Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phosphoCaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.

Section: Cmentioning

confidence: 99%

PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade

Rotfeld¹,

Hillman²,

Ickowicz³

et al. 2014

“…Semen obtained from two to three males was suspended in Hanks' balanced salt solution (HBSS) containing 1.25 mmol/l of CaCl 2 and 0.1% (v/v) of proctodeal gland secretion. We decided to include CaCl 2 in the sperm extender because it is reported that the induction of AR by homogenized PL was almost negligible in the absence of Ca 2C (Ashizawa et al 2006). We added proctodeal gland secretion into the incubation mixture since it inhibits an agglutination of quail spermatozoa.…”

Section: Semen Collection and Preparationmentioning

confidence: 99%

Induction of sperm acrosome reaction by perivitelline membrane glycoprotein ZP1 in Japanese quail (Coturnix japonica)

Sasanami

Murata²,

Ohtsuki³

et al. 2007

The extracellular matrix surrounding avian oocytes, called the perivitelline membrane (PL), consists of at least two major glycoproteins, ZP3 and ZP1. Our previous study using Japanese quail had demonstrated that the PL obtained from the preovulatory follicles was incubated in vitro with spermatozoa, and perforations were observed. This result indicated that the PL might contain a constituent that possesses activity to initiate the acrosome reaction (AR) in quail. In order to elaborate upon our previous findings, we evaluated the effects of ZP3 and ZP1 on the induction of sperm AR in Japanese quail. Ejaculated sperm were incubated with or without the purified PL glycoprotein, and their acrosome status was determined based on the presence or absence of the acrosome. Treatment of spermatozoa with increasing doses of the purified monomeric ZP1 led to a concentration-dependent stimulation of AR. The purified dimeric ZP1 had similar effect. Moreover, we found that the ZP1-induced AR was significantly blocked by the digestion of the PL protein with PNGaseF. In contrast, the addition of purified ZP3 failed to induce AR at any doses tested. These results indicate that N-linked glycans on ZP1 play an important role in triggering the AR in Japanese quail.

“…mechanism is supported by Guan et al (2007) work that shows a direct activation of the S/T phosphatase PP2A by PRKCA which result in PI3K inhibition by dephosphorylation of PI3K. The S/T protein phosphatases PPP1 and PPP2 are found in sperm cells and participate in the capacitation and acrosome reaction processes (Furuya et al 1993, Ashizawa et al 2006, Chakrabarti et al 2007. Following this information, we hypothesized that the PRKCA-mediated PI3K inhibition during sperm capacitation occurs through PRKCA-dependent activation of PPP1 and/or PPP2.…”

Section: Regulation Of Pi3k In Spermmentioning

confidence: 84%

“…The S/T phosphatases PPP1 and PPP2 are present in sperm cells, and were found to participate in the capacitation and acrosome reaction processes, and sperm motility (Furuya et al 1993, Ashizawa et al 2006, Chakrabarti et al 2007). In the mammalian phylum, four PPP1C isoforms were identified: PPP1CA (PP1ca), PPP1CB (PP1cb), PPP1CC1 (PP1cg1), and PPP1CC2 (PP1cg2), of which the latter two are alternative splice variants of the PPP1CC gene.…”

Section: Introductionmentioning

confidence: 99%

Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

Rotman¹,

Etkovitz²,

Spiegel³

et al. 2010

In order to acquire fertilization competence, spermatozoa have to undergo biochemical changes in the female reproductive tract, known as capacitation. Signaling pathways that take place during the capacitation process are much investigated issue. However, the role and regulation of phosphatidylinositol 3-kinase (PI3K) in this process are still not clear. Previously, we reported that short-time activation of protein kinase A (PRKA, PKA) leads to PI3K activation and protein kinase Ca (PRKCA, PKCa) inhibition. In the present study, we found that during the capacitation PI3K phosphorylation/activation increases. PI3K activation was PRKA dependent, and down-regulated by PRKCA. PRKCA is found to be highly active at the beginning of the capacitation, conditions in which PI3K is not active. Moreover, inhibition of PRKCA causes significant activation of PI3K. Similar activation of PI3K is seen when the phosphatase PPP1 is blocked suggesting that PPP1 regulates PI3K activity. We found that during the capacitation PRKCA and PPP1CC2 (PP1g2) form a complex, and the two enzymes were degraded during the capacitation, suggesting that this degradation enables the activation of PI3K. This degradation is mediated by PRKA, indicating that in addition to the direct activation of PI3K by PRKA, this kinase can enhance PI3K phosphorylation indirectly by enhancing the degradation and inactivation of PRKCA and PPP1CC2.