Contribution of epididymal factors to sperm maturation and storage

Summary.  In fertile men, the majority of epididymal spermatozoa acquire the potential to fertilize (assessed with sperm function assays) on passage into the corpus and cauda regions of the epididymis. Although secretions of the epididymal epithelium are clearly important for sperm maturation and survival, their role in this process has yet to be fully determined. Alterations in epididymal sperm membranes may result from the incorporation of protein, sugar and lipid determinants. Most probably, factors of epididymal origin act in concert with constitutional changes to spermatozoa, which together permit full sperm function. Epididymal spermatozoa incubated with epididymal epithelial cell cultures can undergo some maturation in vitro , which can lead to the development of sperm fertilizing capacity. Co-incubations of human sperm with epididymal epithelial cultures, at 37 °C with medium replenished every other day, led to 50% of spermatozoa retaining motility after 8 days. In one case, a few spermatozoa survived for 17 days, the inherent maximal survival time of human spermatozoa in situ. An important aspect of coculture experiments is that close interactions between spermatozoa and epithelial cells can be examined in detail. This coculture technique may yield important information related to epididymal sperm maturation and storage.     

Novel phenotype of mouse spermatozoa following deletion of nine β-defensin genes

β-defensin peptides are a large family of antimicrobial peptides. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. Despite their inducible presence at mucosal surfaces, their main site of expression is the epididymis. Recent evidence suggests that a major function of these peptides is in sperm maturation. In addition to previous work suggesting this, work at the MRC Human Genetics Unit, Edinburgh, has shown that homozygous deletion of a cluster of nine β-defensin genes in the mouse results in profound male sterility. The spermatozoa derived from the mutants had reduced motility and increased fragility. Epididymal spermatozoa isolated from the cauda region of the homozygous mutants demonstrated precocious capacitation and increased spontaneous acrosome reactions compared with those from wild-types. Despite this, these mutant spermatozoa had reduced ability to bind to the zona pellucida of oocytes. Ultrastructural examination revealed a disintegration of the microtubule structure of mutant-derived spermatozoa isolated from the epididymal cauda region, but not from the caput. Consistent with premature acrosome reaction and hyperactivation, spermatozoa from mutant animals had significantly increased intracellular calcium content. This work demonstrates that in vivo β-defensins are essential for successful sperm maturation, and that their disruption alters intracellular calcium levels, which most likely leads to premature activation and spontaneous acrosome reactions that result in hyperactivation and loss of microtubule structure of the axoneme. Determining which of the nine genes are responsible for the phenotype and the relevance to human sperm function is important for future work on male infertility.     

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     

Review article: Structure and function of the epididymis

Summary Testicular spermatozoa are functionally immature in that they cannot fertilize ova. It was first demonstrated by Young [171, 172] that spermatozoa undergo certain changes as they migrate through the epididymis. He proposed that spermatozoa ripen during epididymal transit. It is now known that specific maturational changes occur in spermatozoa during epididymal transit which result in their developing the ability to fertilize ova. Concomitant with this functional maturity are changes in spermatozoal morphology, motility, chemistry, permeability, density and metabolism. It is apparent that in some way not understood these changes are necessary for sperm to achieve the ability to complete the fertilization process. When these mechanisms are understood, we may be able to effectively treat conditions such as necrospermia or abnormally low sperm motility. Furthermore, with the development of the hamster-egg penetration test a new type of male infertility has become evident in recent years; the inability of otherwise normal sperm to penetrate an ovum. It is during epididymal transit that this ability is normally acquired. Thus, any insight into how sperm attain the capacity to penetrate an ovum could lead to an effective treatment of patients whose sperm do not have this ability. In addition, the epididymis holds significant promise as the site of action for a male contraceptive. Thus, it is the purpose of this review to describe the structure and function of the mammalian epididymis with particular emphasis on the factors regulating sperm maturation.     

Orchestration of occludins, claudins, catenins and cadherins as players involved in maintenance of the blood-epididymal barrier in animals and humans

Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility. （Asian J Androl 2007 July; 9： 463- 475）     

Epididymal protein CRISP1 plays different roles during the fertilization process

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.     

Secretion of macromolecules by the rat epididymis

Sperm maturation in the rat epididymis is dependent on the secretion of specific proteins by the epididymal epithelium and subsequent interaction of these proteins with spermatozoa. Evidence has shown that fertility and motility development of epididymal spermatozoa may be impaired by interfering the interaction of these proteins with spermatozoa. When the spermatozoa reach the cauda epididymidis, they are fully mature but their longevity is maintained by being stored in a quiescent state in the cauda. The unique ionic medium therein (low Na⁺, low Ca²⁺, high K⁺ and low pH) suppresses sperm motility and hence reserving energy for the vital processes of capacitation and fertilization. During ejaculation, when the spermatozoa are mixed with the copious secretion from the accessory glands they burst into vigorous motility. This results from an influx of sodium coupled to efflux of K⁺ and H⁺ across the mature sperm membrane. In the presence of a peptide secreted by the cauda epididymidis, these ionic events activate the already mature but otherwise inactive spermatozoa to full motility.     

The epididymis re-visited: a personal view

The sperm maturation and storage functions of the epididymis are important determinants of ejaculate quality, and perhaps provide an avenue to male contraception. In the last 50 years, the creation of epididymal fertility profiles in laboratory animals was followed by recognition of new sperm maturation-related parameters (organization of the acrosome, of the sperm plasmalemma, and –S–S– -based structural change) which made it possible to confirm that a similar pattern of sperm maturation obtains in man. The novel sperm storage function of the cauda epididymidis in therian mammals is regulated by androgen, usually in conjunction with the low temperature of the scrotum. The temperature-dependence of the scrotal cauda is reflected in the secretory and ion transport functions of the epithelium, in its duct dimensions and so in sperm storage capacity. Moreover, a variety of indirect evidence suggests that an elevated temperature of the cauda created by clothing may be compromising its function in man. The pattern of change in the sperm plasmalemma involving sterols, and also glycosylphosphatidylinositol-linked macromolecules as spermatozoa enter the cauda region, may underlie the need for their capacitation subsequently in the female tract. Further, in a variety of taxa the anatomy of the scrotum, together with the U-shaped configuration of the epididymis/vas deferens, suggests that the cauda''s storage function may also underlie the evolution of the scrotum. Finally, despite the still relative paucity of comparative evidence, we can consider now why the epididymis has come to be organized as it is.At the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, USA in 1959, I joined the laboratory of MC Chang, which was focused on in vitro fertilization, cross-species fertilization, sperm capacitation, sperm transport in the female, the Fallopian tube, and implantation of the embryo. At that time, Chang''s group had no specific interest in the epididymis, which was something of a scientific backwater compared to the focus then on the testis. I first became involved with the epididymis in asking whether, as spermatozoa are gaining the ability to fertilize there, they require the same period of capacitation as those from the cauda region – a point answered later for the pig, whose upper-corpus spermatozoa are capacitated more rapidly than those from the cauda.1 However, that question led me to others having to do with sperm maturation and sperm storage in the epididymis.     

Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of gamma-glutamyl-transpeptidase in sperm maturation

Sperm thiol oxidation during sperm maturation is important for sperm component stabilization, the acquisition of sperm motility, and fertilizing ability. A correct degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage. The pathways involved in physiologic sperm thiol oxidation in the epididymis are not completely understood. The nonprotein thiol glutathione (GSH), in addition to playing a major role as an antioxidant and in eliminating toxic compounds, has been implicated in prooxidation processes in various cells, via gamma-glutamyl-transpeptidase (gamma-GT)-dependent catabolism. Little information is available on the dynamics of nonprotein thiols (NPSHs) and disulfides (NPSSNPs) in spermatozoa and epididymal fluid (EF) during sperm passage in the epididymis. It is not clear whether NPSHs and NPSSNPs are involved in sperm protein thiol (PSH) oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm PSH oxidation. In the present study, we used the thiol fluorescence labeling agent monobromobimane to analyze NPSHs and nonprotein disulfides (NPSSRs) (R, nonprotein or protein) in spermatozoa and EF in the rat caput and cauda epididymis. NPSH levels are shown to be significantly higher in the caput than in the cauda (spermatozoa and fluid). GSH in the caput lumen is subject to high gamma-GT activity. A marked loss of sperm GSH and a shift to an oxidized state (resulting in a significantly higher concentration of glutathione disulfides [GSSRs] than GSH) occur during the passage of spermatozoa from the caput to the cauda epididymis. Caput EF and extracellular NPSSNPs induce sperm thiol oxidation. The results suggest that epididymal NPSH/NPSSNP participates in sperm PSH oxidation and that some reactions of GSH in the gamma-GT pathway (in the epididymis) provide oxidizing power, leading to physiologic sperm thiol oxidation.     

Epididymosomes: A potential male fertility influencer

During transit and storage in epididymis, spermatozoa undergo final maturation, acquire motility, functional competence and the ability to fertilise an oocyte. Epididymal secretions contain a complex biochemical milieu of diverse inorganic ions, proteins, metabolites and other molecules. Since it is believed that spermatozoa are translationally silent, proteins appearing in them are thought to be synthesised elsewhere, including epididymis, and then incorporated to the cells. One of the important mechanisms suggested to be involved in transfer of epididymal secretions to spermatozoa is through exosomes called epididymosomes. Epididymosomes released from the epididymal epithelium contain proteins, noncoding RNAs and distinct set of lipids that are transferred to spermatozoa while they pass through the different epididymal regions. Owing to the importance of these molecules for sperm maturation and fertilising ability, research on epididymosomes has gained increasing attention during the last decade. This review is focused on epididymosomes, with emphasis on recent advances in the understanding of mechanisms of epididymosomal cargo transfer to spermatozoa and potential roles of epididymosomes in sperm function and beyond. Possibilities of utilising the molecular signatures of epididymosomes as a tool for male fertility assessment are also discussed.     

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.     

Cathepsin D in human reproductive tissues: cellular localization in testis and epididymis and surface distribution in different sperm conditions

Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.     

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.     

附睾特异性基因的表达与调控

哺乳动物的附睾是一条高度卷曲的管道系统,附睾上皮是执行附睾功能的重要组成部分。附睾上皮蛋白质的合成与分泌,为精子提供了一个特殊的、不断改变的管腔液体环境,使其在附睾内运行过程中获得运动能力并最终达到功能成熟。附睾特异性表达的基因具有高度区域化的特征,受雄激素和(或)睾丸因子的调控,在出生后发育中呈现时空特异性的表达模式,这些都提示它们在附睾中发挥着重要而独特的作用。     

Influence of epididymal maturation on cyclic AMP levels in hamster spermatozoa

Whilst in their native epididymal fluid, sperm from the caput epididymis of the rat and hamster contain significantly (P less than 0.01) greater amounts of cAMP than do sperm from the cauda epididymis. The cAMP levels in both cell types from these species underwent a rapid increase concomitant with dilution to a density of 20 x 10(6)/ml. Further analyses in the hamster indicated that this increase was calcium-dependent, and could be enhanced by treatment with the calmodulin antagonist, calmidazolium. Dilution of hamster sperm to a concentration of 1 x 10(6)/ml was not associated with a rapid rise in cAMP levels. This effect was shown to be due to the dilution of a component in epididymal plasma. When incubated at this lower density, the cAMP content of hamster caput sperm remained low over a 3 h period, whilst similarly treated caudal sperm exhibited a progressive rise in cAMP levels. Thus, in contrast to other species, cAMP does not appear to play a pivotal role in acquisition of the capacity for movement during epididymal maturation in the rat and hamster. However, this nucleotide may be involved in the post-ejaculatory modifications of motility which accompany the terminal stages of capacitation.     

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.     

Redox regulation of mammalian sperm capacitation

Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility.     

Gene expression in the dog epididymis: a model for human epididymal function

The physiology of the epididymis is an integral part of the maturation process by which human spermatozoa acquire the abdity to reach and fertilize an oocyte. Because of the high degree of species specificity exhibited by the epididymal proteins involved in sperm maturation, we have assessed tissue from several alternative species for their suitability as a model for human epididymal physiology. Of these, the dog appears to offer an appropriate system. Northern hybridization using cDNA probes specific for human epidldymal genes established that, irrespective of dog breed, the canine equivalents of the epididymis-specific HE1, HE4 and HE5 mRNAs were expressed highly in the canine epididymis. cDNA cloning and sequencing confirmed that the canine gene products, CE1, CE4 and CE5 were indeed true structural homologues of their human counterparts. Finally, tissue culture conditions were established wherein all three specific canine genes remained up-regulated after 5 days of culture. Thus, the prerequisite criteria for the development of a system which models human epididymal physiology are to a large degree fulfilled by this canine culture system.     

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.     

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。