapy and prognosis for patients presenting with this condition.     

The presence of α-glucosidase, glycerophosphocholine and carnitine in the epididymis and ejaculate of the vervet monkey (Cercopithecus aethiops) and the Chacma baboon (Papio ursinus)

Summary.  The epididymis is the site of post-testicular sperm maturation in the male genital tract. Studies on human epididymides are hampered by the practical inaccessibility of epididymides of healthy men in their reproductive years. The limited use of laboratory animals therefore seems unavoidable. The objective was to establish baseline values of the epididymal markers α-glucosidase, glycerophosphocholine (GPC) and carnitine in the lumen of the caput, corpus and cauda epididymidis and in the ejaculate of adult male Chacma baboons and vervet monkeys. In both primates, α-glucosidase was found throughout the epididymis and in the ejaculate; values did not vary significantly. In monkeys, the highest concentration of GPC was found in the cauda epididymidis, but smaller amounts were found in the other regions and the ejaculate. In baboons, GPC was absent from the caput, but present in the other regions, including the ejaculate. Carnitine concentrations increased significantly from the caput to the cauda in monkeys and from the caput to the corpus in baboons. With this study, the relative concentration ranges in which these markers are present in the epididymides of these primates have been established. In future studies, changes in concentrations of these substances would probably indicate changes in epididymal function.     

Monoclonal antibodies to epididymis-specific proteins using mice rendered immune tolerant to testicular proteins

Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.     

Role of epididymis in sperm maturation

One hundred ninety patients with obstructive azoospermia caused by bilateral epididymal blockage have been followed up for four years or longer after undergoing "specific tubule" vasoepididymostomy. When anastomosis was required in the corpus epididymis, the "patency" rate was 78 percent, and the overall pregnancy rate was 56 percent. The pregnancy rate for "patent" cases was 72 percent, indicating that a high fertility rate can be obtained with sperm that have not transited the full length of corpus epididymis. By contrast, with vasoepididymostomy to the caput epididymis there was a 73 percent "patency" rate, but the overall pregnancy rate was only 31 percent. The pregnancy rate for "patent" cases was 43 percent. Sperm from the corpus epididymis have a higher rate of fertility than sperm from the caput epididymis, but sperm from proximal areas of the corpus have no less fertility than sperm from the distal corpus epididymis. The most remarkable observation is that in almost half the cases sperm that have never journeyed beyond the caput epididymis seem to be capable of causing pregnancy.     

Loss of sperm surface sialic acid induces phagocytosis: an assay with a monoclonal antibody T21, which recognizes a 54K sialoglycoprotein

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200; (1990a): Biol Reprod 42:151-160; (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues.     

大鼠精子在附睾成熟过程中肉毒碱量变化的研究

肉毒碱被认为是与精子在附睾中成熟有关的成熟因子。本文报告测定大鼠附睾头部,体部及尾部精子中的肉毒碱。结果表明精子在循附睾头、体、尾运行过程中,精子中肉毒碱含量逐步增加,在附睾体一头部,附睾尾一头部的精子内的肉毒碱量呈现十分显著性差异。结果提示附睾体部或体一头部交界部位可能是精子在附睾中成熟的关键部位。     

流式细胞术测定睾丸和附睾中生精细胞DNA含量

目的 :观察睾丸和附睾各段组织管腔内生精细胞DNA含量的变化。　方法 :利用流式细胞术 (FCM) ,对15例有生育能力、意外死亡的青年捐献者的右侧新鲜睾丸和附睾 (头、体、尾 )组织管腔内生精细胞的DNA含量进行测定。　结果 :从睾丸至附睾尾均存在单倍体 (1n)、二倍体 (2n)和四倍体 (4n) 3种细胞。 1n细胞由 (2 4 .87±7.2 8) %增至 (96 .33± 1.5 8) % ,其中睾丸至附睾每段之间的差异有极显著性 (P <0 .0 1) ,附睾体与附睾尾之间也有明显差别 (P <0 .0 5 )。 2n和 4n细胞的比例分别由 (6 3.0 7± 8.96 ) %、(9.4 3± 3.83) %下降至 (2 .4 7± 0 .93) %、(1.17± 0 .95 ) %。睾丸至附睾每段之间 2n细胞的比例差异有极显著性 (P <0 .0 1) ,附睾体与附睾尾之间也有明显差别(P <0 .0 5 ) ;除睾丸与附睾头之间的 4n细胞变化不明显外 (P >0 .0 5 ) ,其他各段之间的 4n细胞比例差异也有显著性 (P <0 .0 5 )。　结论 :未成熟生精细胞比例在附睾转运过程中逐渐减少     

Effect of gossypol on the concentration of sodium and potassium in the rat epididymis

The effects of administration of gossypol acetic acid (7.5 mg/kg daily for 4 weeks) on the concentration of Na+ and K+ in the rat epididymis was assessed. Epididymal fluid samples, collected by micropuncture, from the caput, corpus, proximal cauda and distal cauda epididymis from gossypol-treated and control animals were analysed for Na+ and K+ concentrations. Gossypol-treated males failed to impregnate healthy females, presumably because their sperm were immotile. In gossypol-treated rats, Na+ levels decreased significantly (P less than 0.01) in the caput, corpus, proximal and distal cauda epididymis. In contrast, the K+ concentration was increased significantly (P less than 0.05) only in the caput and corpus epididymis. This altered electrolyte milieu may be responsible, to some extent, for immotility and hence infertility.     

【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。     

Attractin蛋白在大鼠睾丸和附睾中的免疫组织化学定位

目的 :探讨Attractin蛋白在成熟大鼠睾丸和附睾组织中的分布。　方法 :健康成年雄性SD大鼠 2 0只 ,灌流取睾丸和附睾组织固定 ,石蜡包埋和冰冻切片。用免疫组化法和间接免疫荧光方法检测Attractin蛋白在成熟大鼠睾丸和附睾组织中的表达。　结果 :睾丸组织间质细胞、睾丸精曲小管管周肌样细胞和各级生精细胞 (精原细胞、初级精母细胞和精子细胞 )、支持细胞呈阳性反应 ,主要表达于胞膜及胞质。睾丸间质细胞表达略强于生精细胞。附睾头、体、尾均未见表达。　结论 :Attractin蛋白在成熟大鼠睾丸组织间质细胞和生精细胞中有较强的表达 ,其生理功能尚有待进一步探讨。     

Circulating and luminal testicular factors affect LRP-2 and Apo J expression in the epididymis following efferent duct ligation

Apolipoprotein J (clusterin or sulfated glycoprotein-2) has been shown to be secreted by the epididymal principal cells, whereupon it binds to sperm in the lumen. Apolipoprotein J also is endocytosed by principal cells along the epididymis. Recently, it has been demonstrated that low-density lipoprotein receptor-related protein-2 (LRP-2) mediates the endocytosis of Apo J and is present in the epididymis. The purpose of the present study was to determine the factors regulating the synthesis of these 2 proteins in various experimentally treated animals. The epididymides of adult rats were fixed with Bouin's fluid and examined with anti-Apo J and anti-LRP-2 antibodies by a light microscope immunocytochemical method. In normal adult animals, expression of Apo J was evident in principal cells of all epididymal regions except the proximal initial segment. Diffuse cytoplasmic staining indicated Apo J secretion. Reactive apical vesicles, presumably endosomal in nature, suggested endocytosis of Apo J. Lipoprotein receptor-related protein-2 expression was solely apical in nature and was seen as an intense apical band in principal cells of all regions except the proximal and distal initial segment and distal caput regions of the epididymis. Hypophysectomy, up to 28 days after the procedure, did not affect expression of Apo J or LRP-2 in principal cells along the entire epididymis. Orchidectomy, with or without testosterone replacement at all time intervals examined, also did not affect LRP-2 expression along the entire epididymis. This also was noted for Apo J expression in all regions except the proximal initial segment. Thus, expression of these 2 proteins does not appear to be regulated by testicular or pituitary factors. In contrast, bilateral as well as unilateral (intact and ligated sides) efferent duct ligation resulted in dramatic differences in LRP-2 and Apo J expression in principal cells in the various epididymal regions. In the case of LRP-2, a complete absence of reaction was noted in principal cells along the entire epididymis. As for Apo J, expression in the distal initial segment, intermediate zone, and caput region remained unchanged compared with that in normal adult animals, whereas in the corpus and cauda epididymides, results of cytoplasmic staining were negligible. These results suggest that under conditions of efferent duct ligation, a circulating factor emanates from the testis to inhibit expression of LRP-2 and Apo J in these epididymal regions. Furthermore, because Apo J was affected in a region-specific manner, unlike the case for LRP-2, different factors appear to be involved for each protein. These factors may be produced to inhibit proteins from being synthesized by the epididymis in the absence of luminal testicular input and may exist in cases of congenital and pathologic epididymal tubule blockages as well as after vasectomy. In the case of immunostaining for Apo J in the proximal initial segment only, normally unreactive principal cells in control adult animals became intensely reactive after orchidectomy as well as bilateral and unilateral (ligated side only) ligation. As this was not the case for hypophysectomized animals and the intact side of unilateral efferent duct-ligated animals, it is suggested that a testicular factor entering via the lumen of the efferent ducts serves to inhibit Apo J expression in this area. The present data also reveal that after efferent duct ligation, there are circulating factors that inhibit Apo J expression in a region-specific manner (corpus and cauda) and that inhibit LRP-2 expression along the entire epididymis and that these are derived from the testis. Furthermore, the data reveal that a testicular luminal factor appears to inhibit Apo J expression in the proximal initial segment of normal adult animals. Key words: Principal cells, orchidectomy, glycoprotein 330, clusterin, sulfated glycoprotein-2.     

Alterations in the testis and epididymis associated with loss of function of the cystatin-related epididymal spermatogenic (CRES) protein

Cystatin-related epididymal spermatogenic protein (CRES) or cystatin 8 (Cst8 gene) is a member of the cystatin superfamily of cysteine protease inhibitors. It differs from typical cystatins because it lacks consensus sites for cysteine protease inhibition and exhibits reproductive-specific expression. In the present study, we examined CRES expression within the testes, efferent ducts, and epididymides of normal mice by light microscope immunolocalization. Alterations to these tissues in male mice lacking the Cst8 gene (Cst8(-/-2)) were also characterized by histomorphometry and electron microscopy. In the normal testis, CRES was localized exclusively in mid and late elongating spermatids. In the efferent ducts, CRES was localized to the apical region of the epithelial cells suggestive of localization in the endosomes. In the initial segment of the epididymis, principal cells showed supranuclear and luminal reactions. In the cauda region, CRES was present exclusively as aggregates in the lumen and was detected in clear cells. Compared with wild-type mice (Cst8(+/+)), older (10-12 months) Cst8(-/-) mice had modest but statistically significant reductions in tubular, epithelial, and/or luminal profile areas in the testis and epididymis. By electron microscopy, some Cst8(-/-) tubules in the testis were normal in appearance, but others showed a vacuolated seminiferous epithelium, degenerating germ cells, and alterations to ectoplasmic specializations. In the epididymal lumen, abnormally shaped sperm heads and tails were noted along with immature germ cells. In addition, principal cells contained numerous large irregularly shaped lysosomes suggestive of disrupted lysosomal functions. In both the testis and epididymis, however, these abnormalities were not apparent in younger mice (4 months), only in the older (10-12 months) Cst8(-/-) mice. These findings suggest that the altered testicular and epididymal histology reflects a cumulative effect of the loss of CRES and support a role for CRES in maintaining the normal integrity and function of the testis and epididymis.     

大鼠附睾尾部的精子参数与灌服昆明山海棠乙醇提取物后的改变及其抗生育效果观察

本文报告３７只Ｗｉｓｔａｒ成年大鼠附睾尾部的三项精子参数值及季节等因素对精子参数的影响，并与每日灌服昆明山海棠乙醇提取物ＴＨ５（１１６ｍｇ／ｋｇ）３０天后的２ｌ只大鼠的改变及生育力进行比较。结果表明：对照组大鼠左、右侧附睾尾部的三项精子参数值虽各不相等但无明显差异（Ｐ＞０．０５），与ＴＨ５组停药２０天后剖检的大鼠相比，上述各项精子参数值间有极显著差异（ｐ＜０．０１），且所有服药大鼠完全丧失生育力；对照组于４～６月剖检的大鼠其精子明显低于１～３和９～１２月剖检的大鼠；用于生殖研究的大鼠的体重＞４００ｇ或睾丸容积＞３．５ｍｌ、＜１．０ｍｌ均可对精子质量有不良影响；至于停服ＴＨ５７０天后剖检的大鼠，其精子参数值和生育力与对照组无异。