Philosophiae Doctor - PhD / Introduction: Typha capensis, commonly referred to as bulrush also called "love reed'' growing in Southern Africa's wetlands, is one of South Africa indigenous medicinal plants that are traditionally used to treat male fertility problems and various other ailments. Previous studies revealed that T. capensis has indeed a beneficial effect on male reproductive functions and aging male symptoms. The T.capensis rhizomes are used in traditional medicine during pregnancy to ensure easy delivery, for venereal diseases, dysmenorrhea, diarrhoea, dysentery, and to enhance the male potency and libido. Typha genuses contain flavones and other phenolic compounds, which exhibit anti-oxidative capacity. Materials and Methods: This study encompasses three parts (part 1: Exposure of different cell lines to crude aqueous extracts of T. capensis rhizomes; part 2: HPLC analysis of Typha capensis crude rhizome extract and exposure of different cell lines to the F1 fraction of the summer season; part 3: Compound identification by means of NMR spectrometric analysis and exposure of different cell lines to bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes. Part 1: TM3-Leydig cells and LNCaP cells incubated with different concentrations of crude aqueous extract of T. capensis rhizomes (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology and viability (determined by means of MTT assay). Part 2: The crude extract HPLC profiles were obtained by preparing the extracts for different seasons (Autumn, Winter, Spring, Summer). TM3-Leydig cells, LNCaP cells and PWR-1E cells incubated with different concentrations T. capensis rhizomes extract F1 fraction of the summer season (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology was observed and recorded, viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding), DNA fragmentation (determined by means of the TUNEL assay). Part 3: NMR spectrometric analysis was performed on a 13C spectra were recorded at 400 MHz. TM3-Leydig cells and LNCaP cells incubated with different concentrations of bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes, for acute exposure (24, 96 hours) and chronic exposure (96 hours), after incubation, the following parameters were evaluated: cell morphology and viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding) and DNA fragmentation (determined by means of the TUNEL assay). Results: Part 1: for TM3-Leydig cells the results reveal no observable morphological changes and no significant influence on cell viability except at highest concentration indicating cellular stress. However, LNCaP cells showed a decline in cell viability at the incubation period 96 hours (-82.4%) more than 24 hours (-64.7%) indicating more cell death. Part 2: HPLC data showed that the most effective fraction was the F1 fraction from the summer harvest. Results revealed that the T. capensis rhizome extract F1 fraction of the summer season significantly enhanced testosterone production in TM3 cells and was more toxic towards cancer cells (LNCaP cells ) compared to the normal cell lines (TM3-Leydig, PWR-1E cells). Part 3: NMR data showed 2 bioactive compounds which were identified as Quercetin and Naringenin. The assays showed that LNCaP cells are more sensitive to the cytotoxic effects and apoptosis induction of both compounds, whereas, the assays resulted in weak effects toward TM3-Leydig cells. However, testosterone production in TM3-Leydig cells was significantly enhanced at low concentrations of Quercetin and Naringenin at all exposure types (acute and chronic) testosterone beak significantly at around 0.100 and 0.125 μg/ml (P<0.0001), stimulatory activity in a dose-dependent manner. Conclusion: Typha capensis enhanced the production of testosterone and might be useful to treat male infertility and aging male problems. Results further reveal that the F1 fraction from the summer harvest had highest biological activity. This study, for the first time, investigated the effects of bioactive compounds (Quercetin and Naringenin) yeilded from aqueous extraction of Typha capensis rhizomes in cell lines investigating male reproductive functions. Active compounds present in the rhizomes have caused an increased production of testosterone level in TM3-Leydig cells. Furthermore, the active compounds of Typha capensis rhizomes in the high dose had a negative effect on the percentage of DNA fragmentation in LNCaP cells. When compared to the effect of the low dose, the two compounds induced significant apoptosis in cancer cell line (LNCaP) compared with the normal cell line (TM3-Leydig). The isolated compounds are significantly selective towards the cancer cells than the normal cell compared with the exposure of bioactive compounds used in this study.

La spermatogenèse est un processus complexe qui dépend de la coopération de nombreux gènes. Son produit final le spermatozoïde, est un sujet d’étude idéal car il renferme à la fois des indices d’événements passés ainsi que des informations qui seront transmises à l'ovocyte lors de la fécondation. L'identification de nouveaux acteurs de la spermatogenèse, des modifications spécifiques de l'ADN du sperme ou la présence de transcrits spécifiques pourraient servir comme biomarqueurs dans le diagnostic de l’infertilité. Cette thèse avait pour but d’analyser le génome, le transcriptome et l’épigénome de spermatozoïdes dans le contexte de l'infertilité masculine. Nous avons identifié de nouvelles causes génétiques et confirmé la présence d'anomalies de méthylation dans le sperme d'hommes infertiles. Nous avons découvert 20 mutations dans le gène SOX8, chez des patients atteints de trouble du développement sexuel ou d'infertilité masculine ou féminine, qui apparaît comme un régulateur du développement et de la fonction gonadique. Par séquençage d’exome, une mutation dans le gène ATAD2 modeleur de la chromatine spécifique de la lignée germinale mâle fut également identifiée. Par RNA-seq et MeDIP-chIP du sperme d’hommes fertiles et infertiles, nous avons caractérisé la signature transcriptionnelle du sperme. La majorité des ARNs spermatiques humain est remarquablement conservée chez les mammifères placentaires suggérant des fonctions ancestrales importantes. Enfin, nos données transcriptomiques et épigénétiques tendent à indiquer qu’une expression et une régulation adéquates des gènes impliqués dans le remodelage de la chromatine constituent un facteur clé pour la fertilité masculine. / Spermatogenesis is a complex process which depends on the cooperation of many genes. The end-product, the spermatozoon, is an ideal subject for study since it carries both clues of the past events and information which will be transmitted to the oocyte at fertilization. The identification of main actors of spermatogenesis, specific modifications of sperm DNAs or sperm specific isoforms could improve our understanding of a such complex mechanism and could serve as a determination of biomarkers or diagnostic tools for fertility. The aim of the project was to go further three omes: genome, epigenome and transcriptome of mature human sperm in the context of male infertility. We identified new genetic causes of male infertility and confirmed the presence of methylation abnormalities in sperm cells of infertile men. Firstly, SOX8 gene was found mutated in a cohort of 20 patients with disorder of sex development and male or female infertility. Similarly, to NR5A1, SOX8 appears to be a novel regulator of gonadal development and function. Then by exome-sequencing, we identified a homozygous nonsense mutation in the male germline-specific chromatin modeler ATAD2. Furthermore, RNA-seq and MeDIP-chIP of sperm from fertile and infertile men along with bioinformatics analyzes of the generated data, enabled us to characterize more deeply the normal sperm transcriptional signature. We also found that the majority of human sperm RNAs are remarkably preserved in placental mammals suggesting crucial ancestral functions. Finally, proper expression and regulation of chromatin remodelers seem to be critical for male fertility, as revealed by both the transcriptomic and the epigenetic data.

L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le séquençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologiquePour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’ERO chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de sperme. / Male infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved in spermatogenesis and cellular components involved in morphogenesis, whilst signature genes of asthenozoospermia were enriched by genes implicated in ribosome and cilium assembly.We complemented this work by a parallel epigenetic analysis of the global epigenetic landscape in infertile men. We compared the levels of reactive oxygen species (ROS), DNA integrity and global epigenetic parameters in sperm from 33 infertile subjects with abnormal semen parameters compared to fertile individuals. We pointed out that infertile men are characterized by strikingly high levels of reactive oxygen species (ROS) which were in part negatively correlated with the global DNA methylation, and positively correlated with the levels of 5-hydroxymethylcytosine and 5-formylcytosine (active demethylation intermediates). These findings suggest that male infertility associated with oxidative stress shapes the sperm epigenetic landscape. In summary, this original work yielded a transcriptional portrait of sperm abnormalities and provided valuable resources that would further elucidate sperm pathologies.

Background: The trend of male infertility is increasing and it correlates with industrial development which has introduced various industrial contaminants in the environment and human life. PFASs are one of those contaminants. Humans are exposed to PFAS through various routes and sources, especially through food and diet. On the other hand, some foods are beneficial for the male reproductive system. Aim: The study aim is to investigate the role of per-or poly-fluoroalkyl substances (PFASs) contaminated food in male infertility. Method: This study has used scoping review to assess the current knowledge concerning the aim that what has been done in this field. A systematic search was done in three databases namely PubMed, Web of Science, and Scopus, as well as manual search of related articles and reference lists during March-April 2023, and found 68 articles. Inclusion criteria included all articles written in English, related to humans, and PFAS exposure to adult males through food worldwide. Articles related to only the female population, other non-human species, human exposure to PFAS in fetal life, molecular level information, information about transport mechanisms of PFAS in food, other environmental contaminants and with no food contamination, toxicological, or health impact information were excluded. Titles, abstracts, and selected full-length articles were read and evaluated. CEBMa checklist for critical appraisal of the cross-sectional study, CASP checklist for systematic review, and randomized control trial were used as quality assessments of articles. Results: In total 68 abstracts were initially found, of which 15 were included in the review. Majority of the studies were either from Europe (European panel data from 16 different European countries) (n=2) or any European country (Italy=2, Georgia=1, Denmark=1, Sweden=1, Spain=1, Denmark + Sweden=1, Denmark + USA + Faroe Islands=1) and the rest were from USA (n=2), Australia (n=2) and Peru (n=1). The articles were analyzed according to the concept combinations of PFAS, food, or male fertility. Out of 15 included studies 3 deals with Pfas in food, 8 with Pfas and male fertility, 2 with male infertility related to PFAS in food, 1 with diet and human PFAS levels, and 1 with relation of food to male fertility. The articles included were of either good or moderate quality. Studies related to the direct impact of PFAS through food on male fertility are scarce. According to most of studies, PFASs have a significant impact on male hormones and sperm morphology but there was no significant association was found between PFAS levels to other seminal parameters. The combination of different PFAS and the combination of PFAS with other contaminants may alter the impact on male fertility. PFAS may have a long-term effect on human spermatogonial cells. Mediterranean diet is associated with high serum levels of PFAS in humans. Fish is a major source of PFAS exposure through food. 8 oz. of fish is recommended without causing health hazards, provided thatthe levels of PFAS contaminations in fish are not high. Conclusion: More studies especially longitudinal studies concerning PFAS exposure through food and its impact on male fertility are needed. No studies are found from the African continent and low-income countries. There is a need to check the levels of contamination of PFAS in food so that the health impacts on male fertility can be avoided.

Background: Human life without sperm is not possible. Therefore, it is alarming that the fertilizing ability of human spermatozoa is continuously decreasing. The reasons for that are widely unknown, but there is hope that metabolomics-based investigations may be able to contribute to overcoming this problem. This review summarizes the attempts made so far. Methods: We will discuss liquid chromatography–mass spectrometry (LC-MS), gas chromatography (GC), infrared (IR) and Raman as well as nuclear magnetic resonance (NMR) spectroscopy. Almost all available studies apply one of these methods. Results: Depending on the methodology used, different compounds can be detected, which is (in combination with sophisticated methods of bioinformatics) helpful to estimate the state of the sperm. Often, but not in all cases, there is a correlation with clinical parameters such as the sperm mobility. Conclusions: LC-MS detects the highest number of metabolites and can be considered as the method of choice. Unfortunately, the reproducibility of some studies is poor, and, thus, further improvements of the study designs are needed to overcome this problem. Additionally, a stronger focus on the biochemical consequences of the altered metabolite concentrations is also required

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Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-18T13:22:41Z No. of bitstreams: 1 BARBARA MARIOTTO BORDIN.pdf: 616329 bytes, checksum: 1c5d8ba717c3f2049b989263d9efa19f (MD5) / Made available in DSpace on 2016-08-18T13:22:41Z (GMT). No. of bitstreams: 1 BARBARA MARIOTTO BORDIN.pdf: 616329 bytes, checksum: 1c5d8ba717c3f2049b989263d9efa19f (MD5) Previous issue date: 2009-03-16 / Oestrogen Receptor (ER ) gene plays an important role in the regulation of fertility in both males and females. The RsaI polymorphism in exon 5 of ER has been shown to be associated with male infertility in Caucasian patients. The aim of this study was to investigate the frequency of this polymorphism in the etiology of idiopathic male infertility and to correlate with smoking and ethylism habits, xenobiotic contact and mumps. We analyzed 287 Brazilian men, including 161 infertile and 126 fertile men to evaluate the association of RsaI polymorphism in male infertility. The RsaI variant alleles (AA, AG or GG) of the patients were determined by allele-specific polymerase chain reaction. Compared with a control group (normozoospermic men), the frequency of the heterozygous RsaI AG-genotype was four times higher in infertile men (9,94 vs. 2,38%; P = 0,01), five times higher in azoospermic men (11,36 vs. 2,38%; P = 0,02) and seven times higher in teratozoospermic men ( 17,79 vs. 2,38%; P = 0,001). The frequency of the heterozygous RsaI AG-genotype was three times higher in infertile smokers (23,8 vs. 7,4%; P = 0,038) compared with infertile nonsmokers and nine times higher in azoospermic smokers (66,7 vs. 6,9%; P = 0,035), compared with azoospermic nonsmokers. The RsaI polymorphism in the ER gene may have modulating effects on human spermatogenesis. There seems to be consistent association between RsaI polymorphism and smoking habits in infertile men. / O gene Receptor de estrogênio (RE ) desempenha um papel importante na regulação da fertilidade tanto em homens e mulheres. O polimorfismo RsaI no éxon 5 do RE tem-se mostrado associada com infertilidade masculina em caucasianos. O objetivo deste estudo foi investigar a freqüência deste polimorfismo na etiologia da infertilidade idiopática masculina e sua correlação com o tabagismo, etilismo, contato com xenobióticos e caxumba. Nós analisamos 287 brasileiros, incluindo 161 inférteis e 126 homens férteis para avaliar a associação do polimorfismo RsaI do gene RE com a infertilidade masculina. Os alelos variantes do polimorfismo RsaI (AA, AG ou GG) foram determinadas pela reação em cadeia da polimerase alelo-específica. Em comparação com um grupo controle (homens normozoospérmicos), a freqüência do genótipo heterozigoto RsaI-AG foi quatro vezes maior em homens inférteis (9,94 vs. 2,38%, P = 0,01), cinco vezes maior em azoospérmicos (11,36 vs. 2,38% , P = 0,02) e sete vezes maior em teratozoospérmicos (17,79 vs. 2,38%, P = 0,001). A freqüência do genótipo heterozigoto RsaI-AG foi três vezes maior nos fumantes inférteis (23,8 vs. 7,4%, P = 0,038) em comparação com não fumantes inférteis e nove vezes maior em fumantes azoospérmicos (66,7 vs. 6,9%, P = 0,035), comparado com não fumantes azoospérmicos. O polimorfismo RsaI no gene RE pode ter efeitos sobre a modulação da espermatogênese humana. Parece haver uma associação consistente entre o polimorfismo RsaI e tabagismo em homens inférteis.

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.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-10-28T13:15:45Z No. of bitstreams: 3 Dissertação - Roberta Machado de Oliveira Frota Curado - 2015.pdf: 1412350 bytes, checksum: 3488f3ea9d285b268adb991ca619df94 (MD5) anexos - dissertacao - roberta m de o f curado.pdf: 1349907 bytes, checksum: d9adb8c0cfdeca8fe004bc23cde29b9b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-28T14:56:17Z (GMT) No. of bitstreams: 3 Dissertação - Roberta Machado de Oliveira Frota Curado - 2015.pdf: 1412350 bytes, checksum: 3488f3ea9d285b268adb991ca619df94 (MD5) anexos - dissertacao - roberta m de o f curado.pdf: 1349907 bytes, checksum: d9adb8c0cfdeca8fe004bc23cde29b9b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-28T14:56:17Z (GMT). No. of bitstreams: 3 Dissertação - Roberta Machado de Oliveira Frota Curado - 2015.pdf: 1412350 bytes, checksum: 3488f3ea9d285b268adb991ca619df94 (MD5) anexos - dissertacao - roberta m de o f curado.pdf: 1349907 bytes, checksum: d9adb8c0cfdeca8fe004bc23cde29b9b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-05 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Male infertility affects about half of couples with infertility history and is considered a multifactorial syndrome, including a broad spectrum of diseases. Chromosomal abnormalities are a major cause of human infertility and interfere with spermatogenesis. Infertility in patients with Klinefelter's syndrome (KS) is a consequence of degeneration of germ cells and that affects about 4% of infertile men. Objective: To investigate the presence of chromosomal abnormalities in infertile men with azoospermia or oligozoospermia seen at the Human Reproduction Laboratory of the Hospital das Clinicas (LabRep -HC) of the Federal University of Goiás, in 2013. Methodology: Descriptive study. Metaphases were analyzed in GTG bands obtained from lymphocytes cultures of 20 infertile men idiopathic causes. Results: The patients' ages ranged from 26-59 years and the design attempts ranged on average of 5 (± 5.02) years. In 3/20 (15%) patients were found karyotype 47, XXY (SK) and the rest, 17/20 patients had a normal karyotype. Conclusion: Genetic testing can help identify which patients would benefit from the technical reproduction. These studies are relevant because the assisted reproduction techniques ignore the process of natural selection and some classic chromosomal abnormalities end some deleterious mutations that could through generations. Thus, genetic assessment can lead to genetic counseling and hence the primary and secondary prevention of congenital defects in offspring of patients with male infertility. This study helps to assess the prevalence of chromosomal abnormalities in some men treated at LabRep - HC UFG. / A infertilidade masculina afeta cerca da metade dos casais com histórico de infertilidade e é considerada uma síndrome multifatorial, incluindo um amplo espectro de doenças. As anormalidades cromossômicas são uma das principais causas de infertilidade humana e interferem na espermatogênese. A infertilidade em indivíduos com a síndrome de Klinefelter (SK) é uma consequência da degeneração de células germinativas e acomete cerca de 4% dos homens com infertilidade. Objetivo: Investigar a presença de anormalidades cromossômicas de homens inférteis com azoospermia ou oligozoospermia, atendidos no Laboratório de Reprodução Humana do Hospital das Clínicas (LabRep -HC) da Universidade Federal de Goiás, no ano de 2013. Metodologia: Estudo descritivo. Foram analisadas metáfases em bandas GTG obtidas a partir de cultura de linfócitos de 20 homens inférteis de causas idiopáticas. Resultados: A idade dos pacientes variou de 26-59 anos e as tentativas de concepção variaram em média de 5 (± 5,02) anos. Em 3/20 (15%) pacientes foi encontrado cariótipo 47,XXY (SK) e o restante, 17/20 pacientes, apresentaram cariótipo normal. Conclusão: Os testes genéticos podem ajudar a identificar quais pacientes poderiam ser beneficiados com as técnicas de reprodução. Estes estudos são relevantes, pois as técnicas de reprodução assistida ignoram o processo de seleção natural e algumas anormalidades cromossômicas clássicas ou algumas mutações deletérias que poderiam ser herdadas. Desta forma, a avaliação genética pode levar ao aconselhamento genético e, consequentemente, à prevenção primária e secundária dos defeitos congênitos nos descendentes dos pacientes com infertilidade masculina. O presente estudo contribui para avaliar a prevalência de anormalidades cromossômicas de alguns homens atendidos no LabRep - HC da UFG.

Parmi les couples avec un projet parental, le facteur masculin d’infertilité est responsable d’environ 20%. Malgré de longues années d’activités d’assistance médicale à la procréation, un nombre important de cas reste idiopathiques. Considérant le nombre élevé des gènes potentiellement impliqués dans la gamétogenèse, il est fort probable que la majorité des formes ‘idiopathiques’ sont d’origine génétique. Dans l'étude présente, nous avons d’identifier deux nouveaux gènes impliqués dans une infertilité masculine. Nos données suggèrent que la mutation dans TEX15 puisse corréler avec une diminution du nombre de spermatozoïdes au fil du temps. Un test diagnostique identifiant la mutation chez un patient pourrait fournir une indication d’organiser au plus tôt une cryopréservation du sperme. On a aussi identifié MAGEB4 liées à l’X comme un nouveau gène impliqué dans une infertilité masculine héritée. Cette étude fournit le premier indice sur la fonction physiologique d'une protéine MAGE. / Among couples with a desire for a child, male factor is responsible approximately 20%. Despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Considering the high predicted number of genes involved in male gametogenesis, it is likely that most ‘idiopathic’ forms may have a genetic origin. In the present study, we have defined two new genes implicated in male infertility. Our data suggested that a nonsense mutation in TEX15 correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age. We also identified MAGEB4 as a new X-linked gene involved in an inherited male infertility. This study provides the first clue on the physiological function of a MAGE protein.