Viability and Protein Phosphorylation Patterns of Boar Spermatozoa Agglutinated by Treatment With a Cell‐Permeable Cyclic Adenosine 3′,5′‐Monophosphate Analog

Abstract

Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in the head. The aim of the present study is to examine viability and protein phosphorylation patterns of cAMP-dependently agglutinated boar spermatozoa. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, a cell-permeable cAMP analog) at 38.5 degrees C up to 180 minutes. Aliquots of the sperm suspensions were recovered after various incubation periods and then used to examine the state of agglutination, the viability by SYBR14-PI staining and motility assay, and the state of protein phosphorylation by Western blotting and indirect immunofluorescence. In the control samples incubated without cBiMPS for 180 minutes, less than 30% of the total spermatozoa were agglutinated with one another at the heads, and more than 70% of the agglutinated spermatozoa were propidium iodide (PI)-positive (dead). However, the incubation with cBiMPS rapidly increased the percentages of head-to-head agglutinated spermatozoa to approximately 60% within 30 minutes, but did not significantly change them thereafter. In the samples incubated with cBiMPS for 180 minutes, moreover, the percentages of PI-positive cells of the agglutinated spermatozoa (approximately 30%) were significantly lower than those obtained in the control samples (more than 70%). This result was supported by the observation that the percentages of motile cells of the agglutinated spermatozoa were much higher in the samples incubated with cBiMPS for 180 minutes than in the control samples incubated without cBiMPS. As revealed by Western blotting and indirect immunofluorescence, cBiMPS-induced serine/threonine phosphorylation of the proteins (eg, >220 kd, 220 kd, 180 kd, 84 kd, and 54 kd) appeared mainly in the connecting and principal pieces of both agglutinated and free spermatozoa within 30 minutes, and additional phosphorylation occurred in the middle piece later than 30 minutes. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kd, 190 kd, 93 kd, 59 kd, 54 kd, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. These findings are consistent with the following suggestions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; and slow (later than 30 minutes) protein tyrosine phosphorylation in th...

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

“…After incubating in capacitating conditions, we detected a quantitative increase in the intensity of several bands, which indicates that ram sperm capacitation is also associated with protein Ser/Thr phosphorylation (Fig. 2) as occurs in other species such as humans (Naz 1999, O'Flaherty et al 2004, hamsters ( Jha & Shivaji 2002b), boars (Harayama 2003, Harrison 2004 and mice ( Jha et al 2006). All controls carried out in this study proved signal specificity.…”

“…2A and C). These observations would suggest that ram spermatozoa might possess dual specificity kinases that would phosphorylate in more than one residue, as already described in other mammalian species such as humans (Naz 1999, Thundathil et al 2002, Ficarro et al 2003, hamsters ( Jha & Shivaji 2002b) and boars (Harayama 2003, Harrison 2004. Acrosomal exocytosis induced by calcium ionophore A23187 has been used in ram sperm to investigate the signaling pathways involved in this process (Roldan & Fragio 1993, Garde & Roldan 2000.…”

“…Although very little is known about serine/threonine phosphorylation, several studies have shown a timedependent increase in serine and/or threonine phosphorylation during sperm capacitation in species such as humans (Naz 1999, O'Flaherty et al 2004, hamsters ( Jha & Shivaji 2002b), boars (Harayama 2003) and mice ( Jha et al 2006). Besides the well-characterized protein kinase A (PKA), other Ser/Thr kinases and the ERKs have been involved in sperm capacitation and acrosome reaction (Luconi et al 1998, du Plessis et al 2001, Liguori et al 2005.…”

Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

“…The cell-permeable cAMP analog cBiMPS is a powerful activator of PKA [36] and could enhance the Ser/Thr phosphorylation states of AGC family kinase protein substrates in the principal pieces of mouse spermatozoa (Fig. 1), as has been shown in boar spermatozoa [43]. This analog was also able to change the flagellar movement from the progressive type to the hyperactivation-like type (Fig.…”

“…However, there are only limited data regarding cAMP-dependent Ser/Thr phosphorylation in sperm proteins (chum salmon [42], boar [43,44], sea urchin [45], human [46] and mouse [47]). In this study, incubation with cBiMPS (0.1 mM) induced Ser/Thr phosphorylation of the sperm flagellar proteins, including the >250-kDa, 170-kDa, 155-kDa, 140-kDa and 42-kDa substrate proteins of AGC family kinases (Fig.…”