Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.

No description available.

On Mesoscale Population Study cruises during summers 2004 and 2005 aboard the sailboat Summer Breeze, researchers from the Sperm Whale Seismic Study (SWSS) surveyed for sperm whales along the continental slope of the northern Gulf of Mexico. SWSS scientists tracked 35 groups of whales during these two summers, recording locations where they did and did not encounter whales. Whales were encountered during both summers at approximately the same frequency (19 groups in 38 survey days in 2004; 16 groups in 29 survey days in 2005), but fluke photo-identifications indicated that 85% of individuals encountered during summer 2005 had never been previously identified in the Gulf throughout 10 years of cetacean research. Composition and distribution of these groups also varied between summers. Oceanographic conditions at the edge of the continental shelf differed between 2004 and 2005, which may have modified the usual trophic cascade that begins with near-surface primary production to create local aggregations of prey at the depths where sperm whales forage. Sperm whales are apex, mesopelagic predators, but have been shown to associate with surface primary productivity over large spatial scales and time scales of months to years. The purpose of this thesis was to look for relationships between sperm whale presence and surface oceanography on smaller spatial and shorter temporal scales. Surface ocean color from NASA’s Moderate Imaging Spectroradiometer (MODIS) and surface dynamic height from NASA’s Earth orbital altimeters were evaluated to assess habitat occupied by sperm whales. Passive acoustic monitoring along transect lines for sperm whale clicks permitted determination of sperm whale presence and absence. Sperm whale encounters were in general associated with negative sea surface height and enhanced sea surface chlorophyll (SSC), especially in or near areas where local SSC anomaly was produced by cyclone induced upwelling of nutrients or from coastal water advected off-margin. During summer 2004, SSC was generally high all along the upper continental slope whereas summer 2005 saw relatively low SSC along the upper continental slope. Whales encountered in this study were most highly correlated with SSC two weeks after the initial development of locally highest-SSC anomalies.

The overall objective of this thesis was to evaluate the effects of apoptosis-like membrane and DNA changes in bull sperm, and to relate these changes to a bull’s fertility potential. This thesis hypothesis is that apoptosis-like changes occurring in fresh or cryopreserved bull sperm have a negative effect on a bull’s fertility potential. Two studies were conducted, the objectives of study 1 were to confirm the relationship of apoptosis-related membrane and nuclear changes in bull sperm with fertility, to predict the fertility of beef bulls used for natural mating; and to evaluate the effect of sperm with nicked-DNA on cleavage and blastocyst formation in vitro. In Experiment 1, phosphatidylserine (PS) translocation, from the inner to the outer plasma membrane, and DNA nicks in the sperm from 50 dairy bulls were determined using Annexin-V/PI and TUNEL assays, respectively. Relationships between the parameters of the assays and the known fertility levels of the bulls were calculated. In Experiment 2, fertility levels of 15 beef bulls used for natural mating were estimated using a regression model of DNA nicks developed in Experiment 1. In Experiment 3, the effect of DNA nicked sperm on cleavage and blastocyst rates were evaluated in in vitro produced embryos, using high and low sperm concentrations (30,000 and 300,000 sperm per IVF droplet) to fertilize the mature oocytes. In Experiment 1, there were significant relationships of fertility with live sperm (P<0.05) and necrotic sperm (P<0.01) (Annexin-V/PI assay), and with DNA-nicked sperm (P<0.001) (TUNEL assay). In Experiment 2, the fertility level of bulls used for natural breeding was estimated and ranged from -7.3 to 2.4. In Experiment 3, the cleavage rate was significantly affected by the number of sperm with nicked DNA, regardless of sperm concentration. At the low sperm concentration, blastocyst rate was significantly lower when higher DNA nicked sperm were used (51% vs 32%; high vs low DNA nicks) (P<0.05). Blastocyst rate was non significant at the higher sperm concentration regardless of DNA nicks. The second objective of this study was to evaluate the effect of apoptosis inhibitors added to post-thaw sperm samples on their longevity, to increase the availability of viable sperm to oocytes for fertilization. Frozen semen from seven bulls was used; six straws from each bull were pooled. Samples included, untreated control (sperm remaining in extender), treated control (washed sperm), and four treatments (inhibitors) each at two concentrations. Apoptosis inhibitors assessed included; Bax channel blocker, z-VAD-FMK, Coenzyme Q10, and XIAP. Motility related characteristics were evaluated using computer assisted sperm analysis (CASA). Membrane intactness and normal acrosomes were evaluated using fluorescein isothiocyanate-peanut agglutination (FITC-PNA)/propidium iodide (PI) assay. Mitochondrial membrane potential was evaluated using Mitotracker Deep Red (MtDR). Sperm parameters were evaluated at 0, 3, 6, and 12 hours of incubation. Our results showed, no significant effect of apoptosis inhibitors on post-thaw sperm motility and structural characteristics. The decline in sperm motility and structural characteristics at 6 h of incubation was lower (P<0.05) in treated control and treatment groups than untreated control group. In conclusion, the presence of nicked DNA in sperm may be used as an estimate of the fertility level of a breeding bull. The levels of sperm with DNA nicks have a negative effect on cleavage rates and subsequent blastocyst development. The second conclusion indicates that the addition of an apoptosis inhibitor post-thaw to semen samples does not improve longevity or fitness, in any of the parameters evaluated. The simple removal of extender showed to be beneficial to sperm longevity and fitness. Further studies are needed to evaluate the cleavage and blastocyst rate of embryos fertilized with a single sperm known to carry DNA nicks. As well, the effect of the addition of apoptosis inhibitors before cryopreservation of bull semen needs to be evaluated.

No description available.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 109-141).

No description available.

Current equine sperm cryopreservation methods fail to reliably prevent damages to important cellular structures such as the cell membrane and DNA. The objective of this study was to determine the effects of supplementing a stallion semen extender with 1 or 10 mM resveratrol on post-thaw sperm characteristics. Results showed that sperm death was increased with 10 mM compared to both the control and 1 mM (P < 0.05). DNA fragmentation was increased in the 1 mM treatment compared to the control (P < 0.05). ROS activity was reduced the most in the 10 mM with differences between all groups (P < 0.05). Membrane integrity was not different between groups (P > 0.05). Motility of the control was higher than the treatment groups (P < 0.05). Resveratrol was able to reduce ROS but was unable to preserve motility or viability at the concentrations tested.

<p>Numerous studies on mammalian spermatozoa have reported large variations in the dimensions of the main sperm structural components, namely the head, midpiece and flagellum. These variations in sperm architecture are believed to be adaptations for functioning of spermatozoa in complex environments outside the male reproductive system. The midpiece of the mammalian&nbsp / permatozoon contains a varied number of mitochondria, but the reason for the marked difference in the size and structure of this sperm component is not clear. This study&nbsp / confirmed the variations in the sperm morphometry of seven selected mammalian species and revealed unique features of the sperm midpiece and sperm mitochondria of these seven species. Evaluation of several sperm kinematic parameters revealed the unique swimming characteristics of the different spermatozoa. The importance of using standardized motility&nbsp / parameters was highlighted as well as the assessment of different subpopulations of spermatozoa in order to produce more reliable and comparable data. Investigating the role of sperm mitochondria in human sperm&nbsp / metabolism indicated that these organelles are related to sperm function in terms of sperm motility. Furthermore, it was suggested that glycolysis and mitochondrial respiration are linked processes and that both are important for the maintenance of human sperm motility. By optimizing and employing standardized experimental procedures and analysis techniques, this study was&nbsp / able to confirm the species specificity of almost all the sperm parameters evaluated, while also elucidating the phylogenetic relatedness of the non-human primate species. In conclusion, the present study has confirmed that the various midpiece morphometry parameters are related to the remaining sperm morphometry parameters as well as to the sperm kinematic parameters.&nbsp / These proposed associations between the various sperm parameters were used to explain the sperm velocity of two hypothetical and morphologically different sperm structures. Therefore, the results of the current study support the idea of co-evolution between sperm components in mammalian spermatozoa and propose that the midpiece morphometry parameters that are selected for in these spermatozoa are midpiece volume, total number of mitochondrial gyres, thickness of the mitochondrial sheath and mitochondrial height.</p>

<p>Numerous studies on mammalian spermatozoa have reported large variations in the dimensions of the main sperm structural components, namely the head, midpiece and flagellum. These variations in sperm architecture are believed to be adaptations for functioning of spermatozoa in complex environments outside the male reproductive system. The midpiece of the mammalian&nbsp / permatozoon contains a varied number of mitochondria, but the reason for the marked difference in the size and structure of this sperm component is not clear. This study&nbsp / confirmed the variations in the sperm morphometry of seven selected mammalian species and revealed unique features of the sperm midpiece and sperm mitochondria of these seven species. Evaluation of several sperm kinematic parameters revealed the unique swimming characteristics of the different spermatozoa. The importance of using standardized motility&nbsp / parameters was highlighted as well as the assessment of different subpopulations of spermatozoa in order to produce more reliable and comparable data. Investigating the role of sperm mitochondria in human sperm&nbsp / metabolism indicated that these organelles are related to sperm function in terms of sperm motility. Furthermore, it was suggested that glycolysis and mitochondrial respiration are linked processes and that both are important for the maintenance of human sperm motility. By optimizing and employing standardized experimental procedures and analysis techniques, this study was&nbsp / able to confirm the species specificity of almost all the sperm parameters evaluated, while also elucidating the phylogenetic relatedness of the non-human primate species. In conclusion, the present study has confirmed that the various midpiece morphometry parameters are related to the remaining sperm morphometry parameters as well as to the sperm kinematic parameters.&nbsp / These proposed associations between the various sperm parameters were used to explain the sperm velocity of two hypothetical and morphologically different sperm structures. Therefore, the results of the current study support the idea of co-evolution between sperm components in mammalian spermatozoa and propose that the midpiece morphometry parameters that are selected for in these spermatozoa are midpiece volume, total number of mitochondrial gyres, thickness of the mitochondrial sheath and mitochondrial height.</p>