Role of cyclic adenosine 3′,5′-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa
Authors: Harayama Hiroshi , Miyake Masashi , Kato Seishiro 
Published: January 2006

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.