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

Abstract

Abstract. Protein serine/threonine phosphorylation in mammalian sperm flagella has been considered to play important roles in regulation of motility. Protein phosphorylation state reflects balance of enzymatic activities between protein phosphatases and protein kinases [predominantly protein kinase A (PKA)]. The aims of this study were to disclose roles of protein phosphatases in the regulation of sperm motility and to provide evidence for suppression of PKA full activation by protein phosphatases in sperm flagella. Mouse epididymal spermatozoa were incubated with a cell-permeable protein phosphatase 1 (PP1)/protein phosphatase 2A (PP2A) inhibitor (calyculin A: 25-125 nM) at 37.5 C. After incubation, they were used for immunodetection of phosphorylated proteins, PKA and PP1γ2, assessment for motility and co-immunoprecipitation of PP1γ2 with PKA. Incubation with calyculin A enhanced the phosphorylation states of several proteins (>250 kDa, 170 kDa, 155 kDa, 140 kDa and 42 kDa for serine/threonine phosphorylation and 70 kDa for tyrosine phosphorylation) and PKA catalytic subunits [at the autophosphorylation residue (Thr-197) for its full enzymatic activation] in the flagella. Coincidently, this incubation induced changes of sperm flagellar movement from the progressive type to the hyperactivation-like type. Indirect immunofluorescence and co-immunoprecipitation showed that PKA was co-localized with PP1γ2 in the principal pieces of sperm flagella. These findings suggest that calyculin A-sensitive protein phosphatases (PP1/PP2A) suppress full activation of PKA as well as enhancement of the phosphorylation states of other flagellar proteins in sperm flagella in order to prevent precocious changes of flagellar movement from the progressive type to hyperactivation. Key words: Cyclic adenosine 3',5'-monophosphate (cAMP), Hyperactivation, Protein kinase, Protein phosphatase 1 (PP1), Protein phosphorylation (J. Reprod. Dev. 55: [327][328][329][330][331][332][333][334] 2009) ammalian spermatozoa are differentiated from spermatogonia in the testicular seminiferous tubules and then transferred through the duct of the epididymis. By arrival in the caudal epididymides, they have acquired the potentials to move in a forward direction, bind with the zona pellucida and fertilize oocytes [1], but they are temporally quieted in the epididymis by the influence of the acidic-base status of the luminal fluid [2,3] and interaction with membrane stabilizing factors [4,5]. After ejaculation into the female reproductive tract, the spermatozoa quickly initiate flagellar beating, which is regulated by the protein serine [Ser (S)]/threonine [Thr (T)] phosphorylation in the flagella [6]. Some of the phosphorylated proteins have previously been identified as dynein [6,7] and axokinin [8]. Subsequently, they gradually undergo a series of changes in their intracellular spaces as well as on their cell surfaces (capacitation), including dissociation of decapacitation factors (e.g., cholesterol and acrosomal stabilizing factors), elevation of th...

Regulation of hyperactivation by PPP2 in hamster spermatozoa

“…Several studies have reported that PPP1CC2 is involved in the regulation of motility activation and hyperactivation in mammalian sperm (Smith et al 1996, Huang et al 2005, Chakrabarti et al 2007, Goto & Harayama 2009). On the other hand, it has been reported that PPP3 is also associated with the regulation of sperm motility and acrosome reaction (Tash et al 1988, Tash & Bracho 1994, Ashizawa et al 2004.…”

“…Previous studies (Tash et al 1988, Tash & Bracho 1994, Smith et al 1996, Si & Okuno 1999b, Ashizawa et al 2004, Huang et al 2005, Chakrabarti et al 2007, Goto & Harayama 2009) suggested that PPP1, PPP2, and PPP3 were associated with the regulation of sperm motility and hyperactivation. Moreover, PPP1CC was detected in sperm (Smith et al 1996, Huang et al 2005, Chakrabarti et al 2007, Goto & Harayama 2009).…”

“…Moreover, PPP1CC was detected in sperm (Smith et al 1996, Huang et al 2005, Chakrabarti et al 2007, Goto & Harayama 2009). At first, we examined which PPPs are present in hamster sperm.…”

“…On the other hand, it has been suggested that protein dephosphorylation is also associated with the regulation of hyperactivation (Fujinoki et al 2006). It has been suggested that inhibitors of protein phosphatase 1/2A (PPP1/PPP2), which are okadaic acid and calyculin A, enhanced hyperactivation and tyrosine phosphorylation in rodent sperm (Si & Okuno 1999b, Goto & Harayama 2009). Moreover, Furuya et al (1992Furuya et al ( , 1993 reported that okadaic acid induced rapid acrosome reaction of mouse sperm, and that calyculin A induced rapid acrosome reaction and enhanced phosphorylation of phosphoproteins of human sperm.…”

Ppp2ca knockout in mice spermatogenesis

“…Previous studies have specifically linked dysregulation of the catalytic subunit with several pathologies, including Alzheimer's disease, cancer, diabetes, and sperm capacitation. For example, SFK-mediated PP2AC phosphorylation inactivates PP2A and leads to mammalian sperm capacitation (Goto & Harayama 2009, Krapf et al 2010.…”