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.

Characterisation of the testicular transcriptome in stallions with age-related testicular degeneration.

BACKGROUND: Age-related testicular degeneration can be defined as the progressive deterioration of the testis that typically occurs in middle-aged or older males and that leads to diminished testicular function and subfertility. In the equine breeding industry, genetically valuable males maintain their value as breeding animals well into old age. Because testicular degeneration is common in middle-aged and older stallions, the disease often has a significant negative impact on a stallion's breeding career and leads to economic losses in the horse breeding industry. OBJECTIVE: Because testicular degeneration is a tissue autologous disease in the horse, the objective of this study was to use whole-transcriptome sequencing to compare the testicular transcriptomes of normal, fertile stallions to those of stallions affected by age-related testicular degeneration in order to better understand the pathophysiology of the disease. STUDY DESIGN: Cross sectional. METHODS: Testicular tissue samples from clinical castrations or euthanasia were collected from normal healthy (n = 3) or older subfertile (n = 4) stallions. Samples were processed and sequenced on an Illumina HiSeq™ 2000 Sequencing System. Bioinformatic analysis of the data was performed in R/RStudio, and the transcriptomes were compared between the two groups. Genes were considered to be differentially expressed between healthy and diseased tissue if they demonstrated at least a ±1.5× fold change difference and had a false discovery rate-adjusted P value <0.05. Gene ontology analysis was performed using Ingenuity® IPA. RESULTS: Analyses of differential expression of individual genes, as well as computer-based gene ontology analysis, identified upregulation of cytokine-mediated inflammatory pathways in testes from stallions affected with testicular degeneration. This upregulation of inflammation was associated with upregulation of cell survival pathways, inhibition of apoptotic pathways and increases in collagen formation. MAIN LIMITATIONS: There are unavoidable confounding factors (e.g. differences in breed, management, environment, age) that could create non disease-related genetic variation between our normal and affected samples. In addition, there are practical limitations to applying computer-based gene ontology analysis to equine samples. Gene ontology software relies on published information (mostly non-equine), and some biological processes (e.g. apoptosis and inflammation) are more commonly studied than others and so are over-represented in the literature and therefore more likely to be identified by computer algorithms. Caution should be taken when interpreting the data, as alterations in gene expression can be the cause of disease processes or can be the result of disease processes. CONCLUSIONS: These results suggest that chronic, low-grade inflammation may be involved in the pathophysiology of age-related testicular degeneration in stallions.

Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†.

Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.

Evaluation of the effect of laboratory methods on semen analysis and breeding soundness examination (BSE) classification in stallions.

The stallion Breeding Soundness Examination (BSE), as proposed by the Society for Theriogenology, recommends that a stallion produce a minimum of one billion progressively motile, morphologically normal sperm (PMMNS) in the second of two ejaculates collected 1 h apart to be classified as a Satisfactory Prospective Breeder. With this in mind, the first objective of this study was to determine if the classification outcome of the traditional BSE differs depending on the methods used to evaluate sperm motility, morphology and concentration. We hypothesized that application of Computer Assisted Sperm Motion Analysis (CASA) and Differential Interference Contrast (DIC) microscopy to stallion semen evaluation would yield a more conservative estimate of the number of PMMNS. If this hypothesis is correct, then the use of CASA and DIC microscopy for semen evaluation would result in significantly fewer stallions meeting the historical standards for classification as a Satisfactory Prospective Breeder. Additionally, we determined whether the use of these modern technologies resulted in more accurate prediction of the actual fertility of a stallion compared to the use of more traditional technologies. Our results support the hypothesis that modern semen analysis techniques (including CASA and DIC microscopy) result in more conservative estimates of the number of PMMNS when compared to standard semen analysis techniques. As a result, the choice of methods used for semen analysis may impact the outcome of the traditional BSE. However, none of the methodologies used in this study reliably predicted different levels of fertility among this group of moderately to highly fertile stallions within the context of the traditional BSE. Additionally, the only individual semen measure that was significantly correlated with fertility was the percentage of morphologically normal sperm as determined using DIC microscopy. These results caution against strict use of the traditional 'cutoff' of 1 billion PMMNS for classification of breeding potential, particularly when attempting to differentiate between moderately and highly fertile stallions and regardless of the laboratory methods employed for semen analysis.

Declining testicular function in the aging stallion: Management options and future therapies.

Declining fertility in association with declining testicular function is commonly seen as stallions age and can be the cause of significant economic losses in the equine breeding industry. This manuscript describes how to clinically recognize the signs of age-related declining testicular function (testicular degeneration) and also provides mare and stallion management strategies for improving reproductive outcomes. Finally, the current understanding of the pathophysiology of the disease is presented, including the results of recent studies that are beginning to uncover the underlying causes for age-related declines in testicular function in stallions. These new findings provide a basis for possible future treatments that could delay the effects of aging on the 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.

Physical, behavioral, endocrinologic, and cytogenetic evaluation of two Standardbred racehorses competing as mares with an intersex condition and high postrace serum testosterone concentrations.

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.

Xenografting restores spermatogenesis to cryptorchid testicular tissue but does not rescue the phenotype of idiopathic testicular degeneration in the horse (Equus caballus).

Spermatogenesis from many mammalian species occurs in fragments of normal testis tissue xenografted to mice. Here we apply xenografting to the study of testicular pathology. Using the horse model, we investigated whether exposure to a permissive extratesticular environment in the mouse host would rescue spermatogenesis in cryptorchid testicular tissue or in tissue affected by idiopathic testicular degeneration (ITD). In cryptorchid tissue, where the extratesticular environment is abnormal, xenografting induced spermatogenesis up to meiosis in a subpopulation of seminiferous tubules. Thus, spermatogonia survive and partially retain their potential to differentiate in cryptorchid horse testes. In contrast, the primary defect in equine ITD is hypothesised to be tissue autologous. In support of this, xenografting did not restore spermatogenesis to tissue affected by ITD, thus confirming that the testis itself is primarily diseased. This outcome was not affected by supplementation of exogenous gonadotropins to the mouse host or by reconstitution of a normal reproductive regulatory axis supplied by functional porcine testicular xenografts. These studies demonstrate the usefulness of xenografting for the study of testicular pathology.

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.

Differential ubiquitination of stallion sperm proteins: possible implications for infertility and reproductive seasonality.

Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.