Role of cyclic adenosine 3′,5′-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa

Abstract

It has previously been shown that when boar spermatozoa are incubated in a modified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs in many cells. The aim of the present study was to investigate the effects of cyclic adenosine 3′,5′-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination. Spermatozoa were collected from four mature boars, washed and incubated in mKRB. After a 1-h incubation, a sample of each sperm suspension was smeared gently on a separate glass slide, dried and stained in a phosphate-buffered solution of Giemsa to assess the percentage of head-to-head agglutinated cells in each suspension. In the samples incubated in mKRB, approximately 50% of the spermatozoa were agglutinated with one another at the acrosome. However, the percentages of head-to-head agglutinated spermatozoa were greatly reduced by a lack of calcium chloride in mKRB, but were recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) in a dose-dependent manner between 1 and 1000 M . Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 M) instead of dbcAMP also significantly increased the percentages of head-to-head agglutinated spermatozoa. Moreover, the effects of adding dbcAMP were attenuated by treatment with Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylamine salt (0.25–1.0 mM, a cAMP antagonist) or H-89 (5 M, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculin A (500 nM) (inhibitors of protein serine/threonine phosphatase). In sperm samples incubated in mKRB containing 0.1% polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplemented with 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in a significant decrease in the percentages of head-to-head agglutinated spermatozoa. Addition of porcine serum albumin (PSA, 1–4 mg mL–1) or methyl-β-cyclodextrin (MBC, 5–10 mg mL–1) instead of BSA was as effective as BSA (4 mg mL–1) in enhancing sperm agglutination. However, the effects of BSA (4 mg mL–1) or MBC (5 mg mL–1) were reduced by pre-mixing these reagents with cholesterol 3-sulfate (a cholesterol analogue, 5 g mL–1 for BSA and 375 g mL–1 for MBC). In addition, a protein ‘anti-agglutinin’ inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. The obtained Western blots revealed that sperm-bound anti-agglutinin was detected less in the samples incubated with either BSA (4 mg mL–1) or MBC (5–10 mg mL–1), compared with control samples. Moreover, pre-mixing MBC (5 mg mL–1) with cholesterol 3-sulfate (375 g mL–1) reduced this reagent’s effects on the loss of sperm-bound anti-agglutinin. Additionally, the assay of sperm agglutination and a chlortetracycline staining assay revealed that the percentages of head-to-head agglutinated spermatozoa were positively correlated with those of spermatozoa classified into B pattern (capacitated spermatozoa). These results are consistent with the following suggestions: (i) an adenylyl cyclase-cAMP-protein kinase system mediates a signalling pathway leading to head-to-head agglutination; and (ii) loss of anti-agglutinin from the spermatozoa may be modulated by changes in the plasma membrane induced by actions of serum albumin or MBC contained in a medium.