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Huang, Wenxin, and 黃聞馨. "Sperm fucosyltransferase-5 mediates the sperm-oviductal epithelial cell interaction to protect human sperm from oxidative damage." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196485.
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Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model.
The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx).
Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins.
Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation.
The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle.
In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress.published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy
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Sartini, Becky Lynn. "Characterization of boar sperm plasma membrane candidate oocyte membrane fusion proteins and investigation of oocyte membrane binding sites in boar sperm populations /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Detmar, Jacqueline. "Studies on putative sex chromosome-specific antigens of bovine sperm membrane." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ47320.pdf.
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Richmond, Alissa Gale. "Sperm-Oocyte Membrane Interactions during Fertilization in the Nematode Caenorhabditis elegans." W&M ScholarWorks, 2004. https://scholarworks.wm.edu/etd/1539624377.
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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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Vicente, Carrillo Alejandro. "Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening." Doctoral thesis, Linköpings universitet, Avdelningen för kliniska vetenskaper, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-131862.
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Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
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Slabbert, Marisa. "Investigating alternative sperm preservation methods for assisted reproductive technologies." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/40838.
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Introduction: Cryopreservation of human sperm is considered a routine practice in assisted reproduction laboratories. Semen samples are mainly cryopreserved as a back-up for procedures, donor sperm, and validation of samples from human immunodeficiency virus-positive patients. Human immunodeficiency virus semen samples generally result in a low yield of purified spermatozoa after decontamination. These samples need to be cryopreserved for later use. Unlike conventional cryopreservation, vitrification does not use harmful cryoprotectants, thereby potentially reducing sperm damage. Vitrification is not yet common practice for sperm cryopreservation in assisted reproduction. The aim of this study was to establish the feasibility of utilising vitrification as an alternative to current conventional cryopreservation of spermatozoa.
Methods: Semen samples were collected from human immunodeficiency virus-negative patients seeking diagnostic assistance from the unit. All samples were processed according to the unit’s standard protocol. For Study 1A (n=10) washed samples were divided and cryopreserved using three different cryopreservation media, and two different freezing protocols. In Study 1B (n=15), washed samples were divided and preserved using cryoprotectant-free vitrification in 100 μl, 300 μl and 500 μl volumes. For Study 2 (n=35) washed samples were split and cryopreserved using cryoprotectant-free vitrification (utilizing the volume that resulted in the highest quality spermatozoa in Study 1B) and conventional slow freezing (using the medium and protocol that resulted in superior quality spermatozoa in Study 1A). Post thawing, motility and kinetic parameters (Studies 1 and 2), viability (Study 1), mitochondrial membrane potential (Study 2), and DNA fragmentation (Study 2) of the two groups were compared.
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Results: Study 1A indicated that cryopreserving spermatozoa using Freezing Medium resulted in the highest quality spermatozoa with regards to motility and viability (p<0.05). Comparing the two preservation protocols, no conclusion could be reached on which protocol yielded superior results (p>0.05). The RBL freezing method is shorter, simpler and requires less equipment, and was therefore deemed the preferred method. Study 1B showed that the larger vitrification volumes (300 μl and 500 μl) yielded better spermatozoa in terms of motility and viability (p<0.05). No significant difference was observed with respect to the 300 μl and 500 μl vitrification volume groups. For practical reasons, 300 μl volumes will provide sufficient sperm for any procedure and, the intermediate volume ensures that more than one straw can be preserved. Study 2 found that cryoprotectant-free vitrification resulted in spermatozoa with significantly higher mitochondrial membrane potential and significantly lower apoptosis post thawing (p<0.05).
Discussion: Conventional cryopreservation methods may compromise various sperm parameters and final yield. In this study, cryopreservation and cryoprotectant-free vitrification had equivalent outcomes with respect to sperm motility. However, the latter method yielded superior results in terms of ΔΨ and DNA sperm fragmentation. In conclusion, vitrification is an easy, rapid and more affordable technique that requires no special equipment. Using vitrification for purified sperm samples of patients could potentially result in a better post thaw quality for ART procedures.Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Obstetrics and Gynaecology
unrestricted
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Fußhöller, David [Verfasser]. "The voltage‐gated H+ channel Hv1 and the plasma‐membrane Ca2+ ATPase PMCA4 in human sperm / David Fußhöller." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1113688157/34.
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Yuen, Chung-yat. "Effects of secretions from ampullary gland and ventral prostate on the sperm plasma membrane of golden hamster (mesocricetus auratus) /." [Hong Kong] : University of Hong Kong, 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13447488.
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Blässe, Anne-Kathrin [Verfasser]. "Effects of cryopreservation on osmotic membrane properties, intracellular ion distribution, and ion channels of bovine sperm / Anne-Kathrin Blässe." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2012. http://d-nb.info/1024339556/34.
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Francisco, Júnior Arthur. "Qualidade do sêmen equino criopreservado com L-acetil-cisteina." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/4034.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
This study was conducted to evaluate the effect of adding L-acetyl-cysteine (CIS) as anti oxidant agent to extender used for sperm cryopreservation of equine Mangalarga Marchador. Three semen samples from seven healthy stallions were obtained by artificial vagina, diluted in commercial extender and distributed into two treatments: control without addition and CIS, containing the antioxidant concentration of 2.5 mM. After the stabilization period, the samples were filled into 0.5 mL straws and subjected to cryopreservation protocol manually. Sixty days after the samples were thawed and evaluated in vitro by means of computerized analysis of sperm kinetics (CASA) and the evaluation of the integrity of plasma and acrosomal membranes by fluorescent probes. Regarding the kinetic variables related to sperm was found that in samples supplemented with CIS percentage of total and progressive motility (TM) (MP) was higher (P <0.01) compared to control samples (23.95±3 , 33 X 11 .38±2.35 and 4.66 ± 0.84 X 2.04 ± 0.36, respectively), but variable for straightness (STR) and linearity (LIN) the percentages were highe r (p ≤ 0.01) for the control samples (86.95 ± 1.02 X 82.28 ± 1.02 and 49.85 ± 1.32 X 45.19 ± 1.17, respectively). As those related to the integrity of the membranes of the sperm variables was superior (P <0.05) in the number of sperm with intact acrosome reaction (ICRA) in the samples compared to those CIS Control (4.7 ± 1.6 x 0.8 ± 0.2, respectively). The results suggest that the addition of acetyl-L-cysteine concentration of 2.5 mM in the diluent medium before cryopreservation can improve both the motility as the integrity of the sperm plasma membrane equine Mangalarga Marcher. More studies should be done to confirm this effect in vivo.
Esse estudo foi conduzido com o objetivo de avaliar o efeito da adição de L-acetilcisteína (CIS) como agente anti oxidante ao meio diluente utilizado para criopreservação de espermatozóides de equinos da raça Mangalarga Marchador. Três amostras seminais de sete garanhões hígidos foram obtidas por meio de vagina artificial, diluídas em meio comercial e distribuídas em dois tratamentos: controle, sem adição e CIS, contendo o antioxidante na concentração de 2,5 mM. Após o período de estabilização as amostras foram envasadas em palhetas de 0,5 mL e submetidas ao protocolo de criopreservação manual. Sessenta dias após as amostras foram descongeladas e avaliadas in vitro por meio da análise computadorizada da cinética espermática (CASA) e da avaliação da inte gridade das membranas plasmática e acrosomal por meio de sondas fluorescentes. Em relação às variáveis relacionadas à cinética espermática verificou-se que nas amostras suplementadas com CIS o percentual de motilidade total (MT) e progressiva (MP) foi superior (P<0,01) em relação às amostras controle (23,95±3,33 X 11,38±2,35 e 4,66±0,84 X 2,04±0,36, respectivamente), mas para as variáveis retilinearidade (STR) e linearidade (LIN) os percentuais foram maiores (P≤0,01) para as amostras Controle (86,95±1,02 X 82,28±1,02 e 49,85±1,32 X 45,19±1,17, respectivamente). Quanto às variáveis relacionadas à integridade das membranas do espermatozóide houve superioridade (P<0,05) no número de espermatozóides íntegros com reação acrossomal (ICRA) nas amostras CIS em relação àquelas controle (4,7±1,6 X 0,8±0,2, respectivamente). Os resultados sugerem que a adição de L-acetil-cisteína na concentração de 2,5 mM ao meio diluente antes da criopreservação pode melhorar tanto a motilidade quanto a integridade da membrana plasmática de espermatozóides de equinos da raça Mangalarga Marchador. Mais estudos devem ser feitos para comprovar esse efeito in vivo.
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Kongmanas, Kessiri. "Roles of Seminolipid and Its Associated Membrane Domain in Male Fertility." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32509.
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Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.
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Muhlrad, Paul, Jessica Clark, Ubaydah Nasri, Nicholas Sullivan, and Craig LaMunyon. "SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans." BioMed Central, 2014. http://hdl.handle.net/10150/610393.
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BACKGROUND:The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid.RESULTS:Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background.CONCLUSIONS:These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.
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Gojowsky, Marina [Verfasser]. "Fourier transform infrared spectroscopy studies on freezing-induced membrane phase behavior of stallion sperm in the presence of cryoprotective agents / Marina Gojowsky." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2012. http://d-nb.info/1024421678/34.
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Fonseca, Lucas dos Santos. "Performance, dry matter intake, seminal parameters and proteomics of seminal plasma and sperm membrane of Morada Nova sheep fed the diet cashew nut base." Universidade Federal do CearÃ, 2013. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=11466.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorO presente estudo foi conduzido com o objetivo de avaliar os efeitos da inclusÃo de 13% de farelo de castanha de caju na dieta de carneiros da raÃa Morada Nova sobre o ganho de peso, consumo de matÃria seca, rendimento de carcaÃa, pesos dos ÃrgÃos sexuais, qualidade seminal e proteÃnas seminais e membranares do espermatozoide. Para tanto, vinte carneiros foram divididos em dois grupos alimentados com dietas contendo 13% (GCA, n=10) ou 0% (GCO, n=10) de farelo de castanha de caju. As dietas foram isoproteicas e isoenergeticas, com uma relaÃÃo de 50/50 de concentrado/volumoso (feno de Tifton 85) e Ãgua e sal mineral à vontade. Durante noventa dias, os animais foram mantidos em baias individuais e avaliados quanto ao consumo de matÃria seca e ganho de peso. As coletas seminais por eletroejaculaÃÃo ocorreram semanalmente com a determinaÃÃo do volume ejaculado e, concentraÃÃo, motilidade e morfologia espermÃtica. ApÃs os noventa dias, as proteÃnas seminais e espermÃticas foram analisadas por meio de eletroforese bidimensional em gel de poliacrilamida. Os mapas proteÃcos foram avaliados por meio do aplicativo PDQuest, version 8.0; Bio Rad, USA. Ao final do experimento, os carneiros foram abatidos e, o peso vivo, rendimento de carcaÃa e peso dos ÃrgÃos sexuais foram mensurados. O consumo de matÃria seca total permaneceu sem alteraÃÃes nos dois grupos experimentais durante os primeiros 60 dias do estudo. PorÃm, apÃs 60 dias, houve e reduÃÃo no consumo de matÃria seca no grupo alimentado com castanha (P < 0,05). NÃo houve efeito da inclusÃo do farelo de castanha sobre o ganho de peso, rendimento de carcaÃa, desenvolvimento dos ÃrgÃos sexuais e parÃmetros seminais dos animais. No entanto, a inclusÃo do farelo de castanha de caju esteve associada à expressÃo de proteÃnas seminais e espermÃticas. Sete spots proteicos presentes nos mapas de plasma seminal foram expressos diferencialmente entre os grupos castanha e controle (P < 0,05) e um spot proteico (21,8 kDa; pI: 5) correlacionou-se negativamente com o vigor espermÃtico (r = -0,49; P<0,05). Adicionalmente, sete spots proteicos dos gÃis com proteinas da membrana dos espermatozoides tambÃm diferiram entre os carneiros alimentados com e sem farelo de castanha de caju (P < 0,05). Conclui-se, portanto, que a inclusÃo de 13% de farelo de castanha de caju na dieta altera a expressÃo de proteÃnas seminais e espermÃticas em ovinos.
The cashew nut meal is alternative food for ruminant nutrition which is rich in lipids, proteins and minerals. However, its nutrition influences on male development and reproduction are still unknown. In this context, seminal and sperm protein analysis can show metabolic consequences from cashew nut meal on sheep reproductive efficiency. Therefore, the current dissertation aims evaluate the effects of 13% of cashew nut meal in the ration on the weight gain, food intake, carcass yield, weight of sexual organs, sperm quality and, seminal and sperm membrane proteins of Morada Nova Rams. Twenty rams were divided in two groups: cashew nut and control, which received 13% or 0% of cashew nut meal in the diet, respectively. During ninety days, the animals were kept in individual boxes and evaluated for ration consumption and weight gain. The sperm collections by eletroejaculation were made by weekly and, the volume, sperm concentration and motility were analyzed. After ninety days, the seminal and sperm membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The gels were digitalized, and their images were evaluated by computer software. At the end of the experiment, the rams were slaughtered and, the weight gain, carcass yield and weight of sexual organs were measured. There was not effect of cashew nut meal addition in the diet on the weight gain, carcass yield and weight of sexual organs. But, after sixty days, there was a reduction on food intake in the cashew nut group (P < 0,05). The sperm quality was not influenced by the diet. We observed an effect of cashew nut diet on the expression of seminal and sperm proteins. Seven spots from seminal plasma were expressed differently between cashew nut and control groups (P < 0,05). The spot 2206 (21,82 kDa; pI: 5,04) was negatively correlated with the individual motility score (r = -0,49). Additionally, seven spots from sperm membrane protein also differ between rams from cashew nut and control groups (P < 0,05). In conclusion, the addition of 13% of cashew nut meal in the diet alters the expression of sheep seminal plasma and sperm membrane proteins.
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Kaydos, Emilie Hayes. "Fertility and the sperm membrane biomarker (SP22) are compromised in an additive fashion by priority disinfection by-products of drinking water a validation of enzyme linked immunosorbant assay (ELISA) for SP22 /." NCSU, 2004. http://www.lib.ncsu.edu/theses/available/etd-04132004-133325/.
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Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water known to produce defects in spermatogenesis and fertility in adult rats. Previous work in our laboratory demonstrated that BCA compromises both the fertility of cauda epididymal sperm and SP22, a sperm membrane protein highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether levels of SP22 on sperm and fertility were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22 to replace the tedious two-dimensional (2D) gels quantitation. The initial study consisted of 8 treatment groups and a water vehicle control group, on which animals were exposed by oral gavage daily for 14 days. BCA was administered alone at 1.6, 4, and 8 mg/kg, and DBA was given in equimolar fashion at 2, 5, and 10 mg/kg. BCA and DBA were also given as two binary mixtures: 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. Proximal cauda epididymal sperm membrane proteins (30 ìg) were resolved following concentration/desalting in 2D gels under denaturing conditions (2D SDS-PAGE) and the SP22 protein was quantified. In addition, SP22 was quantified by enzyme-linked immunosorbant assay (ELISA). Full length rat recombinant SP22 (rSP22) was used to generate a standard curve and affinity purified sheep anti-rSP22 was used as primary antibody. The ED50 for the decrease in SP22 quantified by 2D SDS-PAGE for DBA and BCA was 7.15 and 4.61 mg/kg. The ED50 for the decrease in SP22 quantified by ELISA for DBA and BCA was 8.10 and 5.93 mg/kg. The second study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. In this study proximal cauda sperm were also used for in utero insemination to assess fertility. The ED50 for the decrease in fertility for DBA and BCA was 3.5 and 2.7 mg/kg. Immunostaining for SP22 in the testis revealed staining of both round and elongating spermatids and decreased staining in testes exposed to the DBA + BCA mixture. An evaluation of SP22 in testicular parenchyma was also performed by ELISA and Western blotting. Both evaluations revealed a treatment-related decrease in SP22. For either the 2D SDS-PAGE or ELISA quantitation of SP22 on sperm or the fertility of sperm, additivity or synergy is indicated. Finally, the correlation between SP22 levels measured by ELISA versus fertility was r2=0.74, compared to 0.82 for SP22 levels measured by 2D SDS-PAGE versus fertility, suggesting the ELISA could be used to supplant the time-intensive SDS-PAGE.
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Kurz, Anke. "Organisation und Dynamik der Phospholipide in der Zell- und Akrosommembran von Eberspermien während der Kapazitation und Akrosomreaktion." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät II, 2005. http://dx.doi.org/10.18452/15274.
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Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Voraussetzung für diese enge Assoziation diskutiert. Weitere Hinweise auf eine funktionelle Bedeutung lateraler Membrandomänen lieferten die Arbeiten zur Isolation Triton-unlöslicher Lipiddomänen aus der Plasmamembran von Forellenspermien.One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. An indication for the functional meaning of lateral membrane domains in sperm cells was futher deduced from the isolation of Triton-insoluble lipid domains from membranes of trout sperm cells.
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Springsguth, Hans Christopher. "Mechanismen und Bedeutung der aktivierten Apoptosekaskade in humanen Spermatozoen." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-192660.
Full textAbstract:
Andrologische Forschungsarbeiten der letzten Jahre beweisen, dass einzelne, aus
somatischen Zellen bekannte Apoptose-typische Veränderungen bei humanen Spermien
einen negativen Einfluss auf die Fertilität des Mannes haben. Umstritten ist, ob es sich dabei
nur um einen abortiven Zelltod als Zeichen einer Reifungsstörung während der
Spermatogenese handelt oder ob Apoptose auch in reifen Spermien induzierbar ist.
Ziel der vorliegenden Arbeit war es, durch Untersuchungen zur Induzierbarkeit der Apoptose
in reifen und unreifen Spermatozoen die vollständige Funktionalität verschiedener
Signalkaskaden sowie deren Umsetzung in morphologische Veränderungen aufzudecken.
Darüber hinaus sollte die Rolle des intrazellulären Calciumspiegels als möglicher
Interaktionspartner zwischen Akrosomreaktion und Apoptose geklärt werden, um
Informationen über die Zukunft der nicht fertilisierenden Spermien im weiblichen Genitaltrakt
zu erlangen.
In den vorliegenden Versuchsreihen konnte erstmals die gezielte Induktion der Apoptose in
reifen und unreifen Spermatozoen anhand biochemischer und elektronenmikroskopischer
Untersuchungen nachgewiesen werden. Dabei wurde ausführlich die erfolgreiche
Aktivierung mehrerer intrinsischer Apoptosesignalwege in reifen Spermien gezeigt, deren
Initiation entweder auf den Zusammenbruch innerer Mitochondrienmembranen, auf eine
veränderte intrazelluläre Calciumkonzentration oder auf das Einwirken von oxidativem
Stress zurückzuführen war. Zudem konnten Erkenntnisse zum antioxidativen
Schutzmechanismus von Spermien gewonnen werden, welcher die Spermien gegenüber
einer spezifischen Menge an H2O2 vor oxidativem Stress-bedingter Apoptose bewahrt.
Sowohl die Apoptose als auch die Akrosomreaktion waren durch die Zugabe eines
Calciumchelators blockierbar.
Die Initiation des programmierten Zelltodes in Spermien durch einen Anstieg der
intrazellulären Calciumkonzentration erklärt zudem eine weitere wichtige Funktion dieses
Prozesses: das Absterben von akrosomenreagierten Spermien bei nicht erfolgter
Fertilisation im weiblichen Genitaltrakt. Die Theorie einer rein abortiven Apoptose als Folge
einer Spermatogenesestörung ist damit widerlegt.
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Kavak, Ants. "Evaluation of sperm production, testicular measurements and post-thaw sperm quality in Tori and Estonian breed stallions /." Uppsala : Dept. of Obstetrics and Gynaecology, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/9329559.pdf.
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Fierro-Pastrana, Reyna-Carmen. "Etude de l'architecture moléculaire de la membrane plasmique et des membranes acrosomiques du spermatozoïde humain : leurs modifications au cours des phénomènes de capacitation et de la réaction acrosomique." Nancy 1, 1997. https://hal.univ-lorraine.fr/tel-01747360.
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Ce travail a été mené en utilisant la technique de cytofluorométrie et la technique de microscopie électronique. La première partie met en évidence la localisation des résidus N-Acétylglucosamine au niveau de la membrane plasmique, galactose au niveau de la membrane externe de l'acrosome, et mannose et fucose au niveau de la membrane interne de l'acrosome. On a mis au point une technique de triple marquage en utilisant simultanément trois fluorochromes différents (iodure de propidium, GB24 et lectine). Ces résultats ouvrent une orientation vers des applications pratiques pour l'exploration des qualités fonctionnelles du sperme dans le cadre des études des hypofertilités et des indications cliniques des techniques d'assistance médicale à la procréation. La deuxième partie du travail aborde l'étude de l'influence des cytokines sur la fécondance du sperme. Les résultats montrent qu'il existe chez l'homme des corrélations entre la fixation de certaines cytokines sur les spermatozoïdes et des altérations du spermogramme (nombre, mobilité et formes immatures des spermatozoïdes). Les données obtenues permettent une meilleure connaissance des relations cytokine-spermatozoïde et d'envisager des applications à l'exploration de la fécondance du sperme et de ses mécanismes.
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Brandão, Alessandra Cunha. "Efeito do laser diodo sobre as características de motilidade, de integridade das membranas plasmática e acrossomal e de potencial de membrana mitocondial de espermatozóides criopreservados de eqüinos." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-09012009-162214/.
Full textAbstract:
A motilidade espermática depende do consumo de energia. Em algumas espécies, a mitocôndria espermática tem um importante papel na produção de energia para o batimento flagelar. A irradiação com Laser de baixa intensidade aumenta a produção de energia funcionando como uma ferramenta de biomodulação. O objetivo deste estudo foi analisar o efeito da irradiação contínua de 650 nm de comprimento de onda de Laser Diodo, com dose de 6 J/cm2 por 120s, na motilidade, integridade das membranas plasmática e acrossomal e no potencial de membrana mitocondrial em espermatozóides in natura e criopreservados. Cinco ejaculados foram obtidos de cinco garanhões (n=25). O sêmen foi acondicionado em palhetas de 0,5mL com 200x106 cells/mL em diluidor Botu-CrioTM (Biotech-Botucatu-Ltda/ME, Botucatu, Brasil) e congelado utilizando o sistema automático programável (TK3000®, TK Tecnologia em Congelação_Ltda, Uberaba, Brasil). As amostras in natura foram divididas em dois grupos: espermatozóides tratados COM LASER e espermatozóides não tratados - SEM LASER. As amostras congeladas foram divididas em três grupos: espermatozóides tratados COM LASER antes da congelação; espermatozóides tratados COM LASER depois da congelação e espermatozóides criopreservados não tratados SEM LASER. As amostras criopreservadas foram analisadas imediatamente após a descongelação (tempo zero) e duas horas após a descongelação (tempo 2). A motilidade foi avaliada pelo sistema de análise espermática computadorizada (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial foram avaliados pela técnica de citometria de fluxo (FACSaria-Beckton-Dickeson, San Jose, USA). Os dados foram analisados pelo programa SAS, com nível de significância de 5%. A freqüência de batimentos flagelar (BCF) foi alta (P<0,05) para o grupo de espermatozóides in natura tratados COM LASER (34,8 ± 0,7%) quando comparado com o grupo de espermatozóides in natura não tratados SEM LASER (33,4 ± 0,8%). No tempo zero, o potencial de membrana mitocondrial foi baixo no grupo de espermatozóides tratados COM LASER antes da congelação (40,7 ± 1,5%) quando comparados com o grupo de espermatozóides congelados não tratados - SEM LASER (47,4 ± 2,4%) (P<0,05). Duas horas após a descongelação, as membranas plasmática e acrossomal apresentavam maior porcentagem de integridade no grupo de espermatozóides tratados COM LASER antes da congelação (8,3 ± 0,7%) do que no grupo congelado não tratados - SEM LASER (6,2 ± 0,6%) (P<0,05). O grupo de espermatozóides tratados COM LASER depois da congelação não diferiu (P>0,05) dos outros grupos do tempo 2, porém no tempo zero a porcentagem de espermatozóides com motilidade progressiva foi baixa (2,0 ± 0,3%) e diferente (P<0,05) dos outros grupos (6,5 ± 1,3 nos espermatozóides tratados COM LASER antes da congelação e 5,5 ± 1,1% nos espermatozóides congelados não tratados - SEM LASER). Estes resultados indicam que a irradiação de 650 nm de comprimento de onda aumenta a freqüência de batimentos flagelar no sêmen fresco e confere melhor proteção à membrana plasmática e acrossomal ao longo do tempo (após 2h de incubação). Os resultados com sêmen congelado permitem concluir que o melhor momento para a aplicação do laser é antes da criopreservação. Novos estudos com diferentes comprimentos de onda, potência do raio e dosagem devem ser conduzidos para se obter um melhor protocolo visando aprimorar a criopreservação do sêmen eqüino.Sperm motility depends on energy consumption. In some species sperm mitochondria play an important role in the production of energy for tail activity. Low-level laser irradiation increases this production as a modulation tool. The objective of this study was to analyze the effect of a continuous 650 nm wavelength diode laser irradiation, with dose 6 J/cm2 for 120s, in the motility, plasma and acrosomal membrane integrity and mitochondrial membrane potential in fresh and frozen equine spermatozoa. Five ejaculates were obtained from five stallions (n=25). Semen was packaged into 0.5mL straws with 200x106 cells/mL in a Botu-CrioTM (Biotech-Botucatu-Ltda/ME, Botucatu, Brazil) and frozen by automated technique using a programmed machine (TK3000®, TK Tecnologia em Congelação-Ltda, Uberaba, Brazil). Fresh samples were divided in two groups: spermatozoa treated with laser and without laser (non treated spermatozoa), and frozen samples in three groups: spermatozoa treated with laser before freezing; spermatozoa treated with laser after thawing and without laser (cryopreserved spermatozoa). Cryopreserved samples were analyzed immediately after thawing (time 0) and two hours after thawing (time 2). Motility was evaluated by computer assisted sperm analysis (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), plasma and acrosomal membrane integrity and mitochondrial membrane potential were evaluated by flow cytometry (FACSaria-Beckton-Dickeson, San Jose, USA). The data were analyzed by the SAS program, at a 5% level. Beat cross frequency (BCF) test was higher (p<0.05) for fresh semen group treated with laser (34.8 ± 0.7%) as compared to non treated group (without Laser - 33.4 ± 0.8%). At time zero, mitochondrial membrane potential was lower in laser treatment before freezing group (40.7 ± 1.5%); compared to without laser treatment group (cryopreserved spermatozoa - 47.4 ± 2.4%) (P<0.05). Two hours after thawing, plasma and acrosomal integrity was higher (P<0.05) in the group were spermatozoa were treated with laser before freezing (8.3 ± 0.7%) compared with the group without laser treatment (cryopreserved spermatozoa) (6.2 ± 0.6%). The group treated with laser after thawing didn´t show any difference (P>0.05) compared to the others groups at this period. However, at time 0 percentage of progressive motility was lower (2.0 ± 0.3%) (P<0.05) than groups treated with laser before freezing (6.5 ± 1.3%) and cryopreserved without laser (5.5 ± 1.1%). These results indicated that the irradiation of 650 nm wavelength diode laser improves beat cross frequency in the fresh semen and support a long term (2 hours) protection to plasma and acrosomal membrane of equine spermatozoa. Based on the results of frozen semen, the best moment for laser application is before cryopreservation protocol. New studies with different diode laser wavelength, different power and energy doses should be driven in order to improve stallion semen cryopreservation.
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Schröter, Filip [Verfasser]. "Recombinantly expressed spermadhesin AWN and fluorescence based characterization of porcine sperm membranes - prerequisites for functional interaction studies / Filip Schröter." Berlin : Freie Universität Berlin, 2017. http://d-nb.info/1136608710/34.
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Botha, Alma Ester. "Effect of the acidic buffer 2-(N-Morpholino) ethanesulfonic acid on frozen-thawed bull semen." Diss., University of Pretoria, 2009. http://hdl.handle.net/2263/22848.
Full textAbstract:
The aim of the current study was to determine if frozen-thawed bull semen can be treated with the acidic buffer MES (2-[N- morpholino] ethanesulfonic acid) without any detrimental effect on the motility, plasma membrane, acrosomal membrane and longevity of sperm. Frozen bull semen was obtained from a local co-operative. The semen was frozen in 0.25 mL French straws at a concentration of 80 x 106 sperm cells per millilitre. Semen of two different batches from ten bulls of four different breeds was used in this study. Three frozen semen straws of each batch were thawed at 38° C for 25 seconds. The thawed semen was pooled and then split into two aliquots. The one aliquot was used as control, whilst the other was exposed to MES treatment. The motility, plasma membrane integrity, acrosomal membrane integrity and longevity of sperm were evaluated. The effect of MES on motility was minimal as only the percentage of aberrantly motile sperm increased two hours after treatment. Although no effect on the plasma membranes were observed, it can be assumed that some damage did occur due to the fact that the acrosomal membranes were affected significantly. No significant effect was found for longevity of sperm between the control and treated samples, but a significant effect was found for both the control and treated samples over time. Although the detrimental effects caused by MES treatment would render some sperm unable to fertilise an oocyte, it is likely that a sufficient portion of sperm would survive the treatment. It is probable that this treatment would also be effective in frozen-thawed buffalo semen. The following step would be to treat semen of footand-mouth disease positive bulls with MES to establish if treatment with MES will be effective in inactivating foot-and-mouth disease virus in semen of infected bulls. CopyrightDissertation (MSc (Veterinary Science))--University of Pretoria, 2008.
Production Animal Studies
unrestricted
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Bou, Khalil Maroun. "Biophysical and biochemical properties of the mammalian male germ cell specific sulfogalactosylglycerolipid (SGG): Contribution to the structure and zona pellucida affinity of pig sperm raft membranes." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/29079.
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Raft membranes are implicated in cell adhesion. Here, we demonstrated that rafts isolated from hypermotile capacitated pig sperm as low-density Triton X-100 insoluble membranes had the ability to bind specifically to homologous zonae pellucidae (ZP). This binding was dependent on pig ZP3alpha sulfoglycoprotein, a major player in intact sperm binding. The male germ cell specific sulfogalactosylglycerolipid (SGG) was a sperm raft component and participated in sperm raft-ZP binding, since rafts pretreated with anti-SGG IgG/Fab had decreased ability to bind to the ZP dose dependently. SGG may also partake in raft formation. Fourier transform infrared (FTIR) spectroscopic studies of mixed SGG-cholesterol liposomes revealed that the sulfoglycolipid interacted with cholesterol (a significant raft component) via intermolecular hydrogen bonding. Moreover, mixtures of SGG and cholesterol were insoluble in 1--2% Triton X-100. Since cholesterol is significant for raft formation and since sperm capacitation is associated with cholesterol efflux, we determined whether raft levels in capacitated sperm were the same as those in control sperm. Interestingly, our results revealed an increase in raft levels in the capacitated sperm, as well as an enhanced ZP affinity of these membrane domains, compared to those of control sperm. These results corroborated the implication of rafts in cell adhesion and strongly suggested that the enhanced ZP-binding ability of capacitated sperm may be attributed to increased levels of sperm rafts, with a greater affinity for the ZP.
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Rodríguez, Shirley Andrea Flórez. "Efeitos de diferentes diluidores sobre a cinética, membranas, morfologia e cromatina espermáticas durante a refrigeração de sêmen equino." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-06052013-092347/.
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Incrementar a eficiência do processo de refrigeração do sêmen é de grande interesse na reprodução da espécie equina, à medida que a população de equinos cresce, também aumenta a demanda para a comercialização e transporte de sêmen refrigerado. Esse trabalho foi realizado para verificar os efeitos de diferentes diluidores para refrigeração de sêmen equino a 5°C sobre a cinética, membranas, morfologia e cromatina espermáticas durante 12 horas de armazenagem. Foram utilizados quatro ejaculados de quatro garanhões de diferentes raças, colhidos em intervalos semanais. Imediatamente após a colheita, o sêmen in natura foi avaliado quanto ao movimento espermático utilizando-se o sistema computadorizado de análise espermática (CASA), integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial, utilizando-se sondas fluorescentes (PI, H342, FITCPSA e JC-1, por microscopia de epifluorescência), morfologia espermática por microscopia de contraste de interferência diferencial (DIC) e desnaturação da cromatina pela coloração de Azul de Toluidina. Logo após as análises, o sêmen foi diluído usando-se três diluidores diferentes: SMT, à base de leite desnatado e meio de Tyrode (adaptado de: PADILLA; FOOTE, 1991), BSAG, diluidor contendo BSA (adaptado de: GIBB et al., 2011) e SMK, à base de leite desnatado (KENNEY et al., 1975; controle), em seguida foi envasado em bisnagas na concentração de 50 x 106 sptz/mL e refrigerado a 5°C em caixas BotuFLEX® (Botupharma, Botucatu-SP), durante um período de 12 horas. O sêmen foi analisado 5 minutos após a diluição (T0), 4 (T4), 8 (T8) e 12 horas (T12) após a refrigeração quanto ao movimento espermático, integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial, morfologia espermática e desnaturação da cromatina. A análise estatística foi realizada empregando-se o programa computacional Statistical Analysis System (SAS inst. Inc.). Para todas as variáveis foi utilizada a análise de variância (ANOVA). Para comparação das médias, foi utilizado o método de Tukey sendo considerado o nível de significância de 5%. Foram observadas interações entre tempo X tratamento para a maioria das características de cinética espermática. Dentre os efeitos encontrados dos diluidores, destaca-se que SMT e SMK foram superiores na preservação das motilidades total e progressiva e porcentagem deespermatozoides rápidos em detrimento ao diluidor BSAG. Quanto à preservação das membranas o diluidor SMT (56,6±18,7%) foi significativamente superior ao SMK (49,6±18,6%), que por sua vez foi superior ao BSAG (25,8±14,8%) quanto à porcentagem de espermatozoides com membranas plasmática e acrossomal íntegras e alto potencial mitocondrial (PIAIA). Comportamento semelhante foi observado para o percentual de membrana plasmática e de potencial de membrana mitocondrial. Contrariamente, a membrana acrossomal não foi afetada pela refrigeração do sêmen a 5ºC por até 12 horas independente do diluidor. Foi encontrado maior percentual de defeitos totais quando o sêmen foi refrigerado utilizando o diluidor BSAG (50±12,4%) do que SMT (42,6±11,2%) e SMK (41,8±12,4%). Quando se avaliou a cromatina espermática não foram notados efeitos do tempo de refrigeração nem dos diluidores; todavia, ao se comparar com o sêmen in natura notou-se um aumento no percentual de espermatozoides com desnaturação intermediária da cromatina após 12 horas de refrigeração a 5ºC com o diluidor BSAG. Conclui-se que a refrigeração do sêmen equino a 5ºC altera a cinética espermática de forma oscilante entre os diluidores no decorrer do tempo até 12 horas. Os diluidores SMT e o SMK preservam melhor a cinética, integridade de membranas e morfologia espermática do que o diluidor BSAG.To develop the efficiency of cooling semen is the great advantage in the equine reproduction, as according to the equine population to grow up, too increase the demand to market and transport of cooling semen. This experiment was performed to verify the effects of different extenders to equine cooling semen at 5°C on the sperm kinetic, membranes, morphology and chromatin during 12 hours of storage. Were utilized four ejaculates from four stallions of different breeds, collected a week. Immediately after the collection, the in natura semen was evaluated as the sperm movement using the Computer-Assisted Semen Analysis (CASA), integrity of plasma and acrossomal membranes and mitochondrial membrane potential, using the fluorescent probes (PI, H342, FITC-PSA and JC-1, by epifluorescência microscopy), sperm morphology by microscopy of differential interference contrast (DIC) and chromatin denaturation by Toluidine blue. As soon as finished the analyses, the semen was diluted using three different extenders: SMT, skim milk based and Tyrode medium (adapted from PADILLA; FOOTE, 1991), BSAG, extender containing BSA (adapted from GIBB et al., 2011) and SMK, skim milk based (KENNEY et al., 1975; control), following was packed in flasks at 50 x 106sperm/mL concentration and cooling at 5°C in BotuFLEX® (Botupharma, Botucatu-SP) box, during 12 hours. The semen was analyzed 5 minutes after dilution (T0), 4 (T4), 8 (T8) and 12 hours (T12) after cooling about sperm movement, plasma and acrossomal membranes integrity and mitochondrial membrane potential sperm morphology and chromatin denaturation. The statistical analysis was performed using Statistical Analysis System (SAS inst. Inc.) software. To all variables was utilized the analysis of variance (ANOVA). To media comparison was utilized the Tukey method, considering the significant level at 5%. Were observed interactions between times X treatment to majority of sperm kinetic characteristic. Among the found effects of the extenders, it was detached that SMT and SMK were superior to preserve total and progressive motility and percentage of rapid sperm in detriment to BSAG extender. It about the membranes preservation the SMT extender (56.6±18.7%) was significantly superior to SMK (49.6±18.6%), which was superior to BSAG (25.8±14.8%) as percentage of sperm with plasma and acrossomal membranes integrity and high mitochondrial potential (PIAIA). Behavior similar was observed to the plasma membrane integrity and potential mitochondrial membrane percentage. Contrary, The acrossomal membrane was not affected by semen cooling at 5ºC until 12 hours independently of the extender. It was found major percentage of defect total to cooling semen using BSAG (50±12.4%) extender compared to SMT (42.6±11.2%) and SMK (41.8±12.4%). When evaluated the sperm chromatin were not found effect of the cooling time neither of the extenders; however, when compared to in natura semen notice an increase on the percentage of spermatozoa with moderate chromatin denaturation after 12 hours of cooling at 5ºC with BSAG extender. It was concluded that the equine semen cooling at 5ºC change the sperm kinetic oscillating way among extenders during the time until 12 hours. The SMT and SMK extenders are better to preserve the sperm kinetic, membranes integrity and morphology than BSAG extender.
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Gallego, Andrés Mejia. "Avaliação das características da motilidade (CASA), morfologia e funcionalidade da membrana plasmática (HOST) de espermatozóides bovinos sexados por cimetria de fluxo." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-01042011-143106/.
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A pré-seleção do sexo tem grande impacto na produtividade da bovinocultura de corte e leite. A única técnica comercialmente viável para a pré-seleção do sexo, até agora, é a sexagem espermática utilizando citometria de fluxo. Atualmente palhetas de sêmen com espermatozóides sexados vêm sendo utilizadas regularmente em programas de inseminação artificial. Entretanto, ainda existem limitações referentes à taxa de gestação em condições de campo, explicadas em parte pela perda da integridade celular dos espermatozóides. O intuito deste experimento foi avaliar os efeitos da sexagem e o tempo de espera do ejaculado para a sexagem as 0, 3 e 6 hs sobre os parâmetros de motilidade pelo sistema computadorizado da motilidade espermática (CASA), funcionalidade da membrana plasmática pelo teste de resistência osmótica (HOST), morfologia dos espermatozóides pela técnica de contraste de interferência diferencial (DIC) e entre as subespécies taurinas (Bos taurus taurus) e zebuínas (Bos taurus indicus). Espermatozóides sexados apresentaram a maioria dos parâmetros de motilidade superiores aos submetidos à técnica convencional, indicando que existe uma seleção por parte do citômetro de fluxo. O tempo em que o espermatozóide bovino espera (até seis horas) para ser submetido ao processo de sexagem pela técnica de citometria de fluxo alterou a linearidade (LIN). A funcionalidade da membrana plasmática foi afetada quando o espermatozóide bovino passa pelo processo de sexagem.. A sexagem induz o aumento de defeitos menores, provavelmente por alterações da membrana plasmática da cauda dos espermatozóides. Os zebuínos são mais sensíveis às alterações da membrana plasmática da cauda em relação aos taurinos. Finalmente existem fortes indícios que espermatozóides bovinos que passam pela técnica de citometria de fluxo entrem em estado de hiperativação.Sex selecting has a huge impact in dairy and beef production. The only way commercially viable to obtain sex selecting nowadays, is a flow citometry sex sorted sperm technique. Currently sex sorted sperm have been used regularly in Artificial Insemination programs. Meanwhile, there are limitations about non return rate in farm conditions, superficially explained as cellular loss integrity. The objective of this experiment was assessing sex sorted sperm effects and time to wait 0, 3 and 6 hours before sorting in motility parameters by computer assisted sperm analysis (CASA), plasmatic membrane functionality by hiposmotic swelling test (HOST), sperm morphology by differential interference contrast microcopy (DIC) and between taurine subspecies (Bos taurus taurus) zebuine subspecies (Bos taurus indicus). Sex sorted sperm showed best results in the most motility parameters compared with conventional sperm, suggesting a flow citometer selection. The time to wait of the sperm (untis 6 hours) to keep sorting, altered a linearity (LIN). Plasmatic membrane functionality was affected when sperm runs through flow citometer.. Sex sorted induces increase of minor defects in sperm, probably by tail membrane plasmatic alterations. Zebuin sperm are more sensitive for tail plasmatic membrane alterations compared with taurine sperm. Finally, there is strong indication that bovine sperm that runs through flow citometer sign in hiperactivation state.
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Azevedo, Hymerson Costa [UNESP]. "Integridade e funcionalidade dos espermatozóides ovinos submetidos à criopreservação após a incorporação de colesterol, desmosterol, ácido oléico-linoléico e alfa-lactoalbumina." Universidade Estadual Paulista (UNESP), 2006. http://hdl.handle.net/11449/105970.
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azevedo_hc_dr_botfmvz.pdf: 1090855 bytes, checksum: bd7f7a04aa00c86d1200aecb917ca794 (MD5)Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Objetivando testar a ação do colesterol, desmosterol, ácido oléico-linoléico ou Q-Iactoalbumina sobre os espermatozóides ovinos, alíquotas de sêmen de 25 carneiros foram submetidas à criopreservação após tratamento com essas substâncias. O sêmen foi avaliado antes do processamento e após a refrigeração, descongelação e incubação quanto à, cinética pela análise subjetiva e computadorizada (CASA), morfologia, avaliação simultânea da integridade da membrana plasmática (IMP) e do acrossomo (IAC) e do potencial de membrana mitocondrial (PMM) pela associação da aglutinina de Pisum sativum conjugada ao isotiocianato de fluoresceína (FITC-PSA), iodeto de propídio (PI) e JC-1 e, status de capacitação pela c10rtetraciclina (CTC). A influência dos aditivos em poucos parâmetros não foi suficiente para sugerir a superioridade de qualquer um dos tratamentos. A criopreservação provocou prejuízos à cinética, IMP, IAC, PMM, além de induzir a capacitação e reação do acrossomo. Foram observadas diferenças entre grupos de carneiros quanto ao status de capacitação no sêmen in natura, assim como na sensibilidade desse sêmen à crioinjúria e criocapacitação. Após a descongelação, as mudanças semelhantes à capacitação foram maiores nos animais de menor IMP. A segregação dos carneiros de acordo com a crioresistência da membrana plasmática norteou o comportamento de vários outros parâmetros. Conclui-se, que o colesterol, desmosterol, ácido oléico-linoléico e alactoalbumina não protegem o sêmen ovino contra as crioinjúrias, que a congelação-descongelação é mais danosa que a refrigeração e, que existem diferenças entre grupos de indivíduos relacionadas à crioresistência e criocapacitação espermática.
In order to evaluate the proteeting aetion of some substanceson ram spermatozoa, semen samples of 25 rams were colleeted and submitted to cryopreservation after treatment with cholesterol, desmosterol, oleic-linoleic acid or Q-Iactalbumin. Before the processing and after the procedures of cooling, freezing-thawing and incubation, semen was evaluated as to kínetcs by subjective and computerized analyses (CASA), morphology, simultaneously to plasma membrane integrity (PMI), acrosome integrity (ACI) and mitochondrial membrane potential (MMP) by isothiocyanate-conjugated Pisum sativum (FITCPSA), propidium iodide (PI) and JC-1 combination and as to sperm capacitation and acrosome reaction determined by chlortetracycline (CTC) assay. The influence resulted from the additives in a few parameters was not enough to suggest the superiority of any treatments. The cryopreservation process, especially the freezing-thawing, promotes damages to kinetics, PMI, ACI, MMP and induees accelerated eapacitation and acrosome reaction in spermatozoa. Differenees among groups of rams were observed regarding capacitation status in fresh semen as well as the sensibility of their semen to cryoinjury and cryocapacitation. Higher and more accelerated capacitation-like ehanges were observed in groups of rams presenting lower plasmatic membrane cryoresistance after thawing. The segregation of rams in different levels of PMI influenced the behavior of several other parameters. It was concluded that cholesterol, desmosterol, oleic-Iinoleic acid and Q-Iactalbumin do not protect the ram semen against the cryoinjuries as well as freezing-thawing is more harmful than cooling. Important differenees among groups of individuais were noticed as for the spermatozoa resistance to the damages provoked by cryopreservation such as the eryocapacitation susceptibility.
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Labbé, Catherine. "Membrane plasmique du spermatozoide de salmonides : caracterisation biochimique et biophysique : etude des modifications induites par l'alimentation et la temperature d'elevage : relation avec l'aptitude a la congelation du sperme." Rennes 1, 1992. http://www.theses.fr/1992REN10075.
Full textAbstract:
Afin de developper une etude biochimique et biophysique de la membrane plasmique du spermatozoide de truite, nous avons mis au point une technique de preparation de ces membranes par cavitation a l'azote: les spermatozoides sont soumis a une pression d'azote de 900 psi pendant 20 minutes. La decompression provoque l'eclatement des membranes plasmiques qui sont isolees sur gradient lineaire de sucrose. Les phospholipides majoritaires de la membrane sont la phosphatidylcholine (pc) (50%), la phosphatidylethanolamine (pe) (32%), la phosphatidylserine (ps) (9,8%) et le phosphatidylinositol (pi) (3,4%). Par resonance paramagnetique electronique, nous avons montre que la pe et la ps sont localisees majoritairement sur le feuillet interne de la membrane, dans un environnement plus fluide que la pc et la sm qui sont localisees majoritairement sur le feuillet externe. Le degre d'insaturation des acides gras de la pc est faible tandis que celui de la ps est le plus eleve. L'effet de la temperature d'elevage des geniteurs, 8c et 18c, ainsi que l'effet des lipides alimentaires, regime enrichi en acides gras polyinsatures de la serie n-3 (huile de foie de morue) et regime enrichi en acide linoleique (18:2n-6) (huile de mais), sur la composition lipidique de la membrane plasmique des spermatozoides, sur sa fluidite et sur l'aptitude a la congelation des spermatozoides a ete recherchee. Les caracteristiques des acides gras alimentaires sont repercutees sur la composition en acides gras des phospholipides membranaires mais n'ont d'influence ni sur la fluidite de la membrane, ni sur l'aptitude a la congelation des spermatozoides. La temperature d'elevage des geniteurs n'induit qu'une faible modification des acides gras phospholipidiques et ne modifie pas la fluidite membranaire. Par contre, le sperme des animaux eleves a 18c presente une resistance a la congelation 2 fois superieure a celle du sperme des truites elevees a 8c
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Azevedo, Hymerson Costa. "Integridade e funcionalidade dos espermatozóides ovinos submetidos à criopreservação após a incorporação de colesterol, desmosterol, ácido oléico-linoléico e alfa-lactoalbumina /." Botucatu : [s.n.], 2006. http://hdl.handle.net/11449/105970.
Full textAbstract:
Orientador: Sony Dimas BicudoBanca: Rui Machado
Banca: Jairo Pereira Neves
Banca: César Roberto Esper
Banca: Cezinande de Meira
Resumo: Objetivando testar a ação do colesterol, desmosterol, ácido oléico-linoléico ou Q-Iactoalbumina sobre os espermatozóides ovinos, alíquotas de sêmen de 25 carneiros foram submetidas à criopreservação após tratamento com essas substâncias. O sêmen foi avaliado antes do processamento e após a refrigeração, descongelação e incubação quanto à, cinética pela análise subjetiva e computadorizada (CASA), morfologia, avaliação simultânea da integridade da membrana plasmática (IMP) e do acrossomo (IAC) e do potencial de membrana mitocondrial (PMM) pela associação da aglutinina de Pisum sativum conjugada ao isotiocianato de fluoresceína (FITC-PSA), iodeto de propídio (PI) e JC-1 e, status de capacitação pela c10rtetraciclina (CTC). A influência dos aditivos em poucos parâmetros não foi suficiente para sugerir a superioridade de qualquer um dos tratamentos. A criopreservação provocou prejuízos à cinética, IMP, IAC, PMM, além de induzir a capacitação e reação do acrossomo. Foram observadas diferenças entre grupos de carneiros quanto ao status de capacitação no sêmen in natura, assim como na sensibilidade desse sêmen à crioinjúria e criocapacitação. Após a descongelação, as mudanças semelhantes à capacitação foram maiores nos animais de menor IMP. A segregação dos carneiros de acordo com a crioresistência da membrana plasmática norteou o comportamento de vários outros parâmetros. Conclui-se, que o colesterol, desmosterol, ácido oléico-linoléico e alactoalbumina não protegem o sêmen ovino contra as crioinjúrias, que a congelação-descongelação é mais danosa que a refrigeração e, que existem diferenças entre grupos de indivíduos relacionadas à crioresistência e criocapacitação espermática.
Abstract: In order to evaluate the proteeting aetion of some substanceson ram spermatozoa, semen samples of 25 rams were colleeted and submitted to cryopreservation after treatment with cholesterol, desmosterol, oleic-linoleic acid or Q-Iactalbumin. Before the processing and after the procedures of cooling, freezing-thawing and incubation, semen was evaluated as to kínetcs by subjective and computerized analyses (CASA), morphology, simultaneously to plasma membrane integrity (PMI), acrosome integrity (ACI) and mitochondrial membrane potential (MMP) by isothiocyanate-conjugated Pisum sativum (FITCPSA), propidium iodide (PI) and JC-1 combination and as to sperm capacitation and acrosome reaction determined by chlortetracycline (CTC) assay. The influence resulted from the additives in a few parameters was not enough to suggest the superiority of any treatments. The cryopreservation process, especially the freezing-thawing, promotes damages to kinetics, PMI, ACI, MMP and induees accelerated eapacitation and acrosome reaction in spermatozoa. Differenees among groups of rams were observed regarding capacitation status in fresh semen as well as the sensibility of their semen to cryoinjury and cryocapacitation. Higher and more accelerated capacitation-like ehanges were observed in groups of rams presenting lower plasmatic membrane cryoresistance after thawing. The segregation of rams in different levels of PMI influenced the behavior of several other parameters. It was concluded that cholesterol, desmosterol, oleic-Iinoleic acid and Q-Iactalbumin do not protect the ram semen against the cryoinjuries as well as freezing-thawing is more harmful than cooling. Important differenees among groups of individuais were noticed as for the spermatozoa resistance to the damages provoked by cryopreservation such as the eryocapacitation susceptibility.
Doutor
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Losano, João Diego de Agostini. "Efeito do tratamento antioxidante sistêmico em touros Bos taurus taurus submetidos ao estresse térmico e suplementados com dieta rica em ácidos graxos poliinsaturados sobre a capacidade de ligação de espermatozóides à membrana vitelínica de ovos de galinha." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-09102013-101625/.
Full textAbstract:
Diversos estudos indicam que touros europeus (Bos taurus) apresentam uma maior susceptibilidade ao estresse térmico com um consequentemente aumento do estresse oxidativo. Este, por sua vez, leva a uma diminuição na qualidade espermática. O espermatozoide possui, fisiologicamente, uma grande quantidade de ácidos graxos poli-insaturados (PUFAs) em sua membrana. As ligações duplas entre carbonos desses PUFAs confere a membrana plasmática uma maior fluidez, necessária aos processos fisiológicos do espermatozoide como motilidade e fertilização. No entanto essas ligações são mais facilmente oxidadas e, portanto, mais susceptíveis a peroxidação lipídica. Sendo assim, a suplementação com PUFAs promoveria uma melhoria na qualidade espermática, porém, tornaria o espermatozoide ainda mais susceptível à peroxidação lipídica. A vitamina E é um antioxidante chave na prevenção do estresse oxidativo através da interceptação do radical hidroxila, protegendo os PUFAs da membrana espermática contra a peroxidação lipídica. Portanto, uma alternativa para melhorar a qualidade espermática de touros europeus submetidos ao estresse térmico, seria a suplementação com PUFAs associada a um tratamento com vitamina E para prevenir um possível efeito deletério oxidativo desses ácidos graxos. Estudos sugerem uma importante relação entre os testes convencionais de avaliação de sêmen e fertilidade. No entanto, a limitação destes testes também tem sido reconhecida, principalmente em casos de infertilidade idiopática. Estudos demonstram que os testes funcionais de avaliação do sêmen, podem ser uma alternativa importante para a avaliação da capacidade fecundante de uma determinada amostra seminal. A interação entre espermatozóide e zona pelúcida ocorre através da ligação de receptores acrossomais às proteínas ZP3 e ZP2 do oócito. A membrana perivitelínica de ovos de galinha possui uma homologia com essas proteínas, permitindo sua ligação com espermatozóides de diversas espécies. Sendo assim, o teste de ligação espermática a essa membrana parece ser bastante eficiente para avaliar a capacidade fecundante do sêmen. No presente estudo, 16 touros Bos taurus foram submetidos à insulação testicular durante 4 dias e submetidos a um arranjo fatorial 2x2, sendo os fatores: dieta rica em PUFAs (Megalac®, 4 Kg ao dia) e tratamento com Vitamina E (Monovin E®, 3000 UI a cada 13 dias) durante 60 dias. As amostras seminais criopreservadas/descongeladas foram submetidas à avaliação da análise computadorizada da motilidade (CASA; Hamilton Thorne, Ivos 12.3, USA), integridade de membrana (Eosina Nigrosina), integridade acrossomal (Fast Green Rosa Bengala), atividade mitocondrial (33 diaminobenzidina), susceptibilidade ao estresse oxidativo (Substâncias reativas ao ácido tiobarbitúrico - TBARS), integridade de DNA (SCSA) e o teste de ligação de espermatozóides à membrana vitelínica de ovos de galinha, que foi previamente padronizado. O tratamento com vitamina E apresentou efeitos benéficos para algumas características espermáticas, sendo o contrário observado pelo tratamento com PUFAS, quando ambos foram analisados isoladamente. No entanto, a associação entre estes tratamentos não foi eficiente para melhorar a qualidade espermática assim como a capacidade fecundante avaliada através do teste de ligação espermática em membrana perivitelínica de ovos de galinha.Several studies suggest a high susceptibility of European bulls (Bos taurus) to the heat stress, which will lead to an increase on oxidative stress and consequent impairment on sperm viability. Sperm membranes are rich in poly-unsaturated fatty acids (PUFAs). The carbon-carbon double bounds (insaturations) that characterize these PUFAs confer the necessary fluidity to the sperm membrane, essential to motility and fecundation capacity. However, these insaturations are more unstable and, therefore, more susceptible to the lipid peroxidation (i.e., attack of reactive oxygen species to the lipids). Hence, PUFA supplementation would improve certain sperm characteristics but increase the already high susceptibility to the oxidative stress. Vitamin E is a key antioxidante on oxidative stress prevention by intercepting hydroxyl radical and protecting sperm membrane PUFAS against lipid peroxidation. Therefore, an alternative to improve sperm quality in heat stressed european bulls would be a PUFA supplementation associated to a Vitamin E therapy in order to avoid a possible deleterious oxidative effects of these fatty acids. Estudies suggest an important relationship between conventional tests for sperm evaluation and fertility. However, limitations have been also verified, especially in cases of idiopathic infertility. Thus, tests to evaluate sperm functionality may be an interesting alternative to assess fecundation capacity of semen samples. The binding of sperm to the zona pellucida occurs initially by the interaction between acrosomal receptors and oocyte proteins ZP3 and ZP2. The perivitelline membrane of chicken eggs present homologies to zona pellucida proteins, which allows sperm from several species to bind to this membrane. Thus, the sperm binding test to the perivitelline membrane may be an efficient method to asses the fecundation capacity in semen of bulls. In the present study, 16 Bos taurus bulls were submitted to testicular insulation for 4 day. Animals were then submitted to a 2x2 factorial design, with the factor represented by a PUFA oral supplementation (Megalac®, 4 Kg for day) and the treatment with Vitamin E (Monovin E®, 3000 UI, each 13 days) for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables by the computer assisted sperm analysis (CASA; Hamilton Thorne, Ivos 12.3, USA), membrane integrity (Eosin / Nigrosin), acrosome integrity (Fast Green / Bengal Rose), mitochondrial activity (33 diaminobenzidine), susceptibility to the oxidative stress (tiobarbituric acid reactive substances - TBARS), DNA integrity (SCSA) and the sperm binding test to the chicken egg perivitelline membrane, which was previously validated. Results demonstrated a beneficial effect of Vitamin E on some sperm characteristics, with the inverse effect observed for PUFA when both treatments were considered individually. However, the association between both treatments were not efficient to improve sperm quality as well as the fecundation capacity as evaluated by the sperm binding test to the chicken perivitelline membrane.
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Fonseca, Lucas dos Santos. "Desempenho, consumo de matéria seca, parâmetros seminais e proteômica do plasma seminal e da membrana espermática de carneiros Morada Nova alimentados com dieta à base de castanha de caju." reponame:Repositório Institucional da UFC, 2013. http://www.repositorio.ufc.br/handle/riufc/14861.
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FONSECA, L. S. Desempenho, consumo de matéria seca, parâmetros seminais e proteômica do plasma seminal e da membrana espermática de carneiros Morada Nova alimentados com dieta à base de castanha de caju. 2013. 65 f. Dissertação (Mestrado em Zootecnia) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2013.Submitted by Daniel Eduardo Alencar da Silva (dealencar.silva@gmail.com) on 2015-01-14T19:01:01Z No. of bitstreams: 1 2013_dis_lsfonseca.pdf: 641605 bytes, checksum: 04f084807466b026c5bf42f0ceccacd9 (MD5)
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The cashew nut meal is alternative food for ruminant nutrition which is rich in lipids, proteins and minerals. However, its nutrition influences on male development and reproduction are still unknown. In this context, seminal and sperm protein analysis can show metabolic consequences from cashew nut meal on sheep reproductive efficiency. Therefore, the current dissertation aims evaluate the effects of 13% of cashew nut meal in the ration on the weight gain, food intake, carcass yield, weight of sexual organs, sperm quality and, seminal and sperm membrane proteins of Morada Nova Rams. Twenty rams were divided in two groups: cashew nut and control, which received 13% or 0% of cashew nut meal in the diet, respectively. During ninety days, the animals were kept in individual boxes and evaluated for ration consumption and weight gain. The sperm collections by eletroejaculation were made by weekly and, the volume, sperm concentration and motility were analyzed. After ninety days, the seminal and sperm membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The gels were digitalized, and their images were evaluated by computer software. At the end of the experiment, the rams were slaughtered and, the weight gain, carcass yield and weight of sexual organs were measured. There was not effect of cashew nut meal addition in the diet on the weight gain, carcass yield and weight of sexual organs. But, after sixty days, there was a reduction on food intake in the cashew nut group (P < 0,05). The sperm quality was not influenced by the diet. We observed an effect of cashew nut diet on the expression of seminal and sperm proteins. Seven spots from seminal plasma were expressed differently between cashew nut and control groups (P < 0,05). The spot 2206 (21,82 kDa; pI: 5,04) was negatively correlated with the individual motility score (r = -0,49). Additionally, seven spots from sperm membrane protein also differ between rams from cashew nut and control groups (P < 0,05). In conclusion, the addition of 13% of cashew nut meal in the diet alters the expression of sheep seminal plasma and sperm membrane proteins.
O presente estudo foi conduzido com o objetivo de avaliar os efeitos da inclusão de 13% de farelo de castanha de caju na dieta de carneiros da raça Morada Nova sobre o ganho de peso, consumo de matéria seca, rendimento de carcaça, pesos dos órgãos sexuais, qualidade seminal e proteínas seminais e membranares do espermatozoide. Para tanto, vinte carneiros foram divididos em dois grupos alimentados com dietas contendo 13% (GCA, n=10) ou 0% (GCO, n=10) de farelo de castanha de caju. As dietas foram isoproteicas e isoenergeticas, com uma relação de 50/50 de concentrado/volumoso (feno de Tifton 85) e água e sal mineral à vontade. Durante noventa dias, os animais foram mantidos em baias individuais e avaliados quanto ao consumo de matéria seca e ganho de peso. As coletas seminais por eletroejaculação ocorreram semanalmente com a determinação do volume ejaculado e, concentração, motilidade e morfologia espermática. Após os noventa dias, as proteínas seminais e espermáticas foram analisadas por meio de eletroforese bidimensional em gel de poliacrilamida. Os mapas proteícos foram avaliados por meio do aplicativo PDQuest, version 8.0; Bio Rad, USA. Ao final do experimento, os carneiros foram abatidos e, o peso vivo, rendimento de carcaça e peso dos órgãos sexuais foram mensurados. O consumo de matéria seca total permaneceu sem alterações nos dois grupos experimentais durante os primeiros 60 dias do estudo. Porém, após 60 dias, houve e redução no consumo de matéria seca no grupo alimentado com castanha (P < 0,05). Não houve efeito da inclusão do farelo de castanha sobre o ganho de peso, rendimento de carcaça, desenvolvimento dos órgãos sexuais e parâmetros seminais dos animais. No entanto, a inclusão do farelo de castanha de caju esteve associada à expressão de proteínas seminais e espermáticas. Sete spots proteicos presentes nos mapas de plasma seminal foram expressos diferencialmente entre os grupos castanha e controle (P < 0,05) e um spot proteico (21,8 kDa; pI: 5) correlacionou-se negativamente com o vigor espermático (r = -0,49; P<0,05). Adicionalmente, sete spots proteicos dos géis com proteinas da membrana dos espermatozoides também diferiram entre os carneiros alimentados com e sem farelo de castanha de caju (P < 0,05). Conclui-se, portanto, que a inclusão de 13% de farelo de castanha de caju na dieta altera a expressão de proteínas seminais e espermáticas em ovinos.
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Medeiros, Coralia. "Membrane modifications in bovine sperm during capacitation." 1994. http://catalog.hathitrust.org/api/volumes/oclc/33064256.html.
Full textAbstract:
Thesis (M.S.)--University of Wisconsin--Madison, 1994.Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 108-127).
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Ting-wei, Jhan, and 詹庭威. "The possibility of producin treansgenic porcine embryos with membrane-damaged sperm by intracytoplasmic sperm injection." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/30768347202620347473.
Full textAbstract:
碩士國立宜蘭大學
動物科技學系碩士班
97
The method of sperm-mediated gene transfer is a simple manipulation and costs low, however, direct fertilization in vitro with sperm carrying exogenous gene is difficult to achieve the transgenic animals by polyspermy. The ICSI technique could effectively overcomes the obstacle. Making damage on the sperm membrane was benefit to transgenic efficiency because of enhances the binding of exogenous gene, but the damage treatment on sperm would decrease the sperm motility and influence the fertilization ability. It must ensure normal fertilization by ICSI. The present study was to investigate the exogenous DNA-binding site, binding rate and copies in boar sperm. Further we evaluated the effect of making damage on the sperm membrane by frozen-thawed and dithiothreitol（DTT）co-incubation treatment on the exogenous gene-binding amount of sperm. Finally we used the technology of intracytoplasmic sperm injection combined with sperm vector to inject sperm bearing EGFP into the in vitro maturated porcine oocytes. The formation of pronuclear, fertilization and embryos development were observed to estimate the possibility of producing porcine embryos expressing green fluorescence. The highest survival rate (100%) of oocytes was the group of sperm capacitated within 5 hours combined with the oocytes in vitro maturated for 48 hours following ICSI. That was higher than the group of sperm capacitated within 5 hours combined with the oocytes maturated for 44 hours (86.7%) significantly, but there was no significantly difference with other 2 groups. Just for the rate of activation, the group of sperm capacitated within 1 hour combined with the oocytes maturated for 44 hour oocytes is the highest (80.1%) and higher than the group of sperm capacitated within 5 hour combined with in vitro maturated 44 hour oocytes (41.6%) significantly, but there was no significantly difference with other groups. However, the rate of male pronuclear formation and sperm decondensation were not influenced by the time of oocytes maturation and sperm capacitation. The boar sperm was co-incubated with FITC-dUTP DNA probe and observed the fluorescence on post-acrosome under microscope. The sperm bearing exogenous DNA after co-incubated with EGFP were estimated by real time PCR. The result showed that physical treatment of frozen-thawed enhanced EGFP-binding copies but chemical treatment of DTT co-incubation decreased the binding ability significantly. The EGFP fragments were electroporated into the cumulus cells to confirm that expressed normally. Further the oocytes were injected the frozen-thawed sperm that co-incubated with EGFP previously, the rate of male pronuclear formation or decondensed sperm head, normal fertilization were all lower than the group of DTT co-incubation. The rate of cleavage at 48 hour following ICSI were no significantly difference in the two group. We never obtained any porcine embryos of expressing green fluorescence.
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"The role of membrane character in human sperm function." Tulane University, 1994.
Find full textAbstract:
Fertilization is a membrane-dependant event. Due to the involvement of membranes in the fertilization process, the integrity and function of sperm membranes influences their fertilizing ability. These studies investigate sperm membrane character in relation to function and fertilizing capacity The first phase of experiments used hydrogen peroxide to induce peroxidative damage to membrane phospholipids. As compared to controls, an increase in lipid peroxidation and decrease in sperm percent motility were observed following 15 minute treatment with 0.01% or 0.05% H$\sb2$O$\sb2.$ Increased lipid peroxidation was correlated with an observed decrease in the cholesterol:phospholipid ratio. The same treatment did not significantly alter sperm viability In order to further correlate membrane integrity and sperm function, the second phase of experimentation investigated the role of membrane damage in cryopreservation-induced alterations in sperm function. In one set of experiments, cryopreserved sperm samples that were thawed slowly exhibited reduced motility and increased lipid peroxidation compared to quickly thawed samples. In a second set of experiments, platelet activating factor (PAF) and pentoxifylline (PTX), two known sperm motility stimulants, were used as cryoprotective additives. Pre-freeze addition of both PAF and PTX significantly improved post-thaw motility. Lipid peroxidation was not affected by either agent The next phase of research was designed to determine whether protection from peroxidative damage and improved sperm function were related. Sodium nitroprusside (SNP) was used to scavenge oxygen radicals, which are potentially damaging to membrane phospholipids. In the preliminary investigation, 50 and 100 nM concentrations of SNP significantly improved the maintenance of post-thaw percent motility and viability, and sperm velocity for up to six hours. In a follow-up investigation, treatment with 50 nM SNP significantly improved acrosin enzyme activity and the percentage of sperm which completed the acrosome reaction Because preliminary experiments demonstrated an association between membrane integrity and sperm function, a more detailed examination of membrane phospholipid content was pursued in the final phase of experimentation. Mass spectroscopy was performed on membrane phospholipid extracts. Samples were divided into two groups for data analysis: Group A samples exhibited greater than 40% motility maintenance during the freeze-thaw process, Group B samples exhibited less than 40% motility maintenance. Samples in Group A generated spectra with significantly more activity and a different set of major peaks than did samples in Group B. The most significant peak generated by Group A samples was not generated by Group B samples, suggesting that differences in lipid composition affect sperm motion A general discussion of the results of all phases of experimentation, and propositions concerning their potential significance is includedacase@tulane.edu
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Wrench, Nicola. "Effect of season on sperm membrane protein 22 and selected mRNAs in fresh and cryopreserved stallion sperm." 2007. http://www.lib.ncsu.edu/theses/available/etd-11092007-124518/unrestricted/etd.pdf.
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Lin, Ting-Wei, and 林廷維. "Quantitative Phosphoproteomics Profiling of the Mouse Sperm Membrane Protein During Capacitation." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/19675907050990599867.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
96
After ejaculation, sperm are able to move actively but lack fertilizing competence. Capacitation is an important physiological pre-requisite before the sperm cells can acrosome react and fertilize the oocyte. Several studies suggested that capacitation is associated with phosphorylation of membrane proteins. However, the quantitation and qualification of membrane protein phosphorylation during capacitation is still less understood. To identify protein phosphorylation during capacitation, detergent-base partition coupled with tube-gel digestion and tandem mass spectrometry (MS/MS) was performed. In addition immobilized metal affinity chromatography (IMAC) was used to investigate phosphorylation site in whole protein digests before and after capacitated mouse sperm. After the purification of membrane proteins, about 250 proteins were identified and about 80% of the identified proteins were membrane-associated. After the phosphopeptide purification followed by LC-MS analysis, phosphorylation site for the capacitated and noncapacitated sperm proteins were identified and quantified. Using hexokinase 1 as an internal control, about 27 phosphorylation sites were identified to be increased in phosphorylation levels during capacitation. This approach provides a site-specific quantitation result for the protein phosphorylation during sperm capacitation. The results will help to improve our understanding of the pin-point protein phosphorylation that associates with hyperactive motility and redistribution of the membrane components during capacitation.
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Figueiredo, Maria Inês Lopes. "Investigation of Changes in Stallion Sperm Mitochondrial Membrane Potential During Storage." Master's thesis, 2016. https://hdl.handle.net/10216/83887.
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Figueiredo, Maria Inês Lopes. "Investigation of Changes in Stallion Sperm Mitochondrial Membrane Potential During Storage." Dissertação, 2016. https://repositorio-aberto.up.pt/handle/10216/83887.
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Stump, Karen Elizabeth. "Evaluation of Stallion Sperm Membrane Integrity Using Varied Flow Cytometer-Based Methodologies." Thesis, 2013. http://hdl.handle.net/1969.1/149554.
Full textAbstract:
Artificial insemination using cooled, transported semen has become a popular practice in the equine industry. However, equine sperm are assumed to show a decline in their fertilizing ability after 24 to 48 hours of cooled storage. Two measures that are commonly used to estimate the fertility of an ejaculate are sperm motility and sperm membrane integrity (SMI). Recently, it has been suggested that SMI may have a better correlation with fertility of an inseminate than sperm motility. The effect of cooled-storage on sperm quality over an extended time period was evaluated to illustrate changes in sperm characteristics that might be related to an ejaculate’s fertility.
Semen was stored at 4°C in INRA 96 extender containing 10% seminal plasma for a period of 10 days. Data were collected daily on sperm motion characteristics, SMI, mitochondrial membrane potential, and DNA quality. To measure daily changes in SMI in stallion sperm, two fluorescent vital-staining protocols used in flow cytometric analysis were compared – a combination of SNARF-1, Yo-Pro-1, and Ethidium Homodimer 1 (SYE) and a combination of lectin from Pisum sativum and propidium iodide (PSA/PI). We hypothesized that the SYE protocol adapted for use with stallion sperm could detect more subtle, and perhaps earlier, damage to the sperm plasma membrane than the PSA/PI protocol. A combination of SYBR 14, propidium iodide, and JC-1 (SYPIJC) was used to measure mitochondrial membrane potential, as well as SMI. A computer-assisted sperm motion analysis (CASA) instrument was used to evaluate sperm motion characteristics; the sperm chromatin structure assay (SCSA) was used to measure the degree of DNA fragmentation.
In this study, with the exception of sperm motility, the measures of sperm quality retained values consistent with “viability” after 10 days of cooled-storage. This suggests that the fertility of some stallions may last considerably longer than previously assumed, which could ultimately alter the time-table used for artificial insemination using cooled, transported semen.
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Liu, Chih-Chun, and 劉志濬. "Assessment of Viability and Mitochondrial Membrane Sheath in Fresh and Frozen Boar Sperm." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/35653214082796715282.
Full textAbstract:
碩士國立屏東科技大學
畜產系
91
Semen qulity evalution is the most important thing before artificial insemination. The objective of this study was to compare the vital stain of trypan blue and fluorometric technique to assess different living/killed ratio sperm of viability and mitochondrial membran sheath and utilization of fluorometric technique to estimate frozen-thawed sperm and different stage of frozen-thawed sperm viability. The parameters of motility, viability and mitochandrial membrane sheath of three treatments (fresh, killed and 1:1 ration sperm) were estimated by Trypan blue, SYBR-14/PI, Hoechst 33258 and Rhodamine 123. All of the parameters in three treament were significantly different (p<0.05). Trypan blue, Hoechst 33258 and Rhodamine 123 with SYBR-14/PI were positive correlation (0.96, 0.95 and 0.96). Correlation coefficient among motility, Trypam blue, Hoechst 33258, SYBR-14/PI and Rhodamine 123 were 0.87, 0.86, 0.85 and 0.93, respectively. Trypan blue show almost the same pattern with fluorometric technique and those stains can hightly correlation with motility. 5 ml and 0.5 ml straw were used for freezing package, after thawed 0.5ml straw have better frozen-thawed sperm motility comparison with 5 ml straw (p<0.05), but after 3 hr and 6 hr the motility between 5ml and 0.5ml straw were no significant difference. Estimated frozen-thawed sperm cell membrance status by Hoechst 33258, SYBR-14/PI and Rhodamine 123, those were no significant difference. Different stage among sperm motility and viability at frozen-thawed procedure, the sperm motility between fresh diluted and diluted with first stage freeze diluter were no significant difference, however, the motility decrease after diluted second stage freeze diluter (p<0.05). The sperm viability decrease after diluted with first stage freeze diluter, between first and second stage freeze diluter were no significant difference. Storage at 25℃ after 24hr the sperm motility and viability at different stages decreased almost the same with 0hr, but the motility drop obviously. The sperm cell membrane damaged at all stage in frozen-thawed procedure, after 24hr storage sperm motility drop more than membrane integrity. To estimate sperm viability by vital stain trypan blue is unhandy, fluorometric technique may replace it and empolyed for estimating sperm cell membrane integrity, found out that membrane integrity would be injured at each stage in frozen procedure, and result between 5 ml and 0.5 ml straw is similar except the sperm motility of frozen-thawed at 0 hr.
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Foster, Mary L. "Comparison of Methods for Assessing Viability of Equine Spermatozoa and Effects of Seminal Plasma on Viability and Motion Characteristics of Equine Spermatozoa." 2009. http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7469.
Full textAbstract:
Assessment of sperm viability is an important component for evaluating
stallion sperm quality. The flow cytometer is considered the standard in the
assessment of sperm plasma membrane integrity (viability); however, this
instrument is costly to purchase and use, and it requires an experienced
technician to operate it. The growing practice of assisted reproductive
technologies (ARTs) in the equine industry has increased the need for an
accurate but cost-effective means of determining sperm membrane viability.
The NucleoCounter® SP-100TM is reported to be an accurate, easy-to-perform,
and an efficient stallion-side test for sperm membrane viability.
To evaluate usefulness of the NucleoCounter® SP-100TM for assessing
sperm membrane integrity, neat semen was subjected to four treatments with
varying seminal plasma volumes and sperm concentrations. Sperm membrane
viability was assessed immediately, and at 24 and 48 hours after cooled-storage
using three methods: 1) flow cytometer utilizing the fluorescent vital stains SYBR-14/propidium iodide; 2) NucleoCounter® SP-100TM utilizing the
fluorescent vital stain propidium iodide; 3) eosin-nigrosin stained air-dried
smears of semen. Sperm motion characteristics (total and progressive motility)
were assessed using a computer assisted sperm motion analyzer (CASMA) and
results were compared to sperm membrane viability to determine the
relationship between sperm membrane viability and motion characteristics.
Results were compared statistically by: 1) analysis of variance (ANOVA); 2)
linear regression analysis; 3) coefficient of variation on untransformed and
transformed data (arc sine square root); and 3) the agreement of two
instruments, by means of which the difference between measurements of the
two instruments were plotted on the y-axis and the average of measurements
from the two instruments were plotted on the x-axis.
Results obtained with the NucleoCounter® SP-100TM agreed best with the
flow cytometer, and least with eosin-nigrosin staining. Coefficients of variation
were ≤ 5% for the three methods (transformed data). Sperm motion
characteristics and sperm viability were similar among treatments at Time 0. At
Times 24 and 48, sperm motion characteristics decreased at a more significant
rate compared to viability in the treatments containing ≥ 50% seminal plasma,
whereas differences among treatments were only significant at seminal plasma
concentrations above 50% when only sperm membrane viability was
considered.
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Wen, Chia-Wei, and 温家偉. "Using mouse Izumo1 as a model to study the phosphoproteome of sperm membrane after capacitation." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/34387185548412185658.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
98
Mammalian spermatozoa are unable to fertilize eggs immediately after ejaculation until they undergo a regulatory procedure named capacitation. Although many attempts, there are still many molecular mechanisms of capacitaion remain unclear. Since mature spermatozoa could only adjust their physiological condition through post-translational modifications, especially phosphorylation, we applied a quantitative phosphoproteomic platform, including detergent enrichment of membrane proteins, IMAC chromatography enrichment of phosphopeptides, and LC-MS/MS analysis for globally comparing of capacitated and non-capacitated mouse sperm membrane to identify important fertilization related molecules. Our data indicates that at least 11 proteins increased their phosphorylation level more than 2 folds after capacitation. By using GO algorism, all of the 11 proteins are annotated as membrane associated. Two previous known sperm-egg essential proteins, Izumo1 and SPACA1, were found to increase their phosphorylation level after capacitation. The others are known as membrane-integrated enzymes, transporters or receptors, which may play roles in fertilization awaits further investigation. To evaluate our MS data, we also performed in vitro kinase assay with recombinant Izumo1 (GST-Izumo1) in capacitated or non-capacitated mouse sperm lysate. We found it also increased phosphorylation in capacitated sperm lysate and the phosphorylation site was in accord with endogenous Izumo1. Further, we made a mutant Izumo1 devoid of the phosphorylation site. By comparing mutant and wild-type GST-Izumo1, we found that a potential Izumo1 associated protein binding was also weakened as the phosphorylation level decreased. By using multiple sequence alignment, it is found that the phosphorylation sites of mouse Izumo1 are not observed in the rat Izumo1. However, we found that the rat Izumo1 recombinant protein is still phosphorylated in the capacitated mouse sperm lysate and the phosphorylation sites were in accord with the endogenous rat Izumo1. To sum up, our work provided a powerful tool for studying the molecular mechanism of sperm capacitation and fertilization
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Khunsook, Sumpars. "Purification and characterization of human sperm plasma membrane-associated and human seminal fluid [alpha]-L-fucosidases /." Diss., 2001. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3036265.
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Shalaweh, Salem. "Effect of cissampelos capensis rhizome extract on human sperm capacitation and acrosome reaction." 2013. http://hdl.handle.net/11394/3881.
Full textAbstract:
>Magister Scientiae - MScCissampelos capensis, is commonly known by the Afrikaans name ‟dawidjies” or ‟dawidjieswortel”. C. capensis is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis rhizome extracts on sperm function.
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Yu, YANG. "THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION." Thesis, 2008. http://hdl.handle.net/1974/1589.
Full textAbstract:
During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-11-27 15:33:50.226
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Cheng, Chiu-Hung, and 鄭秋虹. "Research of using opposite-sex immunization to screen sex-specific proteins from X- and Y-sperm plasma membrane of pigs." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/61379468661546998575.
Full textAbstract:
碩士國立中興大學
獸醫學系
92
Biotechnology for pre-selecting gender of one’s offspring is much worth business investment. However, there is no effective, convenient and safe means to separate X and Y sperm for pre-selecting gender to date. One of the feasible ways is to use a non-invasive and immunological method to screen X or Y sperm out. Based on the Ohno’s law that sex-specific proteins (SSPs) are evolutionarily more highly conserved than non-SSPs, separation of SSPs from X or Y sperm creates a foundation for further immunological selection of X or Y sperm. Opposite-sex immunizations (antigens: swine sperm membrane crude proteins) of rabbits was applied to produce the corresponding antibodies for each sex. ELISA confirmed the paramount of the raising antibodies. SDS-PAGE and Western blotting analysis revealed comparable membrane proteins patterns in each sex antibody preparations. Sex-specific affinity columns prepared from the antibodies produced by each sex were used to purify SSPs for three cycles. Preliminary results demonstrated that two obvious X-SSPs bands (13.6 and 72.2 kDa) were detected by the one-dimensional electrophoresis analysis. However, these results were not consistently presented under same preparations (n=3). Further, with the application of high resolution two-dimensional electrophoresis and western blotting (antibodies of each sex), purified X-SSPs (app. 102 kDa) obtained from three-cycles affinity column chromatography, was clearly observed. In conclusion, this study has shown the presence of X-SSPs. But, Y-SSps appears to be imperceptible. Nevertheless, the result of present study indicates the feasibility of immunological sperm sexing techniques. Further investigation should be toward on the immunoaffinity enrichment for SSPs and the protein identification.
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Tan, Wenxian active 21st century. "Elucidating the signal cascades induced by progestins that mediate sperm hypermotility in Atlantic croaker (Micropogonias undulatus) and southern flounder (Paralichthys lethostigma)." Thesis, 2013. http://hdl.handle.net/2152/28716.
Full textAbstract:
The overall goal of this research was to verify the involvement of membrane progestin receptor alpha (mPRα) in mediating progestin-stimulated sperm hypermotility in the Atlantic croaker and southern flounder. Sperm motility in Atlantic croaker and southern flounder were tested with both the endogenous progestin, 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) or the selective mPRα agonist, 10-ethenyl-19-norprogesterone (Org OD 02-0). In croaker, the Pi3k/Akt/Pde and ErbB2/Mapk intracellular signaling pathways were examined. The role of mPRα in mediating sperm hypermotility and fertility in southern flounder was also studied. The effects of seasonal hypoxia on sperm motility in croaker were investigated in a field study in the northern Gulf of Mexico in the fall of 2010. Finally, the effects of acidified activator solution (simulating ocean acidification) were studied in the laboratory. In vitro, Org OD 02-0 mimicked the stimulatory actions of 20β-S in inducing sperm hypermotility and intracellular signaling cascades in croaker and flounder sperm, indicating that mPRα is the mediator of progestin signaling in the sperm of these species. In croaker sperm, both the Pi3k/Akt/Pde and ErbB2/Mapk intracellular signaling pathways were shown to be important mediators of progestin-induced sperm hypermotility, suggesting novel functions of G [subscript olf] βγ-subunits in teleost sperm. In flounder sperm, mPRα was shown to be important in mediating sperm hypermotility as only high motility sperm with high expression of mPRα were responsive to progestin stimulation, resulting in higher fertilization success compared to low motility sperm. A single LHRHa injection resulted in increased sperm motility and fertility, associated with an increase in mPRα expression in the sperm plasma membrane. The results also suggest that the mPRα/Acy/cAMP pathway first described in croaker sperm is present in flounder sperm. Field studies of male Atlantic croaker exposed to chronic seasonal hypoxia showed that hypoxia exposure resulted in smaller gonads, lower spermatogenesis, reduced testicular mPRα expression, and in some sites, reduced sperm motility. Studies with croaker sperm using acidified activator solution to simulate ocean acidification indicated that croaker sperm were sensitive to environmental insult. Furthermore, the results suggested that the progestin signaling mechanism is more sensitive to changes in ocean pH levels than the mechanism that controls sperm motility.text
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Canvin, Andrew T. "Factors affecting the fluidity of boar sperm membranes." 1988. http://hdl.handle.net/1993/16621.
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Springsguth, Hans Christopher. "Mechanismen und Bedeutung der aktivierten Apoptosekaskade in humanen Spermatozoen." Doctoral thesis, 2015. https://ul.qucosa.de/id/qucosa%3A14133.
Full textAbstract:
Andrologische Forschungsarbeiten der letzten Jahre beweisen, dass einzelne, aus
somatischen Zellen bekannte Apoptose-typische Veränderungen bei humanen Spermien
einen negativen Einfluss auf die Fertilität des Mannes haben. Umstritten ist, ob es sich dabei
nur um einen abortiven Zelltod als Zeichen einer Reifungsstörung während der
Spermatogenese handelt oder ob Apoptose auch in reifen Spermien induzierbar ist.
Ziel der vorliegenden Arbeit war es, durch Untersuchungen zur Induzierbarkeit der Apoptose
in reifen und unreifen Spermatozoen die vollständige Funktionalität verschiedener
Signalkaskaden sowie deren Umsetzung in morphologische Veränderungen aufzudecken.
Darüber hinaus sollte die Rolle des intrazellulären Calciumspiegels als möglicher
Interaktionspartner zwischen Akrosomreaktion und Apoptose geklärt werden, um
Informationen über die Zukunft der nicht fertilisierenden Spermien im weiblichen Genitaltrakt
zu erlangen.
In den vorliegenden Versuchsreihen konnte erstmals die gezielte Induktion der Apoptose in
reifen und unreifen Spermatozoen anhand biochemischer und elektronenmikroskopischer
Untersuchungen nachgewiesen werden. Dabei wurde ausführlich die erfolgreiche
Aktivierung mehrerer intrinsischer Apoptosesignalwege in reifen Spermien gezeigt, deren
Initiation entweder auf den Zusammenbruch innerer Mitochondrienmembranen, auf eine
veränderte intrazelluläre Calciumkonzentration oder auf das Einwirken von oxidativem
Stress zurückzuführen war. Zudem konnten Erkenntnisse zum antioxidativen
Schutzmechanismus von Spermien gewonnen werden, welcher die Spermien gegenüber
einer spezifischen Menge an H2O2 vor oxidativem Stress-bedingter Apoptose bewahrt.
Sowohl die Apoptose als auch die Akrosomreaktion waren durch die Zugabe eines
Calciumchelators blockierbar.
Die Initiation des programmierten Zelltodes in Spermien durch einen Anstieg der
intrazellulären Calciumkonzentration erklärt zudem eine weitere wichtige Funktion dieses
Prozesses: das Absterben von akrosomenreagierten Spermien bei nicht erfolgter
Fertilisation im weiblichen Genitaltrakt. Die Theorie einer rein abortiven Apoptose als Folge
einer Spermatogenesestörung ist damit widerlegt.
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Bašus, Kryštof. "Monitorování dynamiky proteinových sítí: role FcRL proteinů při interakci membrány spermie a vajíčka." Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-435880.
Full textAbstract:
Sperm--egg membrane interaction and fusion is mediated by various molecules of the different protein network that are located on both egg and sperm membrane. So far, many proteins have been selected to be fusion candidates, some of them (Izumo1, CD9, Juno) were proven to be essential, whereas others were discovered to play an unsuspected new active role (CD46, tetraspanins). After the adhesion of sperm to an egg, Juno located on the oolema associates with monomeric Izumo1 on sperm membrane, which is results in Izumo1 dimerization following quick removal of Juno from the egg surface as described in mouse. It implies that additional receptor on the egg membrane is required to play a role in sperm--egg fusion. To find a human fusogenic receptor for IZUMO1 protein we used one--bead--one--compound (OBOC) assay, a random screening approach. A bead, fulfilling all the requirements when interacting with the human sperm, carried a peptide sequence showing homology with the conserved Ig domain of the human specific Fc receptor--like protein 3 (FcRL3). In general, the ...