Existence and importance of Na+K+-ATPase in the plasma membrane of boar spermatozoa.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218 ± 23% and 245 ± 38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and ß subunits (isoforms α1, α3, ß1, ß2, and ß3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0 h control (33.0 ± 2.6% vs. 19.2 ± 2.6% capacitated sperm, respectively; p = 0.014) and altered the ß2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

Are isoforms of capacitating Na+ K+ -ATPase localized to sperm head rafts?

Capacitation begins in the sperm head plasma membrane (HPM). Membrane rafts could house signaling molecules, but although these specialized microdomains have been microscopically visualized in sperm heads, rafts have been isolated for study only from homogenized whole sperm or tails, never purified HPM. Sodium/potassium ATPase (Na+ K+ -ATPase) is a membrane-bound signaling protein that induces capacitation in bull sperm in response to the steroid hormone ouabain, and its subunit isoforms α1, α3, ß1, ß2, and ß3 are known in HPM. This study hypothesized that rafts exist in the HPM of bull sperm, with Na+ K+ -ATPase subunit isoforms preferentially localized there. Western immunoblotting (WB) of HPM from fresh, uncapacitated bull sperm (n = 7 ejaculates), and detergent-resistant membranes isolated by density gradient centrifugation from this HPM, contained the raft-marker protein Flotillin-1; the non-raft fraction did not. HPM, raft, and non-raft contained all known Na+ K+ -ATPase isoforms including, for the first time, the previously unknown α2 isoform. Quantification (ImageQuant Software) found α3 and ß1 were relatively dominant isoforms in the HPM raft. WB profiles of raft isoforms differed significantly from HPM and non-raft profiles, with unique banding patterns and amounts, hinting that the capacitation signaling in the now-identified HPM rafts may depend on unique sequences within the isoform structure.

Interaction of ouabain and progesterone on induction of bull sperm capacitation.

The endogenous steroid hormone ouabain induces capacitation of bull sperm acting through its receptor Na+/K+-ATPase on the sperm plasma membrane. Progesterone (P4) is believed to act through the sperm membrane P4 receptor (mPR) to induce non-genomic signalling leading to capacitation and/or acrosome reaction (AR) in the sperm of some species, but the exact nature of this receptor molecule on bull sperm is not known. In amphibian oocytes, P4 acts through the low-affinity ouabain binding site on Na+/K+-ATPase to induce signalling highly reminiscent of ouabain's signalling that initiates capacitation. This study hypothesized that ouabain and P4 interact agonistically or antagonistically to induce bull sperm capacitation. Sperm were incubated with 0, 12.5, 25, 50 and 100 µM ouabain, P4 or ouabain + P4 (12.5, 25 and 50 µM each) under capacitating conditions, and capacitation was assessed microscopically looking at the acrosome status of sperm and as the amount of protein tyrosine phosphorylation (Tyr-P). Both steroids stimulated Tyr-P of certain sperm proteins, but ouabain caused tyrosine phosphorylation of more proteins than P4 and stimulated significantly more overall Tyr-P (P < 0.05). Ouabain also was the only steroid to stimulate significant microscopically-evident AR. When sperm were co-incubated with the two steroids, P4 partially inhibited ouabain-induced Tyr-P and AR. These results suggest that P4 and ouabain may both interact with Na+/K+-ATPase, but ouabain is the more effective hormone. Ouabain may, therefore, be the primary physiological inducer of bovine capacitation.

Demonstration of synchrotron x-ray phase contrast imaging computed tomography of infiltrative transitional cell carcinoma of the prostatic urethra in a dog.

Prostatic urethral transitional cell carcinoma with prostatic invasion in a dog was imaged with abdominal radiography and abdominal ultrasonography antemortem. Synchrotron in-line x-ray phase contrast imaging computed tomography (XPCI-CT) was performed on the prostate ex vivo at the Canadian Light Source Synchrotron and compared to histology. XPCI-CT imaging provides greater soft tissue contrast than conventional absorption-based x-ray imaging modalities, permitting visualization of regions of inflammatory cell infiltration, differentiation of invasive versus noninvasive tumor regions, and areas of necrosis and mineralization. This represents the first report of XPCI-CT images of an invasive prostatic urothelial neoplasm in a dog.

Assessment of freeware programs for the reconstruction of tomography datasets obtained with a monochromatic synchrotron-based X-ray source.

Synchrotron-based in-line phase-contrast computed tomography (PC-CT) allows soft tissue to be imaged with sub-gross resolution and has potential to be used as a diagnostic tool. The reconstruction and processing of in-line PC-CT datasets is a computationally demanding task; thus, an efficient and user-friendly software program is desirable. Four freeware programs (NRecon, PITRE, H-PITRE and Athabasca Recon) were compared for the availability of features such as dark- and flat-field calibration, beam power normalization, ring artifact removal, and alignment tools for optimizing image quality. An in-line PC-CT projection dataset (3751 projections, 180° rotation, 10.13 mm × 0.54 mm) was collected from a formalin-fixed canine prostate at the Biomedical Imaging and Therapy Bending Magnet (BMIT-BM) beamline of the Canadian Light Source. This dataset was processed with each of the four software programs and usability of the program was evaluated. Efficiency was assessed by how each program maximized computer processing power during computation. Athabasca Recon had the least-efficient memory usage, least user-friendly interface, and lacked a ring artifact removal feature. NRecon, PITRE and H-PITRE produced similar quality images, but the Athabasca Recon reconstruction suffered from the lack of a native ring remover algorithm. The 64-bit version of NRecon uses GPU (graphics processor unit) memory for accelerated processing and is user-friendly, but does not provide necessary parameters for in-line PC-CT data, such as dark-field and flat-field correction and beam power normalization. PITRE has many helpful features and tools, but lacks a comprehensive user manual and help section. H-PITRE is a condensed version of PITRE and maximizes computer memory for efficiency. To conclude, NRecon has fewer imaging processing tools than PITRE and H-PITRE, but is ideal for less experienced users due to a simple user interface. Based on the quality of reconstructed images, efficient use of computer memory and parameter availability, H-PITRE was the preferred of the four programs compared.