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

Thesis (M.Sc. in Physiology) -- Faculty of Graduate Studies, Mahidol University, 1979. / Financial support by National Research Council.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 85-97).

The process of sperm capacitation and acrosome reaction (AR) are highly dynamic processes essential for the fertilization, including cytoskeleton proteins in the sperm head. The study of the distribution of actin, spectrin and α-tubulin in the head of representative mammalian sperm indicates that before and after the AR there were changes in the distribution of the cytoskeleton structures in the sperm head of all representatives, mainly in the apical part of the acrosome, but also in the equatorial and postacrosome part of the sperm head. The particularly well-studied actin cytoskeleton plays an important role during the capacitation and before and after the AR. Environmental estrogens can interfere with the function of endogenous hormones in very low concentrations. They can interact with specific receptors affecting several signalling pathways leading to sperm capacitation and AR. The effect of 17β-estradiol, estrone, estriol and synthetic 17α-ethynylestradiol was evaluated by the ratio of tyrosine phosphorylation and the state of the acrosome during in vitro capacitation in the sperm head. This study has provided the evidence that estrogens significantly stimulate capacitation progress in a concentration-dependent manner. Estrogens decrease number of sperm after the induced AR too. The raising...

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O presente estudo visou investigar o efeito da refrigeração do sêmen da jaguatirica sobre o Índice de Motilidade Espermática [IME=(%M+MPx5)/2; %M = proporção de espermatozóides móveis; MP = motilidade progressiva], integridade acrossomal (IA) e capacitação espermática; assim como avaliar a eficácia da técnica FITC-PNA/IP na avaliação simultânea da viabilidade espermática (VE) e IA. Sete jaguatiricas foram eletroejaculadas, sendo utilizados apenas ejaculados (n=16) apresentando %M>=60% e MP>=3. Avaliou-se a IA por meio da Coloração Simples. Os ejaculados foram diluídos 1:1 na Variante do Diluente de PLatz e submetidos aos Protocolos de Transporte: Temperatura Ambiente e Refrigeração, - 0,23ºC/min, (Experimento 1); ou apenas Temperatura Ambiente (Experimentos 2 e 3). Após 2h, as alíquotas foram reaquecidas, reavaliando-se os parâmetros observados antes do transporte. Os espermatozóides foram lavados por centrifugação em meio F10 de Ham, ressuspensos nesse meio e processados conforme o experimento: (1) após pré-incubação (38ºC; 5%CO2) durante 0, 1, 2 e 4 horas, foram retiradas alíquotas a cada intervalo para serem incubadas (30 min) na ausência e na presença do cálcio ionóforo A23187 (Ca2+Ion) (1mM), avaliando-se IA e IME; (2) após pré-incubação por 0, 1 e 2h, foram incubadas alíquotas na ausência e presença de 1 e 2mM de Ca2+Ion, avaliado-se IA e IME; (3) pré-incubados por 9h, sendo retiradas alíquotas a cada hora, para as avaliações da IA e VE, (a) separadamente através da Coloração Simples e do IME, ou (b) simultaneamente através da técnica FITC-PNA/IP. A refrigeração causou declínio (p<0,02) da IA (71,0%) e IME (67,1), em comparação aos valores observados antes do transporte (88,5%; 85,4), enquanto a manutenção das amostras à temperatura ambiente não afetou (p>0,1) essas variáveis (84,8%; 76,4). Dentre as amostras refrigeradas, aquelas expostas ao Ca2+Ion sofreram redução (p<0,01) na IA (52,4%) frente ao controle (55,56%). Já nas amostras transportadas à temperatura ambiente, não foi observada diferença (p>0,1) entre os grupos com e sem ionóforo (64,41% vs. 63,87%). Quando analisados os tempos separadamente, o único tratamento em que houve efeito (p<0,05) do Ca2+Ion sobre a IA foi aquele refrigerado e pré-incubado por 2h. Foi verificada redução (p<0,05) nos valores de IME e IA devida à simples incubação, mesmo na ausência do Ca2+Ion. A concentração de 2µM dessa substância foi mais efetiva na indução da reação acrossômica que 1µM. Apesar dos fluorocromos FITC-PNA/IP terem se ligado aos espermatozóides, nas regiões esperadas, a proporção de células marcadas variou aleatoriamente durante pré-incubação, sem correlação (p>0,1) com IME. A IA avaliada pela Coloração Simples apresentou correlação positiva (r=0,77; p<0,0001) com IME, decrescendo (p<0,0001) durante pré-incubação. A refrigeração mostrou-se desvantajosa frente à manutenção do sêmen à temperatura ambiente, pois foi deletéria à função e às membranas dos espermatozóides. A refrigeração tornou-os capazes de responder ao estímulo do Ca2+Ion, característica observada nos espermatozóides capacitados. O ensaio de reação acrossômica induzida pelo Ca2+Ion deve ser aperfeiçoado para permitir avaliação acurada da capacitação espermática na jaguatirica. A Coloração Simples associada à avaliação do IME foi mais eficiente e menos laboriosa, frente á técnica FITC-PNA/IP, na avaliação da IA e VE. / This study aimed to investigate the effect of ocelot semen refrigeration on Sperm Motility Index [SMI=(%M+PMx5)/2; %M = proportion of motile spermatozoa ; PM = Progressive Motility], acrossomal integrity (AI) and sperm capacitation. Another objective was to evaluate the FITC-PNA/IP technique efficacy on evaluating simultaneously sperm viability (SV) and AI. Five ocelots, were electroejaculated, the semen was evaluated and only ejaculates (n=16) presenting %M>=60% and PM>=3 were used. Sperm AI was evaluated using Fast Green / Rose Bengal staining (FGRB). The ejaculates were diluted 1:1 in Platz Diluent Variant and subjected to the transportation protocols: Room Temperature and Cooling, -0.23ºC/min, (experiment 1); or only Room Temperature (experiments 2 and 3). After 2 hours, the aliquots were rewarmed and samples were taken to re-evaluate the parameters observed before the transport. The spermatozoa were washed in Ham’s F10 medium, ressuspended in fresh medium and processed differently, according the experiment: (1) after pre-incubation (38ºC; 5%CO2) during 0, 1, 2 and 4 hours, samples were taken at each time point to be incubated in the absence and presence of 1mM calcium ionophore A23187 (Ca2+Ion), SMI and AI were evaluated; (2) after pre-incubation during 0, 1 and 2h, aliquots were incubated in the absence and presence of 1 and 2 mM Ca2+Ion; SMI and AI were evaluated; (3) after pre-incubation during 9h, aliquots were taken every hour to compare the evaluation of SV and AI (a) separately by the FGRB staining and SMI or (b) simultaneously by the FITC-PNA / IP technique. Cooling caused decline (p<0.02) on AI (71.0%) and SMI (67.1), when compared to values observed before transportation (88.5%; 85.4). Maintenance at room temperature didn’t affect (p>0.1) these variables (84.8%; 76.4). Among cooled samples, spermatozoa exposed to Ca2+Ion showed smaller (P<0.01) AI value (52.4%) compared to the group incubated without that substance (55.56%). For samples transported at room temperature, it wasn’t observed difference (P>0.05) between the groups with and without ionophore (64.41% vs. 63.87%). When time intervals were analysed separately, the only treatment in which there was effect (p<0,05) of Ca2+Ion on AI was the group refrigerated and pre-incubated for 2h. There was a reduction (p<0,05) on SMI and AI due simply to incubation, even in the absence of Ca2+Ion. The 2µM concentration of this substance was more effective to induce acrosome reaction than 1µM. FITC-PNA and IP fluorocromes bound spermatozoa at the expected sites. However, proportion of marked cells varied randomly during pre-incubation, and didn’t correlate (p>0,1) with SMI. IA evaluated by FGRB staining showed positive correlation (r=0,77; p<0,0001) with SMI, decreasing (p<0,0001) during incubation. Cooling was disadvantageous compared to maintaining semen at room temperature, since it was deleterious to spermatozoa membranes and function, and made those cells capable to answer the Ca2+Ion challenge, a characteristic observed in capacitated spermatozoa. Ca2+Ion induced acrosome reaction assay must be improved to allow accurate evaluation of sperm capacitation on ocelots. FGRB staining associated to SMI evaluation was more efficient and easier to perform, than FITC-PNA/IP technique, for AI and SV investigation.

Fertilization is a process that generates the first cell of a new organism. In mammals, fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility. A comprehensive review on the perspectives of male reproduction, spermatogenesis, the structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2. Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47- treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific. The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed. In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms that govern sperm function in clinical cases of non-obstructive male infertility in the human.

O presente estudo visou investigar o efeito da refrigeração do sêmen da jaguatirica sobre o Índice de Motilidade Espermática [IME=(%M+MPx5)/2; %M = proporção de espermatozóides móveis; MP = motilidade progressiva], integridade acrossomal (IA) e capacitação espermática; assim como avaliar a eficácia da técnica FITC-PNA/IP na avaliação simultânea da viabilidade espermática (VE) e IA. Sete jaguatiricas foram eletroejaculadas, sendo utilizados apenas ejaculados (n=16) apresentando %M>=60% e MP>=3. Avaliou-se a IA por meio da Coloração Simples. Os ejaculados foram diluídos 1:1 na Variante do Diluente de PLatz e submetidos aos Protocolos de Transporte: Temperatura Ambiente e Refrigeração, - 0,23ºC/min, (Experimento 1); ou apenas Temperatura Ambiente (Experimentos 2 e 3). Após 2h, as alíquotas foram reaquecidas, reavaliando-se os parâmetros observados antes do transporte. Os espermatozóides foram lavados por centrifugação em meio F10 de Ham, ressuspensos nesse meio e processados conforme o experimento: (1) após pré-incubação (38ºC; 5%CO2) durante 0, 1, 2 e 4 horas, foram retiradas alíquotas a cada intervalo para serem incubadas (30 min) na ausência e na presença do cálcio ionóforo A23187 (Ca2+Ion) (1mM), avaliando-se IA e IME; (2) após pré-incubação por 0, 1 e 2h, foram incubadas alíquotas na ausência e presença de 1 e 2mM de Ca2+Ion, avaliado-se IA e IME; (3) pré-incubados por 9h, sendo retiradas alíquotas a cada hora, para as avaliações da IA e VE, (a) separadamente através da Coloração Simples e do IME, ou (b) simultaneamente através da técnica FITC-PNA/IP. A refrigeração causou declínio (p<0,02) da IA (71,0%) e IME (67,1), em comparação aos valores observados antes do transporte (88,5%; 85,4), enquanto a manutenção das amostras à temperatura ambiente não afetou (p>0,1) essas variáveis (84,8%; 76,4). Dentre as amostras refrigeradas, aquelas expostas ao Ca2+Ion sofreram redução (p<0,01) na IA (52,4%) frente ao controle (55,56%). Já nas amostras transportadas à temperatura ambiente, não foi observada diferença (p>0,1) entre os grupos com e sem ionóforo (64,41% vs. 63,87%). Quando analisados os tempos separadamente, o único tratamento em que houve efeito (p<0,05) do Ca2+Ion sobre a IA foi aquele refrigerado e pré-incubado por 2h. Foi verificada redução (p<0,05) nos valores de IME e IA devida à simples incubação, mesmo na ausência do Ca2+Ion. A concentração de 2µM dessa substância foi mais efetiva na indução da reação acrossômica que 1µM. Apesar dos fluorocromos FITC-PNA/IP terem se ligado aos espermatozóides, nas regiões esperadas, a proporção de células marcadas variou aleatoriamente durante pré-incubação, sem correlação (p>0,1) com IME. A IA avaliada pela Coloração Simples apresentou correlação positiva (r=0,77; p<0,0001) com IME, decrescendo (p<0,0001) durante pré-incubação. A refrigeração mostrou-se desvantajosa frente à manutenção do sêmen à temperatura ambiente, pois foi deletéria à função e às membranas dos espermatozóides. A refrigeração tornou-os capazes de responder ao estímulo do Ca2+Ion, característica observada nos espermatozóides capacitados. O ensaio de reação acrossômica induzida pelo Ca2+Ion deve ser aperfeiçoado para permitir avaliação acurada da capacitação espermática na jaguatirica. A Coloração Simples associada à avaliação do IME foi mais eficiente e menos laboriosa, frente á técnica FITC-PNA/IP, na avaliação da IA e VE. / This study aimed to investigate the effect of ocelot semen refrigeration on Sperm Motility Index [SMI=(%M+PMx5)/2; %M = proportion of motile spermatozoa ; PM = Progressive Motility], acrossomal integrity (AI) and sperm capacitation. Another objective was to evaluate the FITC-PNA/IP technique efficacy on evaluating simultaneously sperm viability (SV) and AI. Five ocelots, were electroejaculated, the semen was evaluated and only ejaculates (n=16) presenting %M>=60% and PM>=3 were used. Sperm AI was evaluated using Fast Green / Rose Bengal staining (FGRB). The ejaculates were diluted 1:1 in Platz Diluent Variant and subjected to the transportation protocols: Room Temperature and Cooling, -0.23ºC/min, (experiment 1); or only Room Temperature (experiments 2 and 3). After 2 hours, the aliquots were rewarmed and samples were taken to re-evaluate the parameters observed before the transport. The spermatozoa were washed in Ham’s F10 medium, ressuspended in fresh medium and processed differently, according the experiment: (1) after pre-incubation (38ºC; 5%CO2) during 0, 1, 2 and 4 hours, samples were taken at each time point to be incubated in the absence and presence of 1mM calcium ionophore A23187 (Ca2+Ion), SMI and AI were evaluated; (2) after pre-incubation during 0, 1 and 2h, aliquots were incubated in the absence and presence of 1 and 2 mM Ca2+Ion; SMI and AI were evaluated; (3) after pre-incubation during 9h, aliquots were taken every hour to compare the evaluation of SV and AI (a) separately by the FGRB staining and SMI or (b) simultaneously by the FITC-PNA / IP technique. Cooling caused decline (p<0.02) on AI (71.0%) and SMI (67.1), when compared to values observed before transportation (88.5%; 85.4). Maintenance at room temperature didn’t affect (p>0.1) these variables (84.8%; 76.4). Among cooled samples, spermatozoa exposed to Ca2+Ion showed smaller (P<0.01) AI value (52.4%) compared to the group incubated without that substance (55.56%). For samples transported at room temperature, it wasn’t observed difference (P>0.05) between the groups with and without ionophore (64.41% vs. 63.87%). When time intervals were analysed separately, the only treatment in which there was effect (p<0,05) of Ca2+Ion on AI was the group refrigerated and pre-incubated for 2h. There was a reduction (p<0,05) on SMI and AI due simply to incubation, even in the absence of Ca2+Ion. The 2µM concentration of this substance was more effective to induce acrosome reaction than 1µM. FITC-PNA and IP fluorocromes bound spermatozoa at the expected sites. However, proportion of marked cells varied randomly during pre-incubation, and didn’t correlate (p>0,1) with SMI. IA evaluated by FGRB staining showed positive correlation (r=0,77; p<0,0001) with SMI, decreasing (p<0,0001) during incubation. Cooling was disadvantageous compared to maintaining semen at room temperature, since it was deleterious to spermatozoa membranes and function, and made those cells capable to answer the Ca2+Ion challenge, a characteristic observed in capacitated spermatozoa. Ca2+Ion induced acrosome reaction assay must be improved to allow accurate evaluation of sperm capacitation on ocelots. FGRB staining associated to SMI evaluation was more efficient and easier to perform, than FITC-PNA/IP technique, for AI and SV investigation.

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.