Objective—To determine whether a homologue of A-kinase
anchor protein 4 (AKAP4) is present and functional
as an AKAP in equine spermatozoa and examine
the effect of semen cooling and cryopreservation
on binding of equine AKAP4 to the regulatory (RII)
subunit of protein kinase-A (PK-A).
Sample Population—Ejaculated semen collected
from 2 fertile stallions, 3 bulls, and 3 humans.
Procedure—Identification of an equine homologue of
AKAP4 was investigated via DNA sequencing. Protein
was extracted from the spermatozoa of each species
for immunoblot analysis to identify AKAP4 and its precursor
protein, pro-AKAP4; immunofluorescence
microscopy was used to localize those proteins in
spermatozoa. Ligand overlay assays were used to
determine whether the identified proteins bound to
the RII subunit of PK-A and whether cooling or cryopreservation
of spermatozoa affected that binding.
Results—The partial genomic sequence of AKAP4
was identified in equine spermatozoa, and
immunoblot analysis confirmed that AKAP4 and pro-AKAP4 are present in equine spermatozoa. Via
immunofluorescence microscopy, these proteins
were localized to the spermatozoal principal piece.
Results of ligand overlay assays indicated that equine
AKAP4 and pro-AKAP4 bind to the RII subunit of PKA
and are AKAPs; AKAP4-RII binding was not affected
by cooling or cryopreservation of spermatozoa.
Conclusions and Clinical Relevance—Results suggest
that equine AKAP4 anchors PK-A to the spermatozoal
flagellum (where the kinase is likely to be
required for the regulation of spermatozoal motility),
but decreases in spermatozoal motility in cooled or
cryopreserved semen are not associated with
decreased binding of AKAP4 and PK-A. (Am J Vet Res 2005;66:1056–1064)