Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid

Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorter time of exposure to capacitating conditions. Washed, frozen-thawed boar spermatozoa were incubated separately with pre-ovulatory isthmic oviductal fluid (EODF), post-ovulatory ODF (MODF), capacitation medium (CM), and noncapacitating medium (NCM) for 60 minutes. Aliquots of spermatozoa were taken at 0, 5, 15, 30, and 60 minutes during incubation and sperm kinematics, intracellular calcium [Ca2⁺]i content, and protein tyrosine phosphorylation (PTP) were studied. The proportion of motile spermatozoa increased significantly after 5 minutes of incubation with EODF. A similar increase was not observed in the other groups. During the initial 5 minutes of incubation, the proportion of spermatozoa with high [Ca²⁺]i decreased significantly in all four groups. The proportion of tyrosine phosphorylated spermatozoa increased from 6.49 ± 1.93% to 15.42 ± 3.58% and 18.41 ± 1.57% in EODF and MODF groups, respectively, at 5 minutes of incubation. Neither CM nor NCM elicited any immediate effect on PTP in spermatozoa. There was a positive and significant correlation between [Ca²⁺]i and sperm motility (P = 0.009). It may be concluded that frozen-thawed boar spermatozoa undergo capacitation-associated changes after a relatively short exposure to EODF, and there are some subpopulations of spermatozoa that undergo PTP despite possessing low [Ca²⁺]i.     

Oviductal fluid modulates the dynamics of tyrosine phosphorylation in cryopreserved boar spermatozoa during capacitation

Following insemination, spermatozoa are retained in the utero‐tubal junction and isthmic region of the oviduct, where essential steps of capacitation are coordinated. Although a majority of the spermatozoa is exposed to similar conditions in the oviduct, the speed of the response varies depending on the individual male and the state of the spermatozoa. The present study evaluated individual boar variations in terms of the ability of spermatozoa to undergo tyrosine phosphorylation in response to isthmic oviductal fluid (ODF). Cryopreserved spermatozoa from four boars were incubated with pre‐ and post‐ovulatory ODF for 6 hr. Sperm kinematics, global protein tyrosine phosphorylation, and dynamics of different phosphorylation patterns were analyzed at hourly interval. The percentage of phosphorylated spermatozoa in the pre‐ovulatory ODF‐treated group was significantly (P < 0.001) higher than in the other treatment groups. Motility, velocity, and protein tyrosine phosphorylation in spermatozoa in response to ODF and control media also showed differences between boars. Spermatozoa from all four boars showed strong expression of a 19‐kDa phosphoprotein while spermatozoa from two boars showed additionally strong expression of a 32‐kDa phosphoprotein when incubated with pre‐ovulatory ODF. While phosphorylation of proteins in the acrosome and the equatorial segment of the sperm were noticed at an early stage during incubation with ODF, tail phosphorylation appeared at a later stage of capacitation. The results indicate individual variation between boars in terms of sperm proteins, including different phosphorylation patterns, in response to ODF, which might be related to fertility.Mol. Reprod. Dev. 79: 525‐540, 2012. © 2012 Wiley Periodicals, Inc.     

Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa

Capacitation is defined as a series of events that render boar sperm competent to fertilize, either in vivo or in vitro. Moreover, preliminary stages of cryopreservation of spermatozoa involving cooling to 5 degrees C have been shown to induce capacitation-like changes in boar spermatozoa. Capacitation of boar spermatozoa is accompanied by protein phosphorylation, however the relationship between both processes is poorly understood. Capacitation status was assessed by chlortetracycline (CTC) staining. Changes in protein tyrosine phosphorylation were examined in pre-cleared whole cell lysates using a specific anti-phosphotyrosine monoclonal antibody. Our results in boar spermatozoa show a significant positive correlation between p32 tyrosine phosphorylation levels and percentage of capacitated (CTC pattern B) spermatozoa. Moreover, incubation of boar spermatozoa with two unrelated tyrosine kinase inhibitors induces a significant reduction in the percentages of capacitated and acrosome-reacted (AR) boar spermatozoa and a reduction in the p32 tyrosine phosphorylation. In our conditions, cooling boar spermatozoa to 5 degrees C and rewarming to 39 degrees C in a noncapacitating medium results in similar CTC staining patterns to those obtained after incubation of boar sperm for 1 or 4 hr at 39 degrees C in a capacitating medium. However, cooled-rewarmed fails to induce an increase in p32 tyrosine phosphorylation in boar spermatozoa. Moreover, CTC staining patterns of cooled-rewarmed spermatozoa do not change after incubation with a tyrosine kinase inhibitor. In conclusion, our results show a direct relationship between capacitation and tyrosine phosphorylation and suggest that p32 tyrosine phosphorylation levels could be used as a marker of the true capacitation changes observed in boar spermatozoa. Moreover, our results show that true capacitation and capacitation-like changes induced after cooling involve alternative intracellular tyrosine phosphorylation pathways in boar spermatozoa.     

Kinetics of protein tyrosine phosphorylation in sperm selected by binding to homologous and heterologous oviductal explants: how specific is the regulation by the oviduct?

Essential steps of the capacitation process take place in the oviductal isthmus. A crucial step in the process of capacitation is the phosphorylation of membrane proteins. The aims of this work were (1) to study the effect of dog sperm binding to oviductal epithelium on tyrosine phosphorylation and (2) to investigate the specificity of regulation of molecular changes by the oviduct of different species by comparing the numbers of canine sperm bound to heterologous (porcine) and homologous epithelium, and the kinetics of tyrosine phosphorylation. Semen was collected from four healthy dogs and washed through a Percoll gradient. Explants, small pieces of epithelium, were cut from porcine and estrous bitch oviducts. During 6 h of coincubation in Tyrode medium, the numbers of bound sperm were counted by microvideographic observation, and the state of tyrosine phosphorylation was determined immunocytochemically after 3, 30, 90, 180 and 360 min. Canine sperm bound in similar numbers to homologous and heterologous explants. Increasing tyrosine phosphorylation of tail proteins and subsequent phosphorylation of sperm head proteins were observed. Binding occurred mainly in sperm with non-phosphorylated heads (approximately 2% phosphorylated), while higher proportions of head-phosphorylated cells were found in unbound populations (approximately 40-60%;P<0.05). The head phosphorylation progressed significantly during incubation in unbound spermatozoa (P<0.05), while it was suppressed in bound suspensions. The rate of tyrosine phosphorylation of sperm tail proteins was higher in cells bound to explants than in unbound cells or in those incubated in control medium. There were no significant differences with respect to the kinetics of tyrosine phosphorylation between the two coincubation systems. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. Tyrosine phosphorylation of sperm head proteins and capacitation are delayed in spermatozoa in close contact with oviductal epithelium. This mechanism appears to be species-independent, as sperm bound similarly to pig and dog oviduct explants, and similar phosphorylation kinetics were observed in both types of tissue.     

Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation.

To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.     

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after “in vitro” capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.     

Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane.

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.     

In vitro capacitation of bull spermatozoa by oviductal fluid and its components

Sperm capacitation is crucial for fertilization. However, debate continues on exactly how, where and when capacitation is elicited in the bovine female genital tract. In this study we used merocyanine-540 and the chlortetracycline (CTC) assay to test how capacitation of bull spermatozoa is affected in vitro by exposure to oviductal fluid (ODF) collected in vivo, various glycosaminoglycans (GAGs) or bicarbonate. Following different durations of exposure, spermatozoa were stained with CTC or merocyanine-540, and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Incubation time did not significantly affect capacitation. Exposure (30-120 min) to ODF capacitated (p < 0.05) bull spermatozoa as measured by either merocyanine-540 or CTC. Hyaluronan was the only GAG that induced a significant increase in B-pattern spermatozoa (capacitated; p = 0.012) compared with controls. Dermatan sulphate also induced capacitation (merocyanine-540 high fluorescence; p = 0.035). Exposure to bicarbonate-enriched media also yielded an increase in merocyanine-540 high fluorescence (p < 0.0001). When bicarbonate was added to the other treatments (ODF or GAGs) an equal increase in merocyanine-540 high fluorescence was noted (p < 0.0001), compared with before addition of bicarbonate and independent of the treatment before exposure. There was no significant difference in the number of B-pattern spermatozoa when bicarbonate was added, but an significant increase in spermatozoa with an acrosome-reacted (AR)-pattern (p < 0.0001) was observed. Exposure of spermatozoa to solubilized zonae pellucidae significantly increased the AR-pattern spermatozoa (p = 0.016). In conclusion, ODF was more potent in inducing capacitation of bull spermatozoa than the individual GAGs. Our results also indicate that bicarbonate is an effector of bull sperm capacitation.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Sperm capacitation in the porcine oviduct

In vitro studies suggests that sperm capacitation occurs in the sperm reservoir (SR) of the pig, with spermatozoa progressing towards the ampullary-isthmic junction (AIJ) around ovulation as a consequence of capacitation/hyperactivation. In contrast, in vivo studies are scarce. Consequently, we determined the degree of capacitation in boar spermatozoa that were retrieved from the SR of sows at well-defined periods of spontaneous standing oestrus, namely pre-, peri- and post-ovulation, using flow cytometry of Merocyanine-540/Yo-Pro-1-loaded spermatozoa. SR-spermatozoa retrieved and incubated in non-capacitating medium (bicarbonate-free mBO [mBO-]) were largely viable (70-85%) and uncapacitated (69-73%), irrespective of the stage of oestrus considered. Those undergoing capacitation were a minor proportion (1-5%) during pre- and peri-ovulation, but they significantly increased (14%) in post-ovulation oestrus. To clarify whether these SR-spermatozoa were able to undergo capacitation under stimuli, sperm aliquots were challenged in vitro either by incubation in a bicarbonate-rich medium (capacitation medium, mBO+), then further in mBO+ with 20% (v/v) of in vivo collected homologous pre-ovulatory isthmic oviductal fluid (IOF), or incubation with hyaluronan (HA, 500 microg/ml). Exposure to mBO+ significantly increased the sub-population of capacitated spermatozoa from the pre- and peri-ovulation SR, indicating that the uncapacitated SR-spermatozoa were responsive to the effector/inducer bicarbonate at levels recorded in peri-ovulatory AIJ/ampulla in vivo. While addition of IOF or HA to SR-spermatozoa incubated in capacitating medium (mBO+) maintained sperm viability without obviously inducing capacitation in pre- or peri-ovulatory SR-spermatozoa, they significantly increased these percentages during post-ovulation, when compared to baseline values of control incubations (mBO-). The results suggest that massive sperm capacitation does not occur in vivo in the porcine SR under spontaneous standing oestrus, particularly during pre- and peri-ovulation, unless spermatozoa are exposed to the effector bicarbonate. Exposure to IOF (and its component HA) under the present experimental conditions, reversed bicarbonate influence during pre- and peri-ovulation and further increased capacitation in post-ovulation, calling for an active role of the intratubal fluid. Furthermore, HA appears to have an active role in the functionality of the SR.     

Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.     

Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC

In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Endogenous redox activity in mouse spermatozoa and its role in regulating the tyrosine phosphorylation events associated with sperm capacitation

We investigated the role of endogenous redox activity in regulating the signal transduction pathway leading to tyrosine phosphorylation in mouse spermatozoa. Endogenous redox activity was monitored using a luminol-peroxidase chemiluminescent probe. Chemiluminescence increased in spermatozoa that were actively undergoing cAMP-mediated tyrosine phosphorylation events associated with capacitation and was inhibited in a dose-dependent manner by addition of catalase or diphenylene iodonium, both of which also inhibited tyrosine phosphorylation within the cell at points downstream of cAMP. Excluding bicarbonate from the incubation medium reduced the redox activity of sperm by 80-90% and dramatically reduced tyrosine phosphorylation. This study provides the first evidence that tyrosine phosphorylation associated with capacitation in mouse spermatozoa is redox regulated by a flavinoid-containing enzyme involving mediation by hydrogen peroxide. Bicarbonate regulated the redox activity of mouse spermatozoa, and this regulation may contribute to the impact of this anion on tyrosine phosphorylation during capacitation of mouse spermatozoa.     

双酚A暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响

双酚A(BPA)是一种人工合成的雌激素性化合物,广泛存在于环境中,对哺乳动物内分泌有干扰作用,影响雄性生殖系统功能。本研究以新鲜猪精子、17 ℃保存猪精子以及小鼠精子为对象,采用体外培养方法,利用蛋白免疫印迹(WB)和免疫荧光技术,分析不同浓度BPA(0、0.1、1、10、100 μmol·L^-1)暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响及分子机制。结果表明: 低中浓度(0.1、1 μmol·L^-1)BPA暴露对新鲜猪获能精子蛋白酪氨酸磷酸化具有显著促进作用,但在高浓度(10、100 μmol·L^-1)BPA暴露下,猪获能精子蛋白酪氨酸磷酸化呈现降低趋势。BPA暴露下,小鼠获能精子蛋白酪氨酸磷酸化随BPA浓度的增加而增强,并且BPA影响猪和小鼠精子获能相关酪氨酸磷酸化修饰的蛋白种类不同。表明BPA暴露对哺乳动物精子的影响具有物种特异性。免疫荧光结果显示BPA对精子蛋白酪氨酸磷酸化的影响主要发生在鞭毛的中段和主段。     

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.