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.

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.

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.

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.

Induction of endogenous genes by peroxisome proliferator activated receptor alpha ligands in a human kidney cell line and in vivo.

The peroxisome proliferator activated receptor alpha (PPARalpha) plays a key role in regulating fatty acid metabolism by regulating expression of genes involved in fatty acid oxidation. To identify endogenous transcripts that could be used as surrogate markers for on-target activity of PPARalpha agonists, we employed a global profiling approach using DNA microarrays. The HK-2 cell line derived from proximal tubules of the human kidney, showed induction of several genes, including pyruvate dehydrogenase kinase 4 (PDK-4) and adipocyte differentiation related protein (ADRP) by PPARalpha ligands. HK-2 cells express detectable levels of PPARalpha and its dimerization partner the retinoid X receptor (RXRalpha) proteins. Induction of PDK-4 in these cells correlates with induction of PDK-4 in the liver of fat-fed hamsters. The magnitude of fibrate induction of PDK-4 in the liver also mirrors the decrease in serum triglyceride levels. Thus, induction of PDK-4 by PPARalpha agonists in the HK-2 cell model closely correlates with its induction in vivo and may represent an early marker for PPARalpha agonist action.