A 32-kDa Tyrosine-phosphorylated Protein Shows a Protease-dependent Increase in Dead Boar Spermatozoa

Abstract

Abstract. 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. Key words: Acrosome, Boar, Peanut agglutinin (PNA), Protease, Sperm, Tyrosine (J. Reprod. Dev. 54: [502][503][504][505][506][507] 2008) n mammalian spermatozoa, a variety of tyrosine-phosphorylated proteins increase during incubation under capacitationsupporting conditions. These proteins have been considered to be involved in regulation of fertilization-related events [1,2]. It is generally believed that the increase in tyrosine-phosphorylated proteins results from activation of protein tyrosine kinases and inactivation of protein tyrosine phosphatases that are controlled by cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) signaling [1,[3][4][5]. However, a cAMP-dependent increase in 32-kDa tyrosine-phosphorylated protein (called TyrP32 or p32) in boar spermatozoa is exceptionally suppressed by inhibition of tyrosine phosphatases and barely affected by inhibition of protein tyrosine kinases [6]. This tyrosine-phosphorylated protein is primarily found as a capacitation-related protein [7][8][9] and has recently been identified as a (pro)acrosin-binding protein [10,11]. We have previously shown that the cAMP-dependent increase in TyrP32 is greater in samples containing many spermatozoa that undergo calcium-dependent changes in acrosomal morphology (acrosome reaction) ...

The calcium‐sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa

“…Finally, in the last experiment we wanted to determine if p32 PY could be related to increased serine protease activity. However, the serine protease inhibitor, PMSF did not abolish the p18/p32 PY induced by NPS2143 or GSK2334470 (Figure ), indicating that the increase observed in p32 PY was not due to enhanced protease activity, as previously reported in frozen‐thawed boar spermatozoa (Tabuchi et al, ).…”

The addition of calcium ion chelator, EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

“…Although several researchers reported similar results in frozen–thawed sperm, including pig (Bravo et al . 2005; Tabuchi et al . 2008), sheep (Pérez‐Pé et al .…”

Voltage-dependent anion channels are a key factor of male fertility

“…The sperm were then fixed with 3.7% paraformaldehyde for 30 minutes at 4 C. After fixation, the cells were washed with Dulbecco's phosphate-buffered saline (DPBS) containing 0.1% Tween 20 (PBS-T) and blocked for 1 hour in blocking solution (5% BSA in PBS-T) at 4 C. The slide was then incubated with rabbit polyclonal primary antibodies for VDAC2 and VDAC3 IgG (Abcam) diluted with blocking solution 1:50, lectin PNA (34,35) conjugates Alexa Fluor 647 (Molecular Probes) diluted with blocking solution 1:100, or mouse polyclonal to a-tubulin IgG primary antibody (Abcam) diluted with blocking solution 1:100 overnight at 4 C. After thorough washing in DPBS, the slides were incubated for 2 hours at room temperature with fluorescein isothiocyanate conjugated goat polyclonal to rabbit IgG secondary antibody (Abcam) diluted with blocking solution 1:100 and mouse IgG secondary antibody (Abcam) diluted with blocking solution 1:100. Sperm were counterstained with DAPI.…”