Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology In the Faculty of Health and Wellness sciences at the Cape Peninsula University of Technology / Oxidative stress (OS) plays a major role in the pathogenesis of different conditions including male infertility. OS is caused by high amounts of reactive oxygen species (ROS) that exceed the antioxidant ability of a system. The sperm membrane is rich in polyunsaturated fatty acids and is prone to damage by ROS. Sperm damage decreases motility, concentration and viability. Testicular oxidative stress impairs Leydig cell function and leads to decreased hormonal control as the cells secrete testosterone. Studies have shown the role of antioxidants in the fight against OS. Recently more studies have been focused on the use of natural antioxidants derived from fruits, vegetables, nuts and oils for this purpose. The effects of Buriti oil supplementation have been investigated in the diet and it had been shown that it is rich in carotenoids and vitamin E. This study explored the antioxidant effects of Buriti oil on testicular tissue, epidymal tissue and hormonal function in male Wistar rats. Experiments were conducted for 6 weeks and male adult Wistar rats (10 weeks) were divided into two groups (n=30) for each group. The control group received standard rat chow and water while the experimental group received Buriti oil, rat chow and water daily. Both groups were exposed to natural physiological OS. The plasma, testicular and epididymal tissue samples of both groups were analysed for various parameters. Testicular weight and epididymal weight of rats fed with Buriti oil were significantly increased compared to the control group. Testicular and epididymal MDA levels were decreased in rats fed with Buriti oil compared to the control group. Superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activities were increased in both epididymal and testicular tissue of the Buriti oil fed group than the control group. Data were expressed in mean ± SEM. In conclusion, our findings suggest that Buriti oil supplementation could prevent OS damage in the male reproductive system.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-06-17T12:51:11Z No. of bitstreams: 1 Maria da Conceicao Carvalho.pdf: 3109620 bytes, checksum: 2f7f0bd4b88adbfe9f8dee53321968f7 (MD5) / Made available in DSpace on 2016-06-17T12:51:11Z (GMT). No. of bitstreams: 1 Maria da Conceicao Carvalho.pdf: 3109620 bytes, checksum: 2f7f0bd4b88adbfe9f8dee53321968f7 (MD5) Previous issue date: 2016-02-25 / Canids are part of the large number of endangered species. The survival of these species depends on the conservation of existing biodiversity. The use of gamete preservation techniques associated with reproductive technologies such as artificial insemination (AI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were developed to help propagate and preserve the genetic potential of canídeos.A testicular tissue cryopreservation might be a method used when ejaculate freezing techniques is not possible. This work set in two experimentos congelamento and vitrificaçãoin vitro, comparing different cryoprotectants (glycerol, dimetisufóxido (DMSO) and trehalose) in testicular tissue of domestic dogs (Canis familiaris). Fragments of 9 adult dogs testes were subjected to a cooling with 4 cryopreservation protocols: slow freezing with glycerol, dimethylsulfoxide (DMSO) or solid surface vitrification using glycerol or DMSO. Fragments of 3mm testicular parenchyma were separated into groups: control, subjected to slow freezing and vitrification. Histological evaluations of the fragments were performed before, after freezing and thawing by light and electron microscopy. Based on morphology and ultrastructure, the testicular tissue of slow freezing, there was no difference between the cryoprotectants. Among the vitrified groups, exposure to DMSO produced a greater structural integrity and architecture when compared to the glycerol group. Comparison of slow freezing and vitrification have shown that vitrification samples revealed more area consisting of tubular compartment, tubular lumen, seminiferous epithelium and conserved membrane. Furthermore, the intertubular compartment Leydig cells showed normal morphology and had characteristics typical of steroidogenic cells. From the results, it was concluded that vitrification DMSO is the most effective method for criopresevar testicular tissue of adult dogs, and can be used as a routine procedure. / Os canídeos fazem parte do grande número de espécies ameaçadas de extinção. A sobrevivência destas espécies depende da conservação da biodiversidade existente. O uso de técnicas de preservação de gametas associadas às tecnologias reprodutivas tais como, inseminação artificial (IA), fertilização in vitro (FIV) e injeção intracitoplasmática de espermatozoides (ICSI) foram desenvolvidas para ajudar a propagar e preservar o potencial genético dos canídeos.A criopreservação de tecido testicular pode vir a ser um método utilizado quando técnicas de congelação do ejaculado não é possível. A presente dissertação configurou-se em dois experimentoscongelamento e vitrificaçãoin vitro, comparando diferentes crioprotetores (glicerol, dimetisufóxido (DMSO) e trealose) em tecido testicular de cães domésticos (Canis familiaris). Fragmentos de testículos de 9 cães adultos foram submetidos a um arrefecimento com 4 protocolos de criopreservação: congelamento lento com glicerol, dimetilsulfóxido (DMSO), ou vitrificação superfície sólida, utilizando glicerol ou DMSO. Os Fragmentos de 3mm do parênquima testicular foram separados em grupos: controle, submetido ao congelamento lento e vitrificação. As avaliações histológicas dos fragmentos foram realizadas antes, após congelação e descongelação por microscopia óptica e eletrônica. Com base na morfologia e ultraestrutura, o tecido testicular de congelação lenta, não houve diferença entre os crioprotetores. Entre os grupos vitrificados, a exposição ao DMSO produziu maior integridade da estrutura e arquitetura quando comparado ao grupo de glicerol. A comparação do congelamento lento e vitrificação demonstraram que a vitrificação revelou amostras com mais área composta por compartimento tubular, luz tubular, epitélio seminífero e membrana conservada. Além disso, o compartimento intertubular mostrou células de Leydig tinham morfologia normal e características típicas de células esteroidogênicas. Diante dos resultados concluiu-se que a vitrificação com DMSO é o método mais eficaz para criopresevar tecido testicular de cães adultos, podendo ser utilizado como procedimento de rotina.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2018 / Diabetes mellitus is defined as a group of metabolic disorders characterised by chronic hyperglycaemia due to insufficient production and/or action of insulin and is regarded as one of the major sources of morbidity, mortality and economic burden to the modern society. A large body of scientific evidence support the fact that oxidative stress is elevated in diabetic conditions. Oxidative stress plays a significant role in the development of secondary complications of diabetes including diabetes-linked male sexual dysfunction. The management of sexual dysfunction as a secondary complication of diabetes relies on the management of the underlying diabetic condition. Glycaemic control and increased antioxidant protection are therefore necessary in the management of diabetes-induced oxidative stress male infertility. Pharmacological management of diabetes in form of various antihyperglycaemic, synthetic drugs has improved the outlook of diabetic patients; however, they are expensive, harbour unfavourable adverse effects and some have done little to prevent secondary complications of diabetes including diabetes-induced male sexual dysfunction. In addition to this, access to basic technologies for the management of diabetes mellitus and its secondary complications is still a challenge in low resource areas. Because of these challenges, there is a need to search for alternative remedies such as medication from natural products which are more affordable, well tolerated by the human body and are easily accessible. Medicinal plants are therefore viewed as an easily accessible and potent source of antioxidants capable of scavenging free radicals and fighting diabetes-induced oxidative stress. This study therefore investigated the effects of vindoline; an alkaloid extractable from Cantharanthus roseus in ameliorating diabetes-induced oxidative stress effects in testicular and epididymal tissues using male Wistar rats. Forty-eight (48), 6-week old male Wistar rats weighing between 190-230g with a conventional microbial status were divided into 6 groups, n=8, and used for this research project. Group 1 was the normal control, group 2 comprised non-diabetic rats treated with vindoline, and group 3 was the non-diabetic group of rats treated with glibenclamide- the standard drug for the treatment of diabetes. Group 4 was the diabetic control, group 5 comprised diabetic rats treated with vindoline and group 6 was the diabetic group of rats treated with glibenclamide. Diabetes was induced in group 4, group 5 and group 6 rats by subjecting them to 10% fructose water over a period of 2 weeks and thereafter, administering a single intraperitoneal injection of 40 mg/kg b.w streptozotocin (STZ). Fasting blood glucose levels were measured 72 hours after STZ injection and hyperglycaemia was confirmed where fasting blood glucose levels were more than 18mmol/l. The diabetic control (group 4) had higher fasting blood glucose levels, lower body weights as well as lower testicular and epididymal weights in comparison to the normal control (group 1). Additionally, the extent of lipid peroxidation in testicular and epididymal tissues of the diabetic control (group 4) was higher in comparison to that of the normal control (group 1). The diabetic control had lower testicular and epididymal antioxidant enzyme activities (superoxide dismutase and catalase) and lower oxygen radical absorption capacity (ORAC) in comparison to the normal control. Ferric reducing antioxidant power (FRAP) in testicular and epididymal tissues of the diabetic control (group 4) were not significantly different from those of the normal control (group 1). Treatment of diabetic rats with vindoline (group 5) for 5 weeks significantly reduced fasting blood glucose levels although the extent of reduction could not restore diabetic blood glucose levels to near-normal levels. Overall, treatment of diabetic rats with vindoline was able to minimise testicular oxidative stress as reflected by reduction in testicular malondialdehyde (MDA) levels. Furthermore, results of this study showed an increase in both testicular and epididymal catalase activities, an increase in epididymal SOD, an increase in testicular ORAC as well as an increase in both testicular and epididymal FRAP levels after 5 weeks of treating diabetic rats with vindoline (group 5). Epididymal lipid peroxidation levels, epididymal ORAC levels and testicular SOD levels of diabetic rats treated with vindoline (group 5) were however not significantly different from those of the diabetic control (group 4). Treatment of diabetic rats with vindoline or glibenclamide could not restore total body weights and testicular weights of group 5 and group 6 rats respectively, to near-normal levels. Furthermore, epididymal weights and testicular SOD activity of diabetic rats treated with vindoline (group 5) were not significantly different from those of the normal control (group 1). In conclusion, findings from this study demonstrated that treatment with vindoline could have protective effects against diabetes-induced oxidative stress in both testicular and epididymal tissues of male Wistar rats. Vindoline can therefore be considered a potential agent for the management of diabetes-induced oxidative stress male sexual dysfunction. Further studies with advanced technologies are however recommended to study the possible efficacy of vindoline in ameliorating diabetes-induced oxidative stress male sexual dysfunction. Furthermore, studies on the dose-dependent effects and long-term effects of vindoline administration on male reproductive function as well as the overall safety of treatment with vindoline are necessary.

Spermatogenesis is a complex and coordinated process of formation of sperms from the precursor spermatogonia, occurring inside the unique environment existing in the seminiferous epithelium. This process of development, characterized by concomitant changes in the cellular morphology, metabolism and differential gene expression, can be divided into 3 distinct phases: i) proliferation of the spermatogonia through mitosis; ii) meiosis or reduction division, which commences with transformation of the type B spermatogonia into primary spermatocytes and their subsequent entry into the meiotic prophase I. These primary spermatocytes, divide to form secondary spermatocytes, and then divide again to form haploid round spermatids; (iii) spermiogenesis or differentiation and maturation of the round spermatids without further division to form the unique spermatozoa (Kerr and De Kretser, 2006, Clermont, 1966, Heller and Clermont, 1964). This complex process of division and differentiation is regulated at three distinct levels: i) The extrinsic level where the gonadotropins and testosterone regulate gene expression in the germ cells sustaining their survival and differentiation (French, 2012); ii) The interactive regulation that involves interactions between the somatic cells such as the Sertoli cells and the germ cells; iii) The intrinsic gene expression associated with each step of development of the germ cells (Eddy, 2002) wherein each stage of differentiation is accompanied by precise stage-specific differential gene expression. (Kleene, 1996, Kierszenbaum et al., 2003, Sassone-Corsi, 2002, Kleene, 2001, Sassone-Corsi, 1997). Any alterations in this gene expression pattern leads to disruption and/or arrest of spermatogenesis at various stages, causing male infertility (Zorrilla and Yatsenko, 2013, Krausz et al., 2015). Many studies have been focused on investigating the underlying molecular mechanisms governing the process of germ cell development such as self-renewal, meiotic recombination and differentiation (Hecht, 1998, Grootegoed et al., 2000, Robles et al., 2017). Analysis of differential gene expression in isolated and purified populations of different germ cells have been very useful in the understanding of the genetic regulation of human spermatogenesis by providing information about the cell type-specific gene expression and regulation. (Meistrich et al., 1973, Bellvé, 1993, Meistrich et al., 1981, Chalmel et al., 2007). However, these methods are limited by the large amounts of tissue required, which is difficult to obtain in the case of humans (Schultz et al., 2003). Large-scale gene expression studies and the “omics revolution” have also helped in identifying some of the regulators of spermatogenesis (Carrell et al., 2016). In spite of advances in the current understanding of the regulation of spermatogenesis, the exact molecular mechanisms of how the genetic and epigenetic alterations affect human spermatogenesis are still unclear (Neto et al., 2016). The present study is an attempt to investigate the human testicular gene expression pattern in the germ cells of patients with various types of azoospermia, and correlate the same to infertility. Comparative analysis of the testicular transcriptomes of infertile individuals (with arrested spermatogenesis) with the control, fertile individuals (with normal spermatogenesis) would allow identification of the cell type-specific altered genes. Analysis of these genes would provide an insight into the genetic regulation of the progress of spermatogenesis as well as allow identification of the crucial genes responsible for the arrest. The first step in this study was to ascertain the exact status of spermatogenesis in patients’ testes. Forty-four azoospermic patients were classified clinically into two major groups – obstructive (OA) and non-obstructive (NOA) azoospermia and further classified using flow cytometric analysis of the germ cells. The patients with OA exhibited presence of the diploid, tetraploid and haploid cells indicating complete spermatogenesis (Group I: DTH). The patients with NOA showed incomplete spermatogenesis with arrest at either the meiotic stage showing the presence of diploid and tetraploid cells, but not the haploid cells (Group II: DT), or at the pre-meiotic stage with only diploid cells (Group III: D). This was further verified by RT-PCR analyses for specific markers for different testicular cells. The Group I patients showed expression of markers specific for the Leydig cell (LHCGR, HSD3B2 and HSD17B3), the Sertoli cell (FSHR, KITL), spermatogonia (KIT), tetraploid cells (CCNA1, LDHC) and haploid cells (PRM1). The Group II patients showed expression of CCNA1 and LDHC, but not of PRM1. The Group III patients did not express any of the haploid or tetraploid specific markers. The germ cell pattern was further confirmed by histology where a clear difference was seen across the groups in accordance with their flow cytometric profiles. Subsequent to grouping of the patient samples based on their testicular germ-cell pattern, microarray analysis was carried out with representative samples from each group leading to identification of diploid-/tetraploid-/haploid-specific (D/T/H) genes. The enrichment, probable pathways and network interactions of these identified genes were analyzed and found to be in agreement with the classification made in this study. Further, based on their network interactions, the genes that were under multiple modes of regulation and the transcription factors that regulated multiple pathways were selected for further analysis. In absence of an in-vitro system to study germ cell differentiation, the importance of the selected genes in the progression of human spermatogenesis was analyzed from the data extrapolated from information available in the literature about expression of each gene in the human testes (wherever available), known function of the genes in various somatic cells, function in developing and adult testes of model organisms and the data from the knockout or transgenic animals where disruption of the gene/s resulted in an arrest or disruption of spermatogenesis. Expression of all the putative crucial genes was analyzed in all the patients including the control patients at the transcript level and three selected genes (one from each group- D, T and H) were further validated at the protein level using immunohistochemistry. All the genes showed a similar pattern of amplification in the different groups of patients to the pattern observed from the microarray. The diploid-specific genes (selected based on the available literature) were mainly the inhibitors or regulators of the cell cycle (CDKN1A, GADD45A, FOXM1) (Xiong et al., 1991, Jin et al., 2002, Laoukili et al., 2005) and regulators of cellular proliferation (KLFs, FOS, SRF, ATFs, SMADs) (Garrett-Sinha et al., 1996, Persengiev and Green, 2003, Angel and Karin, 1991, Ten Dijke et al., 2002). Six diploid-specific genes that were potential regulators of spermatogenesis were identified to be probable causes for the arrest of spermatogenesis at the pre-meiotic stage. CDKN1A showed elevated expression at the transcript level which suggested that DNA-damage induced proliferation check (mediated through CDKN1A) in the diploid cells probably prevented these cells from entering meiosis. This was further verified at the protein level by immuno-staining for CDKN1A. Further, GADD45A, KLF4, FOS, MCL1 and SERPINE1 were identified as genes crucial for transition from the diploid to the tetraploid stage and their aberrant expression correlated to the arrest of spermatogenesis in the Group D patients. Six tetraploid-specific genes and four haploid-specific genes were identified to be potential regulators of the tetraploid-haploid transition and responsible for the meiotic arrest. Over expression of the pro-inflammatory genes such as CCL3, IL1B and IL8 (Guazzone et al., 2009) was seen in the testis of the arrested patients which suggested that there was a potential alteration of the normal testicular micro-environment. Expression of EGR2 (a spermatogonial-maintenance gene controlling mitosis (Joseph et al., 1988)) was seen in the nucleus of spermatocytes in group DT patients which indicated its role in the meiotic arrest. To understand the role of the haploid-specific genes in the context of spermatocyte differentiation, only those genes whose expression are reported in the spermatocytes and persist till the spermatid stage were selected. Lack of expression of CST8 was identified to be potentially responsible for loss of germ cell integrity, and the loss of GGN expression in the Group DT patients seemed to be a significant contributor to the genotoxic stress in these patients. In the arrested patients RFX2 (reported to be master regulator of spermiogenesis (Wu et al., 2016)) was seen to be down regulated at the transcript level which indicated its role in the control of meiosis. This was further confirmed by IHC, where expression of RFX2 was seen to be present in the tetraploid cells of the Group DTH patients while no expression was seen in the tetraploid cells of Group DT patients. Thus, this study identified a role for RFX2 in the regulation of meiosis in humans, similar to the findings reported in rats (Horvath et al., 2009). The study also identified autophagy as a mechanism for the clearance of the arrested cells in NOA patients. IHC data using αLC3B showed that autophagy was up regulated in the arrested patients as compared to the Group DTH patients suggesting its role in cell survival and recycling of nutrients. Further, in-situ TUNEL labeling of tissue sections from the different groups (DTH, DT and D) revealed that there were no difference in the status of apoptosis in the azoospermic patients. The latter observation further corroborated with the elevated expressions of CDKN1A, GADD45A, MCL1, TNFAIP3 (reported to ensure cell survival by negatively modulating apoptosis) as seen in the NOA patients. In conclusion, this study identifies several genes that control the progression of spermatogenesis, including the genes whose alterations contribute towards an arrest in spermatogenesis, especially in azoospermia. These identified genes may be used as novel markers in the diagnosis of male infertility. The study opens up the possibility of using the identified genes as future therapeutic targets using small molecular regulators for treatment of infertility as well as targets for male contraception. The study also identifies a novel role for autophagy in patients with NOA which opens up new avenues for further investigation. Thus, this study is the beginning of understanding the complex events that regulate spermatogenesis.