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1
Cova, Riccardo. "Analisi di dati citofluorimetrici con tecniche di Data Mining." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amslaurea.unibo.it/4774/.
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Il citofluorimetro è uno strumento impiegato in biologia genetica per analizzare dei
campioni cellulari: esso, analizza individualmente le cellule contenute in un campione ed
estrae, per ciascuna cellula, una serie di proprietà fisiche, feature, che la descrivono.
L’obiettivo di questo lavoro è mettere a punto una metodologia integrata che utilizzi tali
informazioni modellando, automatizzando ed estendendo alcune procedure che vengono
eseguite oggi manualmente dagli esperti del dominio nell’analisi di alcuni parametri
dell’eiaculato.
Questo richiede lo sviluppo di tecniche biochimiche per la marcatura delle cellule e
tecniche informatiche per analizzare il dato.
Il primo passo prevede la realizzazione di un classificatore che, sulla base delle feature delle
cellule, classifichi e quindi consenta di isolare le cellule di interesse per un particolare
esame.
Il secondo prevede l'analisi delle cellule di interesse, estraendo delle feature aggregate che
possono essere indicatrici di certe patologie. Il requisito è la generazione di un report
esplicativo che illustri, nella maniera più opportuna, le conclusioni raggiunte e che possa
fungere da sistema di supporto alle decisioni del medico/biologo.
2
Godo, Pla Anna. "Anàlisi del contingut cromosòmic en espermatozoides d’individus portadors de translocacions: relació entre efecte intercromosòmic i segregació." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/318798.
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Els individus portadors de translocacions cromosòmiques equilibrades presenten un risc incrementat de produir gàmetes amb anomalies cromosòmiques, ja sigui per una segregació desequilibrada dels cromosomes reorganitzats o per la formació d’anomalies numèriques derivades de l’efecte intercromosòmic. Es desconeix si existeix una relació entre les anomalies derivades d’ambdós esdeveniments, que podria residir en l’aparició de fenòmens d’heterosinapsi entre els multivalents i altres cromosomes durant la profase I. D’altra banda, l’heterosinapsi s’ha relacionat amb canvis en l’arquitectura nuclear dels cromosomes en espermatozoides, que en última instància també podrien afectar la fertilitat dels individus portadors.
Els objectius d’aquesta tesi han estat: i) Desenvolupar un mètode d’anàlisi seqüencial basat en tècniques d’hibridació in situ fluorescent (FISH) que permeti identificar anomalies cromosòmiques numèriques i determinar el mode de segregació dels cromosomes reorganitzats sobre els mateixos espermatozoides; ii) Caracteritzar els patrons de segregació de diferents reorganitzacions cromosòmiques en espermatozoides no seleccionats i en espermatozoides portadors d’anomalies numèriques; iii) Determinar si existeix una relació entre els diferents modes de segregació dels cromosomes reorganitzats i la producció d’espermatozoides amb anomalies numèriques; iv) Desenvolupar una tècnica d’anàlisi que permeti valorar la disposició tridimensional dels territoris cromosòmics en nuclis d’espermatozoides.
S’han recollit mostres de semen de vuit individus portadors de translocacions recíproques, onze portadors de translocacions Robertsonianes i un portador d’una triple translocació. En la primera ronda de FISH seqüencial s’han detectat espermatozoides amb anomalies numèriques per cinc cromosomes no relacionats amb la reorganització. En la segona ronda s’han analitzat els productes de segregació dels cromosomes reorganitzats en els espermatozoides amb anomalies numèriques detectats prèviament, així com en espermatozoides no seleccionats. L’optimització del sistema d’anàlisi de la disposició dels territoris cromosòmics en el nucli espermàtic ha inclòs la selecció de nuclis segons el seu genotip, la captura d’imatges tridimensionals, la relocalització dels nuclis d’interès, el tractament digital de les imatges, la normalització de les dades i l’obtenció dels valors de posicionament dels centròmers en l’eix longitudinal i radial de l’espermatozoide.
Els resultats de l’anàlisi de segregació en espermatozoides no seleccionats mostren una elevada homogeneïtat dels patrons de segregació entre portadors del mateix tipus de translocació, caracteritzats per una elevada freqüència dels modes de segregacions que impliquen la disjunció a pols cel·lulars oposats dels cromosomes amb centròmers homòlegs. Els resultats de segregació en espermatozoides aneuploides i diploides/múltiples disòmics mostren un patró de segregació alterat respecte els espermatozoides no seleccionats, on s’afavoreixen els modes de segregació desequilibrats, en detriment dels productes de segregació equilibrats. Els espermatozoides amb diferents tipus d’aneuploïdies i amb diferents cromosomes implicats, influeixen per igual al patró de segregació alterat. Els resultats sobre el posicionament tridimensional dels cromosomes han permès validar la metodologia desenvolupada, ja que s’ha obtingut informació sobre la localització preferent dels cromosomes estudiats en nuclis amb diferents genotips.
Els resultats obtinguts demostren una associació entre la presència d’anomalies numèriques i un contingut desequilibrat dels cromosomes reorganitzats. L’acumulació d’anomalies cromosòmiques en els mateixos gàmetes pot ser atribuïble a un efecte bidireccional de l’heterosinapsi, que comportaria un canvi en la localització nuclear dels cromosomes afectats i una alteració en la formació dels quiasmes. Els problemes d’orientació dels cromosomes a la placa metafàsica poden provocar una aturada meiòtica prolongada, que en cas de no corregir-se, es podria resoldre a través de l’evasió del punt de control. Aquest fet afavoriria la producció de gàmetes que podrien acumular anomalies procedents de segregacions desequilibrades, a més d’aneuploïdies d’altres cromosomes. La tècnica desenvolupada per estudiar l’arquitectura nuclear dels espermatozoides permetrà determinar si les anomalies cromosòmiques presents en els gàmetes dels individus portadors de reorganitzacions cromosòmiques afecten l’organització nuclear global dels cromosomes i condicionen la seva fertilitat.Carriers of chromosomal translocations present a high risk of producing chromosomally abnormal gametes, as a consequence of an unbalanced segregation of the rearranged chromosomes, or the presence of numerical chromosomal anomalies derived from an interchromosomal effect. It is not known whether there exists a relationship between anomalies produced by these two events, but it might be based on the occurrence of heterosynapsis at the meiotic prophase I between multivalents and other chromosomes. Moreover, heterosynapsis has been associated related to changes in the nuclear chromosome architecture in sperm, which may also affect the fertility of reorganization carriers. The objectives of this thesis have been: i) To develop a sequential fluorescence in situ hybridization (FISH) protocol in order to detect numerical chromosomal abnormalities and to establish the segregation mode of rearranged chromosomes in the same spermatozoa; ii) To establish the segregation pattern of different chromosomal rearrangements in random sperm and in sperm with numerical abnormalities; iii) To determine whether there exists a relationship between certain segregation modes and the occurrence of numerical chromosome abnormalities; iv) To develop a methodology to evaluate the tridimensional distribution of chromosome territories in sperm nuclei. Semen samples of eight carriers of reciprocal translocations, eleven carriers of Robertsonian translocations and one carrier of a three-way translocation have been included in the study. In the first sequential FISH round, numerical anomalies for five chromosomes unrelated to the rearrangements have been analysed. In the second round, a segregation analysis has been performed both in the numerically abnormal sperm detected in the first round as well as in randomly assessed sperm. The optimization of the analysis of chromosome territories in sperm nuclei has included: nuclei classification according to their genotype, 3D image recording, relocalization of selected nuclei, digital images editing, data normalization, and prediction of the preferred position of each hybridization signal along the longitudinal and radial axis of spermatozoa. The segregation patterns obtained in randomly assessed sperm show high homogeneity among carriers of the same translocation. These patterns involve high frequencies of segregation modes that entail disjunction to opposite cellular poles of chromosomes with homologous centromeres. Data obtained from segregation analysis in aneuploid and diploid/multiple disomic sperm show altered segregation patterns when compared to randomly assessed sperm, in which unbalanced segregation modes are favoured while balanced segregation products decrease. Aneuploid sperm with different types of chromosomal abnormalities or different chromosomes involved in the aneuploidy have the same effect over the altered segregation pattern. The results obtained in the analysis of chromosome territories allow for the validation of the developed methodology. It has allowed the prediction of the preferred positioning of analysed chromosomes in sperm nuclei with different genotypes. In conclusion, data obtained point out that indeed there exists a relationship between the presence of numerical chromosome abnormalities and an unbalanced segregation content in sperm. This accumulation of chromosome anomalies in the same gametes would be driven by a bidirectional effect of heterosynapsis, which could entail changes in the nuclear positioning of the affected chromosomes, as well as alterations in chiasmata formation. The chromosome misalignment at metaphase plate would cause a meiotic arrest. If unresolved, cells could eventually evade this checkpoint favouring the accumulation of both unbalanced segregation products and aneuploidies for other chromosomes in the same gametes. The developed methodology to study the sperm nuclear architecture can be potentially used in carriers of chromosomal rearrangements, in order to assess whether chromosomal abnormalities in sperm affect the global nuclear chromosome organization and disturb their fertility.
3
Bradley, Leanne Teresa. "Cryopreservation of bovine spermatozoa." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ47313.pdf.
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歐陽鎮怡 and Chun-yee Natalie Auyeung. "Fucosyltransferase-5 on human spermatozoa mediates attachment of spermatozoa to oviductal epithelial cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40738620.
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Auyeung, Chun-yee Natalie. "Fucosyltransferase-5 on human spermatozoa mediates attachment of spermatozoa to oviductal epithelial cells." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40738620.
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Williams, Andrew C. "Glucose metabolism in human spermatozoa." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302101.
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Pandolfi, Susan M. "Cryopreservation of microencapsulated bovine spermatozoa." Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-11012008-063722/.
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Casey, Shannon Marie. "Effects of laundering on spermatozoa." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12314.
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Thesis (M.S.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Collection of physical evidence as soon as possible after a sexual assault is important for obtaining the best evidence of the crime. Actions taken following a sexual assault, such as laundering clothing worn during the assault, may occur before evidence collection has begun, and can have an effect on the forensic laboratory analysis. The purpose of this study was to investigate the effects of laundering on spermatozoa. The effects of All®, Woolite® and bleach on sperm removal during washing, on sperm elution of previously laundered evidence samples, and on sperm transfer during washing were examined and compared. The possibility of sperm transfer between laundry loads was also investigated. Bleach was found to have a significant effect on sperm removal during simulated laundering. The use of detergents during washing did not prevent the subsequent elution of sperm with sodium dodecyl sulfate (SDS), and did not affect the amount of sperm transferred during washing. It was also found that sperm transfer between laundry loads can occur. These results should be considered when dealing with sexual assault evidence that has been laundered prior to forensic examination. Complications with DNA analysis on clothing may be possible due to the occurrence of sperm transfer during laundering either before or after a sexual assault occurs.
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Lewis, Beverley Anne. "Cell biology of rat spermatozoa." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23087.
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The purpose of the research presented in this thesis was to investigate the cell biology of rat spermatozoa. An additional aim was to utilise the knowledge obtained to aid the development of in vitro functional tests for the assessment of rat sperm fertility and identify potential markers of normal epididymal maturation. As mammalian spermatozoa migrate through the epididymis, they acquire the potential for fertilisation, characterised by the acquisition of the ability to express co-ordinated movement and the competence to undergo capacitance. The mechanisms by which epididymal maturation confers upon mammalian spermatozoa the potential to capacitate is poorly understood. These studies investigated the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation using the laboratory rat as an animal model, since this signal transduction pathway is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility, both of which are prerequisites for fertilisation. Western Blot and immunocytochemical analysis demonstrated that epididymal maturation is associated with a progressive loss in phosphotyrosine expression located to the acrosomal domain. These differences in phosphotyrosine expression between caput and caudal epididymal spermatozoa appeared to reflect the normal in vivo situation. In addition, epididymal maturation of rat spermatozoa is also associated with an acquired competence to respond to high levels of intracellular cAMP by phoshorylating tyrosine residues on the sperm tail. Epididymal maturation also led to unique differences in the generation of reactive oxygen species (ROS) by spermatozoa obtained from the caput and caudal regions of the epididymis. Spermatozoa from both regions of the epididymis spontaneously generated equal levels of O2 whereas only mature caudal spermatozoa generated significant levels of H2O2. In contrast, although both caput and caudal spermatozoa generated increased O2-. in response to NADPH, induced levels were significantly greater in the immature caput cells.
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Herold, Florian-Cecil. "Influence of equilibration time and freezing diluent on post-thaw motility and acrosomal integrity of epididymal sperm from the African buffalo (Syncerus caffer)." Pretoria : [s.n.], 2008. http://upetd.up.ac.za/thesis/available/etd-10032005-094824/.
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Brooks, Nicole Lisa. "Apoptotic markers in ejaculated human spermatozoa." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&.
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The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P<0.05) were evident between the three groups. No significant differences (P>
0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<
0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.
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Enginsu, Mehmet Engin. "Morphology and function of human spermatozoa." [Maastricht : Maastricht : Rijksuniversiteit Limburg] ; University Library, Maastricht University [Host], 1994. http://arno.unimaas.nl/show.cgi?fid=6615.
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O'Toole, Christine M. B. "The development of motility in spermatozoa." Thesis, London Metropolitan University, 1994. http://repository.londonmet.ac.uk/3229/.
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Mammalian spermatozoa acquire the capacity for motility during passage through the epididymis. This study on rat spermatozoa shows that pH, cAMP and protein kinase C (PKC) all play an important role in the initiation of motility. pH has the most critical role and until the initial pH change in spermatozoa has occurred between the caput and caudal epididymal regions of the rat, second messengers are not effective in stimulating motility, but they are involved once such pH change has occurred. The spermatozoa of Fucus serratus differ from mammalian spermatozoa in that they are released into the sea prior to fertilisation and the motility of these spermatozoa is initiated upon their release into sea water. The ionic composition of sea water plays an important role in this activation and it is evident that the presence of Na+ is vital for the Initiation of motility. This study shows that a Na+/H+ exchanger, a N+-dependent bicarbonate/chloride exchanger and a Na+/K+ pump, which regulate the concentration of Na+, are present in Fucus serratus and integrated activity of these exchangers/pumps causes an increase in intracellular pH (pHi). An elevation in pHi correlates to an increase in motility, mediated through the activation of the dynein ATPase of the flagella. Motility and respiration of these spermatozoa are closely linked, probably because the ATP produced by respiration is used primarily by the dynein ATPase. Second messengers have also been Implicated in the initiation/regulation of motility and respiration. Indirect evidence shows cAMP and PKC are present and regulate motility, possibly through the phosphorylation and thereby activation of key regulatory proteins, such as the Na+/H+ exchanger. A rise in intracellular Ca2+ is also associated with the activation of Fucus serratus spermatozoa but the exact mechanism by which such a rise regulates motility remains unclear.
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Crosby, John. "Interactions between prostaglandins, phospholipids and spermatozoa." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/18806.
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Jamaludin, Nurul Akmal. "Extracellular vesicles mediated intercellular communication between spermatozoa and oviduct : a new paradigm of spermatozoa-oviduct cross talk." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/21192/.
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Klepper, Ludger. "Der Nachweis der Bindung von Spermatozoen-Autoantikörpern an Spermatozoen mittels indirekter Immunfluoreszenz." [S.l.] : [s.n.], 2000. http://archiv.ub.uni-marburg.de/diss/z2002/0126/.
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Murray, George M. "Acrosome size and kinematics of human spermatozoa." Thesis, Link to the online version, 2007. http://hdl.handle.net/10019/1131.
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Lefièvre, Linda. "Studies on phosphodiesterase activities in human spermatozoa." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38498.
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After ejaculation, spermatozoa undergo a series of transformation in the female genital tract, named capacitation, that enables them to bind to the zona pellucida, initiate the acrosome reaction and fertilize the egg. Mammalian sperm motility, capacitation and acrosome reaction are regulated by signal transduction systems involving cAMP and cAMP-dependent protein kinase A (PKA). Intracellular levels of cAMP are regulated by adenylyl cyclase (AC) and phosphodiesterases (PDEs), responsible for its production and degradation, respectively. Of the 11 PDE families that exist, calmodulin (CaM)-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in human spermatozoa. The aims of the present study were to evaluate the role played by some PDEs in human sperm function, to determine which PDEs are present and to evaluate the changes in cAMP, PDE and PKA activities during sperm capacitation and acrosome reaction. PDE activity did not change during either capacitation or acrosome reaction while PKA activity was increased in both phenomena. Moreover, sperm PDEs were associated with the membrane (50--60%) as well as with the particulate fraction (30--50%) and had much more affinity for cAMP than cGMP as substrate. Type-specific PDE inhibitors such as EHNA for cGMP-stimulated PDE (PDE2), milrinone for cGMP-inhibited PDE (PDE3) and rolipram for PDE4 but not sildenafil for cGMP-specific PDE (PDE5), decreased sperm PDE activity suggesting that PDE2, PDE3 and PDE4 but not PDE5 were present both in membrane and particulate fractions. Moreover, EHNA and rolipram increased sperm capacitation while milrinone increased sperm cAMP. Anti-PDE1A and anti-PDE3A antibodies recognized proteins of 67 kDa (PDE1A) and 97 kDa (PDE3A). Immunolocalization indicated that PDE1A was present on the equatorial segment of the sperm head as well as on the mid- and principal pieces of the flagellum and that PDE3A was present on the post-acrosomal segment of the head. Furth
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McLaughlin, Eilenn Anne. "The effect of cryopreservation on human spermatozoa." Thesis, University of Bristol, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358122.
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Nash, Katherine Louise. "Store-operated calcium entry in human spermatozoa." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3260/.
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The contribution of store-operated calcium entry (SOCE) in the response of human sperm to progesterone, a steroid secreted from the cumulus cells surrounding the oocyte, has not yet been elucidated. The aim of this study was to investigate the presence of SOCE proteins in human sperm and examine the effects of pharmacological modulation of SOCE on the progesterone-induced biphasic intracellular calcium concentration ([Ca\(^{2+}\)]i) response. STIM (stromal interacting molecule) and Orai, proteins of the SOCE system were detected in human sperm in a similar location to intracellular Ca\(^{2+}\) stores. 2-aminoethyldiphenyl borate (2-APB; SOCE modulator) altered SOCE in human sperm in a bimodal manner as seen in other cell types. Furthermore, 5\(\mu\)M 2-APB potentiated the initial progesterone-induced [Ca\(^{2+}\)]i transient within the neck and midpiece, but not in the flagellum. In the sustained phase of the progesterone-induced [Ca\(^{2+}\)]i response both 5\(\mu\)M 2-APB and 10\(\mu\)M loperamide (another modulator of SOCE) potentiated the [Ca\(^{2+}\)]i response. Higher doses of 2-APB (50-200\(\mu\)M) didn’t potentiate the transient [Ca\(^{2+}\)]i and inhibited the sustained response consistent with reported actions on SOCE. Ryanodine receptors were localised to the neck/midpiece region which suggested that they may mobilise intracellular Ca\(^{2+}\) stores in response to progesterone, leading to activation of STIM/Orai and initiating SOCE.
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Morales, Garcia Auden Andres. "The response of human spermatozoa to chemoattractants." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/630/.
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The effect of the chemoattractant bourgeonal on [Ca\(^{2+}\)]i and chemotaxis in human sperm was investigated. Burgeonal induced a dose-dependent, slowly-developing tonic elevation in [Ca\(^{2+}\)]i, The response was dependent on capacitation. In low-Ca\(^{2+}\) or EGTA-buffered saline the response to bourgeonal was inhibited. Pretreating spermatozoa with bis-phenol (20μM) to release stored Ca\(^{2+}\) did not alter the response. Thus bourgeonal acts primarily by inducing Ca\(^{2+}\) influx. Treatment of sperm with bourgeonal caused an increase in [cAMP]. When cells were pretreted with bourgeonal in low-Ca\(^{2+}\)saline, subsequent introduction of Ca\(^{2+}\) resulted in a single, large [Ca\(^{2+}\)]i transient in >75% of the cells, indicating that sudden influx of Ca\(^{2+}\) caused closure of the bourgeonal-sensitive Ca\(^{2+}\)- channel. This negative feedback was not modulated by IBMX (1mM) or dbcAMP (1mM), indicating that cAMP was not involved and that a direct action Ca\(^{2+}\) was more likely. Both Ni\(^{2+}\) (10μM) and La\(^{3+}\) (100μM) inhibited the action of bourgeonal on [Ca\(^{2+}\)]i, suggesting a possible role of CNG channels. Exposing sperm to a temporal bourgeonal gradient caused a series of transient [Ca\(^{2+}\)]i elevations in >20% of the cells. A gradient of progesterone (another characterised chemoattractant for human sperm) induced similar Ca\(^{2+}\) oscillations (in >20% of the cells), which increased in amplitude and frequency in response to the increasing progesterone concentration. Human spermatozoa responded chemotactically to a 1nM bourgeonal gradient, Chemotaxis was dependent on capacitation. The response was inhibited in low [Ca\(^{2+}\)]o but was unaltered by TMB-8 (an inhibitor of stored Ca\(^{2+}\) store release), thus showing a dependence on Ca\(^{2+}\) influx similar to the [Ca\(^{2+}\)]i signal.
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Uçar, Ömer. "Acrosome reaction and cryopreservation of dog spermatozoa." Thesis, University of Bristol, 2000. http://hdl.handle.net/1983/60c06232-353d-4d6c-9ca7-3073a2712d2d.
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The use of AI in dogs has been limited by the lack of effective and reliable means of cryopreservation of semen and by the poor correlation between traditional methods of post-thaw assessment of semen quality and fertility. In order to address these problems, the present study focuses upon methods of cold storage and cryopreservation of dog spermatozoa by undertaking comparative evaluations of post-thaw motility and in vitro induction of acrosome reaction. Split-ejaculate protocols were used to compare the effect of storage at +4°C and cryopreservation upon (i) the maintenance of spermatozoa motility and (ii) spontaneous or A23187-induced acrosome reactions during incubation at 39°C (in 5% CO2 in the humidified air) for 60 or 120 min. The assessments of samples were made by Bright-Field, Phase Contrast (PC), Differential Interference Contrast (DIC), Scanning (SEM) and Transmission (TEM) Electron Microscopy. The interaction between the process of glycerolisation and the presence of seminal plasma is one of the key limitors for success in cryopreservation of dog spermatozoa. Interactions between the effects of removal of seminal plasma (by centrifugation), dilution rate, the temperature at which glycerolisation took place and the concentration of glycerol upon survival of spermatozoa at +4°C were studied in a series of split-ejaculate experiments. Spermatozoa were suspended in Tris-fructose-citric acid extender containing 20% (v/v) egg yolk and 8% (v/v) glycerol at +4°C for 48 h. Survival was assessed as the percentage of spermatozoa displaying progressive motility. Survival of spermatozoa was higher (P < 0.05) after glycerolisation at +4°C than at the room temperature. At dilution rates of 1:1 and 1:2 (semen: extender), the survival was higher (P < 0.05) in samples that were centrifuged and glycerolised at +4°C than the samples that were neat and glycerolised at the room temperature. While at the dilution rate of 1:16 it was higher (P < 0.05) in samples that were neat plus glycerolised at +4°C than all samples that were glycerolised at the room temperature. Concentrations of glycerol that were > 2% (v/v) resulted in lower (P < 0.05) survival than at lower concentrations. Following the initial stage of the investigations, the optimisation and validation of a method for in vitro induction of acrosome reactions were required. Suspensions of spermatozoa in TALP medium were incubated in the presence of a logarithmic. series of concentrations of the calcium ionophore, A23187. Induction of acrosome reactions was assessedb y bright-field (using naphthol yellow S/aniline blue stain, NA) and phase contrast (PC) microscopy. Using these methods, it was determined that incubation in the presence of 1 μM/1 A23187 for a period of 30-45 min was optimal for inducing acrosomer eactions in fresh semen. It was also noted that the assessments of acrosome reactions by using NA staining, were highly correlated with PC microscopy. In consequence, the simple procedure of NA staining might be an acceptable alternative to PC microscopy for use in the field. Subsequently, the effect of chilling and glycerolisation upon in vitro induction of acrosome reactions by A23187 was assessed. Acrosome reactions were studied as these have been described in the literature as providing accurate bioassay of spermatozoal functionality in vivo. Acrosome reactions were assessed by using DIC microscopy. The acrosomal integrity was impaired after chilling, which accelerated the A23187-induced acrosome reaction such that a lower concentration (0.1 μM/1) of A23187 was also effective to induce the reaction within 60 min of incubation. However, the presence of 2% glycerol (v/v, final) in standard Tris extender, containing 20% egg yolk, did not significantly affect the sequence of acrosome reaction. The optimal freezing regimen (from +4°C to -120°C) was determined by using a programmable biological freezer in a series of experiments, in which various cooling rates were combined in a Latin square design. Semen was diluted in standard Tris extender containing 20% egg yolk and 2% glycerol (v/v, final) and packed in 0.25 ml French paiettes (straws). The optimal cooling regimen was -0.5°C/min from +4°C to -9°C, -40°C/min to -20°C, -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen. Changes in temperatures within an individual straw were continuously measured and these data were found to be highly correlated with the eventual post-thaw motility of frozen-thawed spermatozoa. Although freezing and thawing resulted in major acrosomal deterioration, there were no significant differences between freezing regimens on the basis of in vitro induction of acrosome reactions, as assessed by DIC microscopy. Finally, ultrastructural studies, using SEM and TEM, upon chilled (as 'ready to freeze') and frozen-thawed spermatozoa subjected to A23187-induced acrosome reaction demonstrated that freeze-thawing provoked the acrosome reaction such that, with TEM (i) the plasma membrane was usually damaged or missing, (ii) the acrosomal changes (including the loss of acrosomal content, as seen by decondensation and swelling) except vesiculation of the acrosomal membranes, exceeded to the equatorial segment and (iii) a further damage occurred to the post-acrosomal region. In summary, these results show that semen should be; (i) centrifuged for dilutions of < 1:8, (ii) diluted at 1:8 in Trisfructose-citric acid extender containing 20% egg yolk, (iii) glycerolised at +4°C at a final concentration of 2% glycerol (v/v), (iv) cooled at -0.5°C/min from +4°C to -9°C, at -40°C/min to -20°C and at -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen for cryopreservation and (i) introduced to 1 μM/1 A23187 in TALP, (ii) incubated for at least 30-45 min for induction of acrosome reaction in vitro and, thereby, demonstrated that optimisation of cryopreservation and in vitro induction of acrosome reaction of dog spermatozoa are possible.
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Ajonuma, Louis Chuwuemeka. "In vitro study of the effects of hydrosalpinx fluid on the motility and velocity of spermatozoa." Thesis, Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25248728.
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Scheltinga, David Michael. "Ultrastructure of spermatozoa of the amphibia : phylogenetic and taxonomic implications /." [St. Lucia, Qld.], 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16839.pdf.
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Biber, Franz Stefan. "Stimulation kryokonservierter Spermatozoen mit Pentoxifyllin." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=97216507X.
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Maravei, David. "Characterization of the perinuclear theca of bovine spermatozoa." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22767.
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The perinuclear theca is a unique cytoskeletal element that encapsulates the nucleus of mammalian spermatozoa except for a narrow zone around the insertion of the sperm tail. It is composed of two distinct regions, a subacrosomal layer or perforatorium and, continuing caudally beyond the acrosomic system, the postacrosomal sheath. Gel electrophoresis revealed that the extracts were composed of several polypeptide bands, of which the 15.5, 25, 28, 32, 36, and 60 kDa bands were most prominent in the bull. Antibodies against the 15.5 and 60 polypeptides of the bull reacted monospecifically with their respective bands on immunoblots and immunogold labeled the perforatorium and the entire perinuclear theca, respectively. Antibodies against the 25, 28, 32, and 36 kDa polypeptides of the bull showed distinctive patterns of cross reactivity with other polypeptides on Western blots, but immunogold labeled exclusively the entire perinuclear theca. Similar manipulation of human spermatozoa revealed that this element was composed of three major polypeptides, including the 14, 15, and 67 kDa bands. The anti-15.5 (bull) antibody, in addition to an antibody against the 15 kDa protein of rat spermatozoa (raised in a previous study) each cross-reacted with the 15 and 67 kDa polypeptides on Western blots of the human perinuclear theca. Conversely, the anti-60 (bull) antibody and an anti-60 (rat) antibody each reacted monospecifically with the 67 kDa polypeptide of the human perinuclear theca. As well, the anti-60 (bull) antibody immunogold labeled the entire perinuclear theca of human spermatozoa. (Abstract shortened by UMI.)
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Williams, Hannah Lauren. "CatSper : characterisation in human spermatozoa and clinical significance." Thesis, University of Dundee, 2016. https://discovery.dundee.ac.uk/en/studentTheses/0d97594f-eb88-4f33-87de-5784ae973865.
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Nordskov, Harder FIE BARBARA. "Effect of Dextrans on Cryopreservation of Human Spermatozoa." Thesis, Malmö universitet, Fakulteten för hälsa och samhälle (HS), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-24295.
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Cryopreservation is a process where a sample, e.g. cells or tissue, is preserved by cooling to sub-zero temperatures, usually -196°C. Upon freezing these materials, ice crystals form, which eventually results in cell death. In order to reduce the formation of ice crystals, cryoprotectants are added to the storage solution. Current cryoprotective agents have several weaknesses, making the development of cryoprotective agents with advanced properties of great interest. In this study, two possible non-penetrating cryoprotectants termed Dextran Sulphate Sodium Salt 80 (DSSS 80) and Dextran Sulphate Sodium Salt (DSSS 140), were developed. The cryopreservative effect of DSSS 80 and DSSS 140 was studied on human spermatozoa regarding cryorecovery, using a simple cryopreservation procedure. Moreover, the new compound’s cryoprotective properties were compared to conventional types of dextrans, which in previous studies have proven effective. The percentage of sperm motility was assayed before freezing by the optical microscopic method using a haemocytometer as counter chamber. After thawing, the cells were stained with trypan blue and post-thaw motility was determined. It was found that all sperm cells lost their motility, regardless cryoprotectant, concentration or in the absence of dextrans, which indicated that the applied cryopreservation was unfitting. Thus, since the method used in study was not optimal for cryopreservation of sperm cells, it was not possible to determine whether DSSS 80 or DSSS 140 display cryoprotective properties or not, nor if they are superior to conventional types of cryoprotectants.
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Das, Lala Meenakshi. "Identification of Endogenously Biotinylated Proteins in Mammalian Spermatozoa." Kent State University Honors College / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1303846044.
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Downie, Sarah Elizabeth. "Detection of chromosomes and chromosomal abnormalities in human sperm." Title page, contents and overview only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phd751.pdf.
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Bibliography: leaves 135-151. A study of chromosomal abnormalities and the localisation of chromosomes in human sperm, especially from men with TSD, using fluorescence in situ hybridization (FISH). The project entailed: 1. development of reliable FISH protocols, 2. determination of basline frequencies of aneuploidy, 3. analysis of chromosomal abnormalities in men with severe TSD and 4. assessment of the localisation of individual chromosomes within the sperm head.
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Van, Zyl Estee Alwelien. "The effect of incubation time and temperature on sperm motility, human sperm DNA and assisted reproductive technologies (ART) outcome." Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/96793.
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Thesis (MMed)--Stellenbosch University, 2015.ENGLISH ABSTRACT: In all Assisted Reproductive Technologies (ART) procedures the semen sample is handled, processed, prepared and manipulated before use in the fertilization process. During these incubation times, the sperm cells are exposed to factors that may inflict damage to the sperm structure and DNA integrity, impair its functional abilities and subsequently lead to fertilization failure and poor ART outcome. Two of the very basic, but important factors that may have an impact on the sperm quality are time and temperature exposure. The primary objective of this study was to prospectively determine the effect of different incubation times and temperatures on motility and the DNA profile of the spermatozoa. Non-processed (n=36) and processed (n=33) semen samples were incubated for different time intervals (before: 20, 40, 60 minutes; after: 30, 60, 90 minutes) and at different temperatures (room temperature [RT] and 37°C). After incubation, sperm parameters were assessed, the CMA3 assay was applied to determine chromatin maturity and compaction and the TUNEL assay to assess the level of DNA fragmentation. The results showed that in the non-processed group, incubation led to a time-dependent, significant decline in the motility. The highest motility was seen at 20 minutes (37°C) and motility declined in a time-dependent manner. Incubation time and temperature did not affect the CMA3 and TUNEL values. Incubation of the processed sample led to a significant time-dependent decrease in the motility; 90 minutes (RT) had the lowest motility. The CMA3 and TUNEL values between the different incubation groups did not differ significantly. The secondary objective was to retrospectively investigate the effect of sperm incubation time after preparation on ART outcome. A total of 901 patient ART cycles (January 2010- December 2012) were included. Fertilization rates, embryo quality and pregnancy rates were examined. The results showed that the sperm incubation time before insemination between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) differed significantly and the incubation time had a significant negative effect on the fertilization rates in IVF, but not in ICSI. Longer incubation times led to an unexpected significant improvement in the quality of day 2 embryos and were significantly associated with pregnancy failure in IVF and ICSI. These combined findings suggest that non-processed semen samples can be incubated at RT or 37°C, but for no longer than 40 minutes and, for IVF, processed samples should not be incubated for longer than 60 minutes at RT or 37°C. The ICSI sample should not be incubated for more than 60 minutes although longer incubation times do not seem to influence the results for IVF. It can therefore also be concluded that sperm incubation time before insemination should be closely monitored, especially in IVF cycles.
AFRIKAANSE OPSOMMING: In kunsmatige voortplantingstegnieke (ART) word die semen-monster geprosesseer, voorberei en gemanipuleer voordat dit vir die bevrugtingsproses gebruik word. Terwyl die monster geïnkubeer word, word die spermselle blootgestel aan verskeie faktore wat die struktuur van die sperm, die DNS integriteit en die sperm se funksionele vermoë negatief kan beïnvloed. Dit kan lei tot swak bevrugting, embriokwaliteit en swangerskapsyfers. Twee basiese, maar belangrike, faktore wat die spermkwaliteit negatief kan beinvloed is die duur van inkubasie en die temperatuur waarby die spermselle geïnkubeer word. Die primêre doel van die huidige studie was om prospektief te ondersoek wat die effek van verskillende inkubasietye en temperature op die motiliteit en DNA profiel van die sperm het. Monsters is voor en na spermvoorbereiding vir verskillende tydsintervalle (voor: 20, 40, 60 minute; na: 30, 60, 90 minute) en verskillende temperature (kamertemperatuur [KT] en 37°C) geïnkubeer. Na elke inkubasie is ’n spermanalise, ’n CMA3- en ’n TUNEL toets gedoen. Die CMA3 toets bepaal die chromatienmaturiteit en -kompaksie en die TUNEL toets vir die vlak van DNS fragmentasie. Die resultate het getoon dat daar in die voor voorbereiding groep ’n beduidende verskil in motiliteit tussen die verskillende inkubasiegroepe was. Die hoogste motiliteit is in die 20 minute/37°C groep gevind. Die motiliteit het oor tyd afgeneem. Die tyd en temperatuur van inkubasie het nie ’n beduidende effek op die CMA3 en TUNEL uitslae gehad nie. Inkubasie nadat die semen voorberei was het weereens tot ’n beduidende verskil in motilieit tussen die groepe gelei. Die laagste motiliteit is waargeneem by 90 minute/KT. Geen beduidende verskil is tussen die inkubasiegroepe vir CMA3 en TUNEL gevind nie. Die sekondêre doel van die studie was om retrospektief te ondersoek wat die effek van sperminkubasietyd na spermvoorbereiding op die bevrugting, embriokwaliteit en swangerskapsyfers is. 901 pasiëntsiklusse is in die studie ingesluit (Januarie 2010 tot Desember 2012). Die resultate het aangedui dat die inkubasietye van die intrasitoplasmatiese inspuiting (ICSI) en in vitro bevrugting (IVB) beduidend van mekaar verskil het. Langer inkubasietye het ’n beduidende negatiewe effek op die bevrugtinguitslae van IVB siklusse gehad, maar geen effek op ICSI siklusse gehad nie. Langer inkubasietye het ook tot ’n onverwagte verhoging in die kwaliteit van dag 2 embrios gelei en was verder beduidend geassosieer met negatiewe swangerskapuitkoms. Hierdie gesamentlike bevindinge dui aan dat semenmonsters voor voorbereiding by KT of 37°C geïnkubeer kan word, maar nie vir langer as 40 minute nie. Na semenvoorbereiding, behoort die IVB semenmonster vir nie langer as 60 minute voor inseminasie geïnkubeer te word nie (KT of 37°C). Die ICSI semenmonster moet verkieslik binne 60 minute na voorbereiding gebruik word, maar dit wil voorkom asof die tyd hier nie so ’n groot rol speel nie. Daar kan verder afgelei word dat sperminkubasietye voor die gebruik vir inseminasie baie goed gemonitor moet word – veral in IVB siklusse.
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Herold, Florian-Cecil. "Influence of equilibration time and freezing diluent on post-thaw motility and acrosomal integrity of epididymal sperm from the African buffalo (Syncerus caffer)." Diss., University of Pretoria, 2003. http://hdl.handle.net/2263/28368.
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The aim of this study was to test whether or not the equilibration time of two different cryodiluents influences the post thaw qualities of epididymal African buffalo (Syncerus caffer') sperm. Diluents and equilibration times were compared by assessing the post thaw spermatozoal motility, longevity and the acrosomal integrity. African buffaloes belong to Africa's "Big Five" and are, therefore, popular animals amongst game farmers, hunters and tourists. They are also asymptomatic carriers of foot-and-mouth-disease (FMD) and considered to be a wildlife reservoir for this plague. Other diseases, that are carried and can be transmitted from the African buffalo (Syncerus caffer') to livestock include tuberculosis, brucellosis and theileriosis or corridor disease (CD). Therefore, the transportation of African buffaloes is highly regulated. Disease-free buffalo populations are currently derived from a small genetic 8 pool and are smaller in their trophy size than the free-ranging animals from the diseased areas of the Kruger National Park (KNP) and the Hluhluwe/Umfolozi National Park. Hence there is a special interest in bringing new genetic material into
the disease-free populations. Epididymal sperm from 11 mature African buffalo bulls was collected, diluted with two different semen extenders (TriiadylTM [Tris egg yolk extender] and AndroMed® [synthetic extender, i.e. fully defined medium]) and frozen. Pre-freezing equilibration times of 2 and 9 hours were tested. Total and progressive motilities, longevities and
acrosomal integrity were measured and compared.
Results show that there were no differences in post-thaw sperm quality when equilibration times between 2 and 9 hr were used. The use of the egg yolk containing extender (TriiadlyTM) resulted in higher percentage of post-thaw motilities than the
use of the synthetic AndroMed®. Because a high percentage of progressive motile spermatozoa is one of the
prerequisites for successful AI it must be concluded that TriladylTM is superior to AndroMed®. As I believe the advantages of higher motility to be bigger than the hygiene risks of a yolk containing extender I conclude that epididymal buffalo sperm
should rather be frozen with TriiadylTM than with AndroMed®.Dissertation (MSc (Production Animal Science))--University of Pretoria, 2003.
Production Animal Studies
unrestricted
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Rößner, Claudia. "Transduktion von Apoptosesignalen ejakulierter Spermatozoen von Diabetikern." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-132595.
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Die Inzidenz des Diabetes mellitus (DM) nimmt weltweit jährlich zu und erlangt somit große Bedeutung für die Entwicklung der globalen Gesundheit. Die WHO rechnet bis zum Jahr 2030 mit ungefähr 366 Millionen erwachsenen Diabetikern. Es ist bekannt, dass Männer mit Diabetes mellitus Typ 1 (DMI) und 2 (DMII) häufiger an Subfertilität leiden, wobei dies möglicherweise auf erhöhte Apoptoseraten und vermehrte DNA-Fragmentierungen auf zellulärer Ebene zurückzuführen ist. Die Bedeutung der ROS als Regulatoren von physiologischen und pathologischen Signaltransduktionswegen ist bekannt. Demzufolge könnte die Aktivität der Stickstoffmonoxid-Synthetase (NOS) in diesem Zusammenhang eine Bedeutung haben. Das Ziel der vorliegenden Untersuchungen war es, die Auswirkungen von Apoptose und DNA-Fragmentierungen sowie die Bedeutung der NOS im Zusammenhang mit der Fertilitätsfähigkeit von Spermienzellen von DMI und DMII Patienten zu erfassen und damit erste Erklärungsansätze zur Pathophysiologie der diabetesassoziierten Subfertilität zu liefern. Samenproben von Normalspendern und Diabetikern wurden durch Dichtegradientenzentrifugation in Subpopulationen separiert und mittels fluoreszenzbasierten Assays zur Analyse von apoptoseassoziierten Parametern wie dem Zusammenbruch des mitochondrialen Membranpotentials (MMP), Aktivierung von Caspase-3 (CP3), DNA-Fragmentierungen und reaktiven Sauerstoffspezies (ROS) im Flowzytometer (FACS) untersucht. Die Ergebnisse zeigen eine signifikante Erhöhung von Apoptosemarkern (gestörtes MMP, aktivierte CP3), ROS und DNA-Fragmentationsraten in Spermien von DMI und DMII Patienten im Vergleich zu gesunden Normalspendern. Der Effekt ist bei DMII Patienten verstärkt ausgeprägt. Alle gemessenen Parameter korrelieren umgekehrt mit dem Fertilitätspotential der Spermien, gemessen anhand etablierter Spermiogramm-Analysen, womit ein möglicher Erklärungsmechanismus für die Subfertilität bei Diabetikern geliefert werden kann.
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Svetlichnyy, Valentin. "Aufnahme von Fettsäuren in Spermatozoenlipide von Sus scrofa domestica und physiologische Auswirkungen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16665.
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Die vorliegende Arbeit beschäftigt sich mit den physiologischen Veränderungen porciner Spermatozoen, die durch einen metabolischen Einbau von Fettsäuren in Spermatozoenlipide hervorgerufen werden. Ziel dieser Arbeit war die Untersuchung der metabolischen Aufnahme von Fettsäuren in die Spermatozoenlipide und die Bewertung des physiologischen Zustandes porciner Spermatozoen mit Hinblick auf die Niedrigtemperaturlagerung. Alle in den porcinen Spermatozoen vorkommenden Lipide wurden mittels GC und MALDI-TOF-MS analysiert. Hauptvertreter der polaren Lipidklassen sind Glycerophospholipide (GPC, GPE). Der Hauptvertreter der neutralen Lipidklassen ist Diacylglycerol (DAG). Die metabolische Aufnahme von Fettsäuren in die Lipide wurde durch die Supplementierung des Flüssigkonservierungsmediums mit [14C]-Octadecadiensäure radiochemisch untersucht. Anhand dieser Experimente wurde gezeigt, dass die Temperatur und die Inkubationsdauer wichtige Faktoren für die metabolische Aufnahme dieser Radiochemikalie in die Spermatozoenlipide sind. Die zugesetzten Fettsäuren werden sowohl in die neutralen (DAG) als auch in die polaren Lipide (diacyl-GPC) der Spermatozoen eingebaut. Nach Supplementierung mit 13C-markierter Octadecadiensäure wurden die Lipide mittels MALDI- und Q-TOF-MS als DAG (18:2/18:2), GPC (16:0/18:2) und GPC (18:2/18:2) charakterisiert. Die gleichen Ergebnisse wurden auch für die in den Spermatozoenlipiden vorkommenden Hexadecen-, Octadecen-, und Octadecatriensäure erhalten. Bei der Untersuchung des physiologischen Zustandes von Spermatozoen wurde gezeigt, dass insbesondere Supplementierungsvarianten mit endogen vorkommenden Fettsäuren zu einer besseren Spermatozoenvitalität und Motilität bei Niedrigtemperaturlagerung führten. Gleichzeitig wurde eine Verminderung des Auftretens von akrosomalen Schäden festgestellt. Damit stellt eine Supplementierung der Spermatozoen mit ausgewählten Fettsäuren eine effektive Maßnahme zur Lagerung von Spermatozoen bei 4 bis 6°C dar.This study examines the metabolic incorporation of selected fatty acids into the lipids of porcine spermatozoa and evaluates the physiological state of spermatozoa subsequent to low temperature storage supplementation with selected free fatty acids. The aim was to understand the role of fatty acids in relation to the (cryo-)preservation of spermatozoa and successful reproduction in more detail. All lipids present in porcine spermatozoa were analysed using gas chromatography (GC) and mass spectrometry (MALDI-TOF-MS). The main representatives of the polar lipid classes are glycerophospholipids (in particular GPC and GPE). The main representatives of the neutral lipid classes are diacylglycerols (DAG). Metabolic incorporation of fatty acids into lipids was radiochemically monitored using [14C]-octadecadienoic acid in the supplied spermatozoa-preservation medium. Temperature and incubation time were shown to be particularly important determinants. The added fatty acids were incorporated into both the spermatozoas’ neutral (DAG) and polar lipids (diacyl-GPC). The affected lipids were characterised by means of MALDI- and Q-TOF-MS subsequent to the supplementation of uniformly 13C-labelled octadecadienoic acid. DAG (18:2/18:2), GPC (16:0/18:2) and GPC (18:2/18:2) could be identified and a de-novo biosynthesis of DAG (18:2/18:2) could be proven. The same results were obtained when spermatozoa were supplemented with hexadecenoic, octadecenoic and octadecatrienoic acids. Finally, it was shown that the physiological state of the spermatozoa, especially those supplemented with endogeneously present fatty acids, led to an enhanced vitality and motility in spermatozoa subsequent to low temperature storage. It was also observed that acrosomal damage was reduced and that hexadecenoic acid significantly stabilised all the vitality parameters. In conclusion, supplementing spermatozoa with selected fatty acids is an effective solution for the storage of spermatozoa at 4 to 6°C.
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Kasimanickam, Vanmathy. "Adding exogenous lipids into boar spermatozoa alters their functions." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ56335.pdf.
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Rota, Alessandra. "Studies on preservation, capacitation and fertility of dog spermatozoa /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1998. http://epsilon.slu.se/avh/1998/91-576-5444-1.gif.
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Shadan, Sadaf. "Cholesterol dynamics in the plasma membrane of mammalian spermatozoa." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615713.
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Fisher, Helen M. "Mechanisms of reactive oxygen species generation by mammalian spermatozoa." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/20503.
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The generation of reactive oxygen species (ROS) is an activity normally associated with phagocytic leucocytes, which employ an enzymatic complex, the NADPH oxidase, to catalyze the univalent reduction of molecular oxygen to superoxide. However, it is now apparent that many other cell types, including mammalian spermatozoa, generate ROS. ROS have been implicated in peroxidative damage associated male sub-fertility, and also more recently, in the regulation of normal sperm function. With the obvious biological importance of ROS generation by spermatozoa in mind, the aims of this PhD have been to identify and characterize the mechanisms, and cellular components involved in the generation of ROS by these cells. Experiments investigating ROS generation by human spermatozoa have shown that the mechanisms involved are functionally similar to those pertaining to the NADPH oxidase of phagocytic leucocytes. ROS generation by human spermatozoa is enzymatic in nature and utilizes NADPH as the electron donor, reducing molecular oxygen to superoxide, which subsequently dismutates to hydrogen peroxide. Further similarities with the NADPH oxidase include the probable involvement of a flavoprotein and a role for protein phosphorylation, as regulated by PKC and protein phosphatases. Biochemical and molecular analyses of human spermatozoa have shown that the cellular components that form the NADPH oxidase of phagocytic leucocytes are not present in the spermatozoon. Spectral analyses of human sperm membranes failed to reveal the presence of the characteristic low potential cytochrome b558, and similarly, western blot analyses of human spermatozoa failed to detect the cytochrome b558 or the presence of the two NADPH oxidase cytosolic components, p47phox and p67phox. ROS generating activity was extracted from human spermatozoa, using the non-ionic detergent n-octyl-β-D-thioglucoside. The solubilized protein extract was resolved by non-denaturing PAGE and shown to contain 6 bands with ROS generating activity. ROS generating activity was then isolated in an active form via 2'-5'ADP affinity chromatography. The individual components of this activity were subsequently separated via SDS-PAGE, which revealed a heterogeneous protein population. One of these proteins was purified, and a polyclonal antibody raised against it.
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Fortuin, Kay Arlene. "Analyses of spermatozoa surface proteins using different separation techniques." Thesis, University of the Western Cape, 2013. http://hdl.handle.net/11394/4607.
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Magister Scientiae (Medical Bioscience) - MSc(MBS)Passage of spermatozoa through the female reproductive tract is essential for the regulation of fertilization, ensuring that healthy sperm reach the oocyte. Previous studies were devoted to morphological selection of sperm cells by the cervical mucus. However, research prove that the loss of integrity of the sperm plasma membrane is associated with infertile men, irrespective of their normal semen parameters. This indicates that the sperm plasma membrane plays an important role in fertilization. Further studies indicated that sperm surface proteins assist penetration through the female reproductive tract and would therefore provide useful insight in understanding other factors associated with male infertility. The aim of this project was to determine if there are any differences between sperm surface proteins of fertile donor samples in relation to infertile patient samples using different separation techniques and different detergents. Three different sperm separation techniques were employed, including wash, swim-up (SU) and Percoll density gradient centrifugation (DGC).Parallel to this, the deoxy-ribose nucleic acid (DNA) fragmentation of these cells were analysed for comparison of the extent of DNA damage induced due to different separation techniques used. This provided evidence that the best separation technique is the DGC as it minimises the amount of DNA fragmentation caused. Four different detergents were used in the process of extracting the membrane proteins from spermatozoa, namely sodium dodecyl sulphate (SDS), saponin,cetyl-trimethyl-ammonium bromide (CTAB), and TWEEN-20. The membrane proteins were then separated on a12% SDS poly-acrylamide gel electrophoresis (PAGE), and analysed by Coomassie blue and silver staining techniques as well as densitometry. Due to the different chemical nature of the detergents that extracted different surface proteins, CTAB (cationic) and SDS (anionic) extracted the most because of its strong solubilising abilities as non-ionic detergents. Common proteins that were extracted in donor samples included; 115, 92.5, 89, 61, 55.5, 51.5, 47, 44.5, 43, 38.5, 34 and 28 kDa proteins. In patients, commonly occurring proteins included; 92.5, 74.5, 70, 60.5, 51.5, 50, 44.5, 43, 36, 29.5, and 25.5 kDa proteins. Marked differences were found between membrane proteins extracted from donor samples in comparison to patient samples. Identification of these proteins was done using the SwissProt database and a literature search. Mostly non-genomic progesterone receptors were identified; others included oestrogen receptor, a phosphotyrosyl protein, P34H, equatorial segment protein, mannose lectin receptor, human guanylylcyclase receptor, epididymal protease inhibitor receptor, PH30 and estradiol binding protein. The function of the membrane surface proteins identified in this study plays a vital role in fertilization. A few of these functions include sperm attachment and binding to the oocyte as well as penetration thereof. Others play a role in signalling events such as capacitation, hyperactivation and acrosome reaction. The absence of these proteins in patient sperm possibly accounts for the functional inability to successfully achieve fertilization suggesting that this provides molecular insight to reasons for infertility amongst men. In addition to this, proteins presented by patient samples that were absent in healthy donors may too account for their infertility status. Estradiol binding protein and PH30 are two proteins presented only in patient samples. Their function plays a role in the inhibition of the acrosome reaction and sperm-egg fusion, respectively. In conclusion, these differences in protein expression between fertile donors and patients may form the molecular basis of infertility amongst men and indicates possibilities for novel proteonomic approaches to improve andrological diagnosis in future.
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SUGANUMA, NOBUHIKO, YOSHIMASA ASADA, YUTAKA TOMODA, ATSUO ITAKURA, and MASANORI YAMAMOTO. "A Live Birth from Intracytoplasmic Injection of a Testicular Spermatozoon." Nagoya University School of Medicine, 1997. http://hdl.handle.net/2237/16749.
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Castillo, Sandra. "Comparison between different freezing and thawing methods for human spermatozoa." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-158842.
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Preservation of cells and tissues by freezing at temperatures below 70°C has led to new possibilities for the storage of germ cells for fertility preservation. During the freezing process problems might occur, the greatest being ice crystallization which can cause membrane destruction and thus cell death. To minimize this risk, solutions that reduce the freezing point can be added to reduce crystallization and increase survival rates. These solutions are called cryoprotectants. The best method for freezing is still not known.The aim of this study was to analyze the effect of various parameters on the survival rate of human semen frozen with liquid nitrogen. The parameters investigated were thawing method (incubator or water bath) and container choice (straw or ampoule). In addition, two different cryoprotectants were tested.The method used was the instruction for preservation with Sperm CryoProtec™ II from Nidacon. In total 16 samples were collected for the first test and 13 samples for the second test. Sperm concentration and motility was measured.There seem to be no significant differences depending on container choice or thawing method leading to the conclusion that the most cost effective method of storage and thawing may be used. A small but significant difference was found in survival after thawing dependent on cryoprotectant p=0.041. However the study sample was limited and further studies might be of value.
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Prieto, Martínez Noelia. "Aquaporins in boar and bull spermatozoa: identification and functional implications." Doctoral thesis, Universitat de Girona, 2017. http://hdl.handle.net/10803/405771.
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Aquaporins (AQPs) are proteins involved in the transport of water and some other solutes across plasma membranes. The fact that mammalian spermatozoa are highly permeable to water suggests the presence of these proteins. Taking into account that AQPs have been poorly studied in male germ cells, the present Thesis Dissertation is focused on the study of the presence, localisation and function of AQP3, AQP7 and AQP11 in the sperm of two major livestock species (porcine and bovine). On the one hand, this Thesis has demonstrated that these three proteins are found in different regions (head, neck or tail) of the ejaculated sperm in both species. On the other hand, this Dissertation has determined the relationship of the relative abundance of AQP3, AQP7 and AQP11 with sperm cryotolerance and in vitro fertilizing ability of frozen-thawed sperm. Following this, AQP3 and AQP7 have been found to be freezability markers in boar sperm, whereas AQP7 and AQP11 have been indentified to be crucial for bull sperm cryotolerance. Altogether, these results contribute to increase our knowledge about the function of these proteins in mammalian sperm and may also allow the selection of those fresh ejaculates that exhibit higher sperm cryotolerance.Les aquaporines són proteïnes relacionades amb el transport d’aigua i altres soluts a través de les membranes plasmàtiques. El fet que l’espermatozoide madur de mamífer sigui una cèl·lula altament permeable a l’aigua suggereix la presència d’aquestes proteïnes. Tenint en compte que les aquaporines s’han estudiat més aviat poc en les cèl·lules germinals masculines, aquesta Tesi Doctoral s’ha centrat en l’estudi de la presència, localització i funció de les aquaporines 3, 7 i 11 en els espermatozoides de dues espècies d’interès productiu (porcina i bovina). D’una banda, aquest estudi ha demostrat que aquestes tres proteïnes es troben en diferents regions (cap, coll o cua) dels espermatozoides d’ambdues espècies. D’altra banda, aquesta Tesi també ha determinat que hi ha una relació entre la quantitat relativa d’aquestes proteïnes, la criotolerància de les ejaculacions i la capacitat fecundant in vitro després de la descongelació, de tal manera que es pot considerar que les AQP3 i AQP7 són marcadors de congelabilitat de l’esperma porcí i les AQP7 i AQP11 ho són de l’esperma boví. En conjunt, tots aquests resultats contribueixen a incrementar el nostre coneixement sobre el paper d’aquestes proteïnes en els espermatozoides de mamífer i permeten una millor selecció de les ejaculacions prèvia a la seva conservació.
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Merkies, Katrina. "Calcium regulation during epididymal maturation of equine and porcine spermatozoa." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ33314.pdf.
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Robertson, LaShonda Shakita. "IDENTIFICATION OF MICRORNAS IN BOVINE SPERMATOZOA WITH IMPLICATIONS OF FERTILITY." MSSTATE, 2009. http://sun.library.msstate.edu/ETD-db/theses/available/etd-07102009-111345/.
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MicroRNAs are small RNA molecules that could possibly play a major role in fertility. In the experiment, spermatozoa were extracted from bovine followed by an extraction of total RNA. Bovine spermatozoa were extracted from two bulls of different fertility, high and low fertility. An expression array was done to compare the expression levels of the microRNAs. It was shown that thousands of microRNAs are present in bovine spermatozoa but only a small amount was significantly expressed. The microRNAs from low fertility bulls were more highly expressed than those in high fertility bulls. A Bioanalyzer gel was used to confirm the results of the microarray data. The microRNAs were present in the bulls spermatozoa at 25 nucleotides. The functions of the significantly expressed microRNAs are not known but there is a great possibility that their functions affect fertility.
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Lampiao, Fanuel. "Measurement of free radicals and their effects on human spermatozoa." Thesis, Link to the online version, 2006. http://hdl.handle.net/10019/212.
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Tam, Vernon Craig Goodheart. "Identification of a glycodelin-C binding molecule on human spermatozoa." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558666.
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Keating, Jean. "Physiological and morphological studies on cryostored and untreated human spermatozoa." Thesis, University of Hull, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363275.
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Gearon, Ceinwen Mary. "Factors affecting fertilisation following the micro-injection of human spermatozoa." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338678.
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Manolakis, Alexis. "A comparative analysis of traditional and fluorescent spermatozoa staining techniques." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12502.
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Thesis (M.S.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The purpose of this study was to compare the traditional KPIC method for staining sperm to modern fluorescent techniques employed by SPERM HY-LITERTM. Factors such as staining time, sperm recovery, and the ability to quickly and accurately count sperm were evaluated. Dilutions ranging from 1:2 to 1:10,000,000 of semen, post-coital swabs and sperm/epithelial cell suspensions were stained with SPERM HY-LITERTM, SPERM HY-LITERTM Express and KPIC. Case work samples were also analyzed with KPIC and SPERM HY-LITERTM. The sperm counts for the more concentrated dilutions of semen and sperm/e-cell samples were statistically similar in both KPIC and SPERM HY-LITERTM. However, post-coital samples stained with SPERM HY-LITERTM were statistically different than samples stained with KPIC, resulting in the observation of fewer cells. Similar results were observed with SPERM HY-LITERTM Express as well as with case work samples stained with SPERM HY-LITERTM. Although SPERM HY-LITERTM has the benefits of quicker and easier scanning due to the fluorescent filters, it is considerably more costly to employ and did not result in greater sperm recovery. Overall, KPIC provided the best results in the shortest amount of time and within a reasonable budget.
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Tsang, Tsz-wai. "A study on the protective action of glycodelin-A on sperm against lymphocyte attack /." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36433858.
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