Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.
CE Green and PF Watson
A Medrano, PF Watson and WV Holt
A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.
JN Garnier, WV Holt and PF Watson
Female wild black rhinoceroses in Zimbabwe were monitored non-invasively using faecal progesterone metabolite analysis and observation of reproductive behaviour. A postpartum period of reproductive inactivity of at least 4 months, followed by a period of 4-7 months of oestrous cyclicity, was detected in six multiparous females. Three-quarters of the oestrous cycles (n = 21) had a total duration (mean +/- SEM) of 26.8 +/- 1 days. Other types of cycle were characterized either by an extended luteal phase, lasting on average twice as long as the normal cycle, or by an extended follicular phase. These extended cycles may have resulted from early embryo loss and heat stress. Female rhinoceroses did not conceive before 8 months after giving birth and some females (n = 2) most likely aborted after 3.0-3.5 months of gestation. The detected period of cyclic oestrus occurred between May and March in females (n = 9), and there was a 3 month extended interoestrous interval in nulliparous females during the period of decreasing daylengths that can be presumed to be the period of poorest fertility for the black rhinoceros under tropical latitudes. In contrast, the period of optimum fertility in the Southern hemisphere coincided with the late spring and early summer, and corresponded to the early rainy season. As a result, a higher incidence of births was detected in the late rainy season, providing the lactating female with the most suitable environment in terms of nutritional requirements.
CE Green, J Bredl, WV Holt, PF Watson and A Fazeli
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.
S Sukardi, RM Elliott, JO Withers, U Fontaine, JD Millar, MR Curry and PF Watson
After an intracellular calcium influx, fusion of the sperm plasma membrane and outer acrosomal membrane (the acrosome reaction) precedes mammalian fertilization in vivo. This study describes the isolation of outer acrosomal membrane from ram spermatozoa and the subsequent characterization of calcium-binding proteins. Pooled ejaculates were diluted, cooled slowly and washed. Incubation with Hyamine 1622 (benzethenium chloride) and subsequent slow centrifugation gently dislodged and concentrated acrosomal membranes, the fragments of which were isolated on a two-step discontinuous sucrose gradient. The acrosomal membrane material stained with Giemsa, whereas spermatozoa from the gradient pellet stained intensely only in the equatorial segment. The acrosomal fraction showed a limited number of polypeptides by SDS-PAGE. Incubation with 45Ca2+ revealed two radioactive bands at 34 and 39 kDa. Extraction in the presence of EGTA implied that these proteins are not peripheral proteins associated with the membrane only in the presence of calcium ions, but are integral membrane proteins. Polyclonal antisera raised to the two bands showed specific binding to the anterior acrosomal region and demonstrated the intracellular location of the proteins. Sequence data of protein A revealed 83% homology with calnexin homologue precursor and 70% homology with annexin XI. Protein B showed 68% homology with protein SP-10 precursor and 64-72% homology with various annexins. However, crossreactivity with a range of commercial annexin antibodies and a specific antibody to a synthetic motif encompassing the annexin calcium-binding site was not demonstrable. It is concluded that the isolated proteins are unlikely to be annexins, but are possibly novel calcium-binding proteins.
EA Carrey, C Dietz, DM Glubb, M Loffler, JM Lucocq and PF Watson
Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.
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.