附睾与精子成熟的研究进展

附睾与精子成熟的研究进展胡向农周性明作者单位：２１０００９南京铁道医学院附属医院泌尿外科哺乳动物的附睾为精子的成熟提供了特殊的内环境。在类固醇激素的调控下，附睾上皮的合成、分泌、转运等功能使精子发生了一系列结构、生化和功能的改变，获得了授精能力和运动...     

人精子在附睾中的成熟

精子在人附睾内转运过程中获得使卵子受精的主要功能。这些功能包括 :相对活率、一致的形态、结合透明带的能力、发生顶体反应的能力、与卵黄和致密染色质融合的能力、体内和体外使卵子受精的能力、维持正常胚胎发育的能力等。已经鉴定了许多附睾分泌的糖蛋白 ,它们与特定的精子膜区域结合。这些蛋白可能与促进精 卵作用有关。作为辅助生殖技术的结果 ,取自附睾近端的“未成熟”精子或睾丸生殖细胞启动胚胎发育的能力依赖于精子在附睾内的转运、在女性生殖道中存活、输送直至被卵子包围。     

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。     

大鼠附睾精子成熟过程中膜流动性变化的研究

精子在附睾内的运行过程中，结构和功能发生变化，最终达到成熟，并贮存于附睾尾部。本文用５－Ｄｏｘｙｌ－ｓｔｅａｒｉｃ ａｃｉｄ和１６－Ｄｏｘｙｌ－ｓｔｅａｒｉｃ ａｃｉｄ为标记，在电子自旋共振波谱仪上研究成年ＳＤ雄鼠附睾头，体，尾各段的精子膜流动性的变化，并用低渗膨胀试验测定精子膜对渗透压的反应能力。     

大鼠附睾精子成熟过程中膜流动性变化的研究

精子在附睾内的运行过程中，结构和功能发生变化，最终达到成熟，并贮存于附睾尾部。本文用５－Ｄｏｘｙｌ－ｓｔｅａｒｉｃａｃｉｄ（５ＤＳＡ）和１６－Ｄｏｘｙｌ－ｓｔｅａｒｉｃａｃｉｄ（１６ＤＳＡ）为标记，在电子自旋共振（ＥＳＲ）波谱仪上研究成年ＳＤ雄鼠附睾头、体、尾各段的精子膜流动性的变化，并用低渗膨胀（ＨＯＳ）试验测定精子膜对渗透压的反应能力。结果表明，从附睾头部到尾部，精子膜膜脂的流动性逐步降低，特别是从体部到尾部更为明显（Ｐ＜０．０１），而精子膜对渗透压的反应能力也逐渐降低，主要表现在从头部移行到体部的精子（Ｐ＜０．０１）。精子成熟过程中的脂质过氧化反应，可能是降低膜脂流动性的原因之一。     

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。     

附睾精子蛋白P34H与男性生殖

哺乳动物精子在附睾转运过程中会获得精 卵相互作用所必需的精子表面蛋白。P34H是由人附睾上皮细胞分泌并定位于精子顶体的精子膜蛋白 ,属于短链脱氢酶 还原酶 (SDR)超家族成员。初步研究表明 ,P34H能够介导精子 卵透明带的结合 ,可作为附睾精子成熟的标志物 ,且低水平的附睾精子蛋白P34H与原发性男性不育显著相关。因此 ,精子表面P34H的水平可以作为男性不育症的一个辅助诊断指标。本文对附睾精子蛋白P34H的分子生物学特性、基因的表达与调控、作用机理及其与男性生殖的关系作一综述     

热休克蛋白70-2基因沉默对大鼠精子附睾成熟的影响

目的 观察热休克蛋白(HSP)70-2基因沉默对大鼠附睾中精子成熟的影响.方法 构建HSP70-2特异的短发夹RNA(shRNA)质粒载体,注入大鼠附睾中,1周后处死大鼠,无菌切取附睾,用逆转录-聚合酶链反应(RT-PCR)方法 检测附睾中HSP70-2 mRNA表达,Western blot观察HSP70-2蛋白表达,应用苏木素-伊红(HE)染色检测附睾管内精子密度.结果 空白对照组HSP70-2蛋白表达量为1.31±0.10,附睾管内成熟精子密度为(63.46±14.43)%.基因沉默组HSP70-2蛋白表达量为0.93±0.13,附睾管内成熟精子密度为(31.51±10.67)%,与对照组比较均显著减少,差异有统计学意义(P＜0.01).结论 HSP70-2基因沉默影响附睾中精子成熟.     

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca²⁺-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca²⁺ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4''s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca²⁺-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.     

Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit

During epididymal transit, spermatozoa acquire new proteins. Some of these newly acquired proteins behave as integral membrane proteins, including glycosylphosphatidylinositol （GPI）-anchored proteins. This suggests that the secreted epididymal proteins are transferred to spermatozoa by an unusual mechanism. Within the epididymal lumen, spermatozoa interact with small membranous vesicles named epididymosomes. Many proteins are associated with epididymosomes and the protein composition of these vesicles varies along the excurrent duct and differs from soluble intraluminal proteins. Some epididymosome-associated proteins have been identified and their functions in sperm maturation hypothesized. These include P25b, a zona pellucida binding protein, macrophage migration inhibitory factor, enzymes of the polyol pathway, HE5/CD52, type 5 glutathione peroxidase, and SPAM 1 or PH-20. The electrophoretic patterns of proteins associated to epididymosomes are complex and some of these proteins are transferred to defined surface domains of epididymal spermatozoa. Epididymosomes collected from different epididymal segments interact differently with spermatozoa. This protein transfer from epididymosomes to spermatozoa is timedependent, temperature-dependent and pH-dependent, and is more efficient in the presence of zinc. Some proteins are segregated to lipid raft domains of epididymosomes and are selectively transferred to raft domains of the sperm plasma membrane. Some evidence is presented showing that epididymosomes are secreted in an apocrine manner by the epididymal epithelial cells. In conclusion, epididymosomes are small membranous vesicles secreted in an apocrine manner in the intraluminal compartment of the epididymis and play a major role in the acquisition of new proteins by the maturing spermatozoa. （Asian J Androl 2007 July; 9： 483-491）     

Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage

Extracellular microvesicles present in the epididymal fluid have been named epididymosomes. Many epididymosome-associated proteins are transferred to spermatozoa during their maturation in the excurrent duct. Epididymosomes are heterogeneous, with their size varying between 50 and 250 nm. Two distinct population of epididymosomes characterized by different protein compositions and diameters have been isolated from the bovine epididymal fluid using different centrifugation protocols. One subpopulation of epididymosomes was characterized by CD9 and other tetraspanin partners. Transfer of proteins from these epididymosomes to maturing spermatozoa in co-incubation experiments was inhibited by antibodies against tetraspanin proteins. This suggests that this subpopulation of epididymosomes is involved in the acquisition of proteins involved in maturation by spermatozoa in the epididymis. The other population of epididymosomes was characterized by ELSPBP1 (epididymal sperm binding protein 1), known for its affinity for the phospholipid choline group. Flow cytometric analyses showed that ELSPBP1-positive epididymosomes only interacted with dying or dead epididymal spermatozoa in a Zn^{2 +}-dependent manner. BLVRA (biliverdin reductase) was identified as a partner of ELSPBP1. This enzyme reduces biliverdin to bilirubin: two molecules with powerful anti-oxidant properties. We hypothesize that BLVRA is involved in an ROS-scavenging mechanism protecting live epididymal spermatozoa against detrimental molecules (ROS) released by dying cells. Therefore, it appears that there are at least two epididymosome population with distinct functions: targeting specific proteins to transiting spermatozoa by tetraspanin-mediated membrane fusion, and protection of epididymal spermatozoa against ROS released from dying cells. Further work is needed to understand functions of epididymosomes in epididymal physiology and sperm maturation and storage.     

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation

Summary.  Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG_2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.     

Function of human epididymal proteins in sperm maturation

Summary.  Human post-testicular proteins were cloned by subtractive screening of epididymal cDNA libraries, employing testis as the primary negative control. This method identified six human epididymal cDNAs, named HE1-HE6, which are derived from abundant epididymal mRNAs. With the exception of HE5, which turned out to be identical to the lymphocyte surface antigen CD52, they represented completely novel human gene products. To date, there is little information on their function and the mechanism of their deposition on the sperm surface. Unlike the sperm coating antigens, CD52 binds firmly to the sperm membrane via its GPI anchor during epididymal passage. Its synthesis is carefully regulated by the epididymal epithelium. From the results of both in vivo and in vitro studies it was concluded that androgen and temperature are principal factors synergistically modulating epididymal CD52 expression. The human counterparts of two well-known major rodent epididymal proteins, secretory epididymal glutathione peroxidase (sGPX) and acidic epididymal glycoprotein (AEG = Protein DE), were not cloned by the subtractive screening approach, but by RT-PCR amplification.     

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.     

The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage

We have examined extracts of articular cartilage from rabbits aged 3-100 weeks for evidence of age-related changes in the structure and content of link protein (LP) in this tissue, with the following findings: (a) Two major molecular weight forms of LP were seen on SDS-PAGE (41 and 48 kDa) and the proportion of these changed markedly with age. The 48 kDa species was predominant in young animals (representing about 78% of the total LP at 5 weeks) whereas the 41 kDa species increased in amount with age (representing 35% of the total LP at 100 weeks). A minor form of about 43 kDa, representing less than 20% of the total, was present only during the growth phase. A small amount of fragmented link protein (less than 5% of the total) of about 25-30 kDa was present in samples from mature and aged rabbits only. (b) The quantitation of LP in guanidinium: HCl extracts of cartilage, by radioimmunoassay with monoclonal antibody 8-A-4, was markedly influenced by the conditions of preparation and pretreatment of samples. Assays of dialyzed guanidine extracts following treatment at 80 degrees C for 15 min in 0.025% (w/v) SDS indicated that immature and mature cartilage contains about 50 and 180 micrograms of LP/g of tissue, respectively. On the other hand, assays following treatment at 100 degrees C for 20 min in 0.1% (w/v) SDS suggested that rabbit cartilage contains about 300 micrograms of LP/g of tissue at all ages; finally, assay of CsCl purified proteoglycan samples under these conditions indicated a content of about 500 micrograms of LP/g at all ages. (c) Calculations based on the analysis of proteoglycan preparations for aggregating monomer and link protein suggest that a LP:aggregating monomer molar ratio of about 0.9 is maintained in the articular cartilage throughout maturation and aging in the rabbit.     

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.