After 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 sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal 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 < or = 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.
CE Green, J Bredl, WV Holt, PF Watson and A Fazeli
A Fazeli, RM Elliott, AE Duncan, A Moore, PF Watson and WV Holt
Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.
Roslyn M A Elliott, Rhiannon E Lloyd, Alireza Fazeli, Edita Sostaric, A Stephen Georgiou, Nana Satake, Paul F Watson and William V Holt
Previous studies have shown that a soluble protein fraction derived from preparations of apical plasma membrane (APM) of the oviductal epithelium enhances the in vitro survival of mammalian spermatozoa. Here, we show that the survival enhancing property of the soluble protein fraction seems to depend significantly upon heat shock 70 kDa protein 8 (HSPA8 previously known as HSPA10). The following findings in the present study enabled us to draw this conclusion: first, using proteomic analysis, we identified a subset of 70 kDa oviductal surface proteins that bound to spermatozoa, one of which was HSPA8. Second, pre-treatment of the soluble protein fraction with anti-HSPA8 antibody reduced the 24 h (at 39 °C) sperm survival enhancement effect normally induced by the presence of 200 μg/ml soluble APM proteins. Third, complementary experiments showed that substituting the soluble protein fraction with bovine recombinant HSPA8 (0.5–2 μg/ml) also elicited the sperm survival effect. Finally, we also tested the effect of bovine recombinant HSPA8 on bull spermatozoa and found similar, dose-responsive, sperm survival promoting effects. The conserved nature of HSPA8 between mammalian species suggests that this protein may represent a common biological mechanism for the maintenance of sperm survival in the oviduct.
Najmeh Moein-Vaziri, Ian Phillips, Simon Smith, Carmen Almiňana, Carolina Maside, Maria A Gil, Jordi Roca, Emilio A Martinez, William V Holt, A Graham Pockley and Alireza Fazeli
The constitutive 70 kDa heat-shock protein, HSPA8, has previously been shown to contribute to the long-term survival of spermatozoa inside the mammalian female reproductive tract. Here, we show that a recombinant form of HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances IVF performance, and decreases sperm mitochondrial activity. Fluorescence recovery after photobleaching revealed that the repair of membrane damage is achieved by an almost instantaneous increase in sperm membrane fluidity. The ability of HSPA8 to influence membrane stability and fluidity, as well as its conserved nature among mammalian species, supports the idea that this protein protects sperm survival through membrane repair mechanisms.
Free Persian abstract
A Persian translation of the abstract is freely available online at http://www.reproduction-online.org/content/147/5/719/suppl/DC1