Due to overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. The potential use of the porcine zona pellucida (pZP) vaccine as a safe and effective means for population control is explored. While potential effects of pZP treatment on social behaviour of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. The ovarian activity of freeranging, pZP-treated African elephant females was monitored non-invasively for one year duration by measuring faecal progestagen concentrations via enzyme immunoassay. Behavioural observations were recorded for comparison with progestagen concentrations and to determine any behavioural changes surrounding the pZP vaccine darting event. Each elephant under study showed progestagen concentrations rising above baseline at some period during the study indicating luteal functionality. Average progestagen concentrations were 1.61 ± 0.46 ìg/g. Within sampled females, 42.9% exhibited oestrous cycles within the range reported for African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average oestrous cycle duration was 14.72 ± 0.85 weeks. Behavioural oestrous coincided with the onset of the luteal phase and a subsequent rise in progestagen concentrations. Focal sampling to determine activity budgets before and after the darting event revealed no significant change in behavioural activities. In the week following immunization, individual progestagen concentrations decreased significantly from overall average concentrations. Average progestagen concentrations positively correlated with rainfall and with herd dominance. No association between average individual progestagen concentrations or cyclicity status with age, lactation, or parity were detected. Earlier determination of efficacy was made indicating reproductive control was established 22 months post-treatment. Results indicate the presence of ovarian activity amongst pZP-treated female African elephants in two years following initial immunization. Further study should be aimed toward studying the long term effects of pZP vaccination on the reproductive function of female African elephants. / Dissertation (MSc)--University of Pretoria, 2010. / Production Animal Studies / unrestricted

The objective of this study was to identify peptide ligands, using phage display techniques, which bind sites on mouse embryos, ovaries, cytoplasmic membranes and/or intracytoplasmic components. Specifically, M13 coliphage 7-mer, 12-mer and 15-mer random peptide libraries were used separately for biopanning. Peptides derived from the amplified pools were sequenced and studied. The phage display for in vivo ovary experiments yielded no pool of peptides after two cycles of biopanning and re-amplification. With the same initial concentration of a random 7-mer or 12-mer library, there were repeating sequences derived after three and four biopanning cycles on mouse embryos and unfertilized ova. The sequences were not distinguishable from a control group. Subsequent experimentation using a random 15-mer library to select for internalized phage-peptides yielded two apparent consensus sequences, RNVPPIFNDVYWIAF (9/32 or 28%) and HGRFILPWWYAFSPS (11/32 or 34%). The 15-mer control group yielded no clones. The deduced peptide sequences were compared to known sequences to ascertain their uniqueness. No significant similarities were found, yielding two possible novel motifs. Through this adapted process of phage display and further research, the phage display technology may be used as a tool in the recognition of specific mouse gamete sites. By identifying binding sites of mouse gametes, the peptides might be exploited as a means of studying the embryo cell surface or cytoplasmic components and mouse sperm-egg interactions. Such peptides may also be used for macromolecule delivery in transfection or transgenesis. / Master of Science

Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) é uma herbácea amplamente encontrada nos trópicos e que possui diversas propriedades biológicas. Seus estudos fitoquímicos haviam demonstrado a presença da pellucidina A, uma rara dinorlignana ciclobutânica, que seria formada por acoplamento oxidativo de 2,4,5- triidroxi-estireno seguido de metilações. Nesse trabalho, foram caracterizados o ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno, 2,4,5-trimetoxi-benzaldeído, dilapiol, 5,6,7-trimetoxi-flavona, sesamina, além da pellucidina A. Estudos de aspectos dinâmicos envolvidos na formação da pellucidina A incluíram a ontogenia e respostas à diferentes tratamentos como estresse hídrico, predação por herbívoros, ácido jasmônico e luz UV360. O tratamento com ácido jasmônico resultou num significativo incremento do dilapiol enquanto que, o tratamento sob luz UV360 resultou no aumento na produção da pellucidina A sugerindo um mecanismo de cicloadição [2+2] para sua biossíntese. A administração de diferentes precursores in vivo revelou que a L-[2-13C]- fenilalanina (0,75%), ácido [8-13C]-cinâmico (1,32%), ácido [8-13C]-ferúlico (0,51%), ácido 2,4,5-trimetoxi-[8-13C]-cinâmico (7,9%) e o 2,4,5-trimetoxi-estireno (13,3%) foram incorporados à pellucidina A. Ensaios de conversão enzimática indicaram a descarboxilação do ácido 2,4,5-trimetoxi-cinâmico em 2,4,5-trimetoxi-estireno enquanto que o 2,4,5-trimetoxi-estireno foi dimerizado em pellucidina A através da reação de cicloadição [2+2] sensibilizada pela presença da 5,6,7-trimetoxi-flavona (18,45%), tal qual a benzofenona (11,15%). Assim, sugere-se a sequência L-fenilalanina, ácido cinâmico, ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno e pellucidina A, sendo a última etapa através de mecanismo fotoquímico tendo como sensibilizador a 5,6,7-trimetoxi-flavona. / Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) is an herbaceous plant that is widespread in the tropics and have several biological properties. Previous reports described the presence of pellucidin A, a rare dinorlignan having the unique cyclobutane moiety, that was supposedly formed by oxidative coupling of the precursor 2,4,5-trihydroxy-styrene followed by methylations steps. In this study, a comprehensive phytochemical study resulted in the description of 2,4,5-trimethoxy-cinnamic acid, 5,6,7-trimethoxy-flavone, 2,4,5-trimethoxy-styrene, 2,4,5-trimethoxy-benzaldehyde, dillapiol and sesamin in addition to pellucidin A. Studies of the dynamic aspects involved in the formation of pellucidin A included changes during ontogeny and responses to different treatments such as drought stress, herbivory, jasmonic acid and UV360 light. The treatment with jasmonic acid resulted in a significant increase in dillapiol whereas treatment under UV360 light resulted in an increase in production of pellucidin A, suggesting that a cycloaddition [2+2] mechanism is involved in its formation. The in vivo administration of different precursors to plants of P. pellucida revealed that L-[2-13C]-phenylalanine (0.75%), [8-13C]-cinnamic acid (1.32%), [8-13C]-ferulic acid (0.51%) 2,4,5-trimethoxy-[8-13C]-cinnamic acid (7.9%) and 2,4,5-trimethoxy-[8-13C]-styrene (13.3%) were incorporated into pellucidin A. The enzymatic conversion assays indicated decarboxylation of 2,4,5-trimethoxy-cinnamic acid into 2,4,5-trimethoxy-styrene while the 2,4,5-trimethoxy-styrene was dimerized in the pellucidin A by cycloaddition reaction [2+2] sensitized by 5,6,7-trimethoxy-flavone (18.45%), as well as by benzophenone (11.15%). Thus, we suggest the sequence L-phenylalanine, cinnamic acid, 2,4,5-trimethoxy-cinnamic acid, 2,4,5-trimethoxy-styrene and pellucidin A, the last step being carried out by a photochemical mechanism having 5,6,7- trimethoxy-flavone as a sensitizer.

Nas últimas décadas houve um grande avanço em pesquisas no desenvolvimento de medicamentos à base de plantas medicinais. Entretanto, constata-se que há pouca preocupação sobre as concentrações de elementos estáveis, bem como as concentrações de atividade de radionuclídeos naturais das séries do 238U e 232Th em plantas com esta finalidade no Brasil. A presença de minerais e radionuclídeos em plantas constitui um meio de migração destes elementos para o organismo humano, através da ingestão de chás ou medicamentos feitos com ervas medicinais. Peperomia pellucida é conhecida popularmente por erva de jabuti e por suas propriedades medicinais, como cicatrizante, analgésica, bactericida e fungicida. As amostras de Peperomia pellucida e de solo analisadas neste trabalho foram coletadas no Jardim Botânico do Rio de Janeiro e em suas proximidades. A concentração elementar de As, Ba, Br, Ce, Cl, Co, Cr, Cs, Eu, Fe, Hf, K, La, Lu, Mg, Mn, Na, Nd, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, Yb e Zn foi determinada nas folhas, partes aéreas, compreendendo folhas e caule, e raiz de Peperomia pellucida, nos extratos obtidos por maceração e infusão e no solo pela técnica da Análise por Ativação Neutrônica Instrumental (AANI). O método analítico utilizado para determinar os elementos Cd, Hg e Pb foi a Espectrometria por Absorção Atômica e as atividades de 226Ra, 228Ra e 210Pb foram determinadas pela contagem alfa e beta total após separação radioquímica. A qualidade dos resultados obtidos foi verificada pela análise dos materiais de referência certificados IAEA-336 Lichen, IAEA-Soil-7, IAEA-326 Radionuclides in soil, NIST 1515 Apples Leaves e NIST 1542 Peach Leaves. Os resultados obtidos nas análises apresentaram boa precisão e exatidão. O óleo essencial e os extratos etanólico e hexânico obtidos das partes aéreas de P. pellucida foram testados in vitro, em placas de Petri, para verificar a atividade antifúngica diante ao fungo Aspergillus flavus. A atividade antifúngica baseou-se na zona de inibição e nos valores de CI50 frente ao patógeno. Também foi determinada a composição química do óleo essencial pela Cromatografia Gasosa acoplada ao Espectrômetro de Massa (GC-EM). / In recent decades, there has been a great advancement in research in developing drugs from plants. Nevertheless, little significant care exists in the literature about the stable concentration of elements, as well as the activity concentration of 238U and 232Th decay products in plants used for this purpose in Brazil. The presence of stable elements and radionuclides in plants constitutes the pathway for their migration to humans, via uptake of tea or remedies made with medicinal herbs. Peperomia pellucida, whose popular name is erva de jabuti, is a plant known by its medicinal usages, such as healing and analgesic properties plus antibacterial and antifungal activities against food fungi. In this study, the elemental composition of Peperomia pellucida and surrounding soil samples collected in the Botanical Garden in Rio de Janeiro was determined; the elemental concentration in the alcoholic extract and infusion processes of dry plants was also determined. The elemental concentration of As, Ba, Br, Ce, Co, Cr, Cs, Eu, Fe, Hf, K, La, Lu, Na, Nd, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, Yb and Zn was determined in the leaves, aerial parts, including leaves, stems and roots of Peperomia pellucida, in their extracts obtained by maceration and infusions and in the surrounding soil by the Instrumental Neutron Activation Analysis (INAA). The analytical methodology used to determine the elements Cd, Hg and Pb was the Atomic Absorption Spectrometry (AAS); the activities of 226Ra, 228Ra and 210Pb were carried out by gross alpha and beta counting, after radiochemical separation. The quality of the obtained results was assured by the analyses of the certified reference materials IAEA-336 Lichen, IAEA-Soil-7, IAEA-326 Radionuclides in soil, NIST 1515 Apple Leaves and NIST 1542 Peach Leaves. The relative standard deviations and the relative errors obtained in these analyses indicated good precision and accuracy of the results. The essential oil, ethanolic and hexane extracts of Peperomia pellucida were tested for antifungal activity against Aspergillus flavus in vitro on Petri plates. The antifungal activity was based on the inhibition zone and IC50 values against the pathogen on Petri plates assays. Also, the essential oil chemical composition was determined by GC-MS.

Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) é uma herbácea amplamente encontrada nos trópicos e que possui diversas propriedades biológicas. Seus estudos fitoquímicos haviam demonstrado a presença da pellucidina A, uma rara dinorlignana ciclobutânica, que seria formada por acoplamento oxidativo de 2,4,5- triidroxi-estireno seguido de metilações. Nesse trabalho, foram caracterizados o ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno, 2,4,5-trimetoxi-benzaldeído, dilapiol, 5,6,7-trimetoxi-flavona, sesamina, além da pellucidina A. Estudos de aspectos dinâmicos envolvidos na formação da pellucidina A incluíram a ontogenia e respostas à diferentes tratamentos como estresse hídrico, predação por herbívoros, ácido jasmônico e luz UV360. O tratamento com ácido jasmônico resultou num significativo incremento do dilapiol enquanto que, o tratamento sob luz UV360 resultou no aumento na produção da pellucidina A sugerindo um mecanismo de cicloadição [2+2] para sua biossíntese. A administração de diferentes precursores in vivo revelou que a L-[2-13C]- fenilalanina (0,75%), ácido [8-13C]-cinâmico (1,32%), ácido [8-13C]-ferúlico (0,51%), ácido 2,4,5-trimetoxi-[8-13C]-cinâmico (7,9%) e o 2,4,5-trimetoxi-estireno (13,3%) foram incorporados à pellucidina A. Ensaios de conversão enzimática indicaram a descarboxilação do ácido 2,4,5-trimetoxi-cinâmico em 2,4,5-trimetoxi-estireno enquanto que o 2,4,5-trimetoxi-estireno foi dimerizado em pellucidina A através da reação de cicloadição [2+2] sensibilizada pela presença da 5,6,7-trimetoxi-flavona (18,45%), tal qual a benzofenona (11,15%). Assim, sugere-se a sequência L-fenilalanina, ácido cinâmico, ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno e pellucidina A, sendo a última etapa através de mecanismo fotoquímico tendo como sensibilizador a 5,6,7-trimetoxi-flavona. / Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) is an herbaceous plant that is widespread in the tropics and have several biological properties. Previous reports described the presence of pellucidin A, a rare dinorlignan having the unique cyclobutane moiety, that was supposedly formed by oxidative coupling of the precursor 2,4,5-trihydroxy-styrene followed by methylations steps. In this study, a comprehensive phytochemical study resulted in the description of 2,4,5-trimethoxy-cinnamic acid, 5,6,7-trimethoxy-flavone, 2,4,5-trimethoxy-styrene, 2,4,5-trimethoxy-benzaldehyde, dillapiol and sesamin in addition to pellucidin A. Studies of the dynamic aspects involved in the formation of pellucidin A included changes during ontogeny and responses to different treatments such as drought stress, herbivory, jasmonic acid and UV360 light. The treatment with jasmonic acid resulted in a significant increase in dillapiol whereas treatment under UV360 light resulted in an increase in production of pellucidin A, suggesting that a cycloaddition [2+2] mechanism is involved in its formation. The in vivo administration of different precursors to plants of P. pellucida revealed that L-[2-13C]-phenylalanine (0.75%), [8-13C]-cinnamic acid (1.32%), [8-13C]-ferulic acid (0.51%) 2,4,5-trimethoxy-[8-13C]-cinnamic acid (7.9%) and 2,4,5-trimethoxy-[8-13C]-styrene (13.3%) were incorporated into pellucidin A. The enzymatic conversion assays indicated decarboxylation of 2,4,5-trimethoxy-cinnamic acid into 2,4,5-trimethoxy-styrene while the 2,4,5-trimethoxy-styrene was dimerized in the pellucidin A by cycloaddition reaction [2+2] sensitized by 5,6,7-trimethoxy-flavone (18.45%), as well as by benzophenone (11.15%). Thus, we suggest the sequence L-phenylalanine, cinnamic acid, 2,4,5-trimethoxy-cinnamic acid, 2,4,5-trimethoxy-styrene and pellucidin A, the last step being carried out by a photochemical mechanism having 5,6,7- trimethoxy-flavone as a sensitizer.

Nas últimas décadas houve um grande avanço em pesquisas no desenvolvimento de medicamentos à base de plantas medicinais. Entretanto, constata-se que há pouca preocupação sobre as concentrações de elementos estáveis, bem como as concentrações de atividade de radionuclídeos naturais das séries do 238U e 232Th em plantas com esta finalidade no Brasil. A presença de minerais e radionuclídeos em plantas constitui um meio de migração destes elementos para o organismo humano, através da ingestão de chás ou medicamentos feitos com ervas medicinais. Peperomia pellucida é conhecida popularmente por erva de jabuti e por suas propriedades medicinais, como cicatrizante, analgésica, bactericida e fungicida. As amostras de Peperomia pellucida e de solo analisadas neste trabalho foram coletadas no Jardim Botânico do Rio de Janeiro e em suas proximidades. A concentração elementar de As, Ba, Br, Ce, Cl, Co, Cr, Cs, Eu, Fe, Hf, K, La, Lu, Mg, Mn, Na, Nd, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, Yb e Zn foi determinada nas folhas, partes aéreas, compreendendo folhas e caule, e raiz de Peperomia pellucida, nos extratos obtidos por maceração e infusão e no solo pela técnica da Análise por Ativação Neutrônica Instrumental (AANI). O método analítico utilizado para determinar os elementos Cd, Hg e Pb foi a Espectrometria por Absorção Atômica e as atividades de 226Ra, 228Ra e 210Pb foram determinadas pela contagem alfa e beta total após separação radioquímica. A qualidade dos resultados obtidos foi verificada pela análise dos materiais de referência certificados IAEA-336 Lichen, IAEA-Soil-7, IAEA-326 Radionuclides in soil, NIST 1515 Apples Leaves e NIST 1542 Peach Leaves. Os resultados obtidos nas análises apresentaram boa precisão e exatidão. O óleo essencial e os extratos etanólico e hexânico obtidos das partes aéreas de P. pellucida foram testados in vitro, em placas de Petri, para verificar a atividade antifúngica diante ao fungo Aspergillus flavus. A atividade antifúngica baseou-se na zona de inibição e nos valores de CI50 frente ao patógeno. Também foi determinada a composição química do óleo essencial pela Cromatografia Gasosa acoplada ao Espectrômetro de Massa (GC-EM). / In recent decades, there has been a great advancement in research in developing drugs from plants. Nevertheless, little significant care exists in the literature about the stable concentration of elements, as well as the activity concentration of 238U and 232Th decay products in plants used for this purpose in Brazil. The presence of stable elements and radionuclides in plants constitutes the pathway for their migration to humans, via uptake of tea or remedies made with medicinal herbs. Peperomia pellucida, whose popular name is erva de jabuti, is a plant known by its medicinal usages, such as healing and analgesic properties plus antibacterial and antifungal activities against food fungi. In this study, the elemental composition of Peperomia pellucida and surrounding soil samples collected in the Botanical Garden in Rio de Janeiro was determined; the elemental concentration in the alcoholic extract and infusion processes of dry plants was also determined. The elemental concentration of As, Ba, Br, Ce, Co, Cr, Cs, Eu, Fe, Hf, K, La, Lu, Na, Nd, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, Yb and Zn was determined in the leaves, aerial parts, including leaves, stems and roots of Peperomia pellucida, in their extracts obtained by maceration and infusions and in the surrounding soil by the Instrumental Neutron Activation Analysis (INAA). The analytical methodology used to determine the elements Cd, Hg and Pb was the Atomic Absorption Spectrometry (AAS); the activities of 226Ra, 228Ra and 210Pb were carried out by gross alpha and beta counting, after radiochemical separation. The quality of the obtained results was assured by the analyses of the certified reference materials IAEA-336 Lichen, IAEA-Soil-7, IAEA-326 Radionuclides in soil, NIST 1515 Apple Leaves and NIST 1542 Peach Leaves. The relative standard deviations and the relative errors obtained in these analyses indicated good precision and accuracy of the results. The essential oil, ethanolic and hexane extracts of Peperomia pellucida were tested for antifungal activity against Aspergillus flavus in vitro on Petri plates. The antifungal activity was based on the inhibition zone and IC50 values against the pathogen on Petri plates assays. Also, the essential oil chemical composition was determined by GC-MS.

Die immunologische Kontrazeption mittels Zona pellucida (ZP) Proteinen gilt als vielversprechender Ansatz für die Reproduktionskontrolle verwilderter Haus- und Wildtierbestände. Da die Applikation von nativer ZP mit Nebenwirkungen verbunden ist, wird die Verwendung einzelner ZP Peptide als Bestandteil kontrazeptiver Vakzine als besonders aussichtsreich erachtet. Das Prinzip dieser nebenwirkungsfreien ZP Immunisierung ist die gezielte Trennung der Entzündungsreaktionen auslösenden T-Zell-Epitope der ZP von den kontrazeptiv wirkenden B-Zell-Epitopen.<br> Niedermolekulare synthetische oder rekombinante Peptide allein sind gering immunogen und können somit keine ausreichende Immunantwort induzieren. Die Verwendung von Peptiden für die immunologische Kontrazeption erfordert daher ein &bdquo;Vakzin-Design&ldquo;, d. h. die gezielte Kombination der Peptide mit immunstimulierenden Substanzen (Liposomen, Carrierproteinen, Adjuvantien). Zielstellung der vorliegenden Arbeit war die Untersuchung des Potentials synthetischer Peptide für die Immunokontrazeption von verwilderten Hauskatzen (Felis catus). Dazu wurden zunächst relevante B-Zell-Epitope des felinen Zona pellucida Proteins, ZPB2, identifiziert und synthetisiert. Zwei der synthetischen Peptide (P3, P6) wurden zur Herstellung von Antikörpern an BSA konjugiert und zusammen mit Freundschem Adjuvans in Ratten verimpft. Die kontrazeptive Relevanz beider Peptide sowie der Ratten Anti-Peptid Antiseren wurde im in vitro Befruchtungssystem der Hauskatze geprüft. Zur Untersuchung der Immunogenität der Peptide in der Zielspezies Hauskatze erfolgte die Entwicklung von Vakzin-Prototypen für die einmalige Applikation. Neben der Eruierung der Stärke und Dauer der Immunantwort wurde durch Verpaarung der Tiere auch das kontrazeptive Potential in vivo abgeschätzt. / Immunological contraception based on zona pellucida (ZP) proteins is regarded as a promising approach for the control of reproduction in feral domestic and wild animals. Because application of native ZP caused adverse reactions, utilization of single ZP peptides as elements of contraceptive vaccines were considered to bear good prospects. The principle of this ZP immunization is based upon a systematic separation of inflammation triggering T-cell epitopes from the contraceptive B-cell epitopes.<br> The present study evaluates the immunogenicity and contraceptive potential of synthetic feline ZPB2 peptides for immunocontraception in cats (Felis catus). First of all, relevant B-cell epitopes were identified and synthesized. In order to generate antipeptide antibodies two peptides (P3, P6) were chosen and coupled to BSA. Rats were immunized with the conjugation product combined with Freund&prime;s complete adjuvant.<br> The contraceptive efficacy of both peptides and of the anti-peptide antibodies generated were determined using in vitro fertilization of feline oocytes (IVF). To evaluate the peptides immunogenicity in the target species (cat), vaccine-prototypes were developed for a single application protocol. The strength and duration of the immune response was analyzed. Additionally, cats were mated to assess the contraceptive potential of the vaccines in vivo.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: CHAPTER 1 provides literature based background information to emphasize the use of a sequential, multistep diagnostic schedule for couples in an assisted reproductive program as well as the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the chapter includes evidence underlining the growing need for the implementation of the physiologically induced acrosome reaction as an important contribution to the assisted reproductive program. The zona pellucida-induced acrosome reaction (ZIAR), sperm-zona interaction as well as computer-assisted semen analyses were investigated. CHAPTER 2 provides detailed experimental protocols of the materials and methods used in the study. CHAPTERS 3-6 each represent a separate study that was prepared as a scientific paper and encompass the experimental research undertaken in the reproductive biology research laboratory at Tygerberg Hospital to address important aspects of human acrosome processes. In the first study, we aimed to evaluate the regulatory role of the Gi-like protein during the AR of normal sperm donors and the role of intact acrosomes during sperm-zona binding. It seems that pertussis toxin-sensitive Gi-like protein in human spermatozoa plays an important regulatory role in the ZIAR and this underlines the importance of intact acrosomes during sperm-zona binding. In the second study, we aimed to evaluate the relationship between the ZIAR and the percentage normal spermatozoa as well as the sperm-ZP binding potential among men referred for a routine semen analysis. ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of the andrology referrals revealed an impaired AR response to solubilized ZP. In the third study, we aimed to evaluate the possible relationships between the sperm morphology, the acrosome responsiveness to solubulized human ZP and the sperm-zona binding potential among consecutive andrology referrals and randomly selected IVF cases. ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test since the results predicted fertilization failure during IVF treatment. In the fourth study, we aimed to evaluate changes in the sperm motion characteristics and the occurrence of hyperactivated motility after exposure to ZP among andrology referrals. Solubilized human ZP induces hyperactivated motility among sperm populations that have been capacitated under laboratory conditions. Capacitated spermatozoa have an elevated percentage hyperactivated cells that correlate with the percentage normal spermatozoa in the ejaculate. CHAPTER 7, the general discussion, is brief and concise to avoid unnecessary repetition, underlines the validity of a sequential, multistep diagnostic approach and concludes with the recommendation that the ZIAR should form part of the diagnostic tools in the assisted reproductive program. / AFRIKAANSE OPSOMMING: HOOFSTUK 1 bestaan uit ’n omvattende agtergrondstudie wat bestaan uit die ontwikkeling van diagnostiese toetse, die hantering van die egpaar in die reproduktiewe ondersteunings-program asook op die kliniese belang van spermmorfologie. Die toenemende behoefte aan die implementering van die fisiologies-geinduseerde akrosoomreaksie, as ’n belangrike bydrae tot die reproduktiewe ondersteuningsprogram, word ook beklemtoon. Die zona pellucida geinduseerde akrosoomreaksie (ZIAR), sperm-zonabinding asook rekenaar-bemiddelde semenanalises is ondersoek. HOOFSTUK 2 dek gedetailleerde eksperimentele protokolle van die materiale en metodes wat in die studie gebruik is. HOOFSTUKKE 3-6 behandel die eksperimentele navorsing wat in die laboratorium van die reproduktiewe biologie-eenheid te Tygerberg hospitaal uitgevoer is en wat as ses afsonderlike wetenskaplike publikasies aangebied word. Die doel van die eerste studie was om die regulerende rol van Gi-proteiene tydens die AR van normale spermdonors asook die rol van intakte akrosome tydens sperm-zonabinding te evalueer. Dit kom voor asof Gj-protei'ene in spermatozoa ’n belangrike regulerende rol in die ZIAR speel. Dit beklemtoon die belangrikheid van intakte akrosome tydens sperm-zonabinding. Die doel van die tweede studie was om die verhouding tussen die ZIAR en die persentasie normale spermatozoa asook die sperm-zonabindingspotensiaal tussen mans wat vir ’n roetine semenanalise verwys is te evalueer. ZIAR-toetsing moet deel uitmaak van die tweede vlak van manlike fertiliteitsondersoeke, d.w.s. funksionele toetsing, aangesien 15% van die andrologie pasiente ’n verswakte AR respons tot opgeloste ZP openbaar. In die derde studie was die doel om die moontlike verhoudinge tussen sperm-morfologie, die ZIAR en die sperm-zonabindingspotensiaal onder opeenvolgende andrologie-pasiente asook lukraak geselekteerde IVB-pasiente te evalueer. Die ZIAR-resultate bied verdere informasie aangaande disfunksionele spermatozoa en kan gebruik word as ’n addisionele diagnostiese toets aangesien hierdie resultate mislukte bevrugting tydens IVB behandeling voorspel. Die vierde studie het ten doel gehad om veranderinge in spermmotiliteitseienskappe asook hiperaktiwiteit na die blootstelling aan opgeloste zona onder andrologiepasiente te evalueer. Daar is afgelei dat opgeloste menslike zona hiperaktiwiteit induseer in spermpopulasies wat onder gunstige laboratoriumkondisies gekapasiteer is. Die gekapasiteerde spermatozoa het ’n verhoogde persentasie gehiperaktiveerde spermatozoa getoon wat met die persentasie normale spermatozoa in die ejakulaat korreleer. In HOOFSTUK 7 word aangetoon dat dit noodsaaklik is om die diagnostiese skedule by die hantering van die onvrugbare egpaar te gebruik asook dat die ontwikkeling van die funksionele toestand belangrik is vir die bepaling van ZIAR.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb. 23, 2010 ). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dr. Zhen Chen, Dissertation Supervisor. Vita. Includes bibliographical references.

Polyspermic fertilization is one of major problems during in vitro fertilization of porcine oocytes. Zona pellucida is an extracellular matrix, that surrounds growing mammalian eggs and is responsible for polyspermy block after fertilization. Porcine zona pellucida is composed of three glycoproteins called ZP2, ZP3 and ZP4. This study is aimed to modifications of ZP3 protein of oocytes with different developmental competence. No significant differences were found in expression of ZP3 protein in oocytes with lower developmental competence (follicular size S), in compared to oocytes with higher developmental competence (follicular size M). However, it was found significant differences in acidification of ZP3 protein of immature oocytes removed from follicles of size S, in compared to oocytes removed from follicles of size M. Moreover, using Western blot analysis, it was detected band in area > 50 kDa, which could play an essential role during fertilization of oocytes.