大鼠精子在附睾成熟中精子膜变化的研究

我们运用Ｐｅｒｃｏｌｌ离心技术对ＳＤ大鼠附睾头、体、尾各段的精子分离纯化后，再用硫代巴比妥酸法、酶法、ＳＤＳ－ＰＡＧＬ电泳技术等对精手膜依次进行唾液酸、甘油－３－磷酸胆碱（ＧＰＣ）及蛋白质含量变化的检测。结果显示：精子膜唾液酸、ＧＰＣ量不断降低且有显著统计学意义（Ｐ＜０．０１）。附睾头、体、尾各段精子膜唾液酸和ＧＰＣ量分别为１０．１８±２．８２、８．４２±３．０７、７．８３±２．７９μｇ／１０８精子；１１２．３１±２８．１４、１０９．３３±３７．１６、７４．５０±２５．１３ｎｍｏｌ／１０８精子（－ｘ±ｓ）。膜蛋白变化主要是由大分子蛋白转变成较小分工的蛋白。大鼠精子附睾转运中精子膜的变化与精子成熟具有十分密切的关系。     

Use of neonatal tolerization and chemical immunosuppression for the production of monoclonal antibodies to maturation-specific sperm surface molecules.

Mammalian sperm acquire functional maturity as they move from the caput to the cauda epididymidis. Changes occur in the protein/glycoprotein composition of the sperm plasma membrane during this time, and may be essential to the maturation process. The production of monoclonal antibody (Mab) probes to the maturation-specific molecules has been difficult since new proteins comprise a minor portion of total membrane proteins. This report describes a protocol for enhancing the production of Mabs to maturation specific molecules. By injecting neonatal mice with caput epididymal sperm plasma membranes, in combination with chemical immunosuppression at adulthood, the mice were made tolerant to the antigens expressed on the caput sperm membranes. Subsequent immunization with cauda epididymal sperm plasma membranes allowed the production of Mabs to the maturation-specific moieties without the necessity for extensive antigen purification procedures. The majority of the resulting Mabs recognize cauda, not caput, epididymal sperm plasma membranes as determined by enzyme-linked immunosorbent assay (ELISA), immunocytochemistry on unfixed cells, and Western blot analyses, even though the protein profile from caput epididymal sperm plasma membranes is very similar to that from cauda membranes. The five Mabs described also recognize cauda fluid antigens, suggesting that the maturational changes on the sperm plasma membranes arise from interactions with the epididymal fluid. Use of the tolerization/immunosuppression protocol has provided Mab tools to assist in the study of sperm maturation during epididymal transit.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Changes in surface protein structure of bull spermatozoa during epididymal maturation

The surface proteins of bull spermatozoa from caput and cauda epididymidis were labelled by lactoperoxidase-catalyzed radioiodination and solubilized and analysed by SDS-PAG-electrophoresis. The surface protein patterns of caput and cauda epididymal spermatozoa resembled each other but some distinct differences could be found. Caput epididymal spermatozoa revealed a protein peak with molecular weight of 15 000 - 18 000 daltons but this peak was not found on cauda epididymal spermatozoa. On caput epididymal epermatozoa the most intensely labelled protein peak was located between 90 000 and 100 000 daltons but on cauda epididymal spermatozoa the corresponding peak was only weakly labelled and had a molecular weight of 80 000–90 000 daltons. Surface protein with molecular weight of 42 000–47 000 daltons was dominating on cauda epididymal spermatozoa. The surface protein structure of cytoplasmic droplets did not drastically differ from that of epididymal spermatozoa.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

非阻塞性输精管滤过装置节育术的精浆中性α-糖苷酶的检测

本文采用ＷＨＯ推荐的改良精浆中性α－糖苷酶的测定方法测定输精管滤过装置节育术（ＩＶＤ组）和输精管结扎术（结扎组）的精浆中性α－糖苷酶活性。术前两组精浆均测出中性α－糖苷酶活性：ＩＶＤ组为４３．５０±２９．０１ｍＵ／每次射精（ｘ±ｓ）；结扎组为４７．８１±３１．２０（ｘ±ｓ）ｍＵ／每次射精；两组间无显著差异（Ｐ＞０．０５）。术后６、１２个月ＩＶＤ组分别有９１．５７％和７９．１７％的精浆测出中性α－糖苷酶活性；而结扎组仅有４．４８％和４．２４％的精浆测出中性α－糖苷酶活性；两组间均有非常显著的差异（Ｐ＜０．００１）。结果提示：ＩＶＤ组术后一年内其大部分受试对象的精浆中仍有附睾液存在。     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures

The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.     

葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有

哺乳动物睾丸中的精子经过“附睾成熟”期,由静止状态转变成为运动状态。该过程中精子从附睾头部向附睾尾部移动,同时精子发生了一系列的形态、生理和生化改变,如蛋白组成和蛋白修饰的改变可能会影响精子获能的潜能。本实验使用基质辅助激光解吸／电离串联质谱（MALDI-MS／MS）法分析仓鼠睾丸头部和尾部精子的蛋白组学,成功发现了113个蛋白质点。对113个蛋白质点进一步对照比较发现30个蛋白质点（对应20个蛋白）的密度发生了显著改变,其中附睾尾部精子5个蛋白的密度增加,11个蛋白密度减少;此外,葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有,而纤维蛋白原样蛋白1为附睾尾部精子所特有。几个蛋白密度增加可能与附睾成熟过程中精子代谢和ATP产生相关。一些蛋白如ERp57,GRP78,GP96,Hsp60,Hsp70和二氢硫辛酰胺S-乙酰转移酶的密度改变通过免疫印迹法得到验证。本研究首次报道了仓鼠精子的蛋白质组学研究,全面展示了仓鼠精子附睾成熟过程中的蛋白结构改变。     

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.     

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。     

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.     

Correlation between changes in rat sperm membrane lipids, protein, and the membrane physical state during epididymal maturation.

An enriched plasma membrane fraction was isolated from caput, corpus, and cauda rat spermatozoa and analyzed for lipid and protein content, thermal phase transition temperature using electron paramagnetic resonance spectroscopy (EPR), and enzymatic assays of calcium-dependent ATPase activity. Based on sperm concentration, total membrane phospholipid, cholesterol, and protein content declined as sperm passed through the epididymis. A more refined analysis of the bulk plasma membrane phospholipid revealed that approximately 56% of the phospholipid consisted of choline (PC) and ethanolamine (PE) phosphoglycerides; the remainder consisted of sphingomyelin (SM), phosphatidylserine (PS), and diphosphatidylglycerol (DPG). The mole percent of PE increased in sperm proceeding from the caput to the corpus epididymis and then declined from the corpus to the cauda epididymis. The phospholipid-bound fatty acids consisted primarily of palmitate (C16:0) and stearate (C18:0), with a significant increase in the mole percent of the docosapentenoyl acyl group (C22:5) in cauda sperm. Arrhenius' plots of the EPR peak height signals using the lipid soluble spin label, 5-doxyldecane, and the calcium-dependent ATPase activity as a function of temperature demonstrated a change in the apparent fluidity of the membrane and energy of activation of the calcium-dependent ATPase associated with the three sperm membrane preparations. These data suggest that the apparent fluidity and biochemical composition of the sperm membrane change during epididymal maturation.     

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.     

正常男性附睾中精子功能的初步研究

目的　检测正常成年男性附睾中不同部位的精子指标 ,为男性附睾精子功能的研究提供实验资料。方法　将附睾切断分成附睾头、附睾尾和附睾体 ,分别纵向剖开 ,置于含有 10 %小牛血清的F10 溶液中 ,37℃孵育 30min ,孵育液作精子常规分析、低渗肿胀试验 (HOS)、精子 -宫颈粘液穿透试验。结果　附睾不同部位精子的形态和功能存在明显差异 ,尾部和体部精子的活率、活动力、HOS试验和CM穿透试验结果明显高于头部精子 ,差异有显著性 (P <0 .0 5或 0 .0 1) ,以尾部精子的各项指标最高 ,与正常排出体外的精子无显著差异 (P >0 .0 5 )。头部附睾未成熟精子、畸形精子比例明显高于尾部和体部附睾的精子 (P <0 .0 1和 0 .0 5 )。结论　附睾精子可应用于辅助生育技术 ,作为治疗梗阻性无精子症的最好选择。     

大鼠附睾管腔液M_r22000蛋白的鉴定研究

目的:鉴定大鼠附睾管腔液中可能参与精子成熟/精子表面修饰的附睾特异蛋白。方法:通过SDS-PAGE或双向(2D)电泳比较正常大鼠附睾头段和尾段以及实验性精索静脉曲张(ELV)大鼠附睾尾段管腔液中差异蛋白成分。选择主要差异斑点进行质谱分析,并采用免疫印迹对目标蛋白作进一步鉴定确认。结果:发现多个蛋白成分明显增高。对其中之一的相对分子质量(Mr)22000蛋白集中研究。经质谱分析及免疫印迹确认Mr22000蛋白为磷脂酰乙醇胺结合蛋白(PBP)。结论:附睾管腔液中的PBP存在多种分子形式包括糖基化修饰。ELV导致大鼠附睾尾管腔液中PBP升高。因此,该分子与精子表面修饰的关系仍需进一步研究。     

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

Summary.  This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotinstreptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.     

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.     

Glutathione-related enzymes in cell cultures from different regions of human epididymis

Protection of maturing sperm from potential endogenous or exogenous harmful substances during their transit throughout the epididymis is a critical event. The authors studied the activity of gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST), and glutathione (GSH) levels in epithelial cell cultures from human caput, corpus, and cauda epididymides. Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Enzymatic activity was measured in conditioned media and cellular fractions. Androgen influence was also evaluated. Both enzymatic activities were found in cellular homogenates and conditioned media from cultures of all epididymal regions. GGT activity was highest in cultures from cauda epididymis, both in conditioned media and cell fractions, while GST activity did not show regional differences in conditioned media, but exhibited higher activity in cell homogenates from cauda cultures than those obtained from corpus and caput epididymis. GSH level showed no regional difference in cell homogenates and it could not be detected in conditioned media by the method used. Presence of different concentrations of dihydrotestosterone (DHT) had no influence neither on the enzymatic activities nor GSH concentration. The results indicate that GGT and GST are present along the human epididymis and a fraction or isoform of these enzymes might be secreted to the luminal fluid to play a detoxificative role in sperm maturation.