Honors College Thesis
 

Sperm acrosome associated 3 protein expression and antigenicity in horses

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  • Sperm acrosome associated 3 protein (SPACA3) is a unique, intra-acrosomal, conventional-type lysozyme-like protein of mammalian spermatozoa. SPACA3 is present in all stages of ovarian follicles and is localized to granulosa cells and ooplasma in humans, mice, cows, dogs, and cats. The objective of this study was to determine if equine ovarian follicles express SPACA3 and to demonstrate that administration of a SPACA3 vaccine would be safe in horses. We hypothesized that SPACA3 would be immunoexpressed in all equine ovarian follicles and immunization against SPACA3 would stimulate a humoral response. Routine immunohistochemistry was performed on formalin fixed paraffin embedded ovarian sections from three adult Quarter Horse type mares. Briefly, sections were deparaffinized and rehydrated. Then, antigen retrieval was accomplished by heat activation in a sodium citrate buffer. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Nonspecific binding was blocked using a serum-free protein block. A polyclonal rabbit anti-SPACA3 primary antibody was applied to slides at a 1:200 dilution. A universal negative rabbit antibody was applied to adjacent sections to serve as a negative control. A horseradish peroxidase-conjugated anti-rabbit polymer was applied undiluted to all slides. Peroxidase activity was detected using a Nova Red kit. Slides were counterstained with hematoxylin, dehydrated, and cover slipped. Digital images were captured at 10X and 40X magnifications. SPACA3 cellular expression was recorded for each follicle present on the section. For vaccine development, a short SPACA3 sequence was synthesized from a mammalian expression system and conjugated to carrier protein. The conjugated peptide was mixed 1:1 with an adjuvant, which was specifically selected for its safety in horses. One gelding was used for this preliminary safety trial and the research was approved by the Oregon State University Institutional Animal Care and Use Committee (#0103). A total of three vaccinations were given 3 weeks apart intramuscularly into the caudal thigh. Jugular venous blood samples were taken before each vaccine was administered and 3 weeks following the last vaccine. Serum samples were subjected to routine western blotting to confirm the presence of SPACA3 antibodies. SPACA3 was immunoexpressed in all pre-granulosa cells of primordial and in all granulosa cells of primary, secondary, and tertiary follicles in equine ovaries. Additionally, no long-term negative side effects or vaccine reactions occurred, and antibodies were successfully raised against the SPACA3 vaccine. The expression of SPACA3 in the pre-granulosa cells surrounding primordial follicles increases the likelihood that a contraceptive vaccine against this target will yield permanent results. Future studies are planned to evaluate the effect of SPACA3 vaccination on follicle production in mature mares.
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  • Poster: Preliminary safety study for an equine (Equus caballus) vaccine against sperm acrosome associated 3 protein
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