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It has previously been shown that when boar spermatozoa are incubated in amodified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs inmany cells. The aim of the present study was to investigate the effects ofcyclic adenosine 3′,5′-monophosphate (cAMP) and serum albumin onsperm agglutination and to discuss a possible mechanism for spermagglutination. Spermatozoa were collected from four mature boars, washed andincubated in mKRB. After a 1-h incubation, a sample of each sperm suspensionwas smeared gently on a separate glass slide, dried and stained in aphosphate-buffered solution of Giemsa to assess the percentage of head-to-headagglutinated cells in each suspension. In the samples incubated in mKRB,approximately 50% of the spermatozoa were agglutinated with one anotherat the acrosome. However, the percentages of head-to-head agglutinatedspermatozoa were greatly reduced by a lack of calcium chloride in mKRB, butwere recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) ina dose-dependent manner between 1 and 1000 M . Addition of3-isobutyl-1-methylxanthine (IBMX, 100 and 500 M) instead of dbcAMP alsosignificantly increased the percentages of head-to-head agglutinatedspermatozoa. Moreover, the effects of adding dbcAMP were attenuated bytreatment with Rp-adenosine 3′,5′-cyclic monophosphorothioatetriethylamine salt (0.25-1.0 mM, a cAMP antagonist) or H-89 (5 M, aprotein kinase-A inhibitor), but were enhanced by treatment with okadaic acid(500 nM) and calyculin A (500 nM) (inhibitors of protein serine/threoninephosphatase). In sperm samples incubated in mKRB containing 0.1%polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplementedwith 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in asignificant decrease in the percentages of head-to-head agglutinatedspermatozoa. Addition of porcine serum albumin (PSA, 1-4 mgmL<emph type="7">-1) or methyl-β-cyclodextrin (MBC,5-10 mg mL<emph type="7">-1) instead of BSA was aseffective as BSA (4 mg mL<emph type="7">-1) in enhancing spermagglutination. However, the effects of BSA (4 mgmL<emph type="7">-1) or MBC (5 mgmL<emph type="7">-1) were reduced by pre-mixing these reagentswith cholesterol 3-sulfate (a cholesterol analogue, 5 gmL<emph type="7">-1 for BSA and 375 gmL<emph type="7">-1 for MBC). In addition, a protein‘anti-agglutinin’ inhibiting sperm agglutination, was extractedfrom spermatozoa incubated with serum albumin or MBC and detected by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and Western blottingtechniques. The obtained Western blots revealed that sperm-boundanti-agglutinin was detected less in the samples incubated with either BSA (4mg mL<emph type="7">-1) or MBC (5-10 mgmL<emph type="7">-1), compared with control samples. Moreover,pre-mixing MBC (5 mg mL<emph type="7">-1) with cholesterol3-sulfate (375 g mL<emph type="7">-1) reduced thisreagent’s effects on the loss of sperm-bound anti-agglutinin.Additionally, the assay of sperm agglutination and a chlortetracyclinestaining assay revealed that the percentages of head-to-head agglutinatedspermatozoa were positively correlated with those of spermatozoa classifiedinto B pattern (capacitated spermatozoa). These results are consistent withthe following suggestions: (i) an adenylylcyclase-cAMP-protein kinase system mediates a signalling pathway leading tohead-to-head agglutination; and (ii) loss ofanti-agglutinin from the spermatozoa may be modulated by changes in the plasmamembrane induced by actions of serum albumin or MBC contained in a medium. |
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