betadex and adenosine-3--5--cyclic-phosphorothioate

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 microM. Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 microM) 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 microM, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculinA (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-beta-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 microg mL(-1) for BSA and 375 microg 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 el

Topics: 1-Methyl-3-isobutylxanthine; Animals; beta-Cyclodextrins; Bucladesine; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclodextrins; Enzyme Inhibitors; Glycoproteins; Isoquinolines; Male; Marine Toxins; Oxazoles; Phosphoprotein Phosphatases; Serum Albumin; Signal Transduction; Sperm Agglutination; Sperm Capacitation; Sperm Motility; Spermatozoa; Sulfonamides; Swine; Thionucleotides