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.

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.