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"April 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 158-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A criopreservação de sêmen humano tem sido amplamente utilizada como método de preservação da fertilidade. A fim de reduzir os danos causados pelo processo de congelamento, diversos estudos utilizaram substâncias antioxidantes e estimulantes, porém, sua eficácia, bem como seu papel no controle funcional dos espermatozoides, não está bem estabelecida na literatura. Assim, o objetivo do estudo foi avaliar o efeito da suplementação de melatonina e cafeína nas características estruturais e funcionais de espermatozoides pré-criopreservação e pós-descongelamento. Para isso, este estudo prospectivo utilizou 30 amostras seminais de pacientes entre 19 e 45 anos de idade da rotina do Laboratório Androscience entre maio de 2013 e maio de 2017. Todas as amostras foram classificadas como normozoospérmicas, segundo análise seminal inicial de acordo com os critérios da Organização Mundial da Saúde (OMS). Em seguida, as amostras foram criopreservadas com Human Tubal Fluid modificado sem suplemento ou com 2mM de melatonina. Após o descongelamento, as amostras foram analisadas ou suplementadas com 2 mM de cafeína, incubadas por 15 minutos. Ao final dos experimentos, obtivemos 4 grupos: Amostras pós-descongelamento sem suplementação (CONT), amostras suplementadas com cafeína (CAF), melatonina (MEL) e cafeína + melatonina (CM). Parâmetros seminais de contagem, motilidade e cinética, analisados pelos critérios da OMS e pelo software SCA®, a atividade mitocondrial, pela coloração por diaminobenzidina, avaliação da taxa de fragmentação de DNA, pelo método SCSA® e a dosagem dos níveis de radicais livres de oxigênio (ROS), pelo método de luminescência, foram realizados pré-criopreservação e pós-descongelamento com ou sem suplementação. Os dados foram analisados pelo teste T de Student pareado e pela análise de variâncias de uma via com medidas repetidas, seguida pelo pós-teste de Holm-Sidak, e adotado um alfa de 5%. Observamos redução significativa concentração espermática (p < 0,001), motilidade total (p < 0,001) e progressiva (p < 0,001), parâmetros cinéticos (p < 0,009) e atividade mitocondrial (p < 0,001) e aumento das taxas de fragmentação de DNA (p=0,046) e ROS (p=0,052) nas amostras CONT quando comparadas com as amostras a fresco. Quando suplementadas com cafeína e melatonina, observamos aumento da motilidade progressiva nos grupos CAF (p=0,005) e CM (p=0,048) e aumento da atividade mitocondrial no grupo CM (p < 0,05). Podemos concluir que a associação da suplementação com cafeína e melatonina nas amostras pré-criopreservação e pós-descongelamento demonstrou ser uma ferramenta importante para ser aplicada em amostras criopreservadas para atuarem como substâncias estimuladoras de motilidade espermática / Human semen cryopreservation has been widely used as a method of preserving fertility. In order to reduce the damages caused by the freezing process, several studies used antioxidant and stimulant substances, however, their efficiency as well as their role in the functional control of spermatozoa have not been well established in literature. Thus, the goal of this study was to investigate the effect of melatonin and caffeine supplementation on the structural and functional characteristics of pre-cryopreservation and post-thaw spermatozoa. For this, this prospective study used 30 seminal samples from patients aged from 19 to 45 years in the routine of the Androscience Laboratory between May 2013 and May 2017. All samples were classified as normozoospermic, according to the initial seminal analysis following criteria of the World Health Organization (WHO). Then, the samples were cryopreserved with modified Human Tubal Fluid without supplement or with 2 mM of melatonin. After thawing, the samples were analyzed or supplemented with 2 mM of caffeine, and incubated for 15 minutes. At the end of the experiments, we obtained 4 groups: Post-thaw samples without supplementation (CONT), samples supplemented with caffeine (CAF), melatonin (MEL) and caffeine + melatonin (CM). Seminal parameters for count, motility and kinetics, analyzed by WHO criteria and by the SCA® software, mitochondrial activity, by diaminobenzidine staining, evaluation of DNA fragmentation rate, by the SCSA® method, and dosage of radical oxygen species (ROS) levels, by the luminescence method, pre-cryopreservation and post-thaw were carried out with or without supplementation. The data were analyzed by the paired Student\'s t-test and by one-way analysis of variance with repeated measurements, followed by the Holm-Sidak post-test, adopting an alpha of 5%. We observed a significant reduction in sperm concentration (p < 0.001), total (p < 0.001) and progressive (p < 0.001) motility, kinetic parameters (p < 0.009) and mitochondrial activity (p < 0.001), and increased rates of DNA fragmentation (p=0.046) and ROS (p=0.052) production in the CONT samples when compared with fresh sample. When supplemented with caffeine and melatonin, we observed an increase in progressive motility in the CAF (p=0.005) and CM (p=0.048) groups, and an increase in mitochondrial activity in CM group (p < 0.05). We can conclude that that the combination of caffeine and melatonin supplementation in pre-cryopreservation and post-thaw samples proved to be an important tool to be applied in cryopreserved samples to act as substances that stimulate sperm motility

<p>Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. </p><p>The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. </p><p>Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.</p>

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

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

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