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1.
本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础。利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定。结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生酪氨酸磷酸化修饰密切相关;获能精子中126、108、79ku的高分子量蛋白磷酸化程度明显高于未获能精子;分子质量约为25、47、50ku的膜蛋白及47ku胞浆蛋白发生酪氨酸磷酸化,其中25、47ku的膜蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05);分子量约为23、37、42～50ku的核蛋白发生酪氨酸磷酸化,获能精子中23ku的核蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05)。结果提示,猪精子细胞不同亚组分中,发生酪氨酸磷酸化修饰的蛋白以膜蛋白及核蛋白为主,同时有少量的胞浆蛋白。  相似文献   

2.
Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4‐bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris‐based egg yolk extender supplemented with additives like taurine (50 mm ) or trehalose (100 mm ) or 4‐bromophenacyl bromide (200 μm ) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen‐thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris‐based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho‐proteins using SDS–PAGE followed by immunoblotting. Monoclonal anti‐phosphotyrosine antibody (Clone pT‐154) was used as primary antibody followed by treatment with HRP‐conjugated secondary antibody. Signals were detected on X‐ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post‐thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.  相似文献   

3.
Several recent studies have indicated the important roles of Ser/Thr protein phosphatase1γ (PP1γ) in regulating the motility and capacitation of mammalian spermatozoa. Here, we report the presence and distribution of PP1γ protein in freshly ejaculated, in vitro capacitated and cryopreserved buffalo spermatozoa. The presence of PP1γ and its distribution were assessed by Western blotting and indirect immunofluorescence techniques, whereas the isoforms of PP1γ and their tyrosine phosphorylation status were identified by using 2D electrophoresis. The number of isoforms and the status of tyrosine phosphorylation of PP1γ were increased in capacitated spermatozoa when compared with freshly ejaculated spermatozoa. Differential pattern of expression and tyrosine phosphorylation of PP1γ were observed in cryopreserved spermatozoa, wherein some isoforms were degraded and some were tyrosine phosphorylated. In addition, immunofluorescence technique revealed that PP1γ was localized to principle, mid‐piece, post‐acrosomal and equatorial regions of buffalo spermatozoa. Differential distribution of tyrosine‐phosphorylated proteins were observed in fresh, capacitated and cryopreserved spermatozoa. The tyrosine phosphorylation of several proteins (20, 37, 38, 52, 60, 79 and 100 kDa) were increased when sperm cells were incubated with PP1γ inhibitor, okadaic acid. Together, our results suggest that buffalo spermatozoa express different isoforms of PP1γ protein. The protein expression and tyrosine phosphorylation of PP1γ were increased during capacitation. Furthermore, the differential pattern of expression and tyrosine phosphorylation of PP1γ were observed in cryopreserved spermatozoa. In addition, the inhibition of PP1γ protein increases protein tyrosine phosphorylation in capacitation.  相似文献   

4.
Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.  相似文献   

5.
To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.  相似文献   

6.
In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time‐dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)‐induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H‐89, PD9809 and GF‐109) and enhancer (dbcAMP, H2O2 and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F‐actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.  相似文献   

7.
To acquire the fertilizing competence, spermatozoa must undergo a cascade of physiological and biochemical changes collectively defined as capacitation. Compelling evidence signifies that the global increase in protein tyrosine phosphorylation is the driving factor for capacitation. In our laboratory, we previously demonstrated that nitric oxide (NO) induces capacitation in buffalo sperm and is associated with an increase in protein tyrosine phosphorylation. The aim of the present study is to identify the proteins undergo tyrosine phosphorylation during NO induced buffalo sperm capacitation using 2-D immunoblotting and mass spectrometry. The percentage of progressively motile and capacitated sperm was more in presence of l-arginine. Along with known tyrosine phosphoproteins like ATP synthase subunit beta, pyruvate dehydrogenase E1 component subunit beta, GST mu 3, F-actin capping protein subunit beta 2, GPD2 and VDAC2, interestingly novel tyrosine phosphoprotein substrates such as actin, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, and glutamine synthetase were also identified which might be specific to the NO induced signaling and also emphasizes the species specificity with respect to tyrosine phosphorylation of proteins during capacitation. In conclusion, this study forms an essential step in delineating the proteins undergo tyrosine phosphorylation in response to NO induced signaling pathways during capacitation of buffalo sperm.  相似文献   

8.
The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane‐associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2′,5′‐dideoxyadenosine was used as a membrane‐associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin‐capacitated spermatozoa was significantly higher than in hyaluronic acid‐treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane‐associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane‐associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin‐capacitated spermatozoa.  相似文献   

9.
为探讨不同浓度Ca2+对马鹿精子体外获能的影响,本研究以塔里木马鹿冻融精子为试验材料,将精子分别悬浮于含不同浓度Ca2+（0、1.1、2.2、3.5、5.0 mmol/L）的台氏液（sp-TALP液）中,在培养0、2、4 h时,采用金霉素（CTC）染色法评价精子获能状态,采用SDS-PAGE分离精子膜蛋白,进行免疫印迹分析,检测酪氨酸磷酸化蛋白的表达水平。结果表明,Ca2+浓度为1.1、2.2 mmol/L有利于精子活力的维持（P<0.05）,精子获能率极显著高于对照组和高浓度组（3.5、5.0 mmol/L;P<0.01）,精子存活时间最长（P<0.01）,但高浓度Ca2+（5.0 mmol/L）对精子活力具有显著抑制作用（P<0.05）,精子获能率极显著低于低浓度组（P<0.01）,精子存活时间最短（P<0.01）;另外,随着培养时间的推移精子发生酪氨酸磷酸化蛋白的表达水平有所不同,培养2、4 h时,1.1 mmol/L组精子蛋白磷酸化水平极显著高于其他各组（P<0.01）,高浓度Ca2+（3.5、5.0 mmol/L）组酪氨酸磷酸化蛋白的表达水平极显著下降（P<0.01）。结果表明,塔里木马鹿精子体外获能所需的适宜Ca2+浓度为1.1 mmol/L,且获能过程中Ca2+的存在是必要的。  相似文献   

10.
陈璇  金一 《中国畜牧兽医》2018,45(9):2486-2491
在精子细胞发生顶体反应并使卵母细胞受精之前,获能是一个重要的生理先决条件。获能是精子在雌性生殖道中进一步成熟的复杂现象,其赋予精子以增强活性的能力,使精子能够与卵母细胞透明带（ZP）相互作用,进行顶体反应并与卵母细胞质膜融合,进而完成受精过程。然而精子获能的分子机制十分复杂,目前还未完全明确,但获能后的精子会有诸多结构及生化方面的变化,如蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化。蛋白质通过磷酸化或去磷酸化调节精子获能和顶体反应等一些重要的现象,这是精子到达、结合、穿透和融合卵母细胞所必需的过程。因此蛋白磷酸化是获能的一个非常重要的过程,尤其是在酪氨酸残基处的磷酸化是获能过程中发生的最重要的事件之一,且酪氨酸磷酸化可能是细胞中信号转导途径的主要甚至是唯一的指标。作者主要针对蛋白磷酸化、精子中酪氨酸磷酸化的发现、作用和定位,以及影响获能过程中酪氨酸磷酸化的几个因素进行阐述。  相似文献   

11.
Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.  相似文献   

12.
The functional and structural integrity of sperm membrane are crucial for the viability of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing sperm membrane measures only its structural integrity. The hypoosmotic swelling test (HOS) originally developed for human sperm ( Jeyendran et al. 1984 ) has been also applied to several species of domestic animals (bull, pig, horse, dog). The test enables to evaluate the functional status of the sperm membrane. The principle of HOS is based on water transport across the sperm tail membrane under hypoosmotic conditions. It has previously been used to assess the semen quality ( Revell and Mrode 1994 ), to analyse fertilizing capacity ( Rota et al. 2000 ; Perez‐Llano et al. 2001 ) and also to detect viable, immotile cells for ICSI (Intra‐cytoplasmic sperm injection) in human ( Zeyneloglu et al. 2000 ). There are two procedures commonly used for sperm capacitation in the pig‐sperm washing and incubation before insemination ( Nagai 1994 ). Capacitation involves several changes like removing molecules coating the sperm head membrane, changes in membrane fluidity and intracellular ion concentration ( Green and Watson 2001 ). Thus the membrane integrity as well as functionality may be affected as shown by Harrison (1996) . The aim of the present study was to analyse changes in sperm membrane integrity after in vitro capacitation by use of the HOS test.  相似文献   

13.
This study was designed to test the hypothesis that sperm‐bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)‐negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA‐positive ejaculates were cryopreserved from each bull post‐immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm2; 62.5–251.1 sperm/mm2), phase‐contrast microscopy (160.5 sperm/mm2; 56.8–397.4 mm2) or their combination (116.4 sperm/mm2; 56.8–249.6 sperm/mm2) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA‐negative and ASA‐positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA‐positive (114.9; 0 to 201.8 sperm/0.1 mm2) than ASA‐negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm2) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG‐bound (p = 0.013) but not IgA‐bound spermatozoa. In conclusion, sperm‐bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.  相似文献   

14.
Heat shock proteins (Hsp)-60, -70 and -90 are important testis chaperones that fulfil several functions during sperm cell maturation. In post-meiotic cells, their expression may change or may be undetectable and in some species it may be evident in mature spermatozoa. The aims of this study were to verify whether Hsp60, -70 and -90 are present in the sperm, and to compare their localization in boar, stallion, cat and dog spermatozoa by immunofluorescence. Hsp-60 immunoreactivity was detected in sperm midpiece in all the species examined. In stallion sperm, Hsp70 signal was localized in the sub-equatorial band, whereas immunoreactivity was evident on the neck of dog spermatozoa and on both neck and sub-equatorial region of cat spermatozoa. In agreement with our previous observations, a triangular fluorescent signal in the equatorial segment of fresh boar sperm was detected. Hsp90 immunoreactivity was present in different portions of sperm tail: in the midpiece of both boar and cat spermatozoa and in the neck and throughout the tail in dog and stallion spermatozoa, respectively. When capacitation and acrosome reaction were induced in boar, stallion and dog spermatozoa, no changes in both Hsp60 and -90 were recorded by either Western blot or immunofluorescence. After induction of acrosome reaction, a Hsp70 redistribution in boar spermatozoa and an increased percentage of stallion spermatozoa showing the post-acrosomal signal were observed although no changes were recorded by Western blot; in dog spermatozoa, no changes in Hsp70 were found by Western blot and immunofluorescence after capacitation and acrosome reaction.  相似文献   

15.
哺乳动物精子获能的分子机制   总被引：2，自引：0，他引：2  
精子获能是精子能够与卵母细胞发生顶体反应和受精的一个重要生理前提。精子获能的分子机制相当复杂,许多报道表明精子获能受到多种细胞信号途径的调控。尽管目前尚未完全明确,但是许多研究表明获能精子发生许多结构和生化变化,包括蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化,这些变化都有助于精子获能的发生。Ca2 和HCO3-通过对cAMP的调控有助于获能完成,葡萄糖、孕酮和肝素作为获能液的重要添加物,通过不同途径促发精子获能。文章从这些方面对获能做一综述,在此基础上提出以后的研究方向。  相似文献   

16.
Summary

The capacitation process of sperm cells involves complex changes in the composition and orientation of molecules at the surface of the sperm cell. Here we focus on the lipid architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid organization in living cells and extremely rapid lipid movements were observed. The orientation of lipids in the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for temperature and also changed upon binding of sperm cells to the zona pellucida. The changes in membrane properties coincided with an activation of protein kinases resulting in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these results may provide a physiological basis for new assays, able to discriminate between functional and non‐physiological sperm cells.  相似文献   

17.
Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport and the different species. The localization of GLUTs after capacitation and acrosome reaction highlighted the possible changes in their distribution because of the different functional moment. Only in dog spermatozoa changes in GLUTs distribution were demonstrated; these changes could be related to the different metabolic needs and modifications occurring in the sperm cell.  相似文献   

18.
Understanding the biochemical processes associated with ovum fertilization and knowledge about the structure and function of individual substances participating in these processes is crucial for the development of biotechnological methods to improve reproduction of animals and humans. Among many components of seminal plasma, proteins and peptides play a specific role in regulation of the fertilization process, particularly through their ability to bind various types of ligands such as polysaccharides, lipids and ions. Heparin-binding proteins regulate capacitation and acrosome reaction processes. Affinity of plasma proteins to mannans of the fallopian tube epithelium facilitates formation of spermatozoa reservoirs in the female reproductive tract. Ability to bind phosphorylcholine is one of the conditions for the coating of the seminal plasma proteins on the sperm membrane and also determines the formation of oligomeric forms of certain proteins. Zinc binding by seminal plasma proteins regulates sperm chromatin condensation state. It also affects motility of these cells and acrosome reaction. The interspecies analysis indicates significant structural and functional similarities, especially for the proteins with low molecular weight. Fertility associated proteins (FAPs) have been determined in the bull, stallion, boar, ram and dog. The contents of these proteins correlate with the indicators of the fertilizing abilities of sperm. In humans, several seminal plasma proteins were found which serve as diagnostic markers of spermatogenesis, seminiferous epithelium state, and azoospermia. To determine the semen ability for preservation, measurement of some seminal plasma protein content may also be used. Addition of specific plasma proteins to a spermatozoa solution undergoing the process of preservation may be used to retain the features of the cells responsible for efficient fertilization.  相似文献   

19.
The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.  相似文献   

20.
In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.  相似文献   

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