Browsing by Subject "Semen"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Biochemical and functional characterization of zonadhesin: a sperm protein potentially mediating species-specific sperm-egg adhesion during fertilization(Texas Tech University, 2002-05) Bi, MingFertilization is an important and unique process in organisms that generate offspring by sexual reproduction. Several fines of evidence support the idea that glycoproteins in the egg zona pellucida (ZP) mediate species-specific sperm-egg adhesion. In contrast, studies on the complementary molecules in spermatozoa remain controversial. Some potential candidate adhesion molecules have been identified, but none of them is supported by unequivocal evidence. A mosaic protein called zonadhesin is one of them. Zonadhesin was first found in pig sperm membrane extracts based on its ability to bind to the native ZP in a species-specific manner. The zonadhesin cDNA sequence reveals that the protein comprises several extracellular domain types found in other molecules known to mediate cell-cell interactions. Orthologs of pig zonadhesin in three other species possess domain structures similar to pig zonadhesin's, but with variations. Our overall hypothesis is that zonadhesin mediates species-specific adhesion between spermatozoa and the egg ZP. In this dissertation, I report biochemical and functional characterization of pig zonadhesin. I observed that zonadhesin forms disulfidebonded oligomers and multimers, but the pl05/p45 monomer displays a preference for binding to the ZP. Zonadhesin undergoes heterogeneous processing and is targeted to more than one physicochemical compartment in developing sperm cells. Posttranslational processing generates at least three zonadhesin components that are glycosylated with different types of oligosaccharides. Zonadhesin's oligomeric status changes during maturation of spermatozoa in the epididymis. Immunoelectron microscopy demonstrated that zonadhesin localizes to the outer acrosomal membrane and the acrosomal matrix. Moreover, I identified zonadhesin protein in eight additional species by Western blotting, immunofluorescence, or both. Zonadhesin from each of these species localizes to the apical sperm head overlying the acrosome, but the MrS of these proteins' polypeptide chains vary significantly. In addition, a bead adhesion assay was developed for future tests of zonadhesin's ability to bind to homologous or heterologous ZP. Finally, preliminary work on expression of zonadhesin domain(s) initiated future loss- and gain-of-function transgenic studies. Potential practical benefits derived from this work may include development of new contraceptives that function by blocking gamete interactions and diagnostic methods for human fertility and farm animal fecundity.Item Breed differences in seminal plasma chemistry; Implications for(2012-12) Yeomans, Glenn; Prien, Samuel D.; Thompson, Leslie D.; Ballou, Michael A.Seminal plasma serves as a nutrient rich medium for spermatozoa to function and survive. Several components of seminal plasma have also been shown to aid in cryopreservation and post-thaw viability. Although functions and roles of seminal plasma have been studied at length, the actual biochemical composition of sperm cells is poorly understood. In the present study, intra-species comparison of lipids, carbohydrates, and proteins present in sperm were examined in the bovine. Experiments were done in an effort to determine how cryopreservation may be affected by altering the level of cryoprotectant present in a sample based on baseline seminal composition of that particular specie. A wide variety of bovine breeds were used, representing both British and continental beef breeds as well as dairy. A portion of each sample was centrifuged to remove the cellular portion of the sample and both aliquots weighed to determine the cellular component’s contribution to weight of the sample. Results demonstrated a significant weight gain in equal volumes of each animal’s seminal plasma once the cellular component was removed (P < 0.001). Samples were then subjected to protein, carbohydrate, and lipid analysis. Each sample was run under spectrophotometric assay and blood chemistry assay cartridges to compare and contrast testing techniques. Differences in testing technique were detected in triglycerides and total protein levels (P = 0.023 and P = 0.018). Further, breed differences were also shown to be significant (α = 0.05) in triglycerides, glucose, total protein, and fructose (P = 0.001, P = 0.001, P = 0.001, and P = 0.009). However, no significant differences were seen between breeds for cholesterol (P = 0.228), as well as no differences detected in testing technique in cholesterol and glucose (P = 0.648 and P = 0.884 respectively). A non-linear correlation was observed between volume weights and total protein and triglyceride levels (P = 0.047 and P = 0.003). Together, these data suggest a difference in seminal component of various breeds and potentially on an individual basis as well. Further study is needed in order to examine how differences in seminal plasma chemistry effect cryopreservation and if it is possible to adjust cryoprotectant level in an effort to improve post-thaw viability of cryopreserved bovine semen.Item Effect of Density Gradient Centrifugation on Quality and Recovery Rate of Equine Sperm(2010-07-14) Edmond, Ann J.Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.Item Improving Semen Parameters Through Modification of Semen collection/extension(Texas Tech University, 2000-12) Johnson, Dustie LeeTo date, the common thread in the use of semen extenders/collection techniques, whether used for fresh extended semen, chilled semen, or cryopreserved semen, is that the extenders have all traditionally added post-collection. The objective of this study was to determine if modifying the method of collection/extension of semen would improve semen parameters by lessening cold and pH shock. Ten canine semen samples were collected with a modified artificial vagina to allow for a true split collection. The treatment half of the sample was collected into warmed extension media. The control half was collected into a dry container and no attempts to maintain temperature were used. Standard semen parameters along with the available sperm pool and number of inseminations were evaluated at specific time intervals and evaluations continued until the samples reached zero percent motility. Data analysis was performed with SPSS using the general linear model, Chi square and appropriate t-tests. There was a difference between the treatment and control groups for Motility (P<.001), Motility by Time (P<.001), Time to Zero Motility (P<.001), Time to Last Full Insemination (P<.03), Forward Progression (P<.001), Acrosome Reaction (P<.001), Acrosome Reaction by Time (P<.017), and Viability (P<.001). There was no difference in morphology between the treatment and control groups (P>.062). Modification of the semen collection/extension procedure resulted in improved semen parameters for extended periods of time post collection. The data suggest the improvement in semen parameters would result in improved quality and longevity of semen used for artificial insemination procedures and infertility treatment, possibly due to decreased effects of cold and pH shock. Further studies resulting in viable offspring will be needed to confirm these observations.