Comparative characteristics between calyculin A-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa.

This study aimed to characterize calyculin A (CL-A)-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa. Hyperactivation was effectively induced by treating with 10 nM CL-A for 60 min in the presence of cyclic AMP analogs, extracellular Ca2+, and albumin or with 12.5 µM thimerosal briefly in the absence of these capacitation-supporting factors. Majority of the spermatozoa exhibiting CL-A-induced hyperactivation were characterized by the 3-dimensional helical movement with head rotation, higher degree of flagellar curvature, and faster beating of the flagella than those exhibiting thimerosal-induced hyperactivation of the 2-dimensional planar movement without head rotation. The CL-A-induced hyperactivation was linked to the activation of cAMP/protein phosphorylation-dependent signaling cascades and to the decreased activity of glycogen synthase kinase-3α (GSK-3α). In contrast, the thimerosal-induced hyperactivation was suppressed by pretreatment with CL-A and cyclic AMP analogs in the absence of CaCl2 to activate cAMP/protein phosphorylation-dependent signaling cascades. Additionally, the intracellular Ca2+ level in live sperm flagella was significantly higher in the CL-A-treated samples than in the thimerosal-treated samples. These results indicate that CL-A-induced hyperactivation of cryopreserved bovine spermatozoa is an extracellular Ca2+-dependent type with the 3-dimensional helical movement, which can be regulated not only by the activation of cAMP/protein phosphorylation-dependent signaling cascades, leading to a large enhancement of the intracellular Ca2+ level, but also by the reduction in GSK-3α activity. Considering the different characteristics of thimerosal-induced hyperactivation, our results suggest that the diversity of sperm hyperactivation arises from different combinations of flagellar bending and head rotation.

Polyvinyl alcohol, but not bovine serum albumin, promotes the induction of full-type hyperactivation in boar cyclic AMP analog-treated spermatozoa.

This study aimed to verify the effects of polyvinyl alcohol (PVA) and bovine serum albumin (BSA) on the induction of full-type hyperactivation in boar spermatozoa treated with a cyclic AMP analog (cBiMPS). Washed spermatozoa were treated with cBiMPS (100 µM) for 180 min. As shown in the assessment of sperm motility, PVA (0.05%-0.4%) significantly promoted the induction of full-type hyperactivation, whereas BSA (0.025%-0.4%) did not affect the induction. In comparative experiments, BSA (0.4%) effectively promoted the induction of full-type hyperactivation in bovine spermatozoa treated with cBiMPS, calyculin A (a protein phosphatase inhibitor), and digoxin (a Na+ /K+ -ATPase inhibitor), while PVA (0.1%) did not affect the induction. Western blotting showed that protein tyrosine phosphorylation states of >50 kDa sperm proteins were effectively enhanced by treatment with cBiMPS in the PVA/BSA-free medium and not affected by the addition of PVA (0.1%). The assessment of plasma membrane integrity indicated that BSA (0.4%) significantly decreased spermatozoa with intact plasma membranes. These results indicate that PVA (0.1%) promotes the induction of full-type hyperactivation and does not influence the protein tyrosine phosphorylation states in boar cBiMPS-treated spermatozoa. They also suggest that BSA should not be added to medium containing cBiMPS for boar spermatozoa.

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation.

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca2+-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca2+]i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca2+-ATPases (SERCAs), "thapsigargin." Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca2+-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.

Calmodulin is involved in the occurrence of extracellular Ca2+ -dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs.

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation "full-type and nonfull-type patterns" can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca2+ and calmodulin antagonists "W-7 and W-5 (W-5; a less potent antagonist)" on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42 mM CaCl2 for 120-180 min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca2+ -dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca2+ -sensing molecule which can function in the full-type hyperactivation.

Characteristics of bull sperm acrosome associated 1 proteins.

An atypical distribution of sperm acrosomal tyrosine-phosphorylated proteins [which include sperm acrosome associated 1 (SPACA1) proteins] may be related to the relatively lesser pregnancy rates when semen of some bulls are used for artificial insemination (AI). There may also be these associations with bull SPACA1 proteins that are translocated from the equatorial segment to the anterior part in the acrosomes during sperm maturation in the normally functioning epididymis. The aim of the present study, therefore, was assessment of the characteristics of bull SPACA1 proteins. Results from immunocytochemical evaluations indicate there were large variations in sperm percentages with typically distributed SPACA1 proteins in acrosomes of cauda epididymal sperm samples (7%-95%). These values were positively correlated with percentages of epididymal spermatozoa with typically distributed acrosomal tyrosine-phosphorylated proteins (r=0.8564, P<0.001). Results indicate there are individual differences in translocation of SPACA1 proteins in the epididymis during sperm maturation, and that SPACA1 protein is one of the main determinants for the typical distribution of acrosomal tyrosine-phosphorylated proteins. In addition, conception rates as a result of AI using cryopreserved spermatozoa tended to be associated with percentages of epididymal spermatozoa with typically distributed SPACA1 proteins. Results from sucrose gradient centrifugation fractionation experiments indicate SPACA1 proteins are sperm membrane raft-associated proteins. Based on these results, it is hypothesized that there is an association between bull subfertility when semen is used for AI and epididymal dysfunctions in the arrangement of membrane lipid rafts during sperm maturation.

Effects of digoxin on full-type hyperactivation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades.

Previous researches of our laboratory reported that addition of cAMP analog cBiMPS and protein phosphatase inhibitor calyculin A (stimulators of cAMP signaling cascades) improved the capacity of incubation medium to induce full-type hyperactivation in bovine ejaculated spermatozoa. However, this modified medium was valid only for samples with relatively good survivability for incubation with stimulators of cAMP signaling cascades. Thus, it is necessary to make further modified medium for evaluation of potentials to exhibit full-type hyperactivation in bovine sperm samples with relatively lower survivability. Na+/K+-ATPase is an integral membrane protein and involved with the regulation of rodent sperm motility. To make further modification of the medium, we examined effects of Na+/K+-ATPase inhibition with digoxin on motility, full-type hyperactivation and protein tyrosine phosphorylation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades and also performed the immunodetection of bovine sperm Na+/K+-ATPase. The addition of Na+/K+-ATPase inhibitor digoxin to the incubation medium containing cBiMPS and calyculin A had the tendency to lessen the decreases in the percentages of motile spermatozoa in all of 12 samples after the incubation for 1-3 h and significantly increased the percentages of full-type hyperactivation in one group of 4 samples (Sample-A1) and another group of 4 samples (Sample-A2) after 1 and 2 h respectively, though it had no significant effects on full-type hyperactivation in the other group of 4 samples (Sample-B). In addition, incubation time-related changes in the sperm protein tyrosine phosphorylation (a good marker for sperm capacitation) were correlated with those in the percentages of full-type hyperactivation in Sample-A1 containing digoxin. Immunodetection showed that Na+/K+-ATPase is present in the middle and principal pieces of the flagella, indicating that Na+/K+-ATPase has possible relations with sperm motility. These results obtained with bull ejaculated spermatozoa with relatively lower survivability indicate that incubation method using digoxin is useful to evaluate potentials of sperm samples to exhibit full-type hyperactivation, that digoxin has effects on suppressing reduction of sperm motility, and that prolonged incubation with digoxin induces reduction of capacitation state which may suppress the maintenance of full-type hyperactivation.

Identification of isoforms of calyculin A-sensitive protein phosphatases which suppress full-type hyperactivation in bull ejaculated spermatozoa.

In bull spermatozoa, extracellular Ca2+-dependent full-type hyperactivation, which is characterized by the asymmetrical beating in whole parts of the middle/principal pieces, is suppressed by calyculin A-sensitive protein phosphatases. The aim of this study was to identify isoforms of these protein phosphatases. Ejaculated spermatozoa were used for the investigation on effects of protein phosphatase inhibitors (calyculin A with high specificity for both of protein phosphatases 1 and 2A, and okadaic acid with relatively higher specificity for protein phosphatase 2A than protein phosphatase 1) on the induction of extracellular Ca2+-dependent full-type hyperactivation by incubation with CaCl2 and cAMP analog (cBiMPS). They were also used for the immunodetection of protein phosphatases 1α, 1ß, 1γ, 2Aα and 2Aß. Percentages of full-type hyperactivated spermatozoa significantly increased after incubation with calyculin A (10 nM) in a concentration-dependent manner of CaCl2 (0-3.42 mM), though only minor increases in the percentages of full-type hyperactivated spermatozoa were observed after incubation with okadaic acid (10 nM). Moreover, the immunodetection of protein phosphatase isoforms showed sperm connecting piece and flagellum included protein phosphatases 1α and 1γ, but did not do the other isoforms. These results suggest that calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (1α and 1γ) are suppressors for the extracellular Ca2+-dependent full-type hyperactivation in bull ejaculated spermatozoa.

Flagellar hyperactivation of bull and boar spermatozoa.

BACKGROUND: In mammals, flagellar hyperactivation is indispensable to sperm fertilization with oocytes in vivo, although there are species differences in regulatory mechanisms for this event. In this study, I reviewed researches regarding hyperactivation of bull and boar spermatozoa, in comparison with those of spermatozoa from other species. METHODS: Recent publications regarding sperm hyperactivation were collected and summarized. RESULTS MAIN FINDINGS: In bull and boar spermatozoa, there are two types of hyperactivation "full-type hyperactivation and nonfull-type hyperactivation" which are equivalent to anti-hock hyperactivation and pro-hock hyperactivation of mouse spermatozoa, respectively, on the basis of the flagellar parts exhibiting asymmetrical beating. Full-type hyperactivation is initiated in response to a rapid increase of cytoplasmic Ca2+ in the connecting/middle and principal pieces by the mobilization of this divalent ion from extracellular space and internal store through cation channels. Regulatory molecules for the increase of cytoplasmic Ca2+ in the connecting/middle pieces are probably different from those in the principal pieces. CONCLUSION: I have proposed a hypothesis on the regulation of full-type hyperactivation by the distinct signaling cascades leading to the increase in cytoplasmic Ca2+ between the connecting/middle and principal pieces of bull and boar spermatozoa.

Reconsideration of the evaluation criteria for bull ejaculated sperm motility in the context of rotation.

Progressive movement of spermatozoa has conventionally been regarded as a good indicator of motility. However, bull spermatozoa exhibit two types of progressive movement: progressive/planar movement without rotation and progressive/helical movement with rotation. The aim of this study was to reconsider the evaluation criteria of bull ejaculated sperm motility in the context of rotation. Here, we compared the movement patterns of ejaculated spermatozoa with relatively high and low protein kinase A (PKA)-mediated signaling activities, because sperm motility is positively regulated by PKA-mediated signaling activities. We prepared sperm samples with high and low PKA-mediated signaling activities by suspending spermatozoa in media containing either the stimulator (NaHCO3) or inhibitor (KH-7) of adenylyl cyclase 10, and we then investigated movement patterns and relative velocities using a microscopic high-speed camera and recording system. In the control medium without NaHCO3 and KH-7, most spermatozoa exhibited round/planar movement without rotation and asymmetrical bends in the principal pieces. NaHCO3 significantly promoted changes in movement patterns from round/planar movement to progressive/planar movement (without rotation) as well as symmetrization of flagellar bends and increased relative velocities. KH-7 significantly increased spermatozoa exhibiting progressive/helical movement (with rotation), decreased relative velocities, and symmetrized flagellar bends with a reduction in their size. These indicate that progressive/planar movement (without rotation) and fast movement characterize the movement patterns of bull ejaculated spermatozoa with high PKA-mediated signaling activities. A sign of reduced PKA-mediated signaling activity is not only slow movement but also helical movement (with rotation). Thus, it is beneficial to add a new parameter of "rotation" to the evaluation criteria of bull ejaculated sperm motility.

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa.

Background: Although artificial insemination (AI) technique is an established biotechnology for bovine reproduction, the results of AI (conception rates) have a tendency to decline gradually. To our annoyance, moreover, AI-subfertile bulls have been occasionally found in the AI centers. To resolve these serious problems, it is necessary to control the sperm quality more strictly by the examinations of sperm molecules. Methods: We reviewed a number of recent articles regarding potentials of bovine sperm proteins as the biomarkers for bull AI-subfertility and also showed our unpublished supplemental data on the bull AI-subfertility associated proteins. Main findings: Bull AI-subfertility is caused by the deficiency or dysfunctions of various molecules including regulatory proteins of ATP synthesis, acrosomal proteins, nuclear proteins, capacitation-related proteins and seminal plasma proteins. Conclusion: In order to control the bovine sperm quality more strictly by the molecular examinations, it is necessary to select suitable sperm protein biomarkers for the male reproductive problems which happen in the AI centers.

Characterization of extracellular Ca2+ -dependent full-type hyperactivation in ejaculated boar spermatozoa preincubated with a cAMP analog.

Ejaculated boar spermatozoa exhibit two types of hyperactivation: full and non-full. Full-type hyperactivation is characterized by the asymmetrical bending of the entire middle piece-principal piece and a twisting/figure-eight-like trajectory, and can be induced by simple incubation with CaCl2 after preincubation with a cAMP analog (Sp-5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole-3',5'-cyclic monophosphorothioate [cBiMPS]). Here, we compared the sperm flagellar motility after treatments with elevators of [Ca2+ ]i (cBiMPS/CaCl2 , thimerosal, procaine, and 4-aminopyridine) to characterize the regulatory mechanism of extracellular Ca2+ -dependent, full-type hyperactivation in ejaculated boar spermatozoa, and examined the possible involvement of Transient receptor potential cation channel subfamily C member 3 (TRPC3) in this event using the specific inhibitor Pyr3. Full-type hyperactivation was induced by a 60-min incubation with CaCl2 following a 180-min preincubation with cBiMPS but without Ca2+ . Thimerosal-treated spermatozoa exhibited full-type hyperactivation in a manner independent of extracellular Ca2+ ; conversely, this was not observed in procaine- or 4-aminopyridine-treated spermatozoa. A 20-min treatment with Pyr3 between preincubation with cBiMPS and incubation with CaCl2 , significantly suppressed the normal phenotype. These observations indicated that mechanisms underlying full-type hyperactivation in spermatozoa incubated with CaCl2 after preincubation with cBiMPS are different from those in the thimerosal-treated spermatozoa. Furthermore, indirect immunofluorescence localized TRPC3 in the upper segment of the middle piece, which bends asymmetrically during full-type hyperactivation but not in non-full-type hyperactivation, suggesting that TRPC3 may be involved in the extracellular Ca2+ -dependent full-type hyperactivation in ejaculated boar spermatozoa.

Variation among individual bulls in the distribution of acrosomal tyrosine-phosphorylated proteins in epididymal and ejaculated spermatozoa.

In Japanese black cattle, AI severely subfertile males have occasionally been found. In order to solve this problem, we previously asserted the need for exact examinations of acrosomal tyrosine-phosphorylated proteins and acrosome morphology in cryopreserved spermatozoa. In the present study, we further investigated acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation and examined possible relationships between these phosphoproteins and acrosome stability. Ejaculated, epididymal and cryopreserved spermatozoa were subjected to examinations of general characteristics (motility, shape and acrosome morphology) and indirect immunofluorescence of acrosomal phosphoproteins. Unlike all general characteristic parameters, the distribution of acrosomal tyrosine-phosphorylated proteins in ejaculated and cauda epididymal spermatozoa varied considerably among bulls and was linked to the maintenance of morphologically normal acrosomes in cryopreserved spermatozoa or ejaculated spermatozoa after 270min incubation. Moreover, the distribution of these phosphoproteins was arranged in the spermatozoa of the proximal epididymides. These findings indicate that acrosomal tyrosine-phosphorylated proteins are distributionally arranged during early process of sperm maturation, that their distribution of cauda epididymal and ejaculated spermatozoa are largely different among bulls, and that varied states of acrosomal phosphoproteins may result in individual differences in acrosome stability among bulls.

Changes of IZUMO1 in bull spermatozoa during the maturation, acrosome reaction, and cryopreservation.

To obtain basic information of bull IZUMO1 (a sperm protein essential for sperm-egg fusion) and disclose possible causes for the impaired fertilizing ability in bull cryopreserved spermatozoa, we investigated this protein in bull spermatozoa collected from various regions of epididymides, freshly ejaculated spermatozoa, acrosome-reacted spermatozoa, and cryopreserved spermatozoa by Western blotting and the triple staining with the anti-IZUMO1 antibody, fluorescein isothiocyanate-peanut agglutinin, and 4',6-diamidino-2-phenylindole. In the cauda epididymal spermatozoa and freshly ejaculated spermatozoa, bull IZUMO1 was detected mainly as a 45-kDa major form. This major form was derived probably from a 52-kDa precursor form in the epididymis. Bull IZUMO1 was immunolocalized along the border between the principal and equatorial segments of the acrosomal region (pattern P1 of IZUMO1) in the most of epididymal and freshly ejaculated spermatozoa with normal acrosomes. In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMO1 in whole equatorial segment (pattern P2 of IZUMO1) significantly increased. These results indicate that bull IZUMO1 undergoes maturation-related changes during sperm transit through the epididymis and that it is translocated to the equatorial segment of acrosomal region during the acrosome reaction. On the other hand, severe damages were observed in the acrosomes of 60% of the cryopreserved spermatozoa. Localization of IZUMO1 in these spermatozoa was pattern P2 (IZUMO1 in whole equatorial segment), P3 (IZUMO1 in whole acrosomal region), or P4 (IZUMO was lost). Moreover, after the incubation to compare the stability of acrosomes and IZUMO1 localization between cryopreserved spermatozoa and freshly ejaculated spermatozoa, much more spermatozoa lost acrosomes and IZUMO1 in the cryopreserved samples compared with freshly ejaculated samples. These findings indicate that impaired fertilizing ability of bull cryopreserved spermatozoa with damaged acrosomes is related partially to the aberrant translocation of IZUMO1 which may be followed by the loss of intact IZUMO1.

Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction.

The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions.

Involvement of cAMP-dependent unique signaling cascades in the decrease of serine/threonine-phosphorylated proteins in boar sperm head.

We previously suggested that protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins may be necessary for the occurrence of acrosome reaction in livestock spermatozoa (Adachi et al., J Reprod Dev 54, 171-176, 2008; Mizuno et al., Mol Reprod Dev 82, 232-250, 2015). The aim of this study was to examine the involvement of the intracellular cAMP signaling cascades in the regulation of the decrease of postacrosomal phosphorylated proteins in boar spermatozoa. Boar ejaculated spermatozoa were incubated with cAMP analogs and then used for the immunodetection of serine/threonine-phosphorylated proteins and assessment of acrosome morphology. The protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins was greatly promoted by the incubation with a cAMP analog Sp-5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS). This decrease was induced before the initiation of acrosome reaction and did not require the millimolar concentration of extracellular Ca(2+) which was necessary for the initiation of acrosome reaction. Moreover, suppression of protein kinase A activity with an inhibitor (H89) had almost no influence on both decrease of phosphorylated proteins and occurrence of acrosome reaction in the spermatozoa incubated with cBiMPS. In addition, the prolonged incubation with a potentially exchange protein directly activated by cAMP-selective cAMP analog (8pM) could only partially mimic effects of cBiMPS on these events. These results indicate that the cAMP-dependent signaling cascades which are less dependent on protein kinase A may regulate the decrease of postacrosomal phosphorylated proteins in boar spermatozoa before the extracellular Ca(2+)-triggered initiation of acrosome reaction.

Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle.

The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.

Distinct segment-specific functions of calyculin A-sensitive protein phosphatases in the regulation of cAMP-triggered events in ejaculated bull spermatozoa.

Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered events-including capacitation-associated changes-than laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of calyculin A. Based on these results, we suggest that calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa.

Expression patterns of the activator type of cAMP-responsive element modulator in testicular germ cells of Japanese Black bulls.

The characterization and quantitative analyses of the key transcription factors for spermiogenesis are necessary in the identification of causal factors for the production of the seemingly normal sperm with dysfunctions in Japanese Black bulls and further elucidation of whole aspect of molecular mechanisms for spermiogenesis in livestock. The objective of this study was to obtain the information regarding the characterization and individual changes of an activator cAMP-responsive element modulator (CREM), which is necessary to the normal progress of spermiogenesis and is required for the transcriptional activity of genes coding essential factors for the sperm fertilization ability in rodents, using testes from 21 Japanese Black bulls with the ability to produce sperm indicating the normal motility and morphology. The bull CREM ταγ (one of activator variants) was detected in testes more strongly than livers by reverse transcription-polymerase chain reaction and Northern blotting. This variant was localized in the nuclei of spermatids as shown by indirect immunofluorescence with the homemade mouse antiserum. The motility and morphology of the cauda epididymal sperm from 16 Japanese Black bulls were examined before the quantitative analyses of testicular activator CREM to confirm the ability to produce sperm with normal motility and morphology in these males. The percentages of the motile sperm, those of the sperm with the normal acrosomes, and those of morphologically normal sperm were 60.0% to 90.0%, 88.0% to 100%, and 83.0% to 97.9%, respectively. The quantitative analyses with real-time polymerase chain reaction using the testicular RNA from the same bulls revealed that the relative expression levels of activator CREM variants in testes varied significantly among these bulls in the range from 0.56 to 1.64 (P < 0.05). These results are consistent with the suggestions that CREM ταγ are involved in the spermiogenesis in the testes of Japanese Black bulls and that the expression levels of the activator CREM variant mRNAs in the testes are varied significantly among individual bulls that have the ability to produce sperm with the normal motility and morphology.

Roles of intracellular cyclic AMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.

It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca²âº in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.

Novel approach for the detection of the vestiges of testicular mRNA splicing errors in mature spermatozoa of Japanese Black bulls.

There is a serious problem with the reduction of male reproductive performance of the livestock in the world. We have a hypothesis that the splicing error-caused derivation of aberrant sperm motility-related proteins may be one of its causal factors. It is thought that fresh testicular tissues are necessary for the detection of splicing errors of the mRNA. However, it is difficult to obtain testicular tissues from a number of agriculturally important bulls by surgical methods, because such procedures may have deleterious effects on bulls' reproductive performance. The aim of this study was to examine the usefulness of mRNA fragments collected from ejaculated spermatozoa as alternative analytical samples for detection of the splicing errors. In the first experiment, we characterized the alternative splicing and splicing error of bull testicular ADCY10 mRNA which coded the synthase of the regulatory molecule for sperm motility "cAMP". In testes, the exon 11-lacking variant coding the truncated ADCY10 was derived by alternative splicing. However, splicing errors, which accompanied the frame shift in the second cyclase domain, were occasionally observed in the exon 11-lacking variant. This aberrant variant retained intronic nucleotides (4 bases, CCAG) connecting the initial part of exon 10 due to splicing errors and consequently yielded the cleavage site for a restriction enzyme (Cac8I) which recognized the nucleotide sequences (GCNNGC). In the second experiment, we recovered residual testicular mRNA fragments from ejaculated spermatozoa and observed the splicing error-caused derivation of the aberrant variant of ADCY 10. Ejaculated spermatozoa conserved mRNA fragments of the exon 11-lacking variant coding exons 9, 10, 12 and 13. Moreover, the above-mentioned aberrant variant of ADCY10 mRNA fragment was detectable by Cac8I digestion treatment using the sperm mRNAs. These results indicate the utility of sperm mRNA fragments for the detection of splicing errors in bull testicular mRNAs.