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Mammalian Spermatozoa (mammalian + spermatozoa)
Selected AbstractsCharacterization 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] Novel identification of peripheral dopaminergic D2 receptor in male germ cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Carola Otth Abstract Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology. J. Cell. Biochem. 100: 141,150, 2007. © 2006 Wiley-Liss, Inc. [source] A cyclic adenosine 3,,5,-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Hiroshi Harayama Abstract An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5°C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa. Mol. Reprod. Dev. 1169,1178, 2006. © 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] A unique mechanism for cyclic adenosine 3,,5,-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Hiroshi Harayama Abstract A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831,842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-,-cyclodextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes. Mol. Reprod. Dev. 69: 194,204, 2004. © 2004 Wiley-Liss, Inc. [source] Mammalian Sperm Energy Resources Management and Survival during Conservation in RefrigerationREPRODUCTION IN DOMESTIC ANIMALS, Issue 2006JE Rodriguez-Gil Contents The present review has as its main aim to present an overview regarding the mechanisms utilized by mammalian sperm to manage its intracellular energy levels. This management will strongly influence the sperm's ability to maintain its overall function during its entire life span. Thus, the precise knowledge of these mechanisms will be of the utmost interest to optimize the systems utilized to conserve mammalian sperm for a medium-to-long time-lapse. Briefly, utilization of hexoses as energy substrates by mammalian sperm is very finely regulated from the very first step of its metabolization. Furthermore, the equilibrium among the separate, monosaccharide metabolization pathways in mammalian sperm depends on many factors. This prevents the possibility to draw a general vision of sperm energy utilization, which explains the results of all mammalian species in all points of the sperm life-cycle. To complicate the matter further, there are separate energy phenotypes among mammalian spermatozoa. The precise knowledge of these phenotypes is of the greatest importance in order to optimize the design of new extenders for sperm conservation in refrigerated conditions. Moreover, sugars can act on sperm not only as passive metabolic substrates, but also as direct function activators through mechanisms like specific changes in the tyrosine phosphorylation status of distinct proteins. Finally, mammalian sperm utilizes non-glucidic substrates like citrate and lactate to obtain energy in a regular form. This utilization is also finely regulated and of importance to maintain overall sperm function. This implies that the exact proportion of glucidic and non-glucidic energy substrates could be very important to optimize the survival ability of these cells in conservation. [source] The zona pellucida-induced acrosome reaction of human spermatozoa involves extracellular signal-regulated kinase activationANDROLOGIA, Issue 6 2001S. S. Du Plessis Summary. Extracellular signal-regulated kinases (ERKs), belonging to the family of mitogen-activated protein kinases (MAPKs), are cytoplasmic and nuclear serine/threonine kinases involved in the signal transduction of several extracellular effectors. Recent evidence indicates the presence of p21 Ras and the phosphorylation of ERK1 and ERK2, suggesting the occurrence of the Ras/ERK cascade in mammalian spermatozoa. The present article describes the biological role of ERK during the acrosome reaction of human spermatozoa on stimulation with zona pellucida (ZP). The mitogen-activated protein-kinase inhibitor PD098059 was used as a pharmacological tool to study the involvement of extracellular signal-regulated kinases in the induction of the acrosome reaction in human spermatozoa. This compound significantly inhibited the acrosome reaction induced by both ZP and the calcium ionophore A23187. These results suggest that ERKs are involved in the signal transduction pathway through which ZP stimulation works during the process of fertilization. [source] | |||||||||||||