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.

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.

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.

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls.

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen-stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell-permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine-phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen-stored spermatozoa were classified as having a high-grade acrosome condition before incubation, sperm tyrosine-phosphorylated proteins, including the 33-kDa tyrosine-phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41- and 33-kDa sperm tyrosine-phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine-phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen-stored sperm were classified as having a low-grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine-phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine-phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP-dependent hyperactivation for the spermatozoa of individual Japanese Black bulls.

Ovsynch plus CIDR protocol for timed embryo transfer in suckled postpartum Japanese Black beef cows.

We compared synchronization and pregnancy rates, and the increase in blood progesterone concentrations during luteal development, between (1) Ovsynch plus an intravaginal controlled internal drug release (CIDR) device protocol followed by timed embryo transfer (timed ET), and (2) a conventional estrus synchronization method using PGF(2 alpha) and ET in suckled postpartum Japanese Black beef cows. Cows in the PGF group (n=18) received a PGF(2 alpha) analogue when a CL was first palpated per rectum at 10-d intervals after 1 to 2 month postpartum. Cows (n=11), which showed estrus (Day 0) within 5 d of the PGF(2 alpha), and had a CL on Day 7, received ET. Cows in the Ovsynch+CIDR group (n=19) underwent the Ovsynch protocol plus a CIDR for 7 d (GnRH analogue and CIDR on Day-9, PGF(2alpha) analogue with CIDR removal on Day-2, and GnRH analogue on Day 0), with ET on Day 7. The ovulation synchronization (100%) and embryo transfer (100%) rates in the Ovsynch+CIDR group were greater (P<0.01) than the estrus synchronization (66.7%) and the embryo transfer (61.1%) rates in the PGF group. The postpartum interval at ET in the Ovsynch+CIDR group (62.5 +/- 2.5 d) was shorter (P<0.01) than in the PGF group (74.9 +/- 3.9 d). The pregnancy rate in the Ovsynch+CIDR group (57.9%) did not differ significantly from that in the PGF group (50.0%). Plasma progesterone concentrations were not significantly different in the two groups on Days 0, 1, 2, 5, 7, 14 and 21. In summary, higher synchronization and transfer rates, and shorter postpartum interval to ET, can be achieved with timed ET following the Ovsynch plus CIDR protocol than after estrus with the single PGF(2 alpha) treatment followed by ET in suckled postpartum recipient beef cows. Pregnancy rates were similar. Also, the increase in blood progesterone concentrations during luteal development following ovulation synchronized by the Ovsynch plus CIDR protocol was similar to that after estrus induced by the PGF(2 alpha) treatment.

Inhibitory effects of CIDR-based ovulation-synchronization protocols on uterine PGF2alpha secretion at the following luteal phase in early postpartum non-cycling beef cows.

We investigated whether CIDR-based ovulation-synchronization protocols inhibit secretion of prostaglandin (PG) F2alpha from the uterus in the following luteal phase in non-cycling beef cows. Ten early (a month) postpartum non-cycling Japanese Black beef cows were treated with (1) Ovsynch (GnRH analogue on Day 0, PGF2alpha analogue on Day 7, and GnRH analogue on Day 9; n=3), (2) Ovsynch+CIDR (Ovsynch protocol plus a CIDR for 7 days from Day 0; n=4), or (3) estradiol benzoate (EB) Ovsynch+CIDR (EB on Day 0 in lieu of the first GnRH treatment followed by the Ovsynch+CIDR protocol; n=3). An oxytocin challenge was administered on Day 24 to examine uterine PGF2alpha secretion. Plasma concentrations of 13,14-dihydro-15-keto- PGF2alpha were lower at 30-120 min after oxytocin administration in the Ovsynch+CIDR group and 75 min after administration in the EB Ovsynch+CIDR group than in the Ovsynch group (P<0.05). Plasma progesterone concentrations were higher from Days 1 to 7 in the Ovsynch+CIDR group and from Days 1 to 5 in the EB Ovsynch+CIDR group than in the Ovsynch group (P<0.05). The progesterone concentrations were higher on Days 27 and 29 in both CIDR-treated groups than in the Ovsynch group (P<0.05). In conclusion, in non-cycling beef cows, CIDR-based ovulation-synchronization protocols inhibit uterine PGF2alpha secretion in the following luteal phase and prevent premature luteolysis as is seen with the Ovsynch protocol.

Relations between plasma IGF-I concentrations during treatment with CIDR-based or Ovsynch protocol for timed AI and conception in early postpartum Japanese black beef cows.

We examined the relations between plasma insulin-like growth factor (IGF) -I concentrations during treatment with CIDR-based or Ovsynch protocol for timed AI and conception and plasma steroid concentrations in early postpartum Japanese Black beef cows. Cows in the control group (Ovsynch; n = 21) underwent Ovsynch protocol (GnRH analogue on Day 0, PGF(2alpha) analogue on Day 7, and GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the Ovsynch+CIDR group (n = 22) received Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Cows in the further treatment group (EB+CIDR+GnRH; n = 22) received 2 mg of estradiol benzoate (EB) on Day 0 in lieu of the first GnRH treatment, followed by the same treatment as in the Ovsynch+CIDR protocol. Plasma IGF-I concentrations were determined on Days -7, 0, 7, 9 and 17. Conception rates were improved in the CIDR-combined groups (both CIDR-treated groups were combined) relative to Ovsynch group (P < 0.05) for cows with low IGF-I concentrations (<1,000 ng/ml) on Days -7, 0, and 7, but improved conception rate produced by the CIDR-based protocols did not occur in cows with a high IGF-I concentration (> or =1,000 ng/ml). Plasma estradiol-17beta concentrations increased from Day 0 to 7 (P < 0.05) and were unchanged from Day 7 to 9 in the Ovsynch group with low IGF-I concentrations on Day 0, while they were unchanged from Day 0 to 7 and increased from Day 7 to 9 (P < 0.05) in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group. Plasma progesterone concentrations in the Ovsynch group with low IGF-I concentrations on Day 0 were higher on Day 14 than in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group (P < 0.05). In conclusion, CIDR-based protocols may improve conception relative to Ovsynch in early postpartum beef cows with lower plasma IGF-I concentrations at the start of the protocols. This improvement is probably due to prevention of premature increases of estradiol-17beta and progesterone concentrations, which occurred in cows with low IGF-I concentrations treated with Ovsynch, by the CIDR treatment.