ResearchAfter mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate spermoviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig spermoviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l -1 . A significant (P р 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l -1 , 50 mmol lactose l -1 and 50 mmol mannose l -1 . These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.
The objective of this investigation was to examine the nature of freeze/thaw-induced plasma membrane damage in an effort to validate hypotheses about cryoinjury in ram spermatozoa. Spermatozoa were loaded with fluorescein diacetate (FDA), a marker for plasma membrane integrity, and cooled (15 degrees C/min) to temperatures between -10 degrees C and -30 degrees C on a cryomicroscope stage. Post-thaw fluorescence intensity measurements of individual cells indicated that freezing to temperatures between -10 degrees C and -15 degrees C did not induce significant membrane permeabilization. However, freezing below -15 degrees C was followed by membrane permeabilization immediately after thawing. A majority (> 60%) of flagellar plasma membranes of cells frozen to -10 degrees C remained ultrastructurally intact during thawing; principal-piece membranes were more robust than middle piece membranes (p = 0.001). Significant middle-piece membrane breakage was, however, induced as the post-thaw temperature increased from +10 degrees C to +30 degrees C (10 degrees C, 64 +/- 12.3% intact membranes [mean +/- SEM]; 30 degrees C, 43 +/- 12.5% intact membranes [mean +/- SEM]; p = 0.0085). Cells frozen to -30 degrees C did not exhibit this thawing effect, although the distinction between middle-piece and principal-piece plasma membranes was evident (p = 0.002). All sperm head plasma membranes were damaged by freezing and thawing to any combination of temperatures. Although acrosomes became swollen after freezing and thawing, the incidence of outer acrosomal membrane vesiculation remained at control (unfrozen) levels with all treatments used. Experimental exposure to the hyperosmotic conditions generated during freezing induced little flagellar membrane permeabilization, but significant damage was caused by restoration of osmotic equilibrium. It is suggested that membranes are initially destabilized during the freezing process, both by low temperature effects and by exposure to high salt concentrations. The resultant post-thaw degeneration of the plasma membrane is caused by a combination of temperature and osmotic effects.
Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P , .05) percentage of total and progressively motile cells, compared with those in egg yolkcontaining samples. Further incubation of thawed samples revealed changes in motility and mitochondrial functionality that otherwise would not have been detected. These results indicated that lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.
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