Relationship between cyclic AMP-dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa.

In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)-protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP-induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP-induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) and CaCl(2) at 38.5°C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl(2) were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS-induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50-100 nM) coincidently promoted hyperactivation and cAMP-induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl(2). Moreover, the addition of W-7 (a calmodulin antagonist, 2-4 µM) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl(2), independently of protein tyrosine phosphorylation. These findings indicate that cAMP-induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP-induced protein tyrosine phosphorylation.

Daily intake of heat-killed Lactobacillus plantarum L-137 enhances type I interferon production in healthy humans and pigs.

We have previously reported that oral administration of heat-killed Lactobacillus plantarum L-137 (HK L-137) stimulates innate immunity for production of type I interferon (IFN) which subsequently augments host defense against influenza A virus infection in mice. We here examined the effect of HK L-137 intake on type I IFN in humans. Sixteen subjects were randomly assigned to receive a tablet containing 10 mg of HK L-137 or a matching tablet for 8 weeks and the serum levels of type I IFN were examined before and after the first or second dose of the trivalent inactivated influenza vaccine. There were no differences in the seroresponse rate, the seroprotection rate and the geometric mean Ab titers after either the first or second dose of vaccine between the HK L-137 group and the control group. On the other hand, the levels of IFN-ß were significantly higher in the HK L-137 group than in the control group before vaccination although the vaccination conferred little additional induction of IFN-ß. We further examined IFN-ß gene expression in the whole blood cells of pigs fed on a diet containing HK L-137 and found that the IFN-ß mRNA levels were significantly higher in the HK L-137 group than in the control group. The finding that daily intake of HK L-137 enhances type I IFN production and host defense against influenza A virus infection in mice may be applied to at least two additional species.

A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa.

Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation.