Dissertations / Theses on the topic 'Sperm'

Human Cytomegalovirus (CMV) is a common herpesvirus found in 60% of the population. Normally, it poses no risk, however it can have consequences for unborn babies. This is of concern when donor sperm is used in assisted conception, as CMV is present in semen. The risk of transmission from a positive donor is unclear, as it is not known if sperm can act as a vector for transmission. Additionally, this raises questions about whether CMV might affect sperm function. The hypothesis for this study is that CMV will interact with human sperm and alter sperm function and that sperm will act as a vector for viral transmission. A survey was conducted to examine how fertility clinics were screening for CMV in sperm donors. This survey found that the majority of UK clinics are screening for CMV in sperm donors in the manner recommended by current guidelines but that the requirement to screen for CMV is causing problems in clinics with regards to sperm donor supply. Fortunately, this thesis has shown that sperm washing by density gradient centrifugation is mostly effective at removing CMV from semen samples infected in vitro, with CMV (AD169) grown in the laboratory, and in naturally infected samples. This presents a possible approach for alleviating some of the problems relating to CMV infection in sperm donors in UK fertility clinics. However, co-incubation with CMV has no effect on any of the sperm function parameters tested in this thesis, including, motility, viability, acrosome reaction, tyrosine phosphorylation and levels of DNA damage. In conclusion, this thesis has highlighted problems with the current approach to screening and managing CMV infection in sperm donors but has provided evidence to show that there could be a simple solution to the problem. No effect on sperm function was observed, but this does not rule out a direct interaction between CMV and sperm. Overall, this thesis shows that fertility clinics should be concerned about CMV infection in sperm donors, but that simple steps could be taken to alleviate the current problems clinics are experiencing.

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 
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 
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 
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 
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 
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. 
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.

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model. The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx). Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins. Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation. The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle. In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress.
published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy

Ca²⁺ signals from activated Ca²⁺ channels (CatSper) and mobilisation of Ca²⁺ stores regulate human sperm cell behaviour as they ascend the female tract. I investigated the effects on human sperm [Ca²⁺]i and behaviour of CatSper channel activation (alkaline pH and progesterone) and Ca²⁺-store mobilisation (4-aminopyridine, thimerosal) using a fluorescence plate reader and CASA. Extracellular alkalinisation raised pH¬i (pHi = 6.9 and 7.2 at pHo7.4 and 8.5 respectively), caused tonic elevation of [Ca²⁺]i, which was partially inhibited by CatSper block and increased the proportion of hyperactivated cells (from 1.8±0.5 to 10.5±1.6%; n=34, P=1x10⁻⁷). Progesterone elevated [Ca²⁺]i but caused negligible hyperactivation. Co-application of these stimuli revealed little, if any, synergistic interaction. Ca²⁺-store mobilisation (4-aminopyridine) caused prolonged [Ca²⁺]i elevation and was associated with strong hyperactivation. Analysis of [Ca²⁺]i and hyperactivation data from 24 different conditions in this study showed a continuous relationship between [Ca²⁺]i and hyperactivation. The strong hyperactivating effect of store mobilisation (compared to CatSper activation) may reflect opening of store-operated channels. Human sperm behaviour assessed over a 180 s recording revealed regular ‘switching’ between progressive and various hyperactivated types. Mobilisation of Ca²⁺ stores potently increased hyperactivated behaviour and suppressed the rate of behavioural switching.

Doctora en Ciencias, Mención Computación
La infertilidad es un problema clínico que afecta hasta a 15% de parejas en edad reproductiva, con implicancias tanto emocionales como fisiológicas. Un análisis de semen es el primer paso en la evaluación de una pareja infértil. El énfasis en identificar no sólo cabezas normales de espermatozoides sino también categorías de cabezas anormales puede tener una significativa utilidad clínica al decidir por un tratamiento de fertilidad. Esta tesis propone una nueva metodología para detectar, segmentar, caracterizar y clasificar cabezas de espermatozoides humanos, con el objetivo de facilitar el posterior análisis morfológico, para diagnósticos de fertilidad, toxicología reproductiva, investigación básica o estudios de salud pública. En la primera parte de este tesis, se ha tratado la detección y segmentación de cabezas de espermatozoides humanos. En este sentido, se propone un gold-standard para segmentación de espermatozoides construido con la cooperación de un experto referente en el área, para comparar métodos para detección y segmentación de espermatozoides. Además, se ha desarrollado un framework para la detección y segmentación de componentes de cabezas de espermatozoides humanos (incluyendo acrosoma y núcleo) que usa tres espacios de color además de técnicas de clustering y análisis estadístico del histograma. La evaluación experimental muestra que el método propuesto mejora el desempeño del estado del arte. Los resultados logran 98% de detección correcta a expensas de un número menor de falsos positivos, comparado con el estado del arte. Así mismo, los resultados de segmentación de cabeza, acrosoma y núcleo muestran más de 80% de solapamiento comparado con las máscaras de segmentación manual del gold-standard. En la segunda parte de esta tesis, el enfoque estuvo en la caracterización y clasificación de cabezas de espermatozoides humanos. Así, se introduce un gold-standard para clasificación de cabezas de espermatozoides humanos, construido con la colaboración de tres expertos referentes en área, y de acuerdo al criterio de la OMS. Además, se ha formulado un nuevo descriptor para cabezas de espermatozoides que, combinado con otros descriptores basados en forma, permite discriminar entre cabezas de espermatozoides normales y anormales, identificando cuatro tipos de cabezas anormales. También se propone un esquema de clasificación, que permite categorizar las cabezas de espermatozoides en 5 clases diferentes, según la OMS. La evaluación experimental muestra que el esquema propuesto tiene mejor desempeño que distintos clasificadores monolíticos, así como varios esquemas de clasificación en cascada que fueron diseñados en el contexto de esta investigación. Los resultados muestran más de 70% de clasificación correcta usando un dataset de total concordancia entre expertos del área.

Magister Scientiae (Medical Bioscience) - MSc(MBS)
The incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).

Sperm cells are guided to the egg by chemoattractants in many species. Sperm cells are propelled in a liquid by the regular beat of their flagellum. In the presence of a concentration gradient of a chemoattractant, they can steer upwards the concentration gradient, a process called chemotaxis. Eggs release chemoattractants to guide the sperm cells to the egg. Sperm chemotaxis is best studied experimentally in the sea urchin. There, specific receptors in the flagellar membrane of the sperm cells are activated upon binding of chemoattractant molecules and trigger a signaling cascade which ultimately changes the activity of the molecular motors which drive the flagellar beat and result in a swimming response. Sea urchin sperm cells swim along circular and helical paths. Sperm cells of the sea urchin and several other species swim along helical paths far from boundary surfaces in the absence of chemoattractant. In a two-dimensional experimental geometry, sperm swimming paths are planar circles. The non-zero curvature of their swimming paths is a direct consequence of an asymmetry of their flagellar beat. In a concentration gradient of chemoattractant, sperm swimming path are drifting circles in two dimensions and bend helices in three dimensions. What is the working mechanism of sperm chemotaxis? In this thesis, we develop a theoretical description of sperm chemotaxis which can be subsumed as follows: While swimming along an approximately circular path in a concentration gradient a sperm cell traces a periodic concentration stimulus from the concentration field that has the frequency of circular swimming. The chemotactic signaling system processes this stimulus and causes a periodic modulation of the curvature of the swimming path which then gives rise to a swimming path which is a drifting circle. The relative direction of the drift with respect to the gradient direction is determined by the phase shift between the stimulus and the curvature oscillations. This picture is in perfect agreement with recent experimental findings. The mechanism is more general and also works in three dimensions for swimming along helical paths. Our results. Our theoretical description of sperm chemotaxis clarifies the concepts underlying sperm chemotaxis. In particular, we derive the role of internal timing of the chemotactic signaling system for sperm chemotaxis. We conclude that sampling a concentration field along circular and helical paths is a robust strategy for chemotaxis that does not require fine-tuning of parameters and which works reliable also in the presence of fluctuations. In a last chapter of this thesis, we discuss sperm chemotaxis in the more general context of an abstract search problem.

Sperm cells are guided to the egg by chemoattractants in many species. Sperm cells are propelled in a liquid by the regular beat of their flagellum. In the presence of a concentration gradient of a chemoattractant, they can steer upwards the concentration gradient, a process called chemotaxis. Eggs release chemoattractants to guide the sperm cells to the egg. Sperm chemotaxis is best studied experimentally in the sea urchin. There, specific receptors in the flagellar membrane of the sperm cells are activated upon binding of chemoattractant molecules and trigger a signaling cascade which ultimately changes the activity of the molecular motors which drive the flagellar beat and result in a swimming response. Sea urchin sperm cells swim along circular and helical paths. Sperm cells of the sea urchin and several other species swim along helical paths far from boundary surfaces in the absence of chemoattractant. In a two-dimensional experimental geometry, sperm swimming paths are planar circles. The non-zero curvature of their swimming paths is a direct consequence of an asymmetry of their flagellar beat. In a concentration gradient of chemoattractant, sperm swimming path are drifting circles in two dimensions and bend helices in three dimensions. What is the working mechanism of sperm chemotaxis? In this thesis, we develop a theoretical description of sperm chemotaxis which can be subsumed as follows: While swimming along an approximately circular path in a concentration gradient a sperm cell traces a periodic concentration stimulus from the concentration field that has the frequency of circular swimming. The chemotactic signaling system processes this stimulus and causes a periodic modulation of the curvature of the swimming path which then gives rise to a swimming path which is a drifting circle. The relative direction of the drift with respect to the gradient direction is determined by the phase shift between the stimulus and the curvature oscillations. This picture is in perfect agreement with recent experimental findings. The mechanism is more general and also works in three dimensions for swimming along helical paths. Our results. Our theoretical description of sperm chemotaxis clarifies the concepts underlying sperm chemotaxis. In particular, we derive the role of internal timing of the chemotactic signaling system for sperm chemotaxis. We conclude that sampling a concentration field along circular and helical paths is a robust strategy for chemotaxis that does not require fine-tuning of parameters and which works reliable also in the presence of fluctuations. In a last chapter of this thesis, we discuss sperm chemotaxis in the more general context of an abstract search problem.

Darbo tikslas: įvertinti skirtingais racionais šertų veislinių bulių spermos kokybę. Darbo uždaviniai: 1. Išnagrinėti ir įvertinti skirtingus veislinių bulių racionus; 2. Nustatyti tiriamųjų bulių spermos kokybę; 3. Įvertinti skirtingų racionų įtaką veislinių bulių spermos kokybiniams rodikliams. Išvados: 1. B fermos buliai reproduktoriai gavo 16,4 MJ AE, 52,35 žaliųjų baltymų, 20,66 g fosforo, 40,9 mg vario, 859,2 mg cinko, 28290 TV vitamino D bei 122700 TV vitamino A daugiau nei A fermos buliai, kur šios medžiagos yra ypač svarbios reprodukcinei sistemai ir spermos kokybei. 2. A fermoje laikomų bulių išskirtas spermos tūris buvo 5,60 (±0,11) ml, koncentracija 1287,61 (±38,04) mln/cm3, gyvybingų spermatozoidų 81,22 (±0,57) proc., tiesiai judančių spermatozoidų 65,73 (±0,76) proc. B fermoje laikomų bulių vidutinis išskiriamas spermos tūris buvo 8,50 (±0,25) ml, koncentracija 1371,54 (±67,07) mln/cm3, gyvybingų spermatozoidų 80,13 (±0,37) proc, tiesiai judančių spermatozoidų 67,81 (±0,47) proc. 3. B fermoje laikomų bulių išskirtas spermos tūris buvo 2,90 ml (P<0,001), koncentracija – 84,92 mln/cm3 (P>0,05), tiesiai judančių spermatozoidų – 2,07 proc. didesnis (P>0,05) nei A fermoje laikomų bulių. Vienintelis priešingai rodęs rodiklis, t.y. gyvybingi spermatozoidai buvo 1,09 proc. (P<0,05) mažesnis už A fermos bulių. 4. Bulių davusių iki 4 ml spermos kiekį, koncentracija vidutiniškai nuo 1351,11(P<0,001) (A buliai) ir 2218,25 (P<0,001) (B buliai) sumažėjo iki 1097,43 (P<0,001)... [toliau žr. visą tekstą]
The aim of this research is evaluate the quality of bull semen, that are fed different diets. Research goal of the paper: 1. Analyze and evaluate different breeding bull rations; 2. Analyze bull’s semen quality; 3. Evaluate the influence of different diets on breeding bull semen quality (semen volume, concentration, percent viable sperm, directly moving spermatozoa percent). The results of different bull’s diet show that bulls of farm B got more mineral and vitamin needed for reproductive system than bulls from farm A. The breeding bulls of farm B were given 16.4 MJ 52.35 crude protein, 20.66 g of phosphorus, 40.9 mg of copper, 859.2 mg of zinc, 28,290 IU of vitamin D and 122 700 IU of vitamin A more than bulls from farm A. This material is particularly important for the reproductive system and sperm quality. Breeding bull’s sperm quality indicators showed that the semen characteristics (volume, concentration, and right-moving sperm) were on average superior to those bulls whose diets were additionally given mineral - vitamin supplements. The semen quality of bulls is changing dependent on different bull semen volume released. It showed that increasing volume of the sperm determine decreasing in sperm concentration mln/cm3.

Thesis (Ph. D.)--University of California, San Diego, 2005.
Title from first page of PDF file (viewed February 28, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Bibliography
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database. The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature; 60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature; 64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins of which only four proteins, namely Tubulin -3C/D chain, Tubulin -2C chain, Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction.
AFRIKAANSE OPSOMMING: Manlike infertiliteit word dikwels geassosieer met verlaagde sperm motiliteit en morfologie (asthenoteratozoospermia) waarvoor daar tot dusver nog geen spesifieke terapeutiese behandeling is nie. Dit het aan die lig gekom dat die modifisering en uitdrukking van menslike sperm proteïene ‘n belangrike rol speel in spermfunksie. In die huidige studie stel ons data voor van proteiene in menslike sperme in die konteks van abnormale spermfunksie. Unieke tegnieke was gebruik om verskille in proteïen uitdrukking op sellulêre vlak suksesvol te isoleer en identifiseer wat verband hou met asthenoteratozoospermia. Tydens die eerste deel van die studie was verskille in proteïen uitdrukking binne die totale spermproteoom tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme van gesonde skenkers (n=23) is geskei in twee spermpopulasies (onvolwasse en volwasse sperme) deur middel van ‘n 3-laag Percoll gradiënt sentrifugasie tegniek. Selle is gewas en sperm motiliteit en morfologie is gemeet deur rekenaar geassisteerde sperm analise (CASA). Vir proteomiese analise is selle geliseer en proteïene geskei deur twee dimensionele gel elektroforese (2D-elektroforese). PD-Quest sagteware is gebruik om statisties beduidende proteïen verskille aan te dui. Die proteïene van belang is uitgesny en onderwerp aan in-gel vertering. Peptiede is geskei met behulp van hoë druk vloeistof chromatografie (HPLC) analise en aminosuurvolgordes is bepaal deur massa spektrofotometrie. Proteïene is geïdentifiseer met behulp van Mascot deur van die Swiss Prot databasis gebruik te maak. Die resultate toon dat die sperm motiliteit (onvolwasse; 26.1±1.75% totale motiele selle vs. volwasse; 60.93±3.24% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 64.1±2.75% normale kop morfologie vs. volwasse; 87.63±3.24% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2Delektroforese is 16 proteïen kolle geïdentifiseer wat beduidend verskil het, tussen die totale sperm proteoom van onvolwasse spermpopulasies en volwasse spermpopulasies. 56% van die proteïene wat beduidend verskil het, was meer uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse sperm populasie. Funksies is toegeskryf aan hierdie proteïene waarvan net vier proteïene naamlik Tubulin -3C/D ketting, Tubulin -2C ketting, Buite digte vesel proteïen 2 en A-Kinase anker proteïen 4 voorloper direk verband hou met sperm motiliteit en morfologie. In die tweede deel van die studie is die uitdrukking van nukluêre proteïene in menslike spermatozoa tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme was van gesonde skenkers (n=156) versamel en verder geskei van seminale plasma deur middel van ‘n PureSperm® gradiënt sentrifugasie tegniek. Vir die proteomiese analise van nukluêre proteïene is selle gefraksioneer in vier verskillende sub-sellulêre proteïen fraksies, in plaas van analise van die totale spermproteoom. Die resultate toon aan dat die sperm motiliteit (onvolwasse; 32.33±0.51% totale motiele selle vs. volwasse; 88.67±0.85% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 13.51±0.87% normale kop morfologie vs. volwasse; 20.89±1.20% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2D-elektroforese is 21 kern proteïen kolle geïdentifiseer wat betekenisvol uitgedruk was tussen onvolwasse en volwasse spermpopulasies. 95% van die nukluêre proteïene wat beduidend verskil het, was minder uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Slegs een kern proteïen naamlik 78kDa Glukose gereguleerde proteïen was meer uitgedruk in die onvolwasse spermpopulasie in vergelyking met die volwasse spermpopulasie. Funksies is toegeskryf aan hierdie proteïene wat direk verband hou met sperm motiliteit, morfologie en energie metabolisme. Ten slotte, in die huidige studie is unieke tegnieke geïmplementeer om proteïen verskille tussen onvolwasse en volwasse spermpopulasies te ondersoek. Uit hierdie resultate is dit duidelik dat proteïen uitdrukking in die totale sperm proteoom en in die kern proteïen fraksie beduidend verskil en onvolledig is in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Op grond van hierdie bevindinge word aanbeveel dat verdere studies op menslike sperme gedoen moet word ten einde die rol van individuele proteïene in sperm funksie te kan bepaal. Proteomika is ‘n ideale tegniek om die iodiopatiese oorsake van manlike infertiliteit te identifiseer, aangesien dit kan help in die identifisering van unieke reseptore (en seintransduksie paaie) wat gebruik kan word om sperm disfunksie te verbeter deur farmaseutiese behandeling.

Sexually reproducing eukaryotes are typically going through a biphasic life cycle with a diploid and a haploid phase. Unlike in plants where selection on haploid pollen genotypes is well established, the possibility of selection occurring in animal sperm is currently not known. One of the main reasons for this lack of knowledge is the general assumption that due to the shortness and the apparent absence of gene expression in haploid sperm, selection during that phase is unlikely to occur. The aim of this thesis was to fill this gap and address some of the main fundamental questions. I investigated the interaction between sperm phenotype and offspring phenotype with a focus on the trans-generational effects of (i) selection on the haploid sperm genotype, (ii) sperm ageing and (iii) sperm-mediated epigenetic effects. For one, we performed several experimental studies to investigate how selection on the sperm phenotype affects offspring performance in two externally fertilizing fishes, Atlantic salmon and zebrafish. We found that in Atlantic salmon, sperm of intermediate post-activation longevity sire offspring that hatch earlier. In zebrafish, longer living sperm sire more viable offspring with a higher fitness than their short-lived sibling sperm. We explored the mechanisms of these trans-generational effects and found that neither intrinsic post-ejaculation sperm ageing (Atlantic salmon and zebrafish) nor pre-ejaculation sperm ageing (zebrafish) affect offspring performance. However, we identified genetic differences between sperm pools that were obtained by selecting different phenotypes within ejaculates of zebrafish males. These results suggest a genetic basis for intra-ejaculate sperm phenotype variation and show that there is potential for haploid selection in sperm. In a separate experiment, we explored the role of sexual selection in shaping sperm-mediated epigenetic effects, and found that short-time changes in male-male competition affect offspring hatching time and survival. In conclusion, this thesis provides evidence that sperm phenotype affects offspring phenotype, and that sperm phenotype is affected by both epigenetic changes influenced by the male environment and differences in the haploid genome of sperm.

Thesis (MScAgric)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The application of assisted reproductive biotechnologies in sheep flocks is hampered by the susceptability of ovine sperm to cryodamage. There is still considerable scope in the improvement of cryopreservation protocols for ovine sperm to minimize the degree of damage to sperm during the cryopreservation process. Pre-cryopreservation processing has a definite effect on the survivability, motility, and fertilizing ability of sperm. Little information is however available on the potential contribution of the genetic make-up of rams, divergently selected for fecundity, on the ability of sperm to offer resistance to the damage caused by cooling, cryopreservation and thawing. The study aimed to investigate the influence of genetic selection for prolificacy (i.e. High Merino Line and Low Merino Line in terms of fecundity) on the ability of ovine sperm to offer resistance to cryodamage. The study investigated the effect of pre-cryopreservation processing by comparing motility and morphometry traits recorded for fresh- and post-thaw Merino ejaculated and epididymal sperm samples obtained form the High and Low lines, respectively. The effect of different sperm concentrations, equilibration periods and the addition or omission of seminal plasma from cryopreserved samples on the viability and morphometrical traits were also investigated. Ejaculate samples were collected by means of the artificial vagina (AV) method from 8 High Line rams and 7 Low Line rams. Epididymal samples were collected from 6 rams of each of the High and Low lines respectively, by recovering the epididymal sperm via aspiration from the cauda epididymides post mortem. Ejaculate samples were subjected to macroscopic and microscopic evaluation, and epididymal samples only to microscopic evaluation, for which the Sperm Class Analyzer® program was used for the evaluation of motility and morphometric measurements. Sperm motility recordings were captured at 100 frames per second. From findings of the study, it was concluded that genotype had no positive influence on the conception rate of the ewes mated to the High or Low Line rams, even though the rams from the two lines differed significantly in terms of their serving capacity. When sperm morphometry was evaluated for fresh ejaculate samples, the two lines differed significantly in terms of the morphometric traits elongation and ellipticity. Epididymal and ejaculated sperm obtained from Low Line rams had broader and rounder heads, compared to sperm obtained from High Line rams. When morphometry was assessed for sperm samples between the two methods of sperm recovery (collected with an AV or recovery via aspiration from the cauda epididymides of sacrificed rams), no morphometrical differences were observed. Significant differences were reported for the majority of the sperm motility traits (i.e. percentage motile, rapid-, medium-, slow swimming, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of the lateral head displacement (ALH)) recorded for ejaculated and epididymal sperm. The motility traits ALH and beat-cross frequency (BCF) analysed for epididymal sperm differed significantly between the two lines. When epididymal sperm were evaluated post-thaw, it became evident that the sperm obtained from the High Line rams had a larger acrosome surface cover when compared to that of the Low Line ram sperm. The addition of seminal plasma to epididymal samples did not result in an improvement of the preservation of sperm motility. It is known from the literature that cryopreservation causes a decrease in sperm head size. Head width was unaffected by cryopreservation with the addition of seminal plasma in this study, indicating a potential benefit with the use of seminal plasma in the cryopreservation protocol of epididymal ram sperm. The study compared two pre-processing techniques, i.e. the more time consuming swim-up technique (SUT) with a more time-efficient ‘flush technique’ (FT) to optimize the pre-processing protocol for motility assessment of sperm samples before cryopreservation of ram sperm. Comparison of the SUT and FT indicated that almost all of the motility parameters measured using the FT compared favourably with those obtained using the SUT. The results indicated that the FT can be used a more time-efficient technique to use for determining the motility of a sperm sample prior to cryopreservation. In conclusion, line differences associated with reproduction were observed in terms of the serving capacity of the rams, with selection for fecundity influencing the morphometric traits elongation and ellipticity for sperm obtained from the two lines. Future studies should be aimed at investigating morphometric traits of ovine sperm, to correlate it with fertilizing ability of sperm post-thaw and ensure optimal cryopreservation processing.
AFRIKAANSE OPSOMMING: Die toepassing van ondersteunende reproduksie tegnieke in skaaptroppe word bemoeilik deur die onvermoë van ram sperme om weerstand teen bevriesingskade te bied. Daar is nog baie ruimte vir die verbetering van die bevriesingsprotokolle vir skaap sperm om die omvang van bevriesingskade te verminder. Voor-bevriesing verwerking het dan 'n besliste uitwerking op die oorlewing, beweeglikheid en bevrugtingsvermoë, van skaap sperme. Min inligting is beskikbaar oor die potensiële bydrae van die genetiese samestelling van ramme wat uiteenlopend op grond van vrugbaarheid geselekteer is, op die vermoë van skaap sperme om weerstand te bied teen die skade wat deur verkoeling, diepbevriesing en ontdooiing, veroorsaak word. Die doelwit van die studie was om die invloed van genetiese seleksie vir fekunditeit (d.i. Hoë Merino Lyn en Lae Merino Lyn in terme van fekunditeit) op die vermoë van skaap sperme om weerstand teen bevriesingskade te bied, te ondersoek. Die studie het getoets wat die bevriesing proses se effek op epididimale sperme is, deur sperm motiliteit en -morfometrie te vergelyk tussen vars gekollekteerde sperme en sperm monsters na ontdooiing. Die effek van verskillende sperm konsentrasies, ekwilibrasie tydperke en die byvoeging of uitsluiting van seminale plasma op die lewensvatbaarheid en morfometriese eienskappe van Merino ramsperme is ondersoek in die studie. Geëjakuleerde monsters is versamel met behulp van 'n kunsmatige vagina (AV) van 8 Hoë Lyn en 7 Lae Lyn ramme. Epididimale monsters is verkry van 6 ramme van elk van die Hoë en Lae Lyne, deur middel van aspirasie van die sperme uit die cauda epididimii nadoods. Geëjakuleerde sperm monsters is met behulp van makroskopiese en mikroskopiese metodes geëvalueer, en epididimale sperm monsters slegs mikroskopies geëvalueer, met behulp van die Sperm Class Analyzer® program wat vir die evaluasie van beweeglikheid en morfometriese afmetings gebruik is. Sperm beweeglikheids opnames is opgeneem teen 100 raampies per sekonde. Die resultate van die studie het aangedui dat genotipe geen effek het op besetting van die ooie gepaar met die Hoë of Lae Lyn ramme gehad het nie, terwyl die dekvermoë aansienlik tussen ramme van die twee lyne verskil het. Wanneer die morfometriese eienskappe van vars geëjakuleerde sperme vergelyk was, het die lyne beduidend in terme van die morfometriese eienskappe van verlenging (elongation) en elliptisiteit verskil het. Die epididimale en geëjakuleerde sperme verkry vanaf die Lae Lyn ramme het ʼn breër en ronder kopvorm getoon as sperme wat verkry is van die Hoë Lyn ramme. Wanneer die morfometriese eienskappe van sperme versamel met die twee verskillende metodes (d.i. kunsmatige vagina of aspirasie vanuit die cauda epididimides) vergelyk was, is geen morfometriese verskille waargeneem nie. Die meeste sperm beweeglikheidseienskappe (d.i. persentasie beweeglike, vinnig-, medium- en stadig-swemmende sperme, VCL, VSL, VAP en ALH) van geëjakuleerde en epididimale sperme het verskil. Die beweeglikheidseienskappe amplitude van die laterale verplasing van die spermkop (ALH) en frekwensie waarmee sperm sy eie pad kruis (BCF), soos bepaal vir epididimale sperme, het beduidend tussen die twee lyne verskil. Met die evaluering van epididimale sperme na ontdooiing was dit duidelik dat sperme verkry van die Hoë Lyn ramme 'n groter mate van akrosoom-oppervlak gehad het, in vergelyking met sperme van die Lae Lyn ramme. Die byvoeging van seminale plasma by epididimale monsters het nie bygedra tot 'n verbetering van spermbeweeglikheid nie. Bestaande literatuur dui aan dat diepbevriesing 'n afname in die kopgrootte van sperme veroorsaak. In hierdie studie het die byvoeging van seminale plasma ʼn verandering in kopgrootte voorkom, wat dui op ʼn potensiële voordeel om seminale plasma in die bevriesingsprotokol van epididimale ramsperme in te sluit. Die studie het twee beweeglikheid bepalingstegnieke vergelyk om te bepaal of die tydrowende “opswem” tegniek (SUT) vervang kan word met 'n meer tyd-doeltreffende "spoel tegniek” (FT) in die voorbevriesing verwerking protokolle van ram sperme. Vergelyking van die twee tegnieke het aangedui dat die meeste van die kinematiese eienskappe van die FT gunstig met die waardes soos verkry met die SUT, vergelyk het. Resultate het getoon dat die FT parameters goed vergelyk met die beweeglikheid parameters van die SUT, dus kan dit aangeneem word dat die FT ʼn meer tyd-doeltreffende tegniek is wat vergelykbare sperm beweeglikheidsinligting oor skaap sperm monsters voor bevriesing sal verskaf. In samevatting is verskille in terme van die dekvermoë en op morfometriese vlak, meer spesifiek die eienskappe van verlenging (elongation) en elliptisiteit, tussen die twee lyne waargeneem. In toekomstige studies moet die morfometriese eienskappe van skaapsperme verder bestudeer word, asook die korrelasie daarvan met die bevrugtingsvermoë na ontdooiing bepaal om sodoende die diepbevriesing protokolle van skaapsperme te optimaliseer.

The acrosome is a large secretory granule that undergoes exocytosis when receptors on the sperm surface bind ligands found on the oocyte's extracellular matrix. Acrosomal exocytosis resembles stimulated neurotransmitter release in neurons in that it is triggered by a rise in intracellular calcium. In neurons, a core complex composed of the SNARE proteins - syntaxin, SNAP-25 and VAMP is involved in synaptic vesicle fusion. Regulation of this protein complex is accomplished through the action of accessory proteins, including complexin, which is thought to stabilize the core SNARE complex prior to fusion. Recent evidence has revealed that isoforms of the SNARE proteins and their accessory proteins are present in mammalian sperm, where they might mediate the exocytosis of the acrosome. It has been hypothesized that the SNARE complex may be regulated as part of the process of capacitation, a set of physiological changes that occur within the female tract, during which the spermatozoon acquires its fertilizing ability. In this thesis, the hypothesis that complexin is involved in the regulation of the acrosome reaction is examined. To do so, streptolysin-O permeabilized sperm will be used to study the effect of recombinant complexin and of an antibody directed against complexin on the rate of the acrosome reaction, as well as on the sensitivity of this fusion machinery to calcium. Regulation may also occur through posttranslational modification or conformational change in one of the SNARE proteins. Syntaxin 2, which is the isoform present in sperm, showed a shift in apparent molecular weight on Western blots with capacitation. Alkaline carbonate extraction and dephosphorylation were used in an attempt to determine the kind of modification syntaxin 2 is undergoing during capacitation. Together, these data will shed light onto the role of complexin in the regulation of the acrosome reactions as well as on the modifications syntaxin 2 undergoes during capacitation.

Thesis (Ph.D.)--Tufts University, 1999.
Adviser: Sara M. Lewis. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 155-171). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Introduction: There is a nationwide shortage of sperm donors and over the last few years this has been evident at Newcastle Fertility Centre (NFC). As the most common cause for rejection of sperm donors is suboptimal semen quality, external factors that may influence semen quality (i.e. season and vitamin D) were studied to investigate their impact (if any) to improve donor recruitment. Methods: A retrospective review of donor sperm treatments at NFC between Jan 2000 and Dec 2010 was performed to investigate sperm donor shortage. A retrospective review (Dec 2006 to Nov 2009) and a longitudinal study (32 sperm donors) of the semen analyses were conducted to investigate seasonal variation in semen parameters. We performed a retrospective review of donor insemination treatments over 6 years to investigate seasonal variation in donor conceptions. The correlation between semen parameters and serum vitamin D was investigated in a cross sectional study (125 participants) and a cohort study, to examine the change in semen parameters with a rise in serum vitamin D level (with vitamin D supplementation and seasonal rise). Results: A significant reduction in the number of sperm donors recruited and a smaller pool of available donors was seen, which lead to fewer patients receiving treatment and a longer wait for treatment. Seasonal variation with improved semen parameters in winter / spring was noted, but was more prevalent in sperm donors than in patients attending NFC. However, there were no variation donor conceptions by the season of original sperm production. In the cross sectional association study there was no significant difference in the semen parameters between men with different serum vitamin D levels, however in the cohort study, semen parameters deteriorated significantly with increased serum vitamin D levels secondary to vitamin D supplementation and also seasonal rise. Conclusions: A significant local problem of sperm donor shortage is confirmed. Despite significant seasonal differences in donor semen parameters (but not donor conceptions), we do not recommend restricting recruitment of sperm donors to winter / iii spring. A negative association between vitamin D and semen parameters is noted; therefore vitamin D supplementation should not be recommended to improve to semen parameters.

The propulsion mechanics driving the movement of living cells constitutes one of the most incredible engineering works of nature. Active cell motility via the controlled movement of a flagellum beating is among the phylogentically oldest forms of motility, and has been retained in higher level organisms for spermatozoa transport. Despite this ubiquity and importance, the details of how each structural component within the flagellum is orchestrated to generate bending waves, or even the elastic material response from the sperm flagellum, is far from fully understood. By using microbiomechanical modelling and simulation, we develop bio-inspired mathematical models to allow the exploration of sperm motility and the material response of the sperm flagellum. We successfully construct a simple biomathematical model for the human sperm movement by taking into account the sperm cell and its interaction with surrounding fluid, through resistive-force theory, in addition to the geometrically non-linear response of the flagellum elastic structure. When the surrounding fluid is viscous enough, the model predicts that the sperm flagellum may buckle, leading to profound changes in both the waveforms and the swimming cell trajectories. Furthermore, we show that the tapering of the ultrastructural components found in mammalian spermatozoa is essential for sperm migration in high viscosity medium. By reinforcing the flagellum in regions where high tension is expected this flagellar accessory complex is able to prevent tension-driven elastic instabilities that compromise the spermatozoa progressive motility. We equally construct a mathematical model to describe the structural effect of passive link proteins found in flagellar axonemes, providing, for the first time, an explicit mathematical demonstration of the counterbend phenomenon as a generic property of the axoneme, or any cross-linked filament bundle. Furthermore, we analyse the differences between the elastic cross-link shear and pure material shear resistance. We show that pure material shearing effects from Cosserat rod theory or, equivalently, Timoshenko beam theory or are fundamentally different from elastic cross-link induced shear found in filament bundles, such as the axoneme. Finally, we demonstrate that mechanics and modelling can be utilised to evaluate bulk material properties, such as bending stiffness, shear modulus and interfilament sliding resistance from flagellar axonemes its constituent elements, such as microtubules.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotype. Morphologically mature sperm exit the testes, but cannot swim or interact with the oocyte without extensive remodeling during epididymal transit; this includes modifications to the lipid composition of the sperm membrane, gain of necessary proteins, and a dramatic shift in sperm RNA content. Epididymal maturation has also been linked to changes in the sperm methylome suggesting that the epididymis might play a broader role in shaping the sperm epigenome. First, we characterized the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. Our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA. Second, given our interest in the small RNA repertoire of sperm we set out to address known bias in sequencing protocols by comparing several small RNA cloning protocols. We found a protocol recently developed by Kathleen Collins’ lab (OTTR) to be superior to commercially available kits in providing an accurate representation of tRNA fragment levels as compared to Northern blotting. These results not only provide a more accurate representation of tRNA fragments, but also more complexity than previously seen allowing us to reassess the true sperm small RNA content. Taken together, these results provide significant insight into the mechanisms and factors modulating sperm epigenomics during post-testicular sperm maturation.

Mestrado em Biologia Molecular e Celular
Oxidative stress (OS) is believed to be an important cause of male infertility, which accounts for about half of all infertility cases. Reactive species (RS)- induced OS is detrimental to spermatozoa, leading to the damage of many biomolecules, such as lipids, proteins and DNA. Several lifestyle factors, such as alcohol and tobacco consumption, are known to induce OS and have been studied for their negative effects on male reproductive system. The aim of this study was to evaluate the impact of acute lifestyle changes, namely alcohol and tobacco consumption, on semen quality, accessory glands function and oxidative balance of sperm cells. Furthermore, the correlation between the OS parameters analyzed and the basic semen parameters was also assessed. Male students, in reproductive age, who participated in the academic festivities, donated a semen sample at three time points: before and one week and three months after the academic festivities. Basic semen analysis was performed and, subsequently, semen samples were processed. Acessory glands function was evaluated and OS was analyzed through measurement of the total antioxidant capacity of the sperm cells and through determination of the expression of antioxidant enzymes glutathione peroxidase 4 and superoxide dismutase 1. The impact of ROS in spermatozoa was also assessed through the determination of the protein carbonyl and 3-nitrotyrosine groups. The results indicate that a decrease in semen quality, demonstrated by a decrease in progressive motility and neutral α-glucosidase concentration and an increase in tail defects, occurs due to lifestyle alterations. The total antioxidant status of sperm cells and variations in protein oxidation levels are dependent on the alcohol and tobacco consumption. Moreover, some correlations were observed between the studied parameters, which may be useful in a clinical perspective. In conclusion, the lifestyle alterations are responsible for a decrease in semen quality and by an increase in protein modifications, which may consequently lead to a decrease in fertilizing potential.
O stress oxidativo (OS) tem sido considerado uma causa importante da infertilidade masculina, que está envolvida em cerca de metade dos casos de infertilidade. O OS induzido pelas espécies reativas (RS) é prejudicial para os espermatozoides, levando a lesões em várias biomoléculas, como os lípidos, proteínas e DNA. Alterações no estilo de vida, como o consumo excessivo de álcool e tabaco, induzem o OS e têm sido extensivamente estudadas devido aos seus efeitos negativos ao nível do sistema reprodutor masculino. O objetivo deste estudo foi analisar o impacto de alterações agudas no estilo de vida, nomeadamente o consumo de álcool e tabaco, na qualidade seminal, na função das glândulas acessórias e no equilibrio oxidativo dos espermatozoides. Para além disso, outro objetivo deste trabalho foi avaliar a possível relação entre os parâmetros de OS e os parâmetros seminais analisados. Estudantes masculinos, em idade fértil, que participaram nas festividades académicas, doaram uma amostra de sémen em três períodos de tempo: antes e uma semana e três meses após as festividades académicas. A análise básica ao sémen foi realizada e, posteriormente, as amostras foram processadas. A função das glândulas acessórias foi avaliada, assim como determinada a capacidade antioxidante total das células, a expressão das enzimas antioxidantes superóxido dismutase 1 e glutationa peroxidase 4 e a presença de grupos carbonilo e 3-nitrotirosina. Os resultados indicam que uma diminuição na qualidade seminal, demostrada por um decréscimo na motilidade progressiva dos espermatozoides e na concentração de α-glucosidase neutra e um aumento nos defeitos da cauda, ocorre devido a alterações no estilo de vida. A capacidade antioxidante total das células e as variações ao nível da oxidação proteica demonstram também ser dependentes do consumo de alcool e tabaco. Foram também verificadas algumas correlações entre os parâmetros analisados que poderão ser importantes numa perspetiva clínica. Concluindo, alterações no estilo de vida são responsáveis pela diminuição da qualidade seminal e pelo aumento de modificações proteicas, o que pode levar consequentemente a um decréscimo do potencial de fertilização.

[EN] Cryopreserved boar sperm is not used extensively for artificial insemination due to poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged when cooled from body temperature to 5 ºC (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could increase their post-thaw survival, similarly to other species that are cold shock sensitive. Cholesterol can be easily added to sperm membranes using cholesterol-loaded cyclodextrins (CLC). Treating sperm from different species susceptible to cold-shock with CLC before cryopreservation improves sperm cryosurvival. Egg yolk and glycerol are common constituents of extenders used for boar sperm cryopreservation. However, conventional freezing extenders could not be the appropriate for CLC-treated sperm. The aim of this Thesis is to evaluate cryosurvival of CLC or cyclodextrin-treated boar sperm in three different conditions: using conventional freezing extenders, using extenders with alternative concentrations of glycerol and egg yolk and using amides as cryoprotectants. CLC or methyl- ß-cyclodextrin treatment (1 mg/120 x 106 sperm) prior to cryopreservation using a conventional freezing extenders provided either slight or no benefit, respectively, to post-thaw sperm plasma membrane integrity (+ 8%; P < 0.05) and motility (P > 0.05). In addition, sperm from both, good and poor freezers, responded similarly to CLC treatment (P > 0.05). Reduction in egg yolk concentration from 20 to 10% was detrimental for post-thaw sperm viability, even in semen treated with CLC (- 12%; P < 0.05). On the other hand, it was observed that traditional concentration of glycerol (3%) was not the appropriate to freeze CLC-treated sperm (- 13% viable sperm compared to control; P < 0.05). Thus, CLC-treated sperm showed a higher tolerance (+ 13 % sperm viability; P < 0.05) to high glycerol concentrations (5%) than non-treated sperm. Regarding the efficacy of amides as cryoprotectants, three of the amides (lactamide, acetamide and formamide) produced deleterious effects in fresh boar sperm (P < 0.05). The other amides (methylformamide, dimethylacetamide and dimethylformamide) efficiently improved post-thaw sperm viability (+ 5 to 15 %; P < 0.05) but negatively affected the sperm motility (- 11 to 16% total motile sperm; P < 0.05) and the sperm fertilizing ability in vitro (dimethylformamide: - 64 % penetration rate; P < 0.05), irrespective of the sperm treatment. On the other hand, CLC-treated samples showed better in vitro fertilizing ability than control samples when glycerol was used as cryoprotectant (+ 2 penetrated spermatozoa/oocyte; P < 0.05). The results obtained in this Thesis suggest that conventional freezing protocols should be optimized for CLC-treated boar sperm in order to obtain the benefit of CLC treatment observed in other species sensitive to cold shock.
[ES] Las inseminaciones artificiales en la especie porcina se realizan habitualmente con semen refrigerado, debido a las bajas tasas de fertilidad obtenidas con el semen congelado-descongelado. La membrana del espermatozoide sufre importantes daños cuando es sometida a la fase de enfriamiento desde la temperatura corporal hasta alcanzar los 5 ºC (choque térmico), así como durante el proceso de congelación y descongelación. El aumento del contenido de colesterol en las membranas de los espermatozoides de cerdo podría mejorar su supervivencia tras la descongelación, como sucede en otras especies sensibles al choque térmico. Este incremento en la cantidad de colesterol se puede realizar fácilmente utilizando ciclodextrinas saturadas de colesterol (CLC). El tratamiento con CLC de espermatozoides de varias especies susceptibles al choque térmico antes de la congelación ha conseguido mejorar su supervivencia tras la descongelación. En los protocolos convencionales de congelación de semen porcino se utilizan habitualmente diluyentes de congelación compuestos por yema de huevo y glicerol, sin embargo, puede que estos diluyentes de congelación convencionales no sean los más apropiados para congelar espermatozoides tratados con CLC. El objetivo de esta Tesis es evaluar la supervivencia a la congelación de los espermatozoides porcinos tratados con CLC o ciclodextrinas utilizando diluyentes de congelación convencionales, utilizando concentraciones alternativas tanto de yema de huevo como de glicerol o utilizando amidas en lugar de glicerol como crioprotectores Utilizando diluyentes convencionales, el tratamiento con 1mg de CLC o de metil-ß-ciclodextrina/120 millones de espermatozoides previamente a la congelación proporcionó una leve mejora de la integridad de la membrana plasmática espermática (+ 8%; P < 0,05) y ningún beneficio sobre la movilidad espermática (P > 0,05). Además, la respuesta al tratamiento con CLC fue similar independientemente de si los espermatozoides procedían de verracos buenos o malos congeladores (P > 0,05). Una reducción de la concentración de yema de huevo de un 20 a un 10% fue perjudicial para la supervivencia de los espermatozoides tras la descongelación, incluidos aquellos que habían sido tratados previamente con CLC (- 12% espermatozoides vivos; P < 0,05). Por otro lado, observamos que las concentraciones de glicerol utilizadas habitualmente (3%) no son las más apropiadas para congelar espermatozoides tratados con CLC (- 13 % viabilidad espermática comparando con las muestras control; P < 0,05), ya que éstos mostraron una mayor tolerancia (+ 13 % espermatozoides vivos; P < 0,05) que las muestras control a las concentraciones de glicerol más altas (5%). Con respecto a la eficacia de las amidas como crioprotectores para semen porcino, tres de las amidas (lactamida, acetamida y formamida) produjeron efectos perjudiciales durante su incubación con semen fresco (P < 0,05). El resto de amidas evaluadas (metilformamida, dimetilacetamida y dimetilformamida) mejoraron eficientemente la viabilidad espermática tras la congelación (+ 5 a 15 %; P < 0,05), sin embargo, afectaron negativamente tanto la movilidad espermática (- 11 a 16% móviles totales; P < 0,05) como la capacidad de fecundación in vitro (dimetilformamida: - 64 % en la tasa de penetración; P < 0,05), independientemente de si el semen fue tratado con CLC o no. Por otro lado, las muestras tratadas con CLC mostraron mejor capacidad de fecundación in vitro que las muestras control cuando se utilizó el glicerol como crioprotector (+ 2 espermatozoides penetrados/ovocito; P < 0,05). Los resultados obtenidos en esta Tesis sugieren que sería necesaria la adecuación de los protocolos de congelación convencionales para congelar semen porcino tratado con CLC con el propósito de alcanzar los claros beneficios obtenidos con dicho tratamiento cuando ha sido evaluado en otras especies sensibles al choque térmico.
[CAT] Les inseminacions artificials en l'espècie porcina es realitzen habitualment amb semen refrigerat, a causa de les baixes taxes de fertilitat obtingudes amb el semen congelat-descongelat. La membrana de l'espermatozoide pateix importants danys quan és sotmesa a la fase de refredament des de la temperatura corporal fins a arribar als 5 ºC (xoc tèrmic), així com durant el procés de congelació i descongelació. L'augment del contingut de colesterol a les membranes dels espermatozoides de porc podria millorar la seva supervivència després de la descongelació, com succeeix en altres espècies sensibles al xoc tèrmic. Aquest increment en la quantitat de colesterol es pot realitzar fàcilment utilitzant ciclodextrines saturades de colesterol (CLC). El tractament amb CLC d'espermatozoides de diverses espècies susceptibles al xoc tèrmic abans de la congelació ha aconseguit millorar la seva supervivència després de la descongelació. En els protocols convencionals de congelació de semen porcí s'utilitzen habitualment diluents de congelació compostos per rovell d'ou i glicerol, però, pot ser que aquests diluents de congelació convencionals no siguin els més apropiats per congelar espermatozoides tractats amb CLC. L'objectiu d'aquesta Tesi és avaluar la supervivència a la congelació dels espermatozoides porcins tractats amb CLC o ciclodextrines utilitzant diluents de congelació convencionals, utilitzant concentracions alternatives tant de rovell d'ou com de glicerol o utilitzant amides en lloc de glicerol com crioprotectors Utilitzant diluents convencionals, el tractament amb 1 mg de CLC o de metil-ß-ciclodextrina / 120 milions d'espermatozoides prèviament a la congelació va proporcionar una lleu millora de la integritat de la membrana plasmàtica espermàtica (+ 8%; P <0,05) i cap benefici sobre la mobilitat espermàtica (P> 0,05). A més, la resposta al tractament amb CLC va ser similar independentment de si els espermatozoides procedien de verros bons o dolents congeladors (P> 0,05). Una reducció de la concentració de rovell d'ou d'un 20 a un 10% va ser perjudicial per a la supervivència dels espermatozoides després de la descongelació, inclosos aquells que havien estat tractats prèviament amb CLC (- el 12% espermatozoides vius; P <0,05) . D'altra banda, observem que les concentracions de glicerol utilitzades habitualment (3%) no són les més apropiades per congelar espermatozoides tractats amb CLC (- 13% viabilitat espermàtica comparant amb les mostres control; P <0,05), ja que aquests van mostrar una major tolerància (+ 13% espermatozoides vius; P <0,05) que les mostres control a les concentracions de glicerol més altes (5%). Pel que fa a l'eficàcia de les amides com crioprotectors per semen porcí, tres de les amides (lactamida, acetamida i formamida) van produir efectes perjudicials durant la seva incubació amb semen fresc (P <0,05). La resta de amides avaluades (metilformamida, dimetilacetamida i dimetilformamida) van millorar eficientment la viabilitat espermàtica després de la congelació (+ 5 a 15%, P <0,05), però, van afectar negativament tant la mobilitat espermàtica (- 11 a 16% mòbils totals; P <0,05) com la capacitat de fecundació in vitro (dimetilformamida: - el 64% en la taxa de penetració; P <0,05), independentment de si el semen va ser tractat amb CLC o no. D'altra banda, les mostres tractades amb CLC van mostrar millor capacitat de fecundació in vitro que les mostres control quan es va utilitzar el glicerol com crioprotector (+ 2 espermatozous penetrats / oòcit; P <0,05). Els resultats obtinguts en aquesta Tesi suggereixen que seria necessària l'adequació dels protocols de congelació convencionals per congelar semen porcí tractat amb CLC amb el propòsit d'assolir els clars beneficis obtinguts amb el tractament quan ha estat avaluat en altres espècies sensibles al xoc tèrmic.
Blanch Torres, E. (2015). Treating boar sperm with cholesterol-loaded cyclodextrins or cyclodextrins prior to cryopreservation: effects on post-thaw in vitro sperm quality of sperm cryopreserved in different freezing extenders [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58773
TESIS

In externally fertilizing fishes, multiple factors of the spawning environment may affect the sperm viability, and thus the fertilization rate. In this thesis, the sperm activation effect of osmolality of non-electrolytes and electrolytes activation media, pH and ion channel inhibitors on Nile tilapia, Oreochromis niloticus, and the effect of environmentally relevant pollutants (cadmium, malathion and rotenone) on sperm fitness (motility and morphology) were investigated. Seminal fluid samples collected from male fishes (200-250g) were subjected to activation treatments, then analyzed for sperm motility using motility score, and motility variables using Hobson sperm tracker for straight line velocity (VSL), beat cross frequency (BCF) and percentage of motile cells (MOT). For the ion channel inhibitors and pollutants, the effect on sperm motility variables of VSL, VCL (curvilinear velocity) and LIN (linearity) were determined. Multivariate analysis was also carried out to determine the effects of ion channel inhibitors and pollutants on sperm subpopulations. The effects of pollutants on sperm morphology were observed using microscopy techniques, namely, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Sperm motility was initiated when the sperm were exposed to hypoosmotic electrolytes and non-electrolytes solution. We also found that sperm show optimal activity at pH range of 6-8 which depicts that the effect of pH on sperm motility is negligible. Lanthanum (calcium channel blocker) and flunarizine (sodium-calcium exchanger pump blocker) were found to inhibit sperm motility at 25 and 5 µM, respectively, suggesting that both ion channels play a significant role in sperm activation in O. niloticus. In contrast amiloride, ouabain and quinine showed no effects on activation, indicating that epithelial sodium channels, sodium-potassium ATPase and voltage gated potassium channels respectively are unlikely to have major roles in sperm activation or motility. The spermatozoa of Oreochromis niloticus were uniflagellate with clearly differentiated oval-shaped head, midpiece and flagellum. Sperm exposed to hypoosmotic shock showed swelling of the midpiece and sleeve structure. The pollutants showed dose- and time-dependent effect on sperm motility of the fast linear sperm subpopulation. Sperm morphology was not affected. Sperm motility was inhibited at 0.44, 0.03 and 0.063 µM, cadmium, malathion and rotenone respectively. Both cadmium and malathion exerted effects very quickly after exposure. The effect of cadmium, which can exert toxicity by calcium antagonism, is consistent with the effects of calcium channel blockes and further supports an important role for calcium in sperm activation and motility. Malathion had effects at relatively low, environmentally relevant concentrations, suggesting the presence of functionally important acetylcholinesterase activity in sperm, and also the presence of activation cytochrome P450 activity. Rotenone, a well known mitochondrial poison, affected motility only after 15 min of pretreatment. The alteration of sperm trajectories in fast linear spermatozoa subpopulation by pollutants at submicromolar concentrations as demonstrated in our study implies potentially serious consequences for fish populations in polluted environments. Furthermore the results indicate that fish sperm motility as assessed by CASA could be an ecologically relevant, sensitive, and ethically acceptable method for toxicity testing in environmental risk assessment.

Within the female reproductive tract mammalian sperm gradually develop hyperactivated motility, triggered by increased flagellar Ca2+ resulting in deep flagellar bends. Two sources of Ca2+ may contribute to the regulation of hyperactivation. Ca2+ influx at the plasma membrane is crucial and the sperm specific CatSpers channels are of great importance. In addition, studies on sperm have provided strong evidence that Ca2+ stored in the region of the sperm neck contributes to regulation of flagellar activity. 4-aminopyridine caused robust and persistent hyperactivation of motility in human sperm. Ca2+ imaging showed that treatment with 4-aminopyridine induced a parallel elevation of [Ca2+]i, which initiated at the sperm neck/midpiece and was associated with asymmetric flagellar bending in this region. This report demonstrates that 4-aminopyridine induced hyperactivation in human sperm is not solely pH/CatSper channel dependent and that mobilisation of stored Ca2+, possibly causing activation of store-operated Ca2+ influx, is essential for hyperactivation in a population of human sperm. 4-aminopyridine induced IP3R and RyR store release in sacroplasmic reticulum and brain microsomal vesicles. Although the physiological factor(s) that activate hyperactivation in vivo remains elusive we can conclude that release of the neck/midpiece intracellular Ca2+ store was sufficient to initiate hyperactivation in a population of human sperm.

Subcellular fractionation revealed that pp97, pp96 and pp64 are head protein whereas pp90 and pp55 are tail proteins. Advanced proteomic analysis (GeLC-MS) identified 37 proteins, including AKAP4, AKAP3, CALI, HSPAlL and HSP70 as candidates for the dephosphorylated proteins. AKAP4 was excluded because it was localised to the tail. Two AKAP3 antibodies showed non-specific binding and a better quality antibody will be needed for further investigations. CALI was excluded because it was localised to the tail fraction. HSP70/72 and HSPA1L were strong candidates for pp64. However, immunoprecipitation of dephosphorylated proteins using phospho (S/T) PKA substrate Ab and HSPA1L Ab was unsuccessful and further work is now required to address this.