BACKGROUND:Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins.RESULTS:We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract.CONCLUSION:Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.

Post-copulatory sexual selection is a strong evolutionary force, affecting morphological and behavioural traits in males and females in species with polyandrous mating systems. Many insects are subject to sperm competition; sperm from rival males compete to fertilise ova. Since sperm are finite, males should allocate them economically, tailoring ejaculate allocation to suit the reproductive potential of individual matings. Theory suggests when sperm competition risk is high, males should increase sperm numbers to achieve greater reproductive success than their rivals, but evidence of this expected fitness consequence of ejaculate allocation is largely lacking. In this thesis, I use Callosobruchus maculatus beetles to investigate the causes of ejaculate allocation patterns, and to examine whether ejaculate allocation does affect male reproductive success. In Chapter 3, I investigate the effect of rival male presence on ejaculate size and find that, while males grouped with rivals as adults produce bigger ejaculates, their increased effort unexpectedly does not lead to increased reproductive success. In Chapter 4, I examine whether larval conditions also affect ejaculate size, and find that, contrary to sperm competition theory, males reared under dense conditions produce smaller ejaculates than those reared solitarily, and that male reproductive success is consequently elevated in males reared at low larval densities compared to those reared at high densities. In Chapter 5, I then demonstrate that ejaculates produced by low density males contain more sperm than ejaculates produced by high density males, suggesting males do not respond to sperm competition level represented by larval density, but instead suffer resource limitation when reared at high density. In Chapter 6, I investigate the effects of water provision on ejaculate size, and find that males given water produce larger ejaculates, and females given water receive smaller ejaculates. Finally, I link my findings with those of other studies, and suggest my most important result is that plasticity of ejaculate allocation cannot be assumed to be an adaptive behaviour; studies directly measuring the fitness effects of male ejaculate allocation are needed, even when observed patterns conform to theory.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This study aimed to evaluate rainy and dry season effect on plasma seminal composition and semen quality of 17 crossbred male goats raised in tropical region and to investigate if the biochemical composition of seminal plasma (SP) is correlated to semen quality in relation to the season of the year. Other objective of this experiment was to evaluate if the phospholipase A2 (PLA2) activity level in the SP affects the ejaculate and epididymal spermatozoa. This study was divided into three experiments: in the first phase, seventeen male goats were used and then eight animals with low or high PLA2 activity in SP were separated to the second and third phases. In the first experiment, the ejaculates were collected in artificial vagina, weekly, the SP was separated to evaluate the biochemical parameters and biweekly for quality semen determination. The sperm was diluted in coconut water added 2,5% of egg yolk (200 x 106 sperm/mL) and stored at 4 oC for 48 hours. The vigor (0-5) and motility (1-100%) were measured at 2, 24 and 48 hours after cooling by thermoresistance test (TRT). Slides of sperm were made during TRT and 200 cells were classified as normal or abnormal cellular morphology. The fructose, citric acid, total proteins, calcium, phosphorus, magnesium level and the PLA2 activity level in the SP were measured. In the second experiment, eight bucks were separated in two groups according to PLA2 activity level in SP, in group I (<6.7U/mL) and group II (> 11.0 U/mL). The semen was obtained by artificial vagina, diluted and separate in two samples: the first aliquot was the control treatment (nonwashed semen) and the second aliquot was centrifuged at 550 g for 10 minutes to remove the SP (washed semen). In the third experiment, the semen of the eight bucks was collected and the SP obtained by centrifugation. The SP of each buck was added to aliquots of epididymal spermatozoa pool collected from another male goat and a ninth aliquot was added only to diluent (control). After that, the final dilution (200 x 106 sperm/mL) was made in all samples. In the second and third experiments, after the treatment and final dilution, the semen samples were stored at 4 oC for 48 hours and an aliquot was incubated at 38 oC and evaluated to vigor and motility at 0, 2, 12, 24 and 48 hours of cooling. Slides of sperm were made during TRT and 200 cells were classified as normal or abnormal morphology. In all experiments, the semen quality decreased with time (p<0,001). In the first experiment, the semen obtained in the rainy season was better quality than semen collected in the dry season of the year. In addition, the fructose, citric acid, total proteins, phosphorus and magnesium levels in the SP were higher in rainy season, while the PLA2 activity was lower in this period. The fructose, citric acid and total proteins concentration showed positive correlations to vigor and motility in both seasons. The total sperm morphology alterations (TSMA) showed negative correlation with fructose level and positive correlations with citric acid, total proteins concentrations and PLA2 activity level. In the second experiment, the treatment (washed semen) improved the seminal quality, mainly in washed semen of the group I. The PLA2 activity level showed no effect on sperm performance in the ejaculate spermatozoa, except at 48h in washed semen and at 24h in the non-washed semen. Finally, in the third experiment, the SP addition on epididymal spermatozoa decreased the seminal parameters and this effect was higher in group II. However, the TSMA were lower when the PLA2 activity was higher. These results indicated that male goats raised in tropical region showed better seminal quality in the rainy season. In addition, the fructose, citric acid, total proteins, phosphorus and magnesium concentration levels in the SP were higher in rainy season, while the PLA2 activity was lower in this period. The correlations between the seminal and biochemical parameters in SP could indicate that the differences in the semen parameters in the rainy and dry season occurred due the biochemical parameter variations. The SP addition during the semen storage at 4 oC decreased the seminal quality. However the PLA2 activity level was not a determinant factor of the seminal quality in the ejaculate spermatozoa. Therefore, it cannot be used as an indicative of the spermatic quality of male goats raised in tropical region / Este trabalho teve por objetivo avaliar o efeito da Ãpoca chuvosa e seca do ano sobre a composiÃÃo do plasma seminal (PS) e os parÃmetros seminais de 17 caprinos sem padrÃo racial definido (SPRD), criados em clima tropical, alÃm de avaliar se a composiÃÃo bioquÃmica do PS apresenta correlaÃÃo com os parÃmetros seminais, em funÃÃo da Ãpoca do ano, assim como, investigar se o nÃvel de atividade da fosfolipase A2 (FLA2) no PS de caprinos pode ser utilizado como indicativo da qualidade espermÃtica e se esse nÃvel exerce influÃncia na preservaÃÃo dos espermatozÃides ejaculados e epididimÃrios quando conservados a 4 ÂC, por 48 horas. O estudo foi realizado em trÃs etapas: na primeira foram utilizados dezessete machos caprinos e a partir desta, oito bodes com diferentes nÃveis de atividade de FLA2 no PS foram selecionados para realizaÃÃo da segunda e terceira etapas. No primeiro experimento durante a Ãpoca chuvosa e seca, os ejaculados dos animais foram coletados semanalmente, em vagina artificial, para avaliaÃÃo bioquÃmica do PS e a cada 14 dias para determinaÃÃo dos parÃmetros seminais. O sÃmen foi diluÃdo em Ãgua de coco-2,5% gema (200 x 106 sptz/mL), resfriado a 4 ÂC por 48 horas e avaliado o vigor e a motilidade Ãs 2, 24 e 48 horas de resfriamento (T2, T24 e T48), pelo teste de termorresistÃncia lento (TTR). EsfregaÃos do sÃmen diluÃdo foram confeccionados durante o TTR e 200 cÃlulas foram avaliadas e classificadas morfologicamente em normais ou anormais. Determinaram-se ainda as concentraÃÃes de frutose, Ãcido cÃtrico, proteÃnas totais, Ca, P, MG e nÃvel de atividade da FLA2. No segundo experimento, oito caprinos divididos, em funÃÃo do nÃvel de atividade da FLA2 no PS em: grupo I (<6,70 U/mL) e o grupo II (>11,00 U/mL). O sÃmen foi coletado, diluÃdo e dividido em duas alÃquota : a primeira constituiu o tratamento controle (sÃmen nÃo lavado) e a segunda foi centrifugada para remoÃÃo do PS (sÃmen lavado). No terceiro experimento, o sÃmen dos oito animais foi coletado e centrifugado para recuperaÃÃo do PS e adicionados a alÃquotas de um Ãnico âpoolâ de espermatozÃides epididimÃrios obtidos de caprinos castrados cirurgicamente. Uma nona alÃquota foi adicionada apenas de diluidor (grupo controle). Em seguida, procedeu-se a diluiÃÃo final em todas as amostras utilizando o mesmo diluidor. No segundo e terceiro experimentos, apÃs a realizaÃÃo dos tratamentos e diluiÃÃo final, o sÃmen foi resfriado a 4 ÂC por atà 48 horas e amostra de cada tratamento foi avaliada pelo TTR Ãs 0 (fresco), 2, 12, 24 e 48 horas de resfriamento. EsfregaÃos do sÃmen foram confeccionados para avaliaÃÃo da morfologia espermÃtica. Em todos os experimentos, a qualidade espermÃtica diminuiu à medida que se prolongou o tempo de preservaÃÃo (p < 0,001). No experimento 1, o sÃmen apresentou qualidade superior na Ãpoca chuvosa e tambÃm as concentraÃÃes de frutose, Ãcido cÃtrico, proteÃnas totais, P e Mg foram maiores nesta Ãpoca. A FLA2 apresentou menor atividade na Ãpoca chuvosa (p < 0,05). A concentraÃÃo de frutose, Ãcido cÃtrico e proteÃnas totais mostraram correlaÃÃes positivas com o vigor e a motilidade e negativas com a TDM, nas duas Ãpocas. Em relaÃÃo Ãs alteraÃÃes morfolÃgicas totais (AMT), a concentraÃÃo de frutose mostrou correlaÃÃes negativas, enquanto as de Ãcido cÃtrico, proteÃnas totais e atividade da FLA2, positivas. Os nÃveis de fÃsforo no PS apresentaram correlaÃÃo com os parÃmetros seminais apenas na Ãpoca seca, sendo negativa para vigor, motilidade e AMT e positiva para TDM. A concentraÃÃo de Mg correlacionou-se positivamente com a motilidade na Ãpoca chuvosa e AMT na seca. No segundo experimento, o tratamento influenciou a qualidade seminal principalmente no grupo I, sendo o desempenho espermÃtico superior no sÃmen lavado. O nÃvel de atividade da FLA2 nÃo afetou a qualidade dos espermatozÃides ejaculados, exceto em T48 do sÃmen lavado e T24 do sÃmen nÃo lavado. Enquanto no terceiro experimento, a adiÃÃo do PS aos espermatozÃides epididimÃrios diminuiu a qualidade espermÃtica e este efeito foi mais pronunciado quando o nÃvel de atividade de FLA2 foi maior. Contudo, no que se refere e AMT, a adiÃÃo do PS foi benÃfica, reduzindo o nÃmero de alteraÃÃes. Os resultados do presente estudo indicam que caprinos criados em clima tropical apresentam melhor qualidade espermÃtica no perÃodo chuvoso, alÃm das concentraÃÃes de frutose, acido cÃtrico, proteÃnas totais, P e Mg serem mais elevados nesta Ãpoca, enquanto a atividade da FLA2 foi menor neste perÃodo. As correlaÃÃes entre os parÃmetros seminais e a composiÃÃo bioquÃmica do PS podem indicar que as diferenÃas detectadas quanto à qualidade espermÃtica entre as Ãpocas do ano deve-se justamente Ãs variaÃÃes na concentraÃÃo destes componentes. A presenÃa do PS durante a conservaÃÃo do sÃmen caprino a 4 ÂC reduziu a qualidade espermÃtica, contudo o nÃvel de atividade da FLA2 nÃo foi um fator determinante da qualidade seminal para os espermatozÃides ejaculados, por isso, nÃo deve ser utilizado como indicativo de qualidade espermÃtica de caprinos criados em regiÃo de clima tropical

Marine and freshwater environments support numerous species of teleost fish with a wide and diverse range of reproductive strategies. Despite the considerable interest in fish reproduction, our knowledge regarding ejaculate traits and factors affecting them is limited. Using computer-assisted sperm analysis (CASA) I measured ejaculate traits (sperm swimming speed, motility, path trajectory, longevity and concentration) from sexually mature chinook salmon (Oncorhynchus tshawytscha) activated in freshwater and ovarian fluid. I also looked at these ejaculate traits in relation to measures of male quality (body condition) and investment into reproduction (relative testes mass). Furthermore, I determined the chemical composition of seminal and ovarian fluid and looked at the effect these fluids have on sperm behaviour. A considerable amount of intraspecific variation existed in all ejaculate traits measured, and investment into reproduction (relative testes mass) was dependent on male body condition, as males in better condition had relatively larger testes. However, these males did not have superior quality ejaculates or ejaculates with a higher density of spermatozoa; hence the potential reproductive advantage of having larger relative testes in this species remains unknown and requires further investigation. In addition, a positive relation between sperm longevity and sperm swimming speed was observed defying the expected trade-off between ejaculate traits according to theory. There was also a weak negative trend in our data between body condition and sperm swimming speed, linearity, and longevity. All sperm traits measured were greatly enhanced when activated in a solution containing ovarian fluid (a viscous fluid which is excreted with the egg batch during spawning) from female chinook salmon. Interestingly, sperm swimming speed activated in fresh water only accounted for < 12% of the observed variation in mean sperm swimming speed in ovarian fluid. This result suggests the sperm traits measured in fresh water are not relevant to those same traits measured in ovarian fluid, so caution should be applied when comparing the potential for individual males to fertilize ova when sperm traits are activated in water, especially in studies of sperm competition in an externally fertilising species. Sperm competition between males is known to strongly influence sperm and ejaculate traits, but less is known about female sperm choice after copulation via a process called cryptic female choice (CFC). In CFC, females may have the ability to favour the sperm of one male over another and bias fertilisation accordingly. To test whether ovarian fluid could act as a mechanism of CFC in an externally fertilising fish species, I measured sperm traits from each male activated in the ovarian fluids from different females. I found that mean sperm swimming speed, longevity, and path trajectory differed significantly among males, but most importantly, the pattern of within-male variation in these traits also varied significantly among males in response to different females’ ovarian fluids. This result suggests that ovarian fluid may be a potential mechanism of CFC whereby females differentially enhance the swimming speed of sperm from different males. In addition, I found that sperm longevity was negatively correlated with variation in [Ca²⁺] and [Mg²⁺] concentration in the ovarian fluid, while percent motility increased with increasing concentration of [Mg²⁺]. These observations provide a possible chemical basis for cryptic female choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males and thus their fertilisation success. This finding is particularly exciting, as we may have uncovered a potential mechanism of CFC in an externally fertilising species, which is poorly understood. In addition, results from this study suggest new directions for genetic studies to provide direct evidence for CFC. For example, does sperm selection via ovarian fluid promote favoured genetic combinations that enhance male reproductive success?

One of the requirements for a successful insemination in horse breeding is the selection of quality stallions which produced ejaculate in enough quantities and of good quality. For this purpose were discovered methods of dealing with the evaluation of semen quality. In this dissertation was ejaculate evaluated by using an objective computerized method CASA, system SCA. Monitoring the quality of sperm was performed on 11 breeding stallions involved in artificial insemination fresh sperm in ZH Písek. 53 samples of ejaculate were evaluated. Volume, sperm concentration, total and progressive motility and sperm vitality were monitored. Volume of ejaculate was from 20 to 127.5 ml. Average values of sperm concentration for individual stallions were from 104.18 to 384.4 M/ml. Average values of total motility were moving by interval from 51.23 to 89.54 %. Average progressive motility was move from 12.99 to 47.12 %. At sperm vitality were discovered average results from 35.16 to 71.21 %. The average value of individual stallions was identical to the Decree of the Ministry of Agriculture, which sets minimum requirements for quality indicators for short-term preservation of semen. Also the quality of the semen of stallions was compared by age. The first group was made by stallions to 10 years old and the second group were older stallions. It was found that the younger stallions to 10 years had higher semen volume and greater sperm concentration. Stallions older over 10 years had higher levels of total and progressive motility and sperm vitality. Next ejaculate quality was evaluate by the time interval between samples. Regular taken from stallions is doing better ejaculate quality but the time interval between samples is important. The best values were found in samples with interval between the sampling 4 or more days. The worst quality of sperm was in the samples with the interval between 1-2 days.

O objetivo do presente estudo foi determinar a cinética e a integridade e funcionalidade da membrana plasmática após a seleção espermática com um filtro de membrana sintética em câmaras de PVC com dois diâmetros diferentes e dois tempos de filtração. Foram utilizados 12 ejaculados de três garanhões. Imediatamente após a coleta, o sêmen foi diluído com leite desnatado (T0) e analisado. Duas câmaras diferentes em PVC foram produzidas com dois joelhos conectados com um tubo dividido ao meio por um filtro de membrana sintética com poros de 5 μm. Uma câmara de 26 mm e a outra de 36 mm de diâmetro interno. Foi colocado leite desnatado a 37° C em um lado da câmara (A). No outro lado da câmara (B) depositou-se uma amostra do sêmen diluído, com um número conhecido de espermatozoides. Após 7 e 15 min, obteve-se uma amostra do lado "A" de cada câmara e calculou-se a concentração espermática e analisou-se o sêmen. A motilidade total, a motilidade progressiva e a integridade da membrana plasmática melhoraram (P <0,05) após a filtração em ambos os dispositivos e tempos de filtração. A concentração espermática foi menor (P <0,05) em ambas as câmaras nos dois tempos em relação ao T0. O dispositivo de filtração demonstra ser uma alternativa prática e fácil para a seleção espermática. A seleção usando as câmaras permite um aumento da cinética e da integridade da membrana e funcionalidade independente do tempo e do diâmetro do dispositivo. / The aim of the present study was to determine the kinetics and plasma membrane integrity and functionality after sperm selection with a synthetic membrane filter in PVC chambers with two different diameters and two filtrations times. A total of 12 ejaculates from three stallions was used. Immediately after collection semen was diluted with skim milk (T0) and analyzed. Two different chambers in PVC were made with two elbows connected with a pipe divided in half by a 5 μm pore synthetic membrane filter. One chamber had an inner diameter of 26 mm and the other 36 mm. Skim milk at 37° C was placed in a side of the chamber (A). In the other side of the chamber (B) a sample of the extended semen, with known number of spermatozoa was deposited. After 7 and 15 min a sample was obtained from the ‘A’ side of each chamber and sperm concentration was calculated and semen analyzed. Total motility, progressive motility and plasma membrane integrity were improved (P<0.05) after filtration in both devices and filtration times. Concentration was lower (P<0.05) in both chambers at all times in relation to T0 semen. The filtration device demonstrates to be a practical and easy alternative for sperm selection. Selection using the chambers allows an increase in kinetics and membrane integrity and functionality independent of time and device diameter.

O objetivo do presente estudo foi determinar a cinética e a integridade e funcionalidade da membrana plasmática após a seleção espermática com um filtro de membrana sintética em câmaras de PVC com dois diâmetros diferentes e dois tempos de filtração. Foram utilizados 12 ejaculados de três garanhões. Imediatamente após a coleta, o sêmen foi diluído com leite desnatado (T0) e analisado. Duas câmaras diferentes em PVC foram produzidas com dois joelhos conectados com um tubo dividido ao meio por um filtro de membrana sintética com poros de 5 μm. Uma câmara de 26 mm e a outra de 36 mm de diâmetro interno. Foi colocado leite desnatado a 37° C em um lado da câmara (A). No outro lado da câmara (B) depositou-se uma amostra do sêmen diluído, com um número conhecido de espermatozoides. Após 7 e 15 min, obteve-se uma amostra do lado "A" de cada câmara e calculou-se a concentração espermática e analisou-se o sêmen. A motilidade total, a motilidade progressiva e a integridade da membrana plasmática melhoraram (P <0,05) após a filtração em ambos os dispositivos e tempos de filtração. A concentração espermática foi menor (P <0,05) em ambas as câmaras nos dois tempos em relação ao T0. O dispositivo de filtração demonstra ser uma alternativa prática e fácil para a seleção espermática. A seleção usando as câmaras permite um aumento da cinética e da integridade da membrana e funcionalidade independente do tempo e do diâmetro do dispositivo. / The aim of the present study was to determine the kinetics and plasma membrane integrity and functionality after sperm selection with a synthetic membrane filter in PVC chambers with two different diameters and two filtrations times. A total of 12 ejaculates from three stallions was used. Immediately after collection semen was diluted with skim milk (T0) and analyzed. Two different chambers in PVC were made with two elbows connected with a pipe divided in half by a 5 μm pore synthetic membrane filter. One chamber had an inner diameter of 26 mm and the other 36 mm. Skim milk at 37° C was placed in a side of the chamber (A). In the other side of the chamber (B) a sample of the extended semen, with known number of spermatozoa was deposited. After 7 and 15 min a sample was obtained from the ‘A’ side of each chamber and sperm concentration was calculated and semen analyzed. Total motility, progressive motility and plasma membrane integrity were improved (P<0.05) after filtration in both devices and filtration times. Concentration was lower (P<0.05) in both chambers at all times in relation to T0 semen. The filtration device demonstrates to be a practical and easy alternative for sperm selection. Selection using the chambers allows an increase in kinetics and membrane integrity and functionality independent of time and device diameter.

O objetivo do presente estudo foi determinar a cinética e a integridade e funcionalidade da membrana plasmática após a seleção espermática com um filtro de membrana sintética em câmaras de PVC com dois diâmetros diferentes e dois tempos de filtração. Foram utilizados 12 ejaculados de três garanhões. Imediatamente após a coleta, o sêmen foi diluído com leite desnatado (T0) e analisado. Duas câmaras diferentes em PVC foram produzidas com dois joelhos conectados com um tubo dividido ao meio por um filtro de membrana sintética com poros de 5 μm. Uma câmara de 26 mm e a outra de 36 mm de diâmetro interno. Foi colocado leite desnatado a 37° C em um lado da câmara (A). No outro lado da câmara (B) depositou-se uma amostra do sêmen diluído, com um número conhecido de espermatozoides. Após 7 e 15 min, obteve-se uma amostra do lado "A" de cada câmara e calculou-se a concentração espermática e analisou-se o sêmen. A motilidade total, a motilidade progressiva e a integridade da membrana plasmática melhoraram (P <0,05) após a filtração em ambos os dispositivos e tempos de filtração. A concentração espermática foi menor (P <0,05) em ambas as câmaras nos dois tempos em relação ao T0. O dispositivo de filtração demonstra ser uma alternativa prática e fácil para a seleção espermática. A seleção usando as câmaras permite um aumento da cinética e da integridade da membrana e funcionalidade independente do tempo e do diâmetro do dispositivo. / The aim of the present study was to determine the kinetics and plasma membrane integrity and functionality after sperm selection with a synthetic membrane filter in PVC chambers with two different diameters and two filtrations times. A total of 12 ejaculates from three stallions was used. Immediately after collection semen was diluted with skim milk (T0) and analyzed. Two different chambers in PVC were made with two elbows connected with a pipe divided in half by a 5 μm pore synthetic membrane filter. One chamber had an inner diameter of 26 mm and the other 36 mm. Skim milk at 37° C was placed in a side of the chamber (A). In the other side of the chamber (B) a sample of the extended semen, with known number of spermatozoa was deposited. After 7 and 15 min a sample was obtained from the ‘A’ side of each chamber and sperm concentration was calculated and semen analyzed. Total motility, progressive motility and plasma membrane integrity were improved (P<0.05) after filtration in both devices and filtration times. Concentration was lower (P<0.05) in both chambers at all times in relation to T0 semen. The filtration device demonstrates to be a practical and easy alternative for sperm selection. Selection using the chambers allows an increase in kinetics and membrane integrity and functionality independent of time and device diameter.

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.