Magister Scientiae (Medical Bioscience) - MSc(MBS) / A functional sperm is critical for successful fertilization in order to deliver an intact genome to the site of fertilization. It is often characterized by high motility and normal morphology. Moreover, sperm hyperactivated motility is imperative for both detachment from the oviductal wall and for penetration into the zona pellucida, subsequently resulting in fertilization. Several semen parameters such as volume, colour, sperm morphology and sperm concentration are used to clinically discriminate between fertile and sub-fertile males. Additionally, several sperm functional tests assess sperm function and a male’s fertility potential. A sperm feature that is not currently assessed clinically, but could possibly discriminate between fertility and infertility, is hyperactivation. The aim of this project was to investigate motility degrees (good, medium and poor) of sperm subpopulations and induce hyperactivation in each subpopulation, as well as to sperm in semen, by addition of caffeine and procaine. This was achieved by separating three sperm subpopulations from a semen sample using the Puresperm density gradient separating technique. Sperm subpopulations were exposed to 5mM caffeine and 2 mM procaine respectively for 15, 30, 60, 90 and 120 minutes. Sperm in semen was exposed to caffeine and procaine using a flush technique and analysed at 0, 5, 15, 30, 45 and 60 minutes. Sperm displaying hyperactivation was determined using cut-offs for curvilinear velocity, linearity and amplitude of lateral head displacement. The results indicate significant differences in overall percentage motility, sperm kinematic parameters and hyperactivation among the three subpopulations (p<0.05). Procaine and caffeine both induced hyperactivation in subpopulations, although the most pronounced effect of procaine was evident after 15-30 minutes compared to caffeine (60-90 minutes) in subpopulations. Maximum hyperactivation of sperm in semen was seen after 15- 30 minutes in both procaine and caffeine. Moreover, caffeine had significantly higher stimulating effect than procaine. The results suggest that the existence and distinct motility characteristics of subpopulations should be considered in future during clinical assessment of male fertility, especially when assessing hyperactivation. The immediate and higher stimulation response of sperm with the flush technique indicates that the technique may be an ideal sperm functional test compared to the separation technique. The separation technique may be used to categorize sperm subpopulation of a patient in terms of motility (high motile or low motile) and to stimulate such subpopulations with chemicals for use in assisted reproduction technologies.

<p>The increasing number of patients undergoing treatment with assisted reproductive techniques (ART) during the past years have led to the need of developing different methods for separation of spermatozoa that can be used for different fertilisation procedures and for freezing. Cryopreservation of spermatozoa includes preparation, freezing, storage and thawing.</p><p>In this study two different cryomedia (Cryo Protec I and Cryo Protec II) for human spermatozoa were compared. The main outcome was spermsurvival rate for spermatozoa after freezing. Sperm viability was assessed using the Hypo-osmotic swelling test which is based on osmolality.</p><p>A total of 86 samples of semen were used in this study (Cryo Protec I=38, Cryo Protec II=48). The survival rate between the two cryomedia did not differ much but Cryo Protectant I showed a small increase in survival for the spermatozoa after freezing. The Hypo-osmotic swelling test also showed similar values of viable spermatozoa for the two cryomedia both before and after freezing.</p>

Orientador: Frederico Ozanam Papa / Resumo: A recuperação de espermatozoides da cauda do epidídimo é uma das principais alternativas nos casos de óbito inesperado, eutanásia, processos obstrutivos ou castração terapêutica. Nessa técnica os espermatozoides não entram em contato com o plasma seminal, não incorporando seus constituintes, que interferem nos processos fisiológicos importantes para fertilização, como a ligação dos espermatozoides ao reservatório da tuba uterina, que aumenta a vida útil do espermatozoide e diminui as chances de poliespermia. Nesse sentido o objetivo do presente trabalho foi comparar a cinética e ligação da tuba uterina aos espermatozoides recuperados da cauda do epidídimo com e sem adição de plasma seminal. Utilizou-se 8 garanhões da raça Minihorse para o resgate de espermatozoides da cauda do epidídimo pela técnica de fluxo retrógrado. Após a colheita foi dividido em 4 grupos, LD apenas com diluente a base de leite desnatado, GO recebeu adição de diluente para congelação a base de gema de ovo, PSB plasma seminal de garanhão com alta fertilidade e capacidade de refrigeração, e PSR plasma seminal de garanhão com fertilidade e capacidade de refrigeração inferiores. Foi avaliado cinética, integridade de membrana espermática e a taxa de ligação à explantes da tuba uterina. Não apresentaram diferença na integridade da membrana nem na taxa de ligação, no entanto, observou-se diferença quanto a cinética espermática. Conclui-se que a adição de plasma seminal de diferentes garanhões interferiu, de for... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sperm retrieval from the tail of the epididymis is one of the main alternatives in cases of unexpected death, euthanasia, obstructive processes, therapeutic castration. In this technique spermatozoa do not come into contact with the seminal plasma, not incorporating their constituents, which interfere in the physiological processes important for fertilization, such as sperm binding to the uterine tube reservoir, which increases sperm life and decreases the chances of polyspermia. In this sense, the objective of the present study was to compare the kinetics and uterine tubal attachment to the spermatozoa recovered from the tail of the epididymis with and without addition of seminal plasma. Eight minihorse stallions were used to retrieve spermatozoa from the tail of the epididymis by the retrograde flow technique. After harvesting was divided into 4 groups, LD only with diluent the skim milk base, GO received addition of diluent for freezing the egg yolk, PSB seminal stallion plasma with high fertility and cooling capacity, and PSR seminal plasma fertility and lower cooling capacity. Kinetics, spermatic membrane integrity and the rate of binding to the explants of the uterine tube were evaluated. There was no difference in membrane integrity or binding rate, however, a difference was observed in spermatic kinetics. It was concluded that the addition of seminal plasma of different stallions interfered, differently in spermatozoa kinetics of the tail of the epididymis increased t... (Complete abstract click electronic access below) / Mestre

O desvio de segregação, processo biológico definido pela formação de progênie cujas proporções genotípicas diferem das predições mendelianas, pode apresentar diferentes causas: a segregação não aleatória dos cromossomos durante a meiose, a seleção gamética e a viabilidade pós-zigótica diferencial. A fim de investigar a distorção de segregação em humanos, estudamos uma família com transmissão preferencial paterna de uma alteração patogênica no gene CAPN3(CAPN3 c.759_761del), previamente associada à distrofia muscular de cinturas do tipo 2A (LGMD2A). Inicialmente, definimos uma região com 2,1 Mb de extensão, presente na maior parte dos indivíduos da família. O sequenciamento direto de 14 genes contidos nesse intervalo não revelou variantes raras que pudessem estar diretamente associadas à vantagem da subpopulação de gametas portadores da alteração em CAPN3. Entretanto, a presença de variantes comuns distribuídas em cinco genes da região pode indicar que a distorção de segregação observada é consequência da interação, em nível proteico, de pequenas alterações genômicas, resultando no desempenho diferencial de tais células gaméticas. Além disso, utilizamos outra abordagem: espermatozoides de indivíduos saudáveis foram selecionados segundo sua motilidade e as frequências alélicas de cada loco em heterozigose foram comparadas entre células lentas e rápidas, a fim de identificarmos regiões suscetíveis ao desvio de segregação. Alguns resultados estatisticamente significantes foram obtidos para regiões que contêm genes envolvidos na reação acrossômica e na motilidade espermática, como PDE1C e PDE4D. Isso nos sugere que tais processos constituem alvos da seleção, o que pode culminar em formação de progênie com proporções que fogem às predições mendelianas. Esses achados são importantes porque possibilitam a ampliação de nossos conhecimentos acerca do desvio de segregação e seu impacto na espécie humana, considerando ainda possíveis efeitos na fertilidade masculina / The transmission ratio distortion (TRD), a biological process defined as a deviation from Mendelian predictions, is caused by different mechanisms: nonrandom segregation of chromosomes during meiosis or gametic and postzygotic viability selection. In order to investigate TRD in humans, we have studied a family with an overtransmitted pathogenic deletion in the CAPN3 gene (CAPN3 c.759_761del), previously associated to limb-girdle muscular dystrophy type 2A (LGMD2A). Initially, it was possible to define a 2.1 Mb region which was present in most of the individuals. The direct sequencing of 14 genes contained in this interval did not reveal rare variants that could be responsible for the advantage of gametes carrying the CAPN3 mutation. Nevertheless, common variants identified in five genes could indicate that the observed transmission distortion is caused by the interaction among proteins with small dissimilarities, resulting in differential performance of these gametic cells. Subsequently we developed another approach: spermatozoa from healthy individuals were sorted according to their motility and allelic frequencies at each locus in heterozygous state were compared in order to identify genomic regions susceptible to TRD. Surprisingly, some statistically significant results were achieved for regions containing genes involved with acrosome reaction and sperm motility, as PDE1 and PDE4, suggesting these processes are targets of the selection that ends in offspring with proportions skewed away from the Mendelian predictions. These results are important for enlarge our knowledge about TRD and its impact in humans, considering possible effects in male fertility

The increasing number of patients undergoing treatment with assisted reproductive techniques (ART) during the past years have led to the need of developing different methods for separation of spermatozoa that can be used for different fertilisation procedures and for freezing. Cryopreservation of spermatozoa includes preparation, freezing, storage and thawing. In this study two different cryomedia (Cryo Protec I and Cryo Protec II) for human spermatozoa were compared. The main outcome was spermsurvival rate for spermatozoa after freezing. Sperm viability was assessed using the Hypo-osmotic swelling test which is based on osmolality. A total of 86 samples of semen were used in this study (Cryo Protec I=38, Cryo Protec II=48). The survival rate between the two cryomedia did not differ much but Cryo Protectant I showed a small increase in survival for the spermatozoa after freezing. The Hypo-osmotic swelling test also showed similar values of viable spermatozoa for the two cryomedia both before and after freezing.

O desvio de segregação, processo biológico definido pela formação de progênie cujas proporções genotípicas diferem das predições mendelianas, pode apresentar diferentes causas: a segregação não aleatória dos cromossomos durante a meiose, a seleção gamética e a viabilidade pós-zigótica diferencial. A fim de investigar a distorção de segregação em humanos, estudamos uma família com transmissão preferencial paterna de uma alteração patogênica no gene CAPN3(CAPN3 c.759_761del), previamente associada à distrofia muscular de cinturas do tipo 2A (LGMD2A). Inicialmente, definimos uma região com 2,1 Mb de extensão, presente na maior parte dos indivíduos da família. O sequenciamento direto de 14 genes contidos nesse intervalo não revelou variantes raras que pudessem estar diretamente associadas à vantagem da subpopulação de gametas portadores da alteração em CAPN3. Entretanto, a presença de variantes comuns distribuídas em cinco genes da região pode indicar que a distorção de segregação observada é consequência da interação, em nível proteico, de pequenas alterações genômicas, resultando no desempenho diferencial de tais células gaméticas. Além disso, utilizamos outra abordagem: espermatozoides de indivíduos saudáveis foram selecionados segundo sua motilidade e as frequências alélicas de cada loco em heterozigose foram comparadas entre células lentas e rápidas, a fim de identificarmos regiões suscetíveis ao desvio de segregação. Alguns resultados estatisticamente significantes foram obtidos para regiões que contêm genes envolvidos na reação acrossômica e na motilidade espermática, como PDE1C e PDE4D. Isso nos sugere que tais processos constituem alvos da seleção, o que pode culminar em formação de progênie com proporções que fogem às predições mendelianas. Esses achados são importantes porque possibilitam a ampliação de nossos conhecimentos acerca do desvio de segregação e seu impacto na espécie humana, considerando ainda possíveis efeitos na fertilidade masculina / The transmission ratio distortion (TRD), a biological process defined as a deviation from Mendelian predictions, is caused by different mechanisms: nonrandom segregation of chromosomes during meiosis or gametic and postzygotic viability selection. In order to investigate TRD in humans, we have studied a family with an overtransmitted pathogenic deletion in the CAPN3 gene (CAPN3 c.759_761del), previously associated to limb-girdle muscular dystrophy type 2A (LGMD2A). Initially, it was possible to define a 2.1 Mb region which was present in most of the individuals. The direct sequencing of 14 genes contained in this interval did not reveal rare variants that could be responsible for the advantage of gametes carrying the CAPN3 mutation. Nevertheless, common variants identified in five genes could indicate that the observed transmission distortion is caused by the interaction among proteins with small dissimilarities, resulting in differential performance of these gametic cells. Subsequently we developed another approach: spermatozoa from healthy individuals were sorted according to their motility and allelic frequencies at each locus in heterozygous state were compared in order to identify genomic regions susceptible to TRD. Surprisingly, some statistically significant results were achieved for regions containing genes involved with acrosome reaction and sperm motility, as PDE1 and PDE4, suggesting these processes are targets of the selection that ends in offspring with proportions skewed away from the Mendelian predictions. These results are important for enlarge our knowledge about TRD and its impact in humans, considering possible effects in male fertility

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Apis mellifera (honey bees) are essential pollinators and thus considered a keystone species. Reproductive success is therefore important to the survival of the species, with drone sperm quality playing a vital role in fertilization success and colony health. Drone sperm quality is determined by multiple aspects including both structural and functional parameters. Particularly, motility, a functional parameter of sperm, plays an important role in reproductive success by determining the ability of sperm to move in the reproductive tract of the queen and to participate in fertilization. However, for the South African honey bee subspecies, the Cape honey bee (Apis mellifera capensis Escholtz), data on sperm quality parameters, are lacking. / 2023

First, a preliminary study was conducted looking for the optimum sperm concentration to be used for analysis with the Computer-Assisted Sperm Analysis (CASA). Results showed that 75x106 sperm cells/mL is the optimum one. Then, the actions of relaxin on sperm motility were evaluated by determining the effect of relaxin on full motility characteristics of spermatozoa during storage, using CASA; then identifying the relaxin receptors on spermatozoa, and finally establishing actions of relaxin in intraspermatic cAMP content. Motile spermatozoa were selected through percoll gradient and incubated for 1 hour with 4 relaxin concentrations at 37°C, during four days. Relaxin affected sperm motility (P<0.05). This action appears associated with the presence of relaxin receptors RXFP1 and RXFP2 that were found in spermatozoa. However, the cAMP levels were not affected by relaxin (P<0.05). This study indicates a beneficial action of relaxin on sperm motility; however, its mechanism of action requires further research.

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