Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight-line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol-oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol-reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2-min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S-S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley-Liss, Inc.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation.

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum.     

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

Computerized analysis of the motility parameters of hamster spermatozoa during maturation

A computer-aided semen analysis system was used for the objective assessment of hamster spermatozoa during epididymal maturation. The caput epididymal spermatozoa were extremely sluggish, achieved very little progression, and the three velocity parameters, namely curvilinear velocity (VCL), progressive velocity (VSL), and path velocity (VAP), were low. These spermatozoa during progressive movement alternated between the linear shape and “U” shape or attained an “S” shape prior to changing to the “U”; shape. The corpus epididymal spermatozoa were faster, displayed greater VSL, VAP, and VCL compared to caput epididymal spermatozoa, and, during forward motility, attained “U,” “C,”; and (or) “?” shape as in the wriggling motility pattern. The proximal cauda epididymal spermatozoa were actively motile and VSL, VAP, and VCL in these spermatozoa were more than 10 times greater compared to the caput epididymal spermatozoa. The proximal cauda epididymal spermatozoa predominantly moved in circles and with time became slower and more circular in their trajectories and exhibited a reduction in LIN (linearity). The distal cauda epididymal spermatozoa were very similar to the proximal cauda epididymal spermatozoa with respect to their fast motility (VSL, VAP, and VCL are similar) and beat cross frequency (BCF), but showed larger values for STR (straightness) and LIN and moved along curved trajectories. The amplitude of lateral head displacement (ALH) was also considerably lower in the distal cauda epididymal spermatozoa compared to the proximal cauda epididymal spermatozoa. Thus, this study provides for the first time data related to seven motility parameters for caput and corpus epididymal spermatozoa of hamster. It also provides additional data with respect to VCL, LIN, BCF, and ALH for proximal and distal cauda epididymal spermatozoa of hamster. © 1994 Wiley-Liss, Inc.     

Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation

Movement characteristics of untreated bovine caudal epididymal spermatozoa were compared by high-speed cinemicrography with those of theophylline-activated caput epididymal spermatozoa with and without added forward motility protein (FMP). Comparison of individual movement characteristics clearly established the importance of FMP in converting the nonprogressive motility of theophylline-activated caput sperm into the progressive swimming of mature caudal sperm. Although the total or curvilinear distance traveled in 1 sec by theophylline-activated caput sperm was not changed by the addition of FMP, the linear progression was doubled and the percentage of progressively motile sperm was tripled by this protein. Untreated caudal sperm were 80% motile and theophylline-activated caput sperm were nearly 50% motile; the percentage of motile sperm that were progressive was the same for theophylline-activated caput sperm with FMP and for untreated caudal sperm. Caput sperm without FMP roll infrequently, if at all, but caput sperm with FMP and caudal sperm roll at 4.7 Hz. The beat frequency increases significantly with the addition of FMP and is even higher for caudal sperm. The hydrodynamic power output rises concomitantly with the beat frequency. Perhaps the most striking difference between caput sperm without FMP and those with it is in the swimming paths they follow. Caput sperm without FMP exhibit frequent reversals in direction, or yawing of the sperm heads as they loop back and cross over their tails in an apparently very flexible bending. Their average swimming paths are circles. Caput sperm with FMP and caudal sperm do not show this behavior, but swim in average paths which are linear. The minimum radius of curvature of the tail of caput sperm without FMP is much smaller than that for the other two cell types. These studies clarify the role of FMP in epididymal development of sperm motility.     

Creatine phosphokinase in domestic cat epididymal spermatozoa

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.     

Effect of dilauroylphosphatidylcholine liposomes on motility, induction of the acrosome reaction, and subsequent egg penetration of ram epididymal sperm

The effects of dilauroylphosphatidylcholine (PC12) on ram epididymal sperm motility, acrosome reaction (AR) induction, plasma membrane permeability, mitochondrial function, and sperm penetration into zona-free hamster eggs were determined. PC12 (50 microM) induced cell motility in caput and cauda sperm, as measured by subjective estimation and automated motility analysis. Motion parameters of treated caput sperm approached those of control ejaculated sperm. Flow cytometric analysis revealed that membrane permeability to propidium iodide and mitochondrial uptake of rhodamine 123 changed during epididymal transit. PC12 induced the AR in sperm from all epididymal regions relative to control incubated sperm (caput 17% vs. control 8%; corpus 29% vs. control 13%; proximal cauda 48% vs. control 4%; distal cauda 51% vs. control 9%). After PC12 treatment, egg penetration by sperm was increased for sperm from the corpus (corpus 7% vs. control 0%) and cauda (proximal 48% vs. control 0%; distal 51% vs. control 0%), but not for caput sperm (caput 0% vs. control 0%). These studies establish that some sperm in each region of the epididymis possess the capacity for movement and the AR. Caput sperm, however, were unique in that they could not penetrate eggs. Additional maturational changes must occur in the caput and/or corpus epididymidis before penetration capacity can be expressed.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.     

Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation

This study was undertaken to determine the role of calcium ion, a key regulator of the intensity and form of motility in mature demembranated sperm, in the development of motility during passage through the bovine epididymis. Cellular calcium levels in bovine caput and cauda epididymal spermatozoa were measured with three different techniques. 45Ca2+ uptake measurements revealed that net calcium uptake and Ca2(+)-Ca2+ exchange in caput spermatozoa were about 2 to 3 times higher than in caudal spermatozoa. Intracellular free calcium determination with the calcium fluorophore Fura 2 showed that the levels were 6 times higher in caput spermatozoa. The values for caput and caudal sperm were 875 +/- 55 nM (n = 15) and 155 +/- 6 nM (n = 24), respectively. Total cellular calcium levels quantitated by atomic absorption were 626 +/- 30 (n = 48) and 304 +/- 19 (n = 46) ng/10(8) sperm in caput and caudal epididymal sperm, respectively. At least one of the reasons for the high calcium content of caput epididymal sperm is the result of a higher rate and extent of mitochondrial calcium accumulation in caput compared to caudal sperm. Mitochondrial calcium uptake rates measured in digitonin permeabilized cells revealed uptake rates 2- to 3-fold higher in caput compared to caudal sperm. However, mitochondrial calcium efflux rates were identical in caput and caudal epididymal sperm. The efflux rates in both cell types were unaffected by external sodium levels but were found to be proportional to pH. Alkalinization or acidification of internal pH of intact sperm resulted in a corresponding lowering or elevation of cytoplasmic free calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠附睾头精子获得与卵子质膜融合能力的物质基础研究

随着精子在附睾中的转运,它们与卵子质膜的融合能力逐渐增加。怩证明２附睾体和附睾尾的精子均具有相当高的膜融合能力,而附睾头中的精了奶少能与卵子质膜融合,这是还说明附睾头中的精子不具备与云透明带卵子融合的物质条件呢？利用附睾结扎留并延长体外获能时间,可使附睾头远端精子的融合能力明显地提高;在精子培养液中加入ＡＴＰ,并延长精卵共培养时间,也可使一少部分附睾头近端的精子获得与卵子质膜融合的能力。这表明附睾     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.     

Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.     

Alterations in primate sperm motility with maturation and during exposure to theophylline

Micropuncture was used to collect pure suspensions of sperm from the caput and cauda regions of chimpanzee epididymides, which were analyzed with a Motion Analysis VP-110. Sperm recovered from the caput region showed no forward motility. Incubation of these sperm with cauda epididymal fluid affected motility in 62%–90% of the sperm. Dilution of cauda sperm into buffer containing >50 mM theophylline resulted in immediate initiation of progressive forward motility. Although this motility was maintained by at least 50% of the sperm for over 5 hr, these “activated” caput sperm did not penetrate zona-free hamster ova. These data show that sperm from the caput epididymis of the chimpanzee have the capacity for normal motility but do not have the capacity to bind to and penetrate an ovum. Cauda epididymal chimpanzee sperm were motile at the time of recovery and this motility was maintained for over 5 hr. These sperm penetrated both hamster zona-free ova and intact chimpanzee ova. These data show that sperm from the cauda epididymis of the chimpanzee have the capacity for normal motility and also have the capacity to bind to and penetrate an ovum. This is the first use of computer assisted analysis to quantify motility in maturing nonhuman primate sperm.     

Regulation of microtubule sliding by a 36-kDa phosphoprotein in hamster sperm flagella

Cyclic AMP has been shown essential for activation of sperm motility. When immotile hamster caudal epididymal spermatozoa were suspended in a Ca2+-deficient solution, they showed a sluggish motility. Spermatozoa were demembranated and transferred to an ATP-containing reactivation solution. Demembranated spermatozoa did not exhibit reactivated flagellar movement unless cAMP was added. Conversely, when the immotile epididymal spermatozoa were suspended in a Ca2+-containing solution, they were immediately activated to display a vigorous motility; demembranated spermatozoa also exhibited reactivated flagellar movement in the reactivation solution without cAMP. Further investigation of microtubule sliding properties revealed that the effects of Ca2+ on live spermatozoa were identical with the effects of cAMP on demembranated spermatozoa both in microtubule sliding velocity and sliding disintegration pattern. Moreover, a 36-kDa flagellar protein was found to be phosphorylated in a cAMP-dependent manner and coupled to the motility activation. A polyclonal antibody against this protein was developed and showed specific immunolocalization and significant inhibitory effects on microtubule sliding disintegration. These results indicate that extracellular Ca2+ owes its effect to triggering intracellular cAMP production, and cAMP-dependent phosphorylation of a 36-kDa phosphoprotein activates hamster sperm motility through regulation of microtubule sliding properties.     

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

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

Selected proteins of "prostasome-like particles" from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull

During epididymal transit, spermatozoa acquire selected proteins secreted by epithelial cells. We recently showed that P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. To further characterize the interactions between PLPCd and epididymal spermatozoa, PLPCd were prepared by ultracentrifugation of bull epididymal fluid, then surface-exposed proteins were biotinylated and coincubated in different conditions with caput epididymal spermatozoa. Western blot analysis revealed that only selected proteins are transferred from PLPCd to spermatozoa. MALDI-TOF analysis revealed that these transferred proteins are closely related. The pattern of distribution of the PLPCd transferred varied from one sperm cell to the other, with a bias toward the acrosomal cap. This transfer appeared to be temperature sensitive, being more efficient at 32-37 degrees C than at 22 degrees C. Transfer of PLPCd proteins to spermatozoa was also pH dependant, the optimal pH for transfer being 6.0-6.5. The effect of divalent cations on PLPCd protein transfer to caput spermatozoa was investigated. Whereas Mg(2+) and Ca(2+) have no effect on the amount of proteins remaining associated with spermatozoa following coincubation, Zn(2+) had a beneficial effect. These results are discussed with regard to the function of PLPCd in epididymal sperm maturation.