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1.
Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule. 相似文献
2.
Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins. 相似文献
3.
O'Rand MG Widgren EE Hamil KG Silva EJ Richardson RT 《Biochemical Society transactions》2011,39(5):1447-1449
Our laboratory has characterized EPPIN [epididymal protease inhibitor; SPINLW1] as a novel gene on human chromosome 20q12-13.2, which encodes a cysteine-rich protein of 133 amino acids with a calculated molecular mass of 15.283?kDa, containing both Kunitz-type and WAP (whey acidic protein)-type four-disulfide core consensus sequences. Eppin is secreted by Sertoli cells in the testis and epididymal epithelial cells; it is predominantly a dimer, although multimers often exist, and in its native form eppin is found on the human sperm surface complexed with LTF (lactotransferrin) and clusterin. During ejaculation SEMG (semenogelin) from the seminal vesicles binds to the eppin protein complex, initiating a series of events that define eppin's function. Eppin's functions include (i) modulating PSA (prostate-specific antigen) enzyme activity, (ii) providing antimicrobial protection and (iii) binding SEMG thereby inhibiting sperm motility. As PSA hydrolyses SEMG in the ejaculate coagulum, spermatozoa gain progressive motility. We have demonstrated that eppin is essential for fertility because immunization of male monkeys with recombinant eppin results in complete, but reversible, contraception. To exploit our understanding of eppin's function, we are developing compounds that inhibit eppin-SEMG interaction and mimic anti-eppin, inhibiting sperm motility. These compounds should have potential as a male contraceptive. 相似文献
4.
Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive. 相似文献
5.
Erick J.R. Silva Marília T.C.C. Patrão James K. Tsuruta Michael G. O'Rand Maria Christina W. Avellar 《Molecular reproduction and development》2012,79(12):832-842
EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine‐rich protein containing both Kunitz and whey acidic protein (WAP)‐type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down‐regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen‐dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility. Mol. Reprod. Dev. 79: 832–842, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
6.
Capacitation of hamster caudal spermatozoa at a density of 1 × 106/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 × 106/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species. 相似文献
7.
Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility. 相似文献
8.
Extracellular calcium at millimolar concentrations inhibits collective motility of ejaculated ram spermatozoa. In untreated cells, or when motility was made dependent upon glycolytic activity, there is very small inhibition, but when motility was made dependent upon mitochondrial respiration there is very high inhibition in motility by increasing extracellular Ca2+ concentration. Quercetin, which inhibits (Ca2+ + Mg2+)-ATPase activity in isolated plasma membranes, also inhibits motility mainly in cells that have been made dependent upon glycolytic activity, but there is also inhibition in untreated cells. When motility was made dependent upon mitochondrial activity, there is no inhibition but rather some stimulation in motility by quercetin. The inhibitory effect of quercetin is enhanced by increasing Ca2+ concentration in the medium. Quercetin also inhibits uptake of calcium into the cells, in a mechanism by which a calcium channel is involved. This inhibition is high only when the glycolysis is inhibited in the cells. The rate of glycolysis is decreased by quercetin or ouabain, but their effects on motility are quite different. Based on these data, it appears that the plasma membrane (Ca2+ + Mg2+)-ATPase or the Ca2+ pump have a functional role in the regulation of spermatozoa motility. This motility regulation is functioning through mechanisms which include glycolytic activity and maintenance of intracellular calcium concentrations. 相似文献
9.
Small-Molecule Inhibition of BRDT for Male Contraception 总被引:1,自引:0,他引:1
MM Matzuk MR McKeown P Filippakopoulos Q Li L Ma JE Agno ME Lemieux S Picaud RN Yu J Qi S Knapp JE Bradner 《Cell》2012,150(4):673-684
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception. PAPERCLIP: 相似文献
10.
It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility. 相似文献
11.
Jin Tao Min Lin Jiahao Sha Gregory Tan Tuck Wah Soong Shengnan Li 《Cellular physiology and biochemistry》2007,19(5-6):303-312
Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects. 相似文献
12.
Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2.5 mM. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa. The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1.7 mM) typical of media used for in-vitro fertilization. The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1-10 mM. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine. We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway. 相似文献
13.
Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol 总被引:1,自引:0,他引:1
P Vainio T Thurén K Wichman T Luukkainen P K Kinnunen 《Biochimica et biophysica acta》1985,814(2):405-408
Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100 microM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent. 相似文献
14.
Andrzej Łukaszyk Małgorzata Kotwicka Anna Jankowska Aldona Kasprzak Marcin Ruciński Karolina Sterzyńska Agnieszka Ziółkowska Piotr Sawiński Marek Ruchala 《Reproductive biology》2012,12(3):293-300
In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10?9 nor 10?6 mol/L concentration. Ghrelin (10?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10?6 mol/L ghrelin increased, while 10?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions. 相似文献
15.
O Porat 《Molecular reproduction and development》1990,25(4):400-408
The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent. 相似文献
16.
Maria J. Bragado Ines M. Aparicio Maria C. Gil Luis J. Garcia‐Marin 《Journal of cellular biochemistry》2010,109(1):65-73
The cAMP‐dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase‐3α (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase‐resistant cell permeable cAMP analogue 8Br‐cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro‐32‐0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM‐ or 8Br‐cAMP‐stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility. J. Cell. Biochem. 109: 65–73, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
17.
Salmon calcitonin inhibits human sperm motility in vitro 总被引:1,自引:0,他引:1
L Gnessi L Silvestroni A Fabbri C Moretti A E Panerai V Bonifacio F Fraioli 《Biochemical and biophysical research communications》1984,125(1):199-204
We have evaluated by a stroboscopic technique the in vitro effect of salmon calcitonin and human calcitonin on the motility of human migrated spermatozoa. We report here that human calcitonin is uneffective while salmon calcitonin is a potent inhibitor of the sperm motility. This salmon calcitonin action is abolished by the preincubation of the peptide with an anti-salmon calcitonin antiserum, demonstrating the specificity of the effect. In addition, we provide evidence that the release of intracellular calcium represents a necessary step for the action of the peptide. In fact, the salmon calcitonin effect is prevented in a dose-dependent way by dantrolene sodium which inhibits the release of calcium from intracellular stores while the calcium channel blocker verapamil is unefficacious. These results suggest a potential role for calcitonin in regulating human sperm motility. 相似文献
18.
The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations. 相似文献
19.
Spermiogenesis in nematodes involves the activation of sessile spherical spermatids to motile bipolar amoeboid spermatozoa. In Caenorhabditis elegans males spermiogenesis is normally induced by copulation. Spermatids transferred to hermaphrodites as well as some of those left behind in the male become spermatozoa a few minutes after mating. Spermiogenesis can also be induced in vitro by the ionophore monensin (G.A. Nelson and S. Ward, 1980, Cell 19, 457-464) and by weak bases such as triethanolamine. Both triethanolamine and monensin cause a rapid increase in intracellular pH from 7.1 to 7.5 or 8.0. This pH increase precedes the subsequent morphological events of spermiogenesis. Triethanolamine or monensin must be present throughout spermiogenesis for all cells to form pseudopods, but once pseudopods are formed the inducers are unnecessary for subsequent motility. The pH induced spermiogenesis is inhibited by drugs that block mitochondria or glycolysis. Protease treatment can also induce spermiogenesis without increasing intracellular pH, apparently bypassing the pH-dependent steps in activation and the requirement for glycolysis. These results show that the initiation of spermiogenesis in C. elegans, like some steps in egg activation and the initiation of sea urchin sperm motility, can be induced by an increase in intracellular pH, but this pH change can be bypassed by proteolysis. 相似文献
20.
L. Keskes V. Giroux-Widemann C. Serres I. Pignot-Paintrand P. Jouannet D. Feneux 《Molecular reproduction and development》1998,49(4):416-425
The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc. 相似文献