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Acrosome Reaction (acrosome + reaction)
Selected AbstractsIn vitro Effect of Zearalenone and , -Zearalenol on Boar Sperm Characteristics and Acrosome ReactionREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2006IA Tsakmakidis Contents This study was conducted to determine the in vitro effects of three different concentrations (125, 187.5 and 250 ,m in diluted semen) of zearalenone (zen) and , -zearalenol (, -zen) on boar sperm. Semen parameters such as motility, viability and spontaneous acrosome reaction were evaluated. From the results it was shown that both zen and , -zen affected the sperm characteristics significantly (p < 0.05), except for , -zen at the low concentration which did not decrease the percentage of live reacted spermatozoa significantly. In conclusion, zen and , -zen are directly toxic when they affect boar semen in vitro and consequently decrease the fertilization ability of the sperm. The higher the concentration of mycotoxin tested, the greater the decline of sperm parameters noticed. The influence of mycotoxins was found to be time- and dose-dependent. [source] In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without GlucoseREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2004JL Albarracín Contents 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°C in a 5% CO2 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. [source] GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tatsuru Togo Abstract The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465,471, 2004. © 2004 Wiley-Liss, Inc. [source] Acrosome reaction in Chlamydia-positive and negative patientsANDROLOGIA, Issue 5 2003A. Jungwirth Summary. Chlamydia trachomatis infections might have a detrimental effect on various sperm functions. Data concerning the effect of C. trachomatis on the capacitation activity of sperms are lacking. The study was undertaken to evaluate whether chlamydial infection influences acromsome reaction (AR). Three groups of men were investigated for ARs - Chlamydia negative (n = 46) and positive (n = 30) patients, and healthy men (n = 53) undergoing vasectomy. The fluorescence technique for the evaluation of AR was applied. The normal range for the induction of AR was assumed ,AR > 12.5% for this technique. Seminal plasma was examined for IgA antibodies against C. trachomatis. There was a significant difference in AR between healthy volunteers, Chlamydia-negative and Chlamydia- positive patients. ,ARs were 15.8 ± 1.6% in healthy volunteers versus 12.15 ± 2.4% in Chlamydia- negative and 9.08 ± 1.8% in Chlamydia- positive patients, respectively (P <0.05). Significant elevated titres of C. trachomatis- specific IgA in seminal plasma showed a negative correlation with the AR of spermatozoa. AR seems to be a valuable marker, especially in couples with idiopathic infertility. [source] Acrosome reaction: methods for detection and clinical significanceANDROLOGIA, Issue 6 2000T. Zeginiadou The present article reviews the methods for detection and the clinical significance of the acrosome reaction. The best method for the detection of the acrosome reaction is electron microscopy, but it is expensive and labour-intensive and therefore cannot be used routinely. The most widely used methods utilize optical microscopy where spermatozoa are stained for the visualization of their acrosomal status. Different dyes are used for this purpose as well as lectins and antibodies labelled with fluorescence. The acrosome reaction following ionophore challenge (ARIC) can separate spermatozoa that undergo spontaneous acrosome reaction from those that are induced, making the result of the inducible acrosome reaction more meaningful. Many different stimuli have been used for the induction of the acrosome reaction with different results. The ARIC test can provide information on the fertilizing capability of a sample. The ARIC test was also used to evaluate patients undergoing in vitro fertilization since a low percentage of induced acrosome reaction was found to be associated with lower rates of fertilization. The cut-off value that could be used to identify infertile patients is under debate. Therapeutic decisions can also be made on the basis of the value of the ARIC test. [source] Na+/Ca2+ exchanger modulates the flagellar wave pattern for the regulation of motility activation and chemotaxis in the ascidian spermatozoaCYTOSKELETON, Issue 10 2006Kogiku Shiba Abstract Ion channels and ion exchangers are known to be important participants in various aspects of sperm physiology, e.g. motility activation, chemotaxis, the maintenance of motility and the acrosome reaction in the sperm. We report here on a role of the K+ -independent Na+/Ca2+ exchanger (NCX) on ascidian sperm. Reverse-transcriptase PCR reveals that the NCX is expressed in the testis while immunoblotting and immunolocalization demonstrate that the NCX exists on the sperm in the ascidian Ciona savignyi and C. intestinalis. A potent blocker of the NCX, KB-R7943 was found to block sperm-activating and -attracting factor (SAAF)-induced motility activation, sperm motility and sperm chemotaxis. We further analyzed the effects of this blocker on motility parameters such as the flagellar waveform, curvature, beat frequency, amplitude and wavelength of the sperm flagella. Inhibition of the NCX caused two distinct effects: a low concentration of KB-R7943 induced symmetric bending, whereas a high concentration of KB-R7943 resulted in asymmetric flagellar bending. These findings suggest that the NCX plays important roles in the regulation of SAAF-induced sperm chemotaxis, motility activation and motility maintenance in the ascidian. This study provides new information toward an understanding of Ca2+ transport systems in sperm motility and chemotaxis. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulationCYTOSKELETON, Issue 2 2006Zoltán Krasznai Abstract A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing ,1 ,M Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3,,4, -dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 ,M, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50 = 2.44 ,M). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role. Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source] Reduced fertility of mouse epididymal sperm lacking Prss21/Tesp5 is rescued by sperm exposure to uterine microenvironmentGENES TO CELLS, Issue 10 2008Misuzu Yamashita Although the acrosome reaction and subsequent penetration of sperm through the egg zona pellucida (ZP) are essential for mammalian fertilization, the molecular mechanism is still controversial. We have previously identified serine protease Tesp5 identical to Prss21 on the mouse sperm surface as a candidate enzyme involved in sperm penetration through the ZP. Here we show that despite normal fertility of male mice lacking Prss21/Tesp5, the epididymal sperm penetrates the ZP only at a very low rate in vitro, presumably owing to the reduced ability to bind the ZP and undergo the ZP-induced acrosome reaction. The ability of Prss21-null sperm to fuse with the egg in vitro was also impaired severely. Intriguingly, the reduced fertility of Prss21-null epididymal sperm was rescued by exposure of the sperm to the uterine microenvironment and by in vitro treatment of the sperm with uterine fluids. These data suggest the physiological importance of sperm transport through the uterus. [source] Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoaINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 6 2009H. W. R. Li Summary Leptin is a polypeptide hormone with important roles in reproduction. It has been detected in human seminal plasma as well as on human ejaculated spermatozoa. This study aimed at studying the possible role of leptin in regulating human sperm functions. Immunofluorescent staining was used to study the expression of leptin and its receptor. The correlation between the concentration of leptin and soluble leptin receptor (ObRs) in seminal plasma as measured by enzyme-linked immunosorbant assay and sperm motility parameters measured by computer-assisted sperm analyais (CASA) was determined. The effects of recombinant leptin on human sperm motility, capacitation and acrosome reaction as measured by chlortetracycline staing were also studied. Leptin immunoreactivity was demonstrated at the equatorial and neck regions of human spermatozoa, whereas that of ObRs was shown up on the tail. After Percoll separation, spermatozoa with high density had more intense leptin immunoreactivity compared with those with low density. No significant correlation was found between seminal plasma concentration of leptin/ObRs and sperm motility parameters. After incubation with recombinant human leptin for either 3 h or overnight, there was no change in all the CASA motility parameters determined and percentages of capacitated and acrosome-reacted spermatozoa. We concluded that leptin does not have a significant effect on motility and capacitation/acrosome reaction in human ejaculated mature spermatozoa. Its role in male reproduction is yet to be determined. [source] Effects of tumour necrosis factor alpha and interleukin-6 on progesterone and calcium ionophore-induced acrosome reactionINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2009F. Lampiao Summary For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-,) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-, and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-, and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR (p < 0.05) in a dose-dependent manner. TNF-, showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-, and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively. [source] Dynamic expression of the prion-like protein Doppel in ovine testicular tissueINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2006Arild Espenes Summary Transgenic knockout of the gene encoding the prion-like protein Doppel (Dpl) leads to male infertility in mice. The precise role of Dpl in male fertility is still unclear, but sperm from Dpl-deficient mice appear to be unable to undergo the normal acrosome reaction that is necessary to penetrate the zona pellucida of the ovum. We have investigated the expression pattern and some biochemical properties of Dpl in sheep testicular tissue and spermatozoa. Neither the Dpl protein nor its mRNA was detected in pre-pubertal sheep testis. This was in contrast to the findings in adult rams where both Dpl mRNA and protein were present. The molecular mass and glycosylation pattern of sheep Dpl were similar to that of mice Dpl. The Dpl protein was detected in the seminiferous epithelium during the two final (7 and 8) and the two initial (1 and 2) stages of the spermatogenic cycle in a characteristic pattern. In stage 8, an intense brim of granular Dpl-immunoreactivity associated with maturation phase spermatids was observed, while after the release of spermatozoa in stages 1 and 2, the Dpl-staining was disseminated more diffusely in the epithelium, reaching the basal lamina. From stage 3 to stage 6, Dpl-immunoreactivity could not be detected, indicating that the Dpl protein had disappeared between stages 2 and 3. Dpl was not detected on ejaculated spermatozoa. These patterns of staining indicate that Dpl is enriched in residual bodies, which are phagocytosed and destroyed by Sertoli cells after release of sperm into the lumen of the seminiferous tubule. [source] Sperm function tests and fertilityINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2006R. J. Aitken Summary Traditionally, the diagnosis of male infertility has depended upon a descriptive evaluation of human semen with emphasis on the number of spermatozoa that are present in the ejaculate, their motility and their morphology. The fundamental tenet underlying this approach is that male fertility can be defined by reference to a threshold concentration of motile, morphologically normal spermatozoa that must be exceeded in order to achieve conception. Many independent studies have demonstrated that this fundamental concept is flawed and, in reality, it is not so much the absolute number of spermatozoa that determines fertility, but their functional competence. In the light of this conclusion, a range of in vitro tests have been developed to monitor various aspects of sperm function including their potential for movement, cervical mucus penetration, capacitation, zona recognition, the acrosome reaction and sperm,oocyte fusion. Such functional assays have been found to predict the fertilizing capacity of human spermatozoa in vitro and in vivo with some accuracy. Recent developments in this field include the introduction of tests to assess the degree to which human spermatozoa have suffered oxidative stress as well as the integrity of their nuclear and mitochondrial DNA. Such assessments not only yield information on the fertilizing capacity of human spermatozoa but also their ability to support normal embryonic development. [source] The significance of platelet-activating factor and fertility in the male primate: a reviewJOURNAL OF MEDICAL PRIMATOLOGY, Issue 1 2005William E. Roudebush Abstract:, Since its discovery nearly 30 years ago platelet-activating factor (PAF) has emerged as one of the more important lipid mediators known. PAF (1- O -alkyl-2- O -acetyl- sn -glycero-3-phosphorylcholine) exists endogenously as a mixture of molecular species with structural variants of the alkyl moiety. PAF is a novel potent signaling phospholipid that has unique pleiotropic biological properties in addition to platelet activation. PAF also plays a significant role in reproduction and is present in the sperm of a number of primate species. PAF content in squirrel monkey sperm is significantly higher during the breeding season than the non-breeding season. PAF content in rhesus sperm has a significant relationship with sperm motility. PAF content in human sperm has a positive correlation with seminal parameters and pregnancy outcomes. The enzymes (lyso-PAF-acetyltransferase and PAF-acetylhydrolase) necessary for PAF activation and deactivation are present in primate sperm. PAF-acetylhydrolase may act as a ,decapacitation factor'. Removal of this enzyme during capacitation promotes PAF synthesis increasing primate motility and fertilization. PAF also plays a significant role in the fertilization process, enhancing the fertilization rates of oocytes. Enhanced embryo development has also been reported in oocytes fertilized with PAF-treated sperm. Exogenous PAF will also significantly improve primate artificial insemination pregnancy outcomes. PAF antagonists inhibit sperm motility, acrosome reaction, and fertilization thus suggesting the presence of receptors for PAF. The PAF-receptor is present on primate sperm, with altered transcript levels and distribution patterns on abnormal cells. Whereas, the exact mechanism of PAF in sperm function and reproduction is uncertain, its importance in normal primate fertility is substantial. [source] Role of glycerol-3-phosphate dehydrogenase 2 in mouse sperm capacitationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2010Venkatesh Kota A tyrosine phosphoproteome study of hamster spermatozoa indicated that glycerol-3-phosphate dehydrogenase 2 (GPD2), is one of the proteins that enables tyrosine phosphorylation during sperm capacitation. Further, enzymatic activity of GPD2 correlated positively with sperm capacitation [Kota et al., 2009; Proteomics 9:1809,1826]. Therefore, understanding the function of GPD2 would help to unravel the molecular mechanism of sperm capacitation. In this study, involving the use of spermatozoa from Gpd2+/+ and Gpd2,/, mice, it has been demonstrated that in the absence of Gpd2, hyperactivation and acrosome reaction were significantly altered, and a few changes in protein tyrosine phosphorylation were also observed during capacitation. Evidence is provided to demonstrate that GPD2 activity is required for ROS generation in mouse spermatozoa during capacitation, failing which, capacitation is impaired. These results imply that GPD2 is involved in sperm capacitation. Mol. Reprod. Dev. 77: 773,783, 2010. © 2010 Wiley-Liss, Inc. [source] Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis-specific isoformMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010Larissa D. Newton Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis-specific ,4 (ATP1A4) isoforms of Na+/K+ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na+/K+ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti-ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post-acrosomal region. To investigate signaling mechanisms involved in oubain-induced regulation of sperm capacitation, sperm preparations were pre-incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na+/K+ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na+/K+ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na+/K+ATPase can fertilize oocytes in vitro. Mol. Reprod. Dev. 77: 136,148, 2010. © 2009 Wiley-Liss, Inc. [source] HongrES1, a cauda epididymis-specific protein, is involved in capacitation of guinea pig sperm,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2009Ya Ni Capacitation requires removal of proteins secreted by the cauda epididymis. Previously, we isolated and cloned the HongrES1 gene from rat cauda epididymis and found that it was exclusively expressed there. Here we report that HongrES1 mRNA is also expressed in the guinea pig cauda epididymis using Northern blot analysis, and the molecular weight of its cognate protein is approximately 48,kDa by Western blot analysis. Therefore, we investigated whether HongrES1 was involved in regulation of sperm capacitation in guinea pig. The results show that HongrES1 antisera (HA) significantly enhances sperm capacitation with maximal stimulation at a dilution of 1:500. Capacitation was reversed when capacitated spermatozoa were re-exposed to HongrES1 protein (HP, 0.25,µg/ml). In other words, HP acted as a decapacitation factor. HA accelerated the onset of capacitation and promoted a sperm hyperactivated motility response. Sperm capacitation was accelerated by HA stimulation of extracellular calcium influx while HP prevented extracellular calcium from influxing. Indirect immunofluorescence staining finds HP localized over the acrosomal anterior region of the sperm head, which exfoliates gradually during capacitation incubation, and completely disappeared after the acrosome reaction. Thus, HongrES1 expressed by the cauda epididymis is a novel molecule that regulates the physiology of guinea pig sperm prior to fertilization. Mol. Reprod. Dev. 76: 984,993, 2009. © 2009 Wiley-Liss, Inc. [source] Heparin-binding proteins of human seminal plasma: purification and characterizationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2008Vijay Kumar Abstract Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity,chromatography on Heparin,Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS,PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. Mol. Reprod. Dev. 75: 1767,1774, 2008. © 2008 Wiley-Liss, Inc. [source] Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian spermMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2007Longmei Zhao Abstract Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis. Mol. Reprod. Dev. 74: 750,757, 2007. © 2006 Wiley-Liss, Inc. [source] Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reactionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Frederick W.K. Kan Abstract Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Effects of 2-hydroxypropyl-,-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2006Hannah L. Galantino-Homer Abstract Porcine sperm are extremely sensitive to the damaging effects of cold shock. It has been shown that cholesterol-binding molecules, such as 2-hydroxypropyl-,-cyclodextrin (HBCD), improve post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. The objective of this study was to determine the effects of HBCD and cholesterol 3-sulfate (ChS) on porcine sperm viability and capacitation following cold shock or incubation under conditions that support capacitation using a defined medium. We report here that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock (10 min at 10°C) when compared to sperm incubated without HBCD or ChS, or with either component alone. Treatment with HBCD plus ChS also completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment or by incubation for 3 hr under conditions that support capacitation. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock. However, 3-hr incubation with HBCD plus ChS or with 1 mM ChS alone decreased the percentage of sperm undergoing the induced acrosome reaction without significantly affecting viability when compared to the control. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2006Daniel Mariappa Abstract 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. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Role of the sperm proteasome during fertilization and gamete interaction in the mouseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2005Consuelo Pasten Abstract In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head. Mol. Reprod. Dev. 71: 209,219, 2005. © 2005 Wiley-Liss, Inc. [source] Cytoplasmic localization during testicular biogenesis of the murine mRNA for Spam1 (PH-20), a protein involved in acrosomal exocytosisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Carlos R. Morales Abstract The Sperm Adhesion Molecule1 (SPAM1) is the most widely conserved sperm antigen with important roles in mammalian fertilization. Light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of Spam1 mRNA in the murine testis. Transcripts were first detected in step 3 round spermatids, gradually increased until step 8 and abruptly decreased between steps 9,11. They were predominantly localized near the ER and were not dispersed throughout the cytoplasm. Immunohistochemistry revealed that Spam1 is present on both the head and tail of sperm in the seminiferous tubules, and provided support for transcriptional regulation of its transcript. Immunocytochemistry confirmed the location of Spam1 on the tail of testicular sperm and demonstrated that it is localized to both the principal piece and the midpiece. Spam1 on epididymal sperm is localized to the midpiece of the tail and changes from a uniform distribution on the head in the caput to a regionalized pattern, first on the posterior and then on the anterior head, in caudal sperm. Spam1 on the surface of caudal sperm was shown to mediate the increase in acrosome reactions induced by the synergistic effects of HA and progesterone, as confirmed in sperm from the Rb(6.16) translocation-bearing mice which are Spam1 mutants. The similar response of human and mouse sperm to these agonists of the acrosome reaction, underscores the usefulness of the mouse as a model to study physiological aspects of SPAM1 in humans where, unlike the mouse, it is the only sperm hyaluronidase. Mol. Reprod. Dev. 69: 475,482, 2004. © 2004 Wiley-Liss, Inc. [source] Polycystins: what polycystic kidney disease tells us about spermMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Abraham L. Kierszenbaum Abstract Experimental evidence indicates that the membrane-associated proteins polycystin-1 and polycystin-2 operate as a receptor-calcium channel complex that regulates signaling pathways essential for modulation of renal tubulogenesis. Polycystic kidney disease is characterized by defective renal tubular structure and results from mutations in either PKD1 or PKD2 genes. Recent data suggest that polycystin-1 and polycystin-2 might localize to primary cilium in principal cells of renal collecting tubules and are thought to act as mechanosensors of fluid flow and contents. Ciliary bending by fluid flow or mechanical stimulation induce Ca2+ release from intracellular stores, presumably to modulate ion influx in response to tubular fluid flow. Polycystins are also emerging as playing a significant role in sperm development and function. Drosophila polycystin-2 is associated with the head and tail of mature sperm. Targeted disruption of the PKD2 homolog results in nearly complete male sterility without disrupting spermatogenesis. Mutant sperm are motile but are unable to reach the female storage organs (seminal receptacles and spermathecae). The sea urchin polycystin-1-equivalent suPC2 colocalizes with the polycystin-1 homolog REJ3 to the plasma membrane over the acrosomal vesicle. This localization site suggests that the suPC2-REJ3 complex may function as a cation channel mediating acrosome reaction when sperm contact the jelly layer surrounding the egg at fertilization. Future studies leading to the identification of specific ligands for polycystins, including the signaling pathways, might define the puzzling relationship between renal tubular morphogenesis and sperm development and function. Mol. Reprod. Dev. 67: 385,388, 2004. © 2004 Wiley-Liss, Inc. [source] GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tatsuru Togo Abstract The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465,471, 2004. © 2004 Wiley-Liss, Inc. [source] Polycystin-2 associates with the polycystin-1 homolog, suREJ3, and localizes to the acrosomal region of sea urchin spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Anna T. Neill Abstract Polycystin-2, the protein mutated in type 2 autosomal dominant polycystic kidney disease, is an integral transmembrane protein with nonselective cation channel activity. Here we report on the sea urchin sperm homolog of polycystin-2 (suPC2). Like other polycystin-2 family members, suPC2 is a six-pass transmembrane protein containing C-terminal cytoplasmic EF hand and coiled-coil domains. The protein localizes exclusively to the plasma membrane over the sperm acrosomal vesicle. This localization coincides with the previously reported localization of the sea urchin PC1 homolog, suREJ3. Co-immunoprecipitation shows that suPC2 and suREJ3 are associated in the membrane. The location of suPC2 sug-gests that it may function as a cation channel mediating the sperm acrosome reaction. The low cation selectivity of PC2 channels would explain data indicating that Na+ and Ca2+ may enter sea urchin sperm through the same channel during the acrosome reaction. Mol. Reprod. Dev. 67: 472,477, 2004. © 2004 Wiley-Liss, Inc. [source] Novel human testis-specific cDNA: Molecular cloning, expression and immunobiological effects of the recombinant proteinMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001Ramasamy Santhanam Abstract A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-,gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122,124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S -transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the ,17 kDa recombinant TSA-1, and a ,24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility. Mol. Reprod. Dev. 60: 1,12, 2001. © 2001 Wiley-Liss, Inc. [source] Progesterone induces activation in Octopus vulgaris spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001Elisabetta Tosti Abstract The purpose of the present study was to determine whether Octopus vulgaris spermatozoa are activated by progesterone stimulation. Spermatozoa were collected from the spermatophores in the Needham's sac of the male (MS) and from the spermathecae of oviducal glands of the female (FS). We used transmission (TEM) and scanning (SEM) electron microscopy to study the morphology of untreated, Ca2+ ionophore A23187 and progesterone‐treated MS spermatozoa, and untreated FS spermatozoa. We showed that ionophore and progesterone stimulation of MS spermatozoa induce breakdown of the membranes overlapping the acrosomal region, exposing the spiralized acrosome. These modifications resemble the acrosome reaction observed in other species. FS stored in the spermathecae did not show the membranes covering the acrosomal region present in the MS spermatozoa. When ionophore and progesterone treatments were performed in Ca2+‐free artificial sea water, no changes were observed, suggesting the role of external calcium in modifying membrane morphology. Lectin studies showed a different fluorescence distribution and membrane arrangement of FS‐untreated spermatozoa with respect to the MS, suggesting that spermatozoa transferred in the female genital tract after mating, are stored in a pre‐activated state. The plasma membrane of the untreated MS and FS spermatozoa was labelled with Progesterone‐BSA‐FITC, indicating the presence of plasma membrane progesterone receptor. Taken together these data suggest that progesterone induces an acrosome‐ like reaction in MS spermatozoa similar to that induced by calcium elevation. In addition progesterone may play a role in the pre‐activation of spermatozoa stored in the female tract, further supporting the hypothesized parallelism between cephalopods and vertebrates. Mol. Reprod. Dev. 59:97–105, 2001. © 2001 Wiley‐Liss, Inc. [source] Identification of the proteins present in the bull sperm cytosolic fraction enriched in tyrosine kinase activity: A proteomic approachPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Claudia Lalancette Abstract Numerous sperm proteins have been identified on the basis of their increase in tyrosine phosphorylation during capacitation. However, the tyrosine kinases present in spermatozoa that are responsible for this phosphorylation remain unknown. As spermatozoa are devoid of transcriptional and translational activities, molecular biology approaches might not reflect the transcriptional pattern in mature spermatozoa. Working directly with the proteins present in ejaculated spermatozoa is the most reliable approach to identify the tyrosine kinases potentially involved in the capacitation-associated increase in protein tyrosine phosphorylation. A combination of tyrosine kinase assays and proteomic identification tools were used as an approach to identify sperm protein tyrosine kinases. Fractionation by nitrogen cavitation showed that the majority of tyrosine kinase activity is present in the cytosolic fraction of bovine spermatozoa. By the use of Poly-Glu:Tyr(4:1)-agarose affinity chromatography, we isolated a fraction enriched in tyrosine kinase activity. Proteomics approaches permitted the identification of tyrosine kinases from three families: Src (Lyn), Csk, and Tec (Bmx, Btk). We also identified proteins implicated in different cellular events associated with sperm capacitation and acrosome reaction. These results confirm the implication of tyrosine phosphorylation in some aspects of capacitation/acrosome reaction and reveal the identity of new players potentially involved in these processes. [source] Effects of Matrix Filtration of Low-Quality Boar Semen Doses on Sperm QualityREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2009E Bussalleu Contents The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 ± 0.5 cm), CM Sephadex (length 5 ± 0.5 cm), glass wool (length 2 ± 0.5 cm) or glass bead (length 10 ± 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca® 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l -lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l -lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction. [source] | ||||||||||||||||