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Capacitation
Kinds of Capacitation Selected AbstractsNa+/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] 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] The Use of HOS Test to Evaluate Membrane Functionality of Boar Sperm Capacitated in vitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2002D Lechniak Contents 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. [source] Changes in expression and activity levels of ecto-5,-nucleotidase/CD73 along the mouse female estrous cycleACTA PHYSIOLOGICA, Issue 2 2010E. Aliagas Abstract Aim:, Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5,-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods:, Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5,-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results:, Ecto-5,-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5,-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion:, The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility. [source] Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: A statistical studyDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2000Fumie Suzuki-Toyota Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density ± SD of large IMP was 379 ± 64 and 266 ± 58/,m2, and that of small IMP was 1450 ± 155 and 672 ± 252/,m2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability. [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] Characterization of human sperm N -acetylglucosaminidaseINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2008S. L. Perez Martinez Summary N -acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed. [source] Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L -arginine and regulated through a cAMP/PKA-independent pathwayINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2008S. C. Roy Summary The aim of the present study was to determine the effect of l -arginine on nitric oxide (NO,) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or l -arginine or N, -nitro- l -arginine methyl ester (l -NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO, both spontaneously and following stimulation with l -arginine and l -NAME quenched such l -arginine-induced NO, production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. l -Arginine (10 mm) was found to be a potent capacitating agent and addition of l -NAME to the incubation media attenuated both l -arginine and heparin-induced capacitation and suggested that NO, is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of Mr 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and l -arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these l -arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that l -arginine induces capacitation of buffalo spermatozoa through NO, synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA. [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] Localizations of intracellular calcium and Ca2+ -ATPase in hamster spermatogenic cells and spermatozoaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2006H.L. Feng Abstract Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37°C for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca2+ -ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca2+ -ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca2+ -ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [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] Bicarbonate-Induced phosphorylation of p270 protein in mouse sperm by cAMP-Dependent protein kinaseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008Masako Kaneto Abstract Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility. Mol. Reprod. Dev. 75: 1045,1053, 2007. © 2007 Wiley-Liss, Inc. [source] Expression of a novel T-complex testis expressed 5 (Tctex5) in mouse testis, epididymis, and spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007Y.B. Han Abstract Expression of T-complex testis expressed 5 (Tctex5), an orthologue of protein phosphatase-1 inhibitor-3 (PPP1R11), was enhanced in mouse testis and was also expressed in epididymis and spermatozoa. There were three transcripts of Tctex5 including one brain specific and two common transcripts dominant in mouse testis. Tctex5 protein isoforms (75, 52, 32, 25, and 14.3 kDa) were identified. Isoforms of 75 and 52 kDa were spermatogenic-specific and were found in protein fraction containing nuclei, mitochondria, and flagellum accessory, and also in protein fraction containing mainly membranes. Tctex5 was localized in nuclei of pachytene spermatocytes, round spermatocytes, cytoplasm of Sertoli cells in testis; cilia, secretion bodies and nuclei of epithelial cells and interstitium smooth muscle cells in epididymis; and head and principal piece of tail in epididymal spermatozoa. The results suggested that Tctex5 might be a specific protein phosphatase-1 inhibitor in sperm; various Tctex5 transcripts and isoforms and cellular locations imply its different roles in spermatogenesis. Nuclei-type isoforms (75 and 52 kDa) might take part in nucleus remodeling during spermatogenesis whilst membrane-type isoform (52 kDa) might be responsible for dephosphorylation of proteins during capacitation. The other isoforms might play general roles for all kinds of cell types. Mol. Reprod. Dev. 74: 1132,1140, 2007. © 2007 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] Surface of human sperm bears three differently charged CD52 forms, two of which remain stably bound to sperm after capacitationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001C. Della Giovampaola Abstract gp20 is a sialoglycoprotein of the human sperm surface with a core peptide homologous to the leukocyte antigen CD52, a GPI-anchored glycosylated protein which is described by the monoclonal antibody CAMPATH-1. Comparative analyses, by means of CAMPATH and anti-gp20, indicated that they describe it in morphologically and functionally different ways, suggesting that the respective epitopes are different but also casting doubt on the immunological identity of the antigen. In the present study, we used immunodepletion to demonstrate that CAMPATH and anti-gp20 interact with the same antigen, but that anti-gp20 has a much higher avidity for the antigen than CAMPATH. Anion exchange fractionation analysis of the antigen revealed three differently charged gp20-CD52 forms, the least charged of which, was largely without a GPI-anchor. All three forms were associated with freshly ejaculated sperm, whereas capacitated sperm only contained the two GPI-anchored, more charged forms, which were also the ones found in the prostasome fraction of seminal plasma and in leukocytes. The two charged, GPI-anchored forms were described as homogeneous by anti-gp20, since they ran as a singlet; the third form ran as a doublet. When tested for insertion into Jurkat T cells, the medium charged form inserted the most readily and the less charged one could not be inserted at all. Mol. Reprod. Dev. 60: 89,96, 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] Effect of Ram Age on Structural and Functional Competence of Frozen,Thawed Spermatozoa in Dairy SheepREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2010AG Lymberopoulos Contents The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen,thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1,2 years (young) or 4,5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of , 2.5 × 109 sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell® medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY581/591, sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ,0.63 to ,0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro. [source] Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010FS Gonçalves Contents Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 ,m,-mercaptoethanol (,-ME) and 50 ,m cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for ,-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage. [source] Identification of RSVP14 and RSVP20 Components by Two-dimensional Electrophoresis and Western-blottingREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2008JA Cardozo Contents We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4,7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (Mr) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases. [source] Can We Increase the Estimative Value of Semen Assessment?*REPRODUCTION IN DOMESTIC ANIMALS, Issue 2006H Rodríguez-Martínez Contents Estimating the fertility of a semen sample or of the male from where it has been collected by simple assessment of in vitro sperm characteristics is still difficult, owing to the variable correlations that laboratory results achieve with in vivo fertility. A major reason behind these variations is the fact that the ejaculate and the artificial insemination (AI)-doses it generates are composed of a diverse sperm population. Such heterogeneity is reflected both in differences of intactness of attributes needed for fertilization, such as motility or morphology, but also in the relative ability of spermatozoa to prevail fertile over time, handling and exposure to different stimuli, all of which account for innate variations in fertilizing ability among doses, ejaculates and sires. However, methods are already available to select sub-populations of intact spermatozoa which can be tested for their degree of competence for fertilization and whose estimative power is promising, allowing the elimination of cases of sub-fertility, particularly in bovine. Examples of these methods are the separation of viable spermatozoa by swim-up or discontinuous gradient centrifugation, followed by testing the ability of the selected spermatozoa to dose-response/time sustain capacitation and acrosome reaction induction. Finding how large a sperm population with non-compensable attributes for fertilization and ability to display and sustain stimuli is, perhaps by a quick screening of membrane integrity and stability by multi-parametric methods, would allow, provided the particular male produces this sub-population in a repeatable manner, for a better estimation of fertility. [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] The Use of HOS Test to Evaluate Membrane Functionality of Boar Sperm Capacitated in vitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2002D Lechniak Contents 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. [source] Vital Aspects of Fallopian Tube Physiology in PigsREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2002RHF Hunter Contents This essay reviews four topical aspects of Fallopian tube physiology that bear on either successful fertilization or early development of the zygote. An initial focus is on glycoprotein secretions of the duct that accumulate as a viscous mucus in the caudal isthmus. Because this is the site of the pre-ovulatory sperm reservoir, an involvement of the secretions is considered in: preventing uterine and ampullary tubal fluids from entering the functional sperm reservoir; removing residual male secretions from the sperm surface; deflecting spermatozoa towards endosalpingeal organelles and reducing flagellar beat before ovulation. The subtle prompting of flagellar movement with impending ovulation is examined in terms of potential reactivation mechanisms, with overall control attributed to increasing secretion of progesterone. The site of full capacitation and the acrosome reaction in a fertilizing spermatozoon is then debated, with strong arguments pointing to completion of these processes in the specific fluids at the ampullary-isthmic junction. Finally, the synthetic activity of cumulus cells released at ovulation as a paracrine tissue in the Fallopian tube is highlighted with reference to steroid hormones, peptides and cytokines. Not only does the suspension of granulosa-derived cells influence the process of fertilization, but also it may amplify oocyte or embryonic signals to the endosalpinx and ipsilateral ovary. [source] | |||||||||||||