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.