Characterization of an A-kinase anchor protein in equine spermatozoa and examination of the effect of semen cooling and cryopreservation on the binding of that protein to the regulatory subunit of protein kinase-A.
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).


METHODS
Ejaculated semen collected from 2 fertile stallions, 3 bulls, and 3 humans.


METHODS
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 PK-A and are AKAPs; AKAP4-RII binding was not affected by cooling or cryopreservation of spermatozoa.


CONCLUSIONS
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.
Turner
Regina M O
RM
Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692, USA.
Casas-Dolz
Raul
R
Schlingmann
Karen L
KL
Hameed
Saima
S
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015925:Cryopreservation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D004586:Electrophoresis / Q000662:veterinary; D006736:Horses / Q000378:metabolism*; D015151:Immunoblotting / Q000662:veterinary; D008297:Male; D008856:Microscopy, Fluorescence / Q000662:veterinary; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011498:Protein Precursors / Q000235:genetics; D012662:Semen Preservation / Q000662:veterinary*; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013082:Sperm Tail / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C485479:Akap4 protein, mouse; D017931:DNA Primers; D011498:Protein Precursors; D017868:Cyclic AMP-Dependent Protein Kinases