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)
Case Description—2 Standardbred racehorses that had been winning races while competing as mares underwent postrace drug testing and had serum testosterone concentrations above the acceptable limit for female racehorses.
Clinical Findings—Initial physical examinations by the referring veterinarian revealed ambiguous external genitalia and suspected intra-abdominally located testes leading to a preliminary diagnosis of male pseudohermaphroditism. Horses were referred for further evaluation of sex. Physical examination of the external genitalia confirmed the findings of the referring veterinarian. Transrectal palpation and ultrasonography revealed gonads with an ultrasonographic appearance of testes. On cytogenetic analysis, both horses were determined to have a 64,XY karyotype and 8 intact Y chromosome markers and 5 SRY gene markers, which were indicative of a genetic male and confirmed an intersex condition. Additionally, both horses had some male-type behavior and endocrinologic findings consistent with those of sexually intact males.
Treatment and Outcome—Taken together, these findings confirmed that both horses were male pseudohermaphrodites. Both horses returned to racing competition as males.
Clinical Relevance—As of October 1, 2008, the Pennsylvania Horse and Harness Racing Commissions implemented a postrace drug testing policy that included analysis of blood samples for anabolic and androgenic steroids and set maximum allowable concentrations of testosterone for racing geldings and females. Within 8 months of initiation of this drug testing policy, the 2 horses of this report were identified as having an intersex condition. This raises the possibility that intersex conditions may be more common in racing Standardbreds than was previously suspected.