Multiple Sex Cord-stromal Tumors in a Standardbred Stallion Testis.

A 12-year-old Standardbred stallion presented with a 5-month history of a growing mass in the left testis as well as an overall decrease in left testicular size. Palpation and ultrasonography of the left testis revealed a firm, hypoechoic, clearly delineated soft tissue mass in the craniolateral portion of the testis that measured 2.5 × 2.3 × 1.9 cm. Two smaller, hypoechoic regions also were visible ultrasonographically in the left testis, suggesting the presence of multifocal/multicentric neoplasia. The affected testis was very small (testicular volume of 40.3 cm3). The right testis was significantly larger (144.3 cm3), and the parenchyma was ultrasonographically normal. Due to the concern that these findings could indicate the presence of a more aggressive tumor type, unilateral orchiectomy was performed. Multiple soft tissue masses were identified grossly, and histopathologic evaluation identified the larger mass as a Sertoli cell tumor and the two smaller masses as mixed sex cord-stromal tumors with Sertoli cell and Leydig cell differentiation. To our knowledge, this the first report of concurrent Sertoli cell and mixed sex cord-stromal tumors in a single descended equine testis.

Transrectal ultrasonographic characterization of the accessory sex glands, pelvic urethra, and ureters in normal geldings.

Transrectal ultrasound of the internal urogenital tract may be used to aid in the diagnosis of reproductive tract and urinary tract pathology in both stallions and geldings. Abnormalities of the accessory sex glands of geldings are uncommon, although prostatic masses have recently been described in adult geldings presenting with dysuria, stranguria, and/or hematuria. The purpose of this study was to describe the normal ultrasonographic features and sizes of the accessory sex glands, caudal ureters, and pelvic urethra in clinically normal geldings. Eleven healthy geldings with no history of urogenital tract pathology were evaluated by a single observer experienced in ultrasound of the stallion accessory sex glands. The ultrasonographic appearance, relative anatomic relationships and sizes of the accessory sex glands, caudal ureters, and pelvic urethra were investigated using both rectal linear array and microconvex array transducers. Summary statistics including mean, standard error, confidence intervals, and range were calculated for each structure. There were no statistically significant differences in measurements between the left and right sides of paired structures or between measurements obtained with different transducers. Fluid was present in the seminal vesicles of 7 of 9 subjects. Midline cysts of the urethra as well as bulbourethral gland and prostatic cysts were identified. The normal reference ranges defined in this study will be useful in the clinical evaluation of geldings with suspected internal urogenital tract pathology.

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion.

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II ß were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility.

Effect of method and clinician on stallion sperm morphology evaluation.

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.

Calmodulin and CaMKII in the sperm principal piece: evidence for a motility-related calcium/calmodulin pathway.

Both cyclic AMP (cAMP)/protein kinase A (PKA) and calcium (Ca(2+)) signaling pathways are known to be involved in the regulation of motility in mammalian sperm. Calmodulin (CaM) is a ubiquitous Ca(2+) sensor that has been implicated in the acrosome reaction. In this report, we identify an insoluble pool of CaM in sperm and show that the protein, in addition to its presence in the acrosome, is found in the principal piece of the flagellum. These findings are consistent with, though not proof of, the presence of a pool of CaM in the fibrous sheath. The Ca(2+)/CaM-dependent protein kinase IIbeta (CaMKIIbeta), which is a downstream target of Ca(2+)/CaM, similarly localizes to the principal piece. In addition, we confirm earlier reports that a CaM inhibitor decreases sperm motility. However, we find that this inhibition can be largely reversed by stimulation of PKA if substrates for oxidative respiration are present in the medium. Our results suggest that the Ca(2+)/CaM/CaMKII signaling pathway in the sperm principal piece is involved in regulating sperm motility, and that this pathway functions either in parallel with or upstream of the cAMP/PKA pathway.

Moving to the beat: a review of mammalian sperm motility regulation.

Because it is generally accepted that a high percentage of poorly motile or immotile sperm will adversely affect male fertility, analysis of sperm motility is a central part of the evaluation of male fertility. In spite of its importance to fertility, poor sperm motility remains only a description of a pathology whose underlying cause is typically poorly understood. The present review is designed to bring the clinician up to date with the most current understanding of the mechanisms that regulate sperm motility and to raise questions about how aberrations in these mechanisms could be the underlying causes of this pathology.

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). 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 PK-A 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.