Spermatozoa of the loach Misgurnus fossilis as a test system for identification of new centromere proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. for identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein gamma-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes has been obtained from the loach spermatozoa. These centrosomes contained gamma-tubulin although they lost their ability to induce polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and on then microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising object for identification of new proteins of the mammalian centrosome.     

Spermatozoa of the Loach Misgurnus fossilis in the Identification of New Centrosome Proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. in the identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein -tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes was obtained from the loach spermatozoa. These centrosomes contained -tubulin, although they had lost their ability to induce the polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and then on microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising means to identify new proteins in the mammalian centrosome.     

A novel short splice variant of the tumour suppressor LKB1 is required for spermiogenesis

LKB1 was discovered as a tumour suppressor mutated in Peutz-Jeghers syndrome, and is a gene involved in cell polarity as well as an upstream protein kinase for members of the AMP-activated protein kinase family. We report that mammals express two splice variants caused by alternate usage of 3'-exons. LKB1(L) is the previously described form, while LKB1(S) is a novel form in which the last 63 residues are replaced by a unique 39-residue sequence lacking known phosphorylation (Ser(431)) and farnesylation (Cys(433)) sites. Both isoforms are widely expressed in rodent and human tissues, although LKB1(S) is particularly abundant in haploid spermatids in the testis. Male mice in which expression of Lkb1(S) is knocked out are sterile, with the number of mature spermatozoa in the epididymis being dramatically reduced, and those spermatozoa that are produced have heads with an abnormal morphology and are non-motile. These results identify a previously undetected variant of LKB1, and suggest that it has a crucial role in spermiogenesis and male fertility.     

Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit

We have identified an 80 kDa protein in ejaculated bull spermatozoa (p80) which is found in acrosomal and post-acrosomal areas of the head. It has a hyaluronidase activity and shares homologies with PH-20, a sperm surface glycoprotein involved in sperm-egg interaction. The aim of the present study was to characterize bull sperm p80 protein at the nucleic and amino acid levels to determine whether it is the bovine PH-20 ortholog. The complete nucleotide sequence determined by RT-PCR, 3' and 5' RACE show that bull p80, displays identity with the PH-20 nucleotide and amino acid sequences. Messenger RNA and protein expressions determined by Northern blot and immunohistochemistry revealed that the protein is testicular (expressed in spermatocytes and spermatids). The localization of p80 on spermatozoa, determined by indirect immunofluorescence using a monoclonal antibody, shows the protein in acrosomal and post acrosomal areas of the head with an increase in the signal intensity as sperm progress through the epididymis. Post-translational modifications of the protein were investigated during the epididymal maturation by Western blot on protein extracts from sperm collected in the caput, corpus and cauda portions of bull epididymis. Glycolysation status of sperm p80 protein on proteins from ejaculated and epididymidal sperm was investigated. Result show that the glycosylation status is modified as spermatozoa migrate through the epididymis. Hyaluronidase activity evaluated in protein extracts from spermatozoa of the three different epididymal sections revealed that the activity is higher at pH 7 than 4 and is not affected by epididymal maturation. These data strongly suggest that p80 is the bovine PH-20.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

哺乳动物精子性别特异膜蛋白的研究进展

在精子形成过程中,存在性染色体特异基因的表达,这是X、Y精子膜蛋白差异形成的基础。尽管精子形成过程中形成的细胞间桥可能使精子细胞间共享基因表达产物,但雄性传递偏移现象和性别偏移现象的发生又证明两类精子间存在非共享蛋白,同时H-Y抗原表位的成功鉴定、精子分离实验结果以及性别特异蛋白的检测结果都肯定了精子膜蛋白差异的存在,只是这种膜蛋白的差异很小。蛋白分离技术的进步及技术间的优化集成,为精子间细微差异膜蛋白的分离提供了可能。     

Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C: A ROLE FOR AMYLOID IN MALE INFERTILITY*

Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.     

Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs

BACKGROUND INFORMATION: In amphibians, the role of sulphated glycans has not been determined in spermatozoa-egg interaction, although they are known to be involved in other systems. In previous studies, it was found that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology to Xenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of binding the spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably two glycoforms of the same protein. RESULTS: In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggest that hydroxy groups of sugars are not directly involved in spermatozoa-vitelline envelope binding. Competition assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidan indicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar competition assays, Le(x) (Lewis(x)) trisaccharide 3'-sulphate inhibited spermatozoa binding to VE in contrast with 3'-sialyl-Le(x) tetrasaccharide. Assays performed with gp75- or gp63-coated beads and spermatozoa treated with fucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa binding to gp75-coated beads, yet not to gp63-coated beads. Finally, solubilized VE digested with N-glycosidase F retains the inhibitory activity in spermatozoa-VE binding assays in contrast with VE treated with alpha-N-acetylgalactosaminidase. CONCLUSION: It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups are present in gp75 glycoconjugates and are probably located in O-linked glycoconjugates.     

Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.     

Delivery of ferric ion to mouse spermatozoa is mediated by lipocalin internalization

The aim of this study was to illustrate the further process of 24p3 protein after association with epididymal spermatozoa. We have previously identified a caput-initiated 24p3 protein, which interacts with the spermatozoa surface in vitro. In the present study, we investigate another role of the 24p3 protein with spermatozoa. Mouse epididymal spermatozoa exhibit the ability to bind spontaneously with exogenous 24p3 protein, a part of which is further internalized into the spermatozoa in epididymal caput. We have now focused on this issue using freshly prepared spermatozoa from caudal region of epididymis. First, the cytosolic fractionation of spermatozoa has revealed that biotinylated 24p3 protein signal could be detected by supplying biotinylated protein under 37 degrees C incubation after 30 min at this experiment. Further, flow cytometric analysis of FITC-protein containing spermatozoa has revealed two distinct types of fluorescent spermatozoa, and microscopical experimentation with fluorescent FITC-24p3 protein has shown that the 24p3 protein did accumulate in the cytosolic portion of spermatozoa. All of these events, which showed protein uptake into the cell, demonstrated time- and temperature-dependence of endocytotic characteristics, these constituting the critical points in the process of endocytosis for spermatozoa as for other cells. Using a fluorometric method, the binding affinities of ferrous ion and ferric ion to 24p3 protein were shown to be (1.5+/-0.2)x10(6) and (3.0+/-0.4)x10(7)M(-1), respectively. We have also determined the internalization of this protein in the transition of iron into spermatozoa. We report here that spermatozoa, from the caudal epididymis, demonstrate the ability to bind with 24p3 protein and further internalize it and deliver the ferric ion to the spermatozoa via protein internalization. We suggest that the 24p3 protein plays a physiological role in spermatozoa in the context of protein-ligand complex internalization.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Region-specific expression and secretion of the fibrinogen-related protein, fgl2, by epithelial cells of the hamster epididymis and its role in disposal of defective spermatozoa

The cauda epididymidis functions in the storage and protection of mature, fertile spermatozoa. We previously identified a region-specific secretory glycoprotein (termed HEP64) of the hamster proximal cauda epididymidis that specifically bound and coated the nonviable, but not the viable, spermatozoa within the epididymal lumen. In this study we employed expression screening of a hamster epididymal cDNA library to obtain the full-length sequence of HEP64 and to identify it as the fibrinogen-like protein fgl2. Northern blot analysis demonstrated that fgl2 mRNA is highly expressed by the proximal cauda epididymidis in comparison to other hamster tissues examined, and, in situ hybridization analysis of the epididymis revealed that fgl2 mRNA exhibited a region- and principal cell-specific expression pattern. Immunohistochemistry confirmed the association of fgl2 with abnormal spermatozoa in the cauda epididymidis and revealed smaller fgl2-containing particles. Immunoelectron microscopy revealed that fgl2 was distributed throughout an amorphous, "death cocoon," complex assembled onto abnormal spermatozoa and that the smaller fgl2 aggregates consisted of the amorphous material with embedded sperm fragments, organelles, and membrane vesicles. A protocol was developed to isolate an enriched death cocoon fraction. SDS-PAGE and microsequence analyses revealed that the Mr 64,000 fgl2 monomer was assembled into two disulfide-linked oligomers of Mr 260,000 and 280,000. These data demonstrate that the epididymis possesses a specific mechanism to identify and envelop defective spermatozoa with a protein complex containing the fibrinogen-like protein fgl2. We propose that this represents an important protective mechanism not only to shield the viable sperm population from potentially deleterious enzymes released by dying spermatozoa but also to prevent the release of sperm proteins that could initiate an immune response if they escaped the epididymal environment.     

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.     

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.     

Freezing injury: the special case of the sperm cell

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice although no convincing evidence of intracellular ice formation has ever been obtained. We demonstrate that the high intracellular protein content together with the osmotic shrinkage associated with extracellular ice formation leads to intracellular vitrification of spermatozoa during cooling. At rapid rates of cooling the cell damage to spermatozoa is a result of an osmotic imbalance encountered during thawing, not intracellular ice formation. The osmotic imbalance occurs at rapid cooling rates due to a diffusion limited ice crystallisation in the extracellular fluid, i.e. the amount of ice forming during the cooling is less than expected from the equilibrium phase diagram. This explanation allows insights into other aspects of the cryobiology of spermatozoa and it is anticipated that this understanding will lead to specific improved methods of conventional cryopreservation for mammalian spermatozoa. It is also likely that this model will be relevant to the development of novel technologies for sperm preservation including vitrification and freeze drying.     

Relative DNA content of normal and sex-ratio distorting spermatozoa of the mosquito,Aedes aegypti (L.)

Scanning microdensitometry inAedes aegypti (L.) has revealed an extended range of DNA levels among the spermatozoa of sex-ratio distorting males compared with their normal counterparts. Fewer than 1% of spermatozoa contain less than the 1C level of DNA but more than 20% include greater quantities. Since this variation is identified with morphologically abnormal spermatozoa, it is considered to be a direct consequence of preferential X-chromosome breakage during male meiosis and hence of meiotic drive.     

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.     

Decreased expression of heat shock protein A4L in spermatozoa is positively related to poor human sperm quality

Heat shock protein A4L (HSPA4L), which is highly expressed in the testis, is correlated with male fertility. However, the relationship between HSPA4L expression and sperm quality remains unknown. In the present study, a systematic characterization of HSPA4L expression on spermatozoa was performed. HSPA4L is highly expressed in the human testis, characterized by abundant localization in testicular spermatocytes and round spermatids. Compared with the testis from young adults (aged 27–36 years old), downregulated expression of HSPA4L in the testis from elderly adults (aged 78–82 years old) was observed. Immunofluorescence quantification demonstrated the localization of HSPA4L in the middle piece of sperm. Compared with mature spermatozoa, a similar lower intensity and localization percentage of HSPA4L in immature and asthenozoospermic spermatozoa were observed, and the consistently decreased expression of HSPA4L in immature and asthenozoospermic spermatozoa was validated by western blot analysis. Functional analysis revealed a correlation between HSPA4L and sperm motility by Spearman correlation analysis and its involvement in sperm–oocyte penetration by the human sperm–hamster egg penetration test. The current study demonstrates that HSPA4L is a promising marker for the assessment of sperm quality and provides clues for exploring biomarkers for the molecular diagnosis and treatment of male infertility.