Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane.

The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization (e.g., lipid rafts). Herein, we determined the effects of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the sperm plasma membrane, using Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit ß), liquid chromatography - mass spectroscopy (LC-MS) analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence were used to describe and compare sperm fluidity, membrane composition, as well as lipid raft composition and distribution among sperm types. Stripped sperm became more fluid following motility activation (40% increase in highly fluid cells characterized by a 2 × increase in fluorescence) and contained lipid rafts restricted to the midpiece. Testicular harvest yielded sperm with characteristics similar to stripped sperm. By contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process. Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decrease in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm. Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to substantially affect the sperm plasma membrane, but not always detrimentally. These results provide multiple potential biomarkers of sperm quality as well as insight into sources of sub-fertility resulting from use of ARTs.

An unusual case of eosinophilic uveitis in a cat.

An 8-year-old female spayed domestic short-haired cat was examined for recurrent unilateral anterior uveitis of 5 month's duration. No underlying cause was found on infectious disease screening. The cat also had a 4-year history of allergic or immune-mediated skin disease that was controlled with corticosteroid injections followed by long-term oral cyclosporine therapy. Medical management with frequent topical anti-inflammatory drugs (prednisolone acetate 1% suspension, diclofenac 0.1% solution) controlled the intraocular inflammation; however, the uveitis would relapse when therapy was discontinued. Eventually, secondary glaucoma developed OD and the eye was enucleated. At the time of surgery, a complete blood count showed a mild eosinophilia. Histopathology revealed a marked panuveitis characterized by an abundant accumulation of mixed inflammatory cells, with a predominantly eosinophilic infiltrate in the anterior chamber, iris, ciliary body, and choroid. No etiologic agents were found on serial sections, and there were no cellular criteria for malignancy noted. Nine months after enucleation, the chronic dermatitis appeared to be in remission despite no further medical management. This is the first known report of a primarily eosinophilic uveitis in a cat with chronic allergic skin disease and may be considered an ocular variant of feline eosinophilic granuloma complex.

Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines. RESULTS: We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested. CONCLUSIONS: These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

Primary central corneal hemangiosarcoma in a dog.

PURPOSE: To report a case of primary central corneal hemangiosarcoma in the dog. METHODS: An 11-year-old, neutered, female, German shepherd mixed breed dog was referred to the Hospital Veterinario Sierra de Madrid (Spain) for evaluation of an enlarging corneal mass of the left eye (OS). The dog was predominantly housed outdoors and was diagnosed with a history of chronic superficial keratitis of both eyes (OU) by the referring veterinarian. The corneal mass was resected by routine superficial keratectomy and submitted for histopathology and Factor VIII immunohistochemical staining. RESULTS: The mass was diagnosed as a corneal hemangiosarcoma with complete excision. Postoperatively, the keratectomy site healed without complication and there was no evidence of recurrence three and a half months postoperatively. Complete systemic evaluation, including abdominal ultrasound and CT scan of the head and thorax, indicated no other detectable neoplasia in the dog. DISCUSSION: Outdoor housing and ultraviolet exposure, breed, and chronic superficial keratitis were all suspected as contributing factors to the development of a primary corneal hemangiosarcoma. Surgical removal and postoperative treatment for chronic superficial keratitis provided effective therapy.