Validation of Pisum sativum agglutinin fluorescent marker for stallion spermatozoal acrosomes with transmission electron microscopy

Abstract

This study was designed to validate the use of a pea lectin to assess changes in acrosomal integrity of equine spermatozoa. The optimal conditions for the induction of the acrosome reaction were determined using ejaculates from three stallions. Either 1 uM or 10 uM A23187 (a calcium ionophore) was added to each ejaculate and incubated for 1,2 and 3 hours at two different temperatures (37C̊ and 22C̊). Raw semen or extender were fixed at time zero to serve as baseline controls. Other untreated samples in extender were incubated at 22C̊ and 37C̊ for 1 or 3 hours. These samples were processed for TEM. Extended spermatozoa were exposed to A23187 and incubated (37 C̊) for various time intervals. The semen was then processed using a filter technique, centrifuge technique and TEM. During the filter technique, the extended semen was drawn through a millipore system and exposed to Dulbecco's phosphate-buffered saline (DPBS), paraformaldehyde, ethanol, blocking solution and fluoresceinated isothiocyanate Pisum sativum agglutinin (FITC-PSA). The same reagents were used for the centrifuge technique. The finished product for both techniques was stallion spermatozoa exhibiting various patterns of acrosomal staining with FITC-PSA conjugated with ethidium homodimer (EthD-1). Spermatozoa were counted and classified according to their staining or micrograph pattern for both of the PSA assays or TEM, respectively. Once grouping for reacted and intact cells was established, the PSA filter and centrifuge technique results were compared to the TEM data. The percentage of acrosome reacted cells from the PSA assays was compared to that of TEM reacted cells. The optimal conditions for the induction of the acrosome reaction include 37C̊ incubation at 2-3 hours using 10 uM A23187. Percentages of acrosome reacted cells from both the PSA filtration and centrifugation methods compared favorably with that of percentage of acrosome reacted cells by TEM. Results of the FITC-PSA staining techniques corresponded to the percentage of acrosome reacted and intact spermatozoa assessed with TEM in the three samples examined. The PSA assay is rapid and evaluates the progression of the acrosome reaction by producing a variety of patterns.