共查询到20条相似文献,搜索用时 15 毫秒
1.
Eun Jeong Lee Un-Jung Yun Kyung Hee Koo Jee Young Sung Jaegal Shim Sang-Kyu Ye Kyeong-Man Hong Yong-Nyun Kim 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2014,1841(1):190-203
Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. 相似文献
2.
Bellyei S Szigeti A Boronkai A Pozsgai E Gomori E Melegh B Janaky T Bognar Z Hocsak E Sumegi B Gallyas F 《Apoptosis : an international journal on programmed cell death》2007,12(1):97-112
AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product
is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol
induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2
protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition
of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2
over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera
toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase—Akt pathway by LY-294002 or wortmannin significantly
decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death
via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation
of PI-3-kinase—Akt cytoprotective pathway. 相似文献
3.
Subcellular fractionation is central to a range of cell biological, biochemical and proteomic studies. Purification of nuclear-enriched fractions is critical for studies on nuclear structure and function. Here we show that detergent-based nuclear isolation methods cause the redistribution of proteins associated with plasma membrane lipid rafts into nuclear fractions. The glycosyl-phosphatidylinositol (GPI)-anchored prion protein (PrP(C)) and a GPI-anchored construct of angiotensin converting enzyme (GPI-ACE), as well as the lipid raft markers flotillin-1 and -2, were present in the nuclear fractions derived using three different subcellular fractionation protocols. Incubation of intact cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves GPI-anchored proteins from the cell surface, significantly reduced the amount of PrP(C) and GPI-ACE in the nuclear fraction. Buoyant sucrose density gradient centrifugation in the presence of Triton X-100 of the nuclear fraction resulted in a significant proportion of the GPI-anchored proteins being recovered in the low density lipid raft fractions. These data indicate that the nuclear fraction isolated using such subcellular fractionation protocols is contaminated with components of plasma membrane lipid rafts and raises questions as to the integrity of the nuclear fraction isolated by such protocols for use in detailed cell biological studies and proteomics analysis. 相似文献
4.
Yulia Evdokimovskaya Yuri Skarga Veronika Vrublevskaya Oleg Morenkov 《Cell biology international》2010,34(10):985-990
The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role. 相似文献
5.
Experimental evidence indicates that the mammalian cell membrane is compartmentalized. A structural feature that supports membrane segmentation implicates assemblies of selected lipids broadly referred to as lipid rafts. In T-lymphocytes, lipid rafts are implicated in signalling from the T-cell antigen receptor (TCR) and in localization and function of proteins residing proximal to the receptor. This review summarizes the current literature that deals with lipid raft involvement in T-cell activation and places particular emphasis in recent studies investigating lipid rafts in autoimmunity. The potential of lipid rafts as targets for the development of a new class of immune-modulating compounds is discussed. 相似文献
6.
Ramírez-Sánchez I Mendoza-Lorenzo P Zentella-Dehesa A Méndez-Bolaina E Lara-Padilla E Ceballos-Reyes G Canto P Palma-Flores C Coral-Vázquez RM 《Biochimie》2012,94(9):1884-1890
Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity. 相似文献
7.
Lipid rafts are membrane microdomains of unique lipid composition that segregate proteins with poorly understood consequences for membrane organization. Identification of raft associated proteins could therefore provide novel insight into raft-dependent functions. Monocytes process antigens for presentation to T cells by ingesting pathogens into calcium-dependent plasma membrane invaginations called "phagosomes" which develop by sequential fusion with the endoplasmic reticulum, early and late endosomes. We investigated the protein composition of Triton X-100 insoluble low density membranes of the monocyte cell-line THP-1 by matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry. The ganglioside GM1 colocalized on the plasma membrane with the raft markers flotillin 1 and 2, which were enriched in low buoyant density fractions containing 52 identifiable proteins, 28 of which have not been reported in rafts, and nine of which are associated with the endoplasmic reticulum (ER). Remarkably, 27 of the 52 proteins are components of phagosomes, including the ER protein calnexin which we demonstrate is phosphorylated on serine 562, a switch controlling calcium homeostasis. The presence of the early and late endosome trafficking proteins Rab-1, and Rab-7 together with the late endosome protein LIMPII, indicate lipid rafts are present throughout endosome maturation. Identification of vacuolar ATP synthase, and synaptosomal-associated protein-23, proteins implicated in membrane fusion, together with the cytoskeletal proteins actin, alpha-actinin, and vimentin, and Rac 1, 2, and 3, regulators of cytoskeletal assembly, indicate monocyte lipid rafts contain the machinery to direct vesicular fusion and actin based vesicular migration throughout phagosome development. 相似文献
8.
Ki‐Bum Kim Bong‐Woo Kim Hyo‐Jung Choo Young‐Chan Kwon Byung‐Yoon Ahn Jong‐Soon Choi Jae‐Seon Lee Young‐Gyu Ko Dr. 《Proteomics》2009,9(9):2373-2382
Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent‐resistant lipid raft proteins from 3T3‐L1 preadipocytes and adipocytes by 2‐DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte‐specific proteins was globular C1q receptor (gC1qR), an acidic 32 kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin‐induced activation of insulin receptor, insulin receptor substrate‐1 (IRS‐1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling. 相似文献
9.
10.
Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout. 相似文献
11.
Ishii H Mori T Shiratsuchi A Nakai Y Shimada Y Ohno-Iwashita Y Nakanishi Y 《Biochemical and biophysical research communications》2005,327(1):94-99
Externalization of phosphatidylserine (PS) takes place in apoptotic cells as well as in viable cells under certain circumstances. Recent studies showed that externalized PS is localized at the lipid raft in viable activated immune cells. We found that lipid rafts and PS existed in a mutually exclusive manner in apoptotic cells. The number of PS-exposing apoptotic cells decreased when lipid rafts were disrupted. BCtheta;, which binds selectively to cholesterol in a cholesterol-rich region, did not effectively recognize lipid rafts of apoptotic cells. Lipid rafts rich in GM1 were successfully prepared from apoptotic cells, but the lipid raft protein LAT was not enriched in the preparation. Furthermore, the amount of PS and phosphatidylethanolamine but not of cholesterol in lipid rafts appeared to change after induction of apoptosis. These results suggest that lipid rafts are structurally modified during apoptosis and, despite being localized differently from PS, are involved in the externalization of PS. 相似文献
12.
Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release 总被引:18,自引:0,他引:18
Broquet AH Thomas G Masliah J Trugnan G Bachelet M 《The Journal of biological chemistry》2003,278(24):21601-21606
Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70. 相似文献
13.
Kim KB Lee JW Lee CS Kim BW Choo HJ Jung SY Chi SG Yoon YS Yoon G Ko YG 《Proteomics》2006,6(8):2444-2453
In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidation-reduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex. 相似文献
14.
《Cryobiology》2018
Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile. 相似文献
15.
In vitro fertilization with cryopreserved inbred mouse sperm 总被引:8,自引:0,他引:8
Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation. 相似文献
16.
Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively). 相似文献
17.
《Cryobiology》2019
As the largest proportion of male infertility population, asthenozoospermia patients often resort to sperm cryopreservation to preserve fertility as well as to enrich motile sperm for assisted reproductive techniques (ART), although it may cause some cryodamage during the freezing–thawing process. The objective of this study was to investigate whether mitochondrial antioxidant Mito-Tempo was effective in preventing cryodamage of asthenozoospermic spermatozoa. Asthenozoospermic semen samples were collected and cryopreserved in media supplemented with different concentrations (0.0, 1.0, 10 and 100 μM) of Mito-Tempo. We measured sperm motility, viability, membrane integrity, DNA fragmentation, mitochondrial membrane potential, oxidation product, and antioxidant enzymes activities. Supplementation of the cryopreservation media with Mito-Tempo (10 and 100 μM) induced a significant improvement in sperm viability, motility, membrane integrity, mitochondrial membrane potential and chromatin integrity (P < 0.05). Significant enhancement of antioxidant enzymes activities accompanied by the decreased formation of oxidation products (ROS and MDA) was also observed in groups supplemented with Mito-Tempo (10 and 100 μM). It is concluded that mitochondria targeted antioxidant Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients after cryopreservation. 相似文献
18.
Apically expressed human MUC1 is known to become endocytosed and either to re‐enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi‐vesicular bodies and the release of exosomes. By using recombinant fusion‐tagged MUC1 as a bait protein we followed an anti‐myc affinity‐based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF‐7 breast cancer cells. MUC1+ lipid rafts were not only found to contain genuine raft proteins (flotillin‐1, prohibitin, G protein, annexin A2), but also raft‐associated proteins linking these to the cytoskeleton (ezrin/villin‐2, profilin II, HSP27, γ‐actin, β‐actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin‐dependent pathways and export via exosomes. 相似文献
19.
Relatively large number of post-synaptic density (PSD) proteins, including Ca2+ /calmodulin-dependent protein kinase II (CaMKII), have the potential to associate with lipid rafts. We in this study demonstrate that the CaMKIIα clusters induced by ionomycin in human embryonic kidney 293 cells, as well as unclustered CaMKIIα (Du F., Saitoh F., Tian Q. B., Miyazawa S., Endo S. and Suzuki T, 2006, Biochem. Biophys. Res. Commun 347, 814–820), were associated with lipid rafts. The CaMKIIα clusters associated with lipid raft fraction became resistant to treatment with methyl-β-cyclodextrin and subsequent cold Triton X-100, which suggests the stabilization of CaMKIIα cluster-associated lipid rafts. Next, we found that PSD-95, which is also a component of lipid raft fraction and does not interact directly with CaMKII, was trapped by stable CaMKIIα cluster-containing structure. Association of PSD-95 with CaMKIIα clusters was also observed in cultured neuronal cells. These results suggest the CaMKIIα clusters associated with the lipid rafts in the cytoplasmic region play a role in the assembly and stabilization of certain PSD proteins that have the potential to associate with lipid rafts. 相似文献
20.
A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa 总被引:7,自引:0,他引:7
After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins. 相似文献