获能期间精子蛋白的酪氨酸磷酸化

哺乳动物精了获能是精子与卵子成功受精的前提。蛋白酪氨酸磷酸化对精子获能十分重要。精了获能期蛋白酪氨酸磷酸化程度增高与sAC/cAMP/PKA途径、受体酪氨酸激酶途径和非受体蛋白酪氨酸激酶途径调节有关。获能过程中酪氨酸磷酸化蛋白分布于精子细胞的不同区域,蛋白的酪氨酸磷酸化与精子功能密切相关。     

双酚A暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响

双酚A(BPA)是一种人工合成的雌激素性化合物,广泛存在于环境中,对哺乳动物内分泌有干扰作用,影响雄性生殖系统功能。本研究以新鲜猪精子、17 ℃保存猪精子以及小鼠精子为对象,采用体外培养方法,利用蛋白免疫印迹(WB)和免疫荧光技术,分析不同浓度BPA(0、0.1、1、10、100 μmol·L^-1)暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响及分子机制。结果表明: 低中浓度(0.1、1 μmol·L^-1)BPA暴露对新鲜猪获能精子蛋白酪氨酸磷酸化具有显著促进作用,但在高浓度(10、100 μmol·L^-1)BPA暴露下,猪获能精子蛋白酪氨酸磷酸化呈现降低趋势。BPA暴露下,小鼠获能精子蛋白酪氨酸磷酸化随BPA浓度的增加而增强,并且BPA影响猪和小鼠精子获能相关酪氨酸磷酸化修饰的蛋白种类不同。表明BPA暴露对哺乳动物精子的影响具有物种特异性。免疫荧光结果显示BPA对精子蛋白酪氨酸磷酸化的影响主要发生在鞭毛的中段和主段。     

镉对小鼠精子蛋白酪氨酸磷酸化修饰的影响及EGTA的保护作用

采用体外培养的方法,利用精子活力分析软件(CASA)、蛋白免疫印迹(WB)及免疫荧光技术, 探讨镉(Cd)对小鼠精子活力参数、蛋白酪氨酸磷酸化修饰的影响,并对小鼠精子酪氨酸磷酸化蛋白进行细胞亚组分定位. 结果表明: Cd对小鼠精子活力具有明显抑制作用,且随着Cd浓度的增加抑制作用增强,当Cd浓度达到1.0 μmol·L^-1时, 小鼠精子活力(MOT)显著低于对照组;同时,Cd促进小鼠精子蛋白酪氨酸磷酸化,当浓度≥1.0 μmol·L^-1时,尤其分子量约为55 kDa的蛋白酪氨酸磷酸化程度显著增强,且免疫荧光结果显示主要集中在小鼠精子中段;当用30 μmol·L^-1 乙二醇二乙醚二胺四乙酸(EGTA)和10 μmol·L^-1 Cd同时培养时,55 kDa蛋白并未发生明显的酪氨酸磷酸化修饰,而且小鼠精子活力变化不显著. 表明Cd可能是通过诱导中段55 kDa蛋白发生酪氨酸磷酸化修饰从而抑制精子活力,EGTA能螯合Cd离子并有效防止其毒性作用. 研究证实,Cd诱导精子特异性蛋白酪氨酸磷酸化增强,进而抑制精子活力. EGTA可以用于体外控制Cd进入细胞的阻断剂,为Cd繁殖毒性分子机制的研究提供了研究手段.     

酪氨酸磷酸化在精子获能中的作用

获能是精子发生顶体反应以及与卵子结合之前所必需的生理过程.研究发现在精子获能过程中伴随有蛋白质的磷酸化特别是酪氨酸的磷酸化.主要对酪氨酸磷酸化蛋白在精子获能过程中的作用及其存在的部位进行归纳总结,为进一步阐明精子获能分子机制奠定基础.     

精子获能及其发展

获能是哺乳动物精子受精前必须经历的一个成熟过程,它所需时间因种而导,并具有非均一怀和可逆性,获能涉及精子质膜去获能因子（ＤＦ）的去除和膜组分的重新分布,包括膜脂成分的改变,蛋白迁移,受体暴露,钙通道激活,胞内ｃＡＭＰ产生和蛋白酪氨酸磷酸化等,获能的终点是发生顶体反应（ＡＲ）并呈现超激活运动（ＨＡＭ）受精取决于上述变化,并可用金霉素染色法,ＡＲ,ＨＡＭ和穿卵率定量检测获能。     

精子获能中HCO-3介导的信号转导途径

获能是哺乳动物精子受精前必须经历的一个生理过程。获能涉及精子膜性质的改变、Ca2 通道活化、胞内cAMP增加,以及蛋白酪氨酸磷酸化(PTP)等。实验证明,HCO3-在该过程中起重要作用。本文旨在介绍HCO3-介导的cAMP信号转导途径。     

调控精子获能的相关因素

获能是精子发生顶体反应以及与卵子结合之前所必需的生理过程,目前精子获能的机制得到初步阐明,获能伴随着质膜重组,离子通道的调节,胆固醇的流失以及许多蛋白磷酸化状态的改变.获能同时受到内在和外在因子的调节,其中胆固醇、HCO3-、Ca2+以及蛋白磷酸化在精子获能过程中发挥着重要作用.     

豚鼠精子信号通路的研究

钙调素(CaM)拮抗剂三氟拉嗪(TFP)在10~100 μmol/L浓度范围内对豚鼠精子获能和顶体反应具有极显著的促进作用,且不依赖于外源Ca²⁺的存在.Forskolin,dbcAMP和咖啡因对非同步培养系统获能早期豚鼠精子的顶体反应有显著的促进作用,但对同步培养系统获能早期精子的顶体反应无显著影响.若在同步培养系统获能培养较长时间(15h)使精子获能,则这3种药物对顶体反应均具有显著的促进作用,且不依赖外源Ca²⁺的存在.蛋白激酶C(PKC)激活剂PMA和PDB对获能前期豚鼠精子的顶体反应无显著影响,但对获能精子的顶体反应有显著的促进作用,且这种促进作用也不依赖于外源Ca²⁺的存在.相反,PKC抑制剂stau-rosporine对顶体反应有显著的抑制作用.     

蛋白质酪氨酸磷酸化在抗失巢凋亡的癌细胞中的失调变化

失巢凋亡是细胞与细胞外基质脱离发生的一种特定的凋亡方式 . 癌细胞抗失巢凋亡或失巢生存能力可以使之在转移过程中生存 . 业已发现癌细胞失巢生存与 PI3K-PKB/Akt 、 MAPK 这两条重要信号途径有关,但是 PI3K-PKB/Akt 、 MAPK 通路的上游酪氨酸激酶途径还不甚清楚 . 为此设计了一种基于 SH2-pTyr 特异性结合特性的功能性筛选方法,以期发现癌细胞失巢生存相关的酪氨酸磷酸化蛋白质,为最终明确酪氨酸激酶途径提供有力的实验依据 . 实验发现, MDCK 细胞悬浮培养后失巢凋亡,但癌细胞可以失巢生存 . 与这一现象相一致的是,悬浮培养后, MDCK 细胞中一系列 SH2 结合的酪氨酸磷酸化蛋白质水平急剧下降,而癌细胞中蛋白质酪氨酸磷酸化水平并不呈锚着依赖性 . 细胞悬浮培养后,随着培养时间的延长, MDCK 细胞中 Abl S SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐降低,在 H460 肺癌细胞中经过短暂下降后升高, H1792 肺癌细胞随着培养时间的延长, Abl SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐增加 . Fyn SH2 和 Crk SH2 结合的蛋白质分别为 FAK 和 p130Cas ,后者是重要的失巢生存信号 . 这些结果提示,酪氨酸磷酸化蛋白质可能赋予肺癌细胞失巢生存能力 . 结果也表明,功能性 SH2 筛查方法可以有效地发现肿瘤细胞中失巢生存相关的酪氨酸磷酸化蛋白质 .     

哺乳动物精子获能过程中信号通路研究进展

哺乳动物精子在雌性生殖道内及体外获能培养过程中伴随着胆固醇外流、质膜重组、离子通道调节及获能相关蛋白磷酸化状态改变等相关生理调节过程,其中信号通路及相应信号分子对精子获能及功能修饰起到重要调节作用,成为精子细胞超激活运动及完成受精作用的关键环节。根据近年来的研究报道,对哺乳动物精子获能过程中已知的信号通路、信号分子及调节因子、离子通道、存在的问题及未来研究主要方向进行综述,为精子体外培养及辅助生殖等提供理论参考。     

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.     

Capacitation-associated protein tyrosine phosphorylation starts early in buffalo (Bubalus bubalis) spermatozoa as compared to cattle

A comparative study was conducted on protein tyrosine phosphorylation events of capacitating sperm of two ruminant species, cattle and buffalo. Ejaculated cattle and buffalo bull spermatozoa were suspended separately in sp-TALP medium at 50 × 10⁶ mL⁻¹ and incubated at 38.5 °C with 5% CO₂ in air in the absence or presence of heparin for a period of 6 h. The extent of sperm capacitation after various periods of incubations was assessed by lysophosphatidyl choline-induced acrosome reaction followed by a triple-staining technique and capacitation-associated tyrosine-phosphorylated proteins were detected by immunoblotting technique using a monoclonal antiphosphotyrosine antibody. In the same media, over a time-period, a significant increase in capacitation percentage was observed even in control group of buffalo spermatozoa as compared to a non-significant increase in that of cattle sperm. In both cattle and buffalo spermatozoa, at 6 h, four proteins of molecular weight 49, 45, 32, and 20 kDa (designated as p49, p45, p32, and p20) were tyrosine phosphorylated. However, in buffalo, two additional proteins of 38 and 30 kDa were also tyrosine phosphorylated. In a time-course study, p20 appeared as early as at 0 h in capacitated buffalo spermatozoa as compared to 4 h in cattle. Further, in heparin-treated buffalo spermatozoa, with a time-dependent increase in tyrosine phosphorylation of some proteins, there was time-dependent dephosphorylation of some other proteins that was never seen in heparin-treated cattle sperm. Thus, the present findings revealed that though buffalo sperm takes more time than cattle for capacitation but its associated protein tyrosine phosphorylation event starts very early as compared to cattle.     

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP‐dependent hyperactivation in the spermatozoa of Japanese Black bulls

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010. © 2010 Wiley‐Liss, Inc.     

Kinetics of protein tyrosine phosphorylation in sperm selected by binding to homologous and heterologous oviductal explants: how specific is the regulation by the oviduct?

Essential steps of the capacitation process take place in the oviductal isthmus. A crucial step in the process of capacitation is the phosphorylation of membrane proteins. The aims of this work were (1) to study the effect of dog sperm binding to oviductal epithelium on tyrosine phosphorylation and (2) to investigate the specificity of regulation of molecular changes by the oviduct of different species by comparing the numbers of canine sperm bound to heterologous (porcine) and homologous epithelium, and the kinetics of tyrosine phosphorylation. Semen was collected from four healthy dogs and washed through a Percoll gradient. Explants, small pieces of epithelium, were cut from porcine and estrous bitch oviducts. During 6 h of coincubation in Tyrode medium, the numbers of bound sperm were counted by microvideographic observation, and the state of tyrosine phosphorylation was determined immunocytochemically after 3, 30, 90, 180 and 360 min. Canine sperm bound in similar numbers to homologous and heterologous explants. Increasing tyrosine phosphorylation of tail proteins and subsequent phosphorylation of sperm head proteins were observed. Binding occurred mainly in sperm with non-phosphorylated heads (approximately 2% phosphorylated), while higher proportions of head-phosphorylated cells were found in unbound populations (approximately 40-60%;P<0.05). The head phosphorylation progressed significantly during incubation in unbound spermatozoa (P<0.05), while it was suppressed in bound suspensions. The rate of tyrosine phosphorylation of sperm tail proteins was higher in cells bound to explants than in unbound cells or in those incubated in control medium. There were no significant differences with respect to the kinetics of tyrosine phosphorylation between the two coincubation systems. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. Tyrosine phosphorylation of sperm head proteins and capacitation are delayed in spermatozoa in close contact with oviductal epithelium. This mechanism appears to be species-independent, as sperm bound similarly to pig and dog oviduct explants, and similar phosphorylation kinetics were observed in both types of tissue.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation.

To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.     

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO₂. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.     

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.