Introduction: Cryopreservation of human sperm is considered a routine practice in assisted reproduction laboratories. Semen samples are mainly cryopreserved as a back-up for procedures, donor sperm, and validation of samples from human immunodeficiency virus-positive patients. Human immunodeficiency virus semen samples generally result in a low yield of purified spermatozoa after decontamination. These samples need to be cryopreserved for later use. Unlike conventional cryopreservation, vitrification does not use harmful cryoprotectants, thereby potentially reducing sperm damage. Vitrification is not yet common practice for sperm cryopreservation in assisted reproduction. The aim of this study was to establish the feasibility of utilising vitrification as an alternative to current conventional cryopreservation of spermatozoa. Methods: Semen samples were collected from human immunodeficiency virus-negative patients seeking diagnostic assistance from the unit. All samples were processed according to the unit’s standard protocol. For Study 1A (n=10) washed samples were divided and cryopreserved using three different cryopreservation media, and two different freezing protocols. In Study 1B (n=15), washed samples were divided and preserved using cryoprotectant-free vitrification in 100 μl, 300 μl and 500 μl volumes. For Study 2 (n=35) washed samples were split and cryopreserved using cryoprotectant-free vitrification (utilizing the volume that resulted in the highest quality spermatozoa in Study 1B) and conventional slow freezing (using the medium and protocol that resulted in superior quality spermatozoa in Study 1A). Post thawing, motility and kinetic parameters (Studies 1 and 2), viability (Study 1), mitochondrial membrane potential (Study 2), and DNA fragmentation (Study 2) of the two groups were compared. vi Results: Study 1A indicated that cryopreserving spermatozoa using Freezing Medium resulted in the highest quality spermatozoa with regards to motility and viability (p<0.05). Comparing the two preservation protocols, no conclusion could be reached on which protocol yielded superior results (p>0.05). The RBL freezing method is shorter, simpler and requires less equipment, and was therefore deemed the preferred method. Study 1B showed that the larger vitrification volumes (300 μl and 500 μl) yielded better spermatozoa in terms of motility and viability (p<0.05). No significant difference was observed with respect to the 300 μl and 500 μl vitrification volume groups. For practical reasons, 300 μl volumes will provide sufficient sperm for any procedure and, the intermediate volume ensures that more than one straw can be preserved. Study 2 found that cryoprotectant-free vitrification resulted in spermatozoa with significantly higher mitochondrial membrane potential and significantly lower apoptosis post thawing (p<0.05). Discussion: Conventional cryopreservation methods may compromise various sperm parameters and final yield. In this study, cryopreservation and cryoprotectant-free vitrification had equivalent outcomes with respect to sperm motility. However, the latter method yielded superior results in terms of ΔΨ and DNA sperm fragmentation. In conclusion, vitrification is an easy, rapid and more affordable technique that requires no special equipment. Using vitrification for purified sperm samples of patients could potentially result in a better post thaw quality for ART procedures. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

Introduction: The desire to have a biological child transcends race, religion and socio-economic status. However for those faced with infertility, the financial resources needed to conceive are often not available. Current research in assisted reproduction has gravitated towards cost reduction to address restricting financial factors, without compromising quality of treatment. One such initiative is the development of a low-cost embryo culture method by The Walking Egg foundation. This method utilizes a standard chemical reaction and simple equipment to equilibrate culture media pH and to regulate temperature; both aspects were investigated in this study. An exploration into the insemination concentration to achieve oocyte fertilization was also undertaken. Methods: Quality control of temperature regulation on six different heating devices, including a comparison of inter- and intra-variations was carried out. The utilization of citric acid and bicarbonate of soda for carbon dioxide production, which subsequently facilitate setting of pH values, was tested by injecting increasing citric acid volumes (1.2 ml – 3.0 ml in 0.2 ml increments) into set volumes of bicarbonate of soda. Further investigation evaluated gas production at various temperatures (37°C, 25°C and 15°C), at increasing intervals (16 – 30 hours) of equilibration and these were compared by measuring pH of the culture media. The influence of altitude on pH was explored by repeating the chemical reaction experiment at five different locations in South Africa. Furthermore, the addition of water to citric acid before gas generation was explored. The minimal insemination concentration needed for fertilization was determined by the addition of decreasing numbers of spermatozoa to non-fertilized bisected oocytes. The experiment was repeated with a selected sperm insemination number in 1 ml or 50 μl culture media to compare the tested culture system with conventional culture. Spermatozoa bound to the hemi-zonae were counted with the aid of an inverted phase contrast microscope. Hemi-zonae with bound sperm were also stained with ethidium homodimer and evaluated using a confocal laser-scanning microscopy system. After removal of hemi-zonae, the spermatozoa in culture were isolated for deoxyribonucleic acid fragmentation analyses and reactive oxygen species presence in the culture media was measured. Additionally, reactive oxygen species generation in simulated culture was measured over time. Results: All the equipment tested bar one, the warming oven, proved useable with the simplified Walking Egg in vitro fertilization culture system. By decreasing the citric acid volumes, it was indicated that 1.8 ml citric acid, diluted with 1.2 ml water, is the optimal volume to facilitate the required culture media pH. Omitting the water dilution from citric acid volumes affected the culture media pH adversely, however reducing the temperature during gas equilibration did not. A change in altitude had no effect on culture media pH. Lower insemination numbers resulted in decreased sperm binding, with 2 x 103 motile sperm insemination providing the lowest number to still obtain sufficient sperm–zona binding (≥20 sperm bound). Incubation in 1 ml vs. 200 μl culture media indicated decrease in sperm bound. Sperm deoxyribonucleic acid fragmentation and the presence of reactive oxygen species in the culture media were similar in both the test and control groups. A comparison over time revealed less reactive oxygen species in 1 ml culture media, from the simplified Walking Egg in vitro fertilization culture system after three days of culture, than 200 μl culture media drops under oil, from conventional culture after 18 hours, however the results were not statistically significant. Discussion: Purpose-made heating devices provide superior stabilization of culture media temperature. When selecting a heating device, intra-variations should be considered. Culture media can be manipulated to the required pH by carbon dioxide production, with meticulous attention paid to the citric acid volumes used. However, if gas generation is performed at room temperature, equilibration time must be increased. In conventional culture, the minimum insemination number can be reduced to 2 x 103 motile sperm. Due to lower binding of sperm in large volumes of culture media, 2 – 5 x 103 motile sperm should be considered for the simplified culture system, depending on a holistic consideration of all sperm parameters. Extended culture for at least three days with the simplified culture system can be performed without increasing reactive oxygen species present in culture media. Further research of this novel culture method should include the application of the culture method in a South African environment. / Dissertation (MSc)--University of Pretoria, 2017. / Obstetrics and Gynaecology / MSc / Unrestricted

This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.

Andrologische Forschungsarbeiten der letzten Jahre beweisen, dass einzelne, aus somatischen Zellen bekannte Apoptose-typische Veränderungen bei humanen Spermien einen negativen Einfluss auf die Fertilität des Mannes haben. Umstritten ist, ob es sich dabei nur um einen abortiven Zelltod als Zeichen einer Reifungsstörung während der Spermatogenese handelt oder ob Apoptose auch in reifen Spermien induzierbar ist. Ziel der vorliegenden Arbeit war es, durch Untersuchungen zur Induzierbarkeit der Apoptose in reifen und unreifen Spermatozoen die vollständige Funktionalität verschiedener Signalkaskaden sowie deren Umsetzung in morphologische Veränderungen aufzudecken. Darüber hinaus sollte die Rolle des intrazellulären Calciumspiegels als möglicher Interaktionspartner zwischen Akrosomreaktion und Apoptose geklärt werden, um Informationen über die Zukunft der nicht fertilisierenden Spermien im weiblichen Genitaltrakt zu erlangen. In den vorliegenden Versuchsreihen konnte erstmals die gezielte Induktion der Apoptose in reifen und unreifen Spermatozoen anhand biochemischer und elektronenmikroskopischer Untersuchungen nachgewiesen werden. Dabei wurde ausführlich die erfolgreiche Aktivierung mehrerer intrinsischer Apoptosesignalwege in reifen Spermien gezeigt, deren Initiation entweder auf den Zusammenbruch innerer Mitochondrienmembranen, auf eine veränderte intrazelluläre Calciumkonzentration oder auf das Einwirken von oxidativem Stress zurückzuführen war. Zudem konnten Erkenntnisse zum antioxidativen Schutzmechanismus von Spermien gewonnen werden, welcher die Spermien gegenüber einer spezifischen Menge an H2O2 vor oxidativem Stress-bedingter Apoptose bewahrt. Sowohl die Apoptose als auch die Akrosomreaktion waren durch die Zugabe eines Calciumchelators blockierbar. Die Initiation des programmierten Zelltodes in Spermien durch einen Anstieg der intrazellulären Calciumkonzentration erklärt zudem eine weitere wichtige Funktion dieses Prozesses: das Absterben von akrosomenreagierten Spermien bei nicht erfolgter Fertilisation im weiblichen Genitaltrakt. Die Theorie einer rein abortiven Apoptose als Folge einer Spermatogenesestörung ist damit widerlegt.

Bnip3 is a BH3-only member of the Bcl-2 family of apoptotic proteins. Our laboratory has previously shown that Bnip3 induces a unique pathway of cardiac myocyte cell death, characterized by mitochondrial dysfunction, cytochrome c release and DNA fragmentation. Bnip3 is induced by hypoxia and the death pathway is activated by concurrent acidosis. We have shown that hypoxia-acidosis creates an environment that is permissive to calpain but not caspase activation and is characterized by enhanced DNase(s) activity as evidenced by genomic DNA fragmentation. This dissertation describes the nuclear consequences of Bnip3 activation by hypoxia-acidosis. Chapter 3 presents my evidence that hypoxia with progressive acidosis in cardiac myocytes results in a biphasic activation of DNases. In phase 1, [pH]o 6.9-6.7, apoptosis-inducing factor (AIF) is released from the mitochondria and translocates to the nucleus. AIF release coincided with the loss of mitochondrial membrane potential and with the release of cytochrome c from the mitochondria. In Phase II, [pH]o 6.3-6.0, DNase II translocates from the cytoplasm to the nuclear compartment. Nuclear localization of DNase II was associated with the collapse of endosomal pH gradients, indicated by diffuse Lysotracker Red staining and with single strand DNA nicks. Both phases of DNase release were independent of Bnip3, the mPTP and calpains. Neither phase involved activation of caspase-dependent DNases. Chapter 4 describes a unique role for Bnip3 in the modulation of histone acetylation. I found that hypoxia with acidosis in cardiac myocytes but not hypoxia alone stimulated a global increase in the acetylation of histones H3 and H4. Acetylation was initiated at [pH]o ~ 6.8 and increased as the pH declined. Histone hyperacetylation was associated with an increase in histone acetyltransferase (HAT) activity but no change in deacetylase (HDAC) activity. Knockdown of Bnip3 protein expression with siRNA dramatically reduced both histone H3 and H4 acetylation levels and HAT activity indicating an essential role for Bnip3 in this process. Components of the hypoxia-acidosis death pathway including the mPTP and calpains are not required for Bnip3-mediated histone hyperacetylation. These results reveal a novel role for Bnip3 in regulating HAT activity and histone acetylation which may lead to altered cardiac gene expression.

Die Inzidenz des Diabetes mellitus (DM) nimmt weltweit jährlich zu und erlangt somit große Bedeutung für die Entwicklung der globalen Gesundheit. Die WHO rechnet bis zum Jahr 2030 mit ungefähr 366 Millionen erwachsenen Diabetikern. Es ist bekannt, dass Männer mit Diabetes mellitus Typ 1 (DMI) und 2 (DMII) häufiger an Subfertilität leiden, wobei dies möglicherweise auf erhöhte Apoptoseraten und vermehrte DNA-Fragmentierungen auf zellulärer Ebene zurückzuführen ist. Die Bedeutung der ROS als Regulatoren von physiologischen und pathologischen Signaltransduktionswegen ist bekannt. Demzufolge könnte die Aktivität der Stickstoffmonoxid-Synthetase (NOS) in diesem Zusammenhang eine Bedeutung haben. Das Ziel der vorliegenden Untersuchungen war es, die Auswirkungen von Apoptose und DNA-Fragmentierungen sowie die Bedeutung der NOS im Zusammenhang mit der Fertilitätsfähigkeit von Spermienzellen von DMI und DMII Patienten zu erfassen und damit erste Erklärungsansätze zur Pathophysiologie der diabetesassoziierten Subfertilität zu liefern. Samenproben von Normalspendern und Diabetikern wurden durch Dichtegradientenzentrifugation in Subpopulationen separiert und mittels fluoreszenzbasierten Assays zur Analyse von apoptoseassoziierten Parametern wie dem Zusammenbruch des mitochondrialen Membranpotentials (MMP), Aktivierung von Caspase-3 (CP3), DNA-Fragmentierungen und reaktiven Sauerstoffspezies (ROS) im Flowzytometer (FACS) untersucht. Die Ergebnisse zeigen eine signifikante Erhöhung von Apoptosemarkern (gestörtes MMP, aktivierte CP3), ROS und DNA-Fragmentationsraten in Spermien von DMI und DMII Patienten im Vergleich zu gesunden Normalspendern. Der Effekt ist bei DMII Patienten verstärkt ausgeprägt. Alle gemessenen Parameter korrelieren umgekehrt mit dem Fertilitätspotential der Spermien, gemessen anhand etablierter Spermiogramm-Analysen, womit ein möglicher Erklärungsmechanismus für die Subfertilität bei Diabetikern geliefert werden kann.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Made available in DSpace on 2015-12-06T23:46:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2014 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / funcionais nos espermatozoides e o nivel seminal de peroxidacao lipidica. Metodo: Um estudo transversal foi realizado incluindo 156 pacientes normozoospermicos. Apos a coleta do semen por masturbacao, uma aliquota foi utilizada para a analise seminal e outra para a avaliacao da atividade mitocondrial, da integridade do acrossoma e da integridade do DNA dos espermatozoides. O volume remanescente de semen foi centrifugado e o plasma seminal sobrenadante foi utilizado para a avaliacao do nivel seminal de peroxidacao lipidica e para a analise proteomica. Posteriormente, os pacientes foram divididos em percentis (15%) para formacao dos grupos experimentais de cada estudo: Estudo 1 - alta (grupo controle) e baixa (grupo alterado) atividade mitocondrial dos espermatozoides, Estudo 2 - alta (grupo controle) e baixa (grupo alterado) integridade do acrossoma dos espermatozoides, Estudo 3 - baixa (grupo controle) e alta (grupo alterado) fragmentacao do DNA dos espermatozoides e Estudo 4 - baixo (grupo controle) e alto (grupo alterado) niveis seminais de peroxidacao lipidica. A analise proteomica foi realizada utilizando LCMS/MS. Os grupos foram comparados por meio de analise univariada (teste t de Student) e analise multivariada (PLS-DA e analise discriminante). As proteinas significantes foram posteriormente submetidas a analise de enriquecimento funcional. Resultados: Nos estudos 1, 2, 3 e 4 foram observadas 506, 493, 474 e 629 proteinas, respectivamente. As funcoes enriquecidas no estudo 1 foram detoxificacao de EROs e ligacao a NADP (controle) e atividade de oxidoredutase intramolecular, catabolismo de aminoglicanos, inibicao de endopeptidases, lisossomos e resposta imune de fase aguda (alterado). As principais funcoes enriquecidas no estudo 2 foram resposta imune (controle) e inibicao de fosfolipase, metabolismo do acido araquidonico, exocitose, resposta inflamatoria aguda, resposta ao peroxido de hidrogenio e transporte lisossomal (alterado). As principais funcoes enriquecidas no estudo 3 foram metabolismo de carboidratos, regulacao de lipoproteinas, regulacao negativa da apoptose, metabolismo de hormonios, atividade de metalopeptidases, ligacao ao NAD e lisossomos (controle) e biossintese de prostaglandinas e ligacao a acidos graxos (alterado). As principais funcoes enriquecidas no estudo 4 foram biossintese de acidos graxos insaturados, atividade de oxidantes e antioxidantes e resposta celular ao estresse termico (alterados). Nos estudos 1, 2, 3 e 4 foram sugeridos 8, 6, 8 e 7 biomarcadores seminais de atividade mitocondrial, integridade acrossoma, fragmentacao de DNA e peroxidacao lipidica, respectivamente. Conclusoes: O perfil proteomico do plasma seminal reflete alteracoes funcionais dos espermatozoides e o nivelseminal de peroxidacao lipidica e diversas funcoes pos-genomicas estao relacionadas as alteracoes estudadas. Proteinas relacionadas as alteracoes funcionais dos espermatozoides e ao nivel seminal de peroxidacao lipidica constituem potenciais biomarcadores seminais para cada alteracao / Objective: To verify if the seminal plasma proteomic profile reflects sperm functional alteration and semen lipid peroxidation levels. Method: A cross-sectional study was performed including 156 normozoospermic patients. After semen retrieval by masturbation, an aliquot was utilized for semen analysis and another for evaluation of sperm mitochondrial activity, acrosome integrity and DNA fragmentation. The remaining semen volume was centrifuged and the supernatant seminal plasma was utilized for semen lipid peroxidation levels evaluation, and proteomic analysis. Patients were divided into percentiles (15%) to form the experimental groups: Study 1 – high (control group) and low (altered group) sperm mitochondrial activity; Study 2 – high (control group) and low (altered group) sperm acrosome integrity; Study 3 – low (control group) and high (altered group) sperm DNA fragmentation; Study 4 – low (control group) and high (altered group) semen lipid peroxidation levels. Proteomic analysis was performed by a LC-MS/MS approach. Groups were compared using univariate (Student’s t test) and multivariate (PLS-DA and discrimant analysis) analyses. Differentially expressed proteins were then utilized for functional enrichment analysis. Results: 506, 493, 474, and 629 proteins were observed in studies 1, 2, 3, and 4, respectively. Enriched functions in study 1 were reactive oxygens species detoxification, and NADP binding (control group), and intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (altered group). In study 2, main enriched functions were acute-phase response (control group), and phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammatory response, response to hydrogen peroxide and lysosomal transport (altered group). In study 3, main enriched functions were carbohydrates metabolism, lipoprotein regulation, negative regulation of apoptosis, hormone metabolism, metalopeptidases activity, NAD binding, and lysosomes (control group), and prostaglandin biosynthesis, and fatty acid binding (altered group). In study 4, enriched functions were unsaturated fatty acid biosynthesis, oxidants and antioxidants activity, and cellular response to heat stress (altered group). In total, 8, 6, 8, and 7 seminal biomarkers were proposed for studies 1 (mitochondrial activity), 2 (acrosome integrity), 3 (DNA fragmentation), and 4 (lipid per oxidation), respectively. Conclusions: The seminal plasma proteomic profile reflects sperm functional alterations and semen lipid perodixation levels, and several post-genomic functions are related to the studied alterations. Proteins related to sperm functional alterations, and semen lipid peroxidation levels constitute potential seminal biomarkers for each alteration. / BV UNIFESP: Teses e dissertações

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Eurycoma Longfolia Jack, also known as Tongkat Ali (TA) is a tropical plant belonging to the family of Simaroubaceae is widely distributed in South East Asian countries. The extracts of TA have been proven to have cytotoxicity, anti-proliferative and aphrodisiac properties. In vitro assays revealed cytotoxicity toward human breast cancer cell lines MCF-7, KB, CaOV-3, RD, DU-145and HepG2 human liver cancer cells and appear promising as a new chemotherapeutic agent against human cervical carcinoma (HeLa) cells. Although, there are extensive studies reported on its cytotoxicity benefits there are none pertaining to LNCaP human prostate cell line. Therefore, this study aimed at testing the effects of TA on LNCaP cells and prostate specific antigen (PSA) production. Materials and Methods This study investigated the effect of different concentrations of TA (0.0025, 0.025, 2.5, 25 and 250 g/ml) TA on LNCaP human prostate cancer cell line for 24 and 96 hours. The following parameters were investigated: morphology, cell viability (MTT), testosterone modulation, Annexin V-CY3 binding (Apoptosis), DNA fragmentation (TUNEL), caspase 3/7 activity (apoptosis), and PSA production. Results When observing the morphological changes of LNCaP cells exposed to TA, a clear increase in detachment and cell death via apoptosis as the concentrations of TA increased. The viability decreased significantly in both 24 and 96 hour treatment of TA at higher dosages (25 and 250 g/ml). The significant inhibitory effects on testosterone stimulated cell proliferation were seen at TA concentrations as low as 0.0025 μg/ml TA. At higher concentrations of TA (25 and 250 μg/ml), for all testosterone dosages a decreasing trend in proliferation was found. vii Testosterone concentrations of 10 nM showed maximum stimulation of cell proliferation for TA dosages up to 2.5 μg/ml. All concentrations of TA showed significant increase in apoptosis of the cells as dosages increased. A higher amount of DNA damage found at the highest dosage (250 μg/ml) of TA. The relative caspase 3/7 activation showed significant (P=0.0043) activation at the highest concentration (250 μg/ml) of TA. Relative PSA production resulted only a 5% increase with no significant difference at all doses indicting that TA does not change the cell PSA production and the decline in PSA concentration is due to LNCaP cells dying as a result of this exposure to TA. Conclusion In summary, the major finding of this study is that Tongkat Ali inhibits the viability of prostate cancer cell lines (LNCaP) through caspase-mediated pathway, as well as increased the level of apoptotic such as DNA fragmentation. In addition, Tongkat Alin also inhibited PSA production. In LNCaP cells, testosterone with the addition of TA does not increase the growth of the cells. However, more in-vitro and in-vivo studies are needed to establish the exact constituents of the extracts and their mechanism of action. Thus, this study opens perspectives on the use of Tongkat Ali preparations in the treatment of aging male symptoms, prostate cancer prevention or as additional treatment to standard prostate cancer therapy.

>Magister Scientiae - MSc / Cissampelos capensis, is commonly known by the Afrikaans name ‟dawidjies” or ‟dawidjieswortel”. C. capensis is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis rhizome extracts on sperm function.