Dissertations / Theses on the topic 'Infertility, Male - Genetic aspects'

Approximately one in twenty men has impaired spermatogenesis due to mutation of genes involved in the establishment or maintenance of fertility. Our understanding of male infertility is complicated by the variable phenotypes produced by similar genetic changes, largely due to the practise of screening a single fertility gene in isolation. This thesis aimed to increase our understanding of the role of synergistic mutations in relation to differences in semen quality. Each sample was analysed for mutation in: CAG trinucleotide repeat variation in the X-linked androgen receptor (AR) gene, micro deletion within the three Y chromosome azoospermic factor (AZF) regions, and CAG trinucleotide repeat variation and exonuclease domain mutation in the nuclear polymerase gamma (POLγ gene. These genes have been associated with reduced semen quality in past research. Each gene region was amplified by polymerase chain reaction (PCR), followed by sequencing. Suspected AZF micro-deletions were confirmed by Southern blot hybridisation. Associations with semen quality were evaluated using either a t-test or Gtest for independence at α=0.05. Yq AZF micro-deletions were observed in 6.6% (14/211) of men with poor semen quality but not in normozoospermic samples (0/104); P<0.001). Micro-deletion frequency was greatest in azoospermic and severely oligoasthenozoospermic individuals (15% and 11.5%, respectively). AR CAG repeat length ranged from 9-38 CAG repeats in the normozoospermic population (n=98) and 13-31 CAG repeats in men with poor semen quality (n= 119). Variation in AR CAG trinucleotide repeat number was not significantly related to poor semen quality (P>0.05). Variation in POLγ CAG repeat number was not significantly different between normozoospermic men (n=93) and men with poor semen quality (n= 182); P>0.05. No nucleotide changes were observed in any of the three POLγ exonuclease motifs (n=83 normozoospermic and 191 non-normozoospermic motif, 61 and 65 motif II, and 60 and 64 motif III). Although most gene regions did not show an association with poor semen quality on their own, there was a general trend towards greater severity of impaired spermatogenesis with the presence of both Yq micro-deletion and mitochondrial DNA substitutions or moderately expanded AR CAG repeats. These results support the idea that male infertility is a complex process, due to many factors, some of which act dominantly and others act in concert.

This dissertation revolves around three main elements: 'male infertility'; existing social science research on infertility; and ethno methodology. The substantive topic 'male infertility' is enclosed in quotation marks for two reasons. First, following the overall form of ethno methodological inquiry, the aim is to explicate how the sense and order of 'male infertility' is constituted through available socially organised procedures; hence, the quotation marks are used to 'bracket' the phenomenon and focus on the methods that make it available. Second, 'male infertility' is a convenient shorthand topic label, a general organising concept, as opposed to a precise label for a tightly defined phenomenon. While this study's approach makes it very different to existing sociological studies of infertility, the difference is not to the extent of isolation - a strong attempt is made to engage with prior studies. Often this engagement takes a critical form, the general argument being that sociological studies which approach phenomena for the way they 'bear the marks' of societal structures, will ignore the incarnate orderliness of social action - that is, the way social action is readily explicable to members, in and as it occurs, using the resources at-hand, with 'no time-out'. Ethno methodology suggests that this ready explicability is based upon taken-for-granted, socially organised sense-assembly practices - thus, this study's argument that the content, the intelligibility of 'male infertility is interdependent with the social scenes and embedded socially organised procedures, with and within which 'male infertility' is found. Form and content stand or fall together. Consistent with this viewpoint, four empirical analyses of the social organisation of 'male infertility' are offered. The specific topics discussed are: the conversational disclosure of infertility; the language of reproduction; humour and infertility; and high rates of non-response by men in studies of infertility. In general, the empirical analyses are 'indifferent' to the topic of study, that is, there is no overriding aim of offering practical correctives or broader socio-political critique. However, in at least one empirical chapter a more critical stand is taken, and, in the concluding chapter, it is argued that an ethno methodological descriptivist approach can have socio-political implications. Overall, the study supports the growing trend for ethno methodological insights to be utilised in the study of substantive topics; and, since the dissertation is a form of writing 'anew', it can be considered to minimally change 'male infertility' as a form of life.

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

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

Studies have discovered higher frequencies of single nucleotide polymorphisms (SNPs) in different mitochondrial genes are associated with subnormozoospermia. However, the frequencies of SNPs in ND1 and ND2 are not unknown. The present research was aimed to determine the frequencies of SNPs in ND1 and ND2 genes of the mitochondrial genome in fertile and subfertile men and whether changes in codon usage was associated with fertility phenotypes. Total genomic DNA from 157 semen samples was extracted using the proteinase K/SDS digestion procedure, followed by phenol/chloroform purification and ethanol precipitation. ND1 and ND2 genes were amplified respectively from 80 and 92 DNA samples from different fertility groups. Each PCR product was sequenced to identify mutations. Codon change resulting from a nucleotide substitution was determined by comparison with a reference mtDNA sequence obtained from the NCBI database. The frequency of codon usage in the reference mtDNA was determined by the computer program MEGA version 2.1. Eleven synonymous nucleotide substitutions and two non-synonymous substitutions were found in this study. Four SNPs were previously characterized; all SNPs were homoplasmic. None of the SNPs were likely to affect the function of the proteins on the basis of the hydrophobicity plots or secondary structure predictions. Sixty two percent of synonymous mutations were found to change from a high to a low relative codon usage values; 37% of synonymous mutations changed from a low to a high relative usage value. Chi-square (χ²) test (χ²= 0.067 with 1 d.f.) showed that there was no significant difference at the 5% level between these changes. Thus, change in codon usage was not related to semen quality in men. Further, there were no statistically significant differences in the observed frequencies of SNPs of fertile and subfertile men. However, the sample size was small and this study was only focused on a single NZ Caucasian population. Further study including larger and more diverse population samples may provide further insight into the functional importance of codon usage and its relevance to fertility

It has been estimated that approximately 15% of couples attempting to attain pregnancy are unable to do so. A female factor is the primary aetiology in nearly 40% of these couples and in another 30% the factors are purely male. A combmation of male and female factors has been implicated for the remaining 30% of cases. Infertility is a common problem among men with testicular cancer. Such men are especially affected by infertility and they often decide to undergo sperm banking (the collection and freezing of sperm) before beginning chemo/radiotherapy.

Microarray technology holds great promise for allowing the global analysis of the testicular transcriptome. The aim of this project is to demonstrate the feasibility and utility of the microarray approach to the study of the spermatogenesis. This thesis details the construction, validation and characterisation of two subtracted cDNA libraries, and their use in microarray analysis of several different models of murine-spermatogenesis. These models consist of the postnatal first wave of spermatogenesis, a series of germ-cell depleted mutant models, a series of models with deletions of the Y chromosome long arm (Yq) and the TM4 juvenile Sertoli cell line under stimulation with follicle stimulating hormone (FSH) and dihydrotestosterone (DHT). Analysis of the first wave has been used to develop a framework of expression patterns associated with different germ cell types within which to analyse other models, allowing better characterisation of the spermatogenic arrest in the germ cell depleted models. Candidate genes (including Xmr, mgclh and the unknown EST AK0059221) have been discovered with regard to the morphological abnormalities exhibited by the spermatozoa in the Yq deletion models. Microarray analysis also leads to a better understanding of the nature of the TM4 cell line, specifically how well it can be said to demonstrate the range of responses of normal Sertoli cells. Selected gene profiles of interest have been validated in this laboratory and others using established techniques of real-time RT-PCR and Northern blotting. The success of microarray technology in analysing these models demonstrates its utility for the study of spermatogenesis, and the utility of the constructed libraries in further microarray experiments. A fuller understanding of genetic events during spermatogenesis may enable therapeutic intervention in cases of male infertility, or yield insights leading to development of novel contraceptives, as well as casting light on the fundamental processes of mitosis, meiosis, fate commitment, cell differentiation and apoptosis.

A espennatogênese é andrógeno-dependente, porém muitos homens com problemas na espermatogênese têm níveis hormonais androgênicos normais. O mau funcionamento do receptor de andrógenos (Androgen Receptor- AR) é a possível causa deste problema. O AR é membro da família dos receptores nucleares e é codificado pelo Gene do Receptor de Andrógenos, que é de cópia única, localizado no cromossomo X e possui oito éxons. O éxon 1 contém um segmento com repetições CAG, que codifica poliglutamina. O trato glutamínico é polimórfico e varia de 1 O a 35 repetições na população normal. Alterações no segmento CAG estão envolvidas na etiologia de doenças neurodegenerativas (repetições CAG > 40) e câncer de próstata (< a 16 repetições). Mutações no Gene do AR estão correlacionadas com a síndrome de insensibilidade aos andrógenos, que varia desde a completa feminilização até homens inférteis com fenótipo normal. O objetivo do presente estudo constituiu em investigar a correlação entre o polimorfismo CAG e a prevalência de mutações nos éxons 5 e 7 e a alteração dos parâmetros seminais na população da Serra Gaúcha. O segmento CAG e a região codificadora dos éxons 5 e 7 foram amplificadas pela técnica de reação da polimerase em cadeia (PCR) e analisadas pela técnica de seqüenciamento automatizado. A média das repetições CAG do grupo de pacientes (n = 45) foi de 20,04±3,94 e a média do grupo controle (n = 45) foi 20,64±3,71. Não há significânc:ia estatística entre elas (p = O, 459). Verificamos correlação entre as repetições CAG e a morfologia seminal (Organização Mundial da Saúde) (p = O, 032; r = O, 349). Porém não se verificou associação com os demais parâmetros seminais: concentração (p =O, 134; r= O, 227), motilidade (p = 0,184; r= 0,202), morfologia (Kruger) (p = 0,213; r= 0,210). Não foram detectadas mutações nos éxons 5 e 7 nos dois grupos de estudo. Nosso estudo sugere que polimorfismo CAG e a presença de mutações nos éxons 5 e 7 não estão correlacionados com alterações seminais no grupo de estudo.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES.
Spermatogenesis is androgen-dependent, but most men with impaired spermatogenisis have normal serum androgens leveis. Malfunction of androgen receptor (AR) may be a possible cause of this problem. AR is a member of the nuclear receptor family. It encodes a single copy gene in the X chromosome. The AR Gene is composed by eigth exons. The exon 1 contains a segment of CAG repeats, translated to polyglutamine. This glutamine repeat tract is polymorphic and its size varies from 10 to 35 in normal population. These changes have clinicai implications for human diseases: neurodegenerative disorders (CAG repeat > 40) and prostate cancer (CAG repeat < 16). Mutations in AR Gene cause a variety of defects related to androgen insensitivity, ranging from complete feminization to phenotypic males with infertilty. The aim of this study was to investigate the relationship between CAG repeat length and the prevalence of mutations in the exon:s 5 and 7 and impaired spermatogenesis in a Serra Gaucha population. The CAG repeat leng1th and the coding region of exons 5 and 7 was amplified by polymerase chain reaction (PCR) and analyzed by direct DNA sequencing. The mean CAG repeat length in the experimental group (n = 45) was 20.04±3.94 and in the control group (n = 45) was 20.64±3.71. No difference was found between patientes and controls in the mean values (p = 0.459). 1We found relationship between CAG repeat and morphology (World Health Organization) (p = 0.032; r = 0.349). However, the correlation was not found between CAG repeat and others seminais parameters: concentration (p = 0.134; r= 0.227), morphology (Kruger) (p = 0.213;. r= 0.210) and motility (p = 0.184; r= 0.202). No mutations were detected in the coding regions of exons 5 and 7 in both groups. Our study suggests that CAG polymorphism and mutations in the exons 5 and 7 are not likely to cause of spermatogenesis abnnormalities in our population.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2015.
Diabetes mellitus (DM) has been reported to be one of the greatest global public health threats. Statistics of the fertility status of modern society has linked increased DM to a decrease in fertility rates. Hyperglycaemia is characteristic of DM that results in a disturbance of proteins, lipids and carbohydrate metabolism leading to an increase production of reactive oxygen species (ROS). In the case where ROS overwhelms antioxidant mechanisms, the body goes into state of oxidative stress (OS). OS plays a vital role in the progression of DM which leads to dysfunction and damage of various organs including that of the reproductive system. Os has shown to cause damage to the sperm membraneby oxidation of polyunsaturated fatty acids (PUFA’s) as the sperm membrane are rich in PUFA’s. This damage contributes to reduced sperm motility, concentration, morphological abnormalities and the sperms ability to fuse with the ZP of the oocyte. DM has been observed to cause testicular degeneration by interrupting sertoli cell production and maintenance thus resulting in a disturbance of the normal functioning of the reproductive system. Experimental studies have targeted more natural sources for treating DM and its complications of the reproductive system. Plants and natural dietary substances have shown to have high antioxidant contents that combat DM induced oxidative stress. This study explored the effect the Hypoxis hemerocallidea (H. hemerocallidea) supplementation on testicular and epididymal tissue, sperm motility and reproductive hormones in male wistar rats. The experiment were conducted for 6 weeks and the rats (230-260 grams) were randomly divided into 5 groups (n=12 per group). Diabetes was induced in 3 of the 5 groups. The first group was the normal control group (A), second the diabetic control group (B), third was the diabetic group treated with 800mg/kg H. hemerocallidea (group C), fourth the diabetic group treated with 200mg/kg H. hemerocallidea (group D) and fifth the non-diabetic group supplemented with 800mg/kg H. hemerocallidea (group E). Blood glucose showed a significant increase in the diabetic group when compared to the normal control and treated groups. H. hemerocallidea showed improvement in sperm motility and sperm morphology more at 800mg/kg when compared to diabetic group and diabetic group treated with 200mg/kg. Body, testicular and epipidymal weights of diabetic control were significantly lower when compared to the other groups. Testicular and epididymal Malondialdehyde levels were decreased in normal control, diabetic groups treated with different doses of H. hemerocallidea and the non-diabetic group supplemented with H. hemerocallideaon comparing with the diabetic control group. Antioxidants such as Superoxide dismutase, Catalase and total Glutathione activity was observed to be dosage dependent in certin groups but most showed a significant increase when compared to the diabetic control group. The total antioxidant capacity was measured using Oxygen radical absorbance capacity (ORAC) and Ferric ion reducing antioxidant power (FRAP); increase was observed when normal control group and treated groups were compared to the diabetic group. Testosterone and estradiol levels were also increased when the normal control group and treated groups were compared to the diabetic control group. Based on our findings it can be concluded that H. hemerocallidea supplementation can potentially be used to counteract deleterious effects of DM on the male reproductive system.

A presente pesquisa teve como objetivo geral compreender a experiência de homens que vivenciam a infertilidade. E, especificamente, enfocou interpretações da masculinidade e infertilidade, contextualizando-as na contemporaneidade; apresentou a perspectiva fenomenológica existencial como possibilidade para tematizar o fenômeno do corpo enquanto expressão da existência; bem como descreveu e compreendeu a experiência de homens, na condição de inférteis, os quais procuram o serviço de Reprodução Humana Assistida do Instituto de Medicina Integral de Pernambuco IMIP. De natureza qualitativa, esta investigação está afinada à perspectiva fenomenológica hermenêutica, privilegiando a compreensão interpretativa fundada na Hermenêutica Filosófica de Gadamer, vinculada às compreensões ontológicas heideggerianas. Para acesso à experiência foi escolhida a narrativa, colhida tanto dos colaboradores, quanto dos registros feitos no diário de bordo da pesquisadora, a partir da sua inserção no lócus da pesquisa. Os relatos dos colaboradores apontaram para dificuldades vividas durante a tentativa de métodos de Reprodução Assistida, as quais levaram a experiências de desconforto, bem como de desesperança frente a burocracia e morosidade dos serviços. Aproximando-se desta via compreensiva, a possibilidade de procriação artificial foi revelada com certa estranheza, ressaltando a supervalorização da parentalidade biológica. Em tal cenário, os interlocutores narraram sua vivência frente aos procedimentos técnicos/médicos, desvelando de um lado a utilidade da técnica no projeto parental e, de outro, o seu domínio na hegemonia do discurso científico, bem como na compreensão do corpo masculino como matéria-prima a ser explorada e aperfeiçoada.
The present research had the aim to understand the experience of infertile men. Specifically, it focused on masculinity and infertility interpretations, contextualizing them in contemporaneity; it also presented an existential phenomenological perspective as a possibility to thematize the body phenomenon as an expression of existence; as well as it described and understood the experience of infertile men who searched for the Assisted Human Reproduction service from the Institute of Integral Medicine of Pernambuco IMIP. This investigation is of qualitative nature, linked to the hermeneutic phenomenological perspective that privileges Gadamers Philosophical Hermeneutic and Heideggerian ontological comprehensions. Narratives from collaborators and from the researchers field journal were used in order to access such experience. Collaborators reports pointed to difficulties during the attempt for Assisted Reproduction methods, which led to uncomfortable experiences, as well as a lack of hope face to the services bureaucracy and slowness. The possibility of artificial procreation was revealed with certain awkwardness, with highlights to the over-valorization of biological parenthood. In such scenario, interlocutors narrated what they have lived regarding technical/medical procedures, unveiling, on one side, the utility of the technique for the parental project and, on the other side, the domain of scientific discourse, as well as the comprehension of mens bodies as raw material to be explored and improved.

Further studies were conducted to define an indispensable role of uterine bicarbonate secretion at pre-implantation for the success of blastocyst implantation. The in vitro implantation experiments showed that only when cultured in bicarbonate-containing medium, the blastocysts exhibited normal rate of attachment and outgrowth level. The forskolin-induced endometrial bicarbonate secretion measured by the Isc on pregnant day 4 was almost abolished by CA inhibitor acetazolamide. The efflux of intracellular bicarbonate, measured by intracellular pH-sensitive dye, was blocked by CFTR inhibitor, NPPB, and SLC26a6 inhibitor, DIDS, indicating their involvement in mediating uterine bicarbonate secretion.
In conclusion, the present findings have demonstrated an important role of CFTR in formation of optimal uterine fluid, in terms of both volume and composition, which is crucial for various reproductive events occurring in the uterus. Deviation from the normal uterine fluid composition and volume due to defects in CFTR function or abnormal regulation under pathological conditions, such as CF and genital bacteria infection, probably leads to infertility. The information obtained may provide insight into regulatory mechanism underlying fertility and infertility, as well as the rationale for development of treatment methods for female infertility and new strategies for female contraception. (Abstract shortened by UMI.)
The last part of the study was to demonstrate possible cause of infertility by disturbance of uterine fluid dynamic due to abnormal expression of CFTR using a model of uterine Chlamydia (C.) trachomatis infection, the most common infection-related sterility with the underlying cause unexplained. Uterine C. trachomatis infection induced up-regulated expression of CFTR with enhanced electrolyte and fluid transport as demonstrated by the increase in the cAMP-dependent Isc and uterine wet weight with obvious fluid accumulation in the lumen at diestrus stage, during which the endometrium normally undergoes a series of changes preparing for blastocyst implantation with minimum CFTR expression and uterine fluid volume. The abnormal uterine fluid accumulation upon uterine C. trachomatis infection significantly reduced implantation rate in uterine C. trachomatis infection mouse model.
The present study was aimed to elucidate the cellular and molecular mechanisms underlying the CFTR-related reproductive events in physiological and pathological conditions by using a variety of techniques, including RT-PCR, Western blot, intracellular and extracellular pH measurements, and the short-circuit current (Isc) measurement, in conjunction with mouse primary culture of endometrial cells and blastocyst, as well as several animal models including CF mouse, mouse uterine infectious model and overyectomized (OVX) mouse, etc.
We first examined dynamic changes in uterine bicarbonate secretion, as indicated by bicarbonate-dependent forskolin-induced Isc and epithelial surface pH measurement, and the expression profile of candidate genes and proteins known to be involved in bicarbonate secretion throughout the estrous cycle in mouse uterus. The results showed that the maximum mRNA and protein levels of CFTR, SLC26a6, carbonic anhydrase (CA)2 and CA12 were observed at proestrus stage and/or estrus stages. Luminal surface pH measured by 5-N-hexadecanoyl-aminofluorescein (HAF) showed that the basal endometrial epithelial surface pH at estrus stage was significantly higher than that in diestrus, which could be reduced significantly by CFTR inhibitor DPC, SLC26a6 inhibitor 4',4'-Diisothiocyanostilbene-2',2' Disulfonic Acid (DIDS) and CA suppressor acetazolamide. In the ovariectimized (OVX) mice and primary culture of endometrial cells, estrogen could induce up-regulation of CFTR, SLC26a6, CA2 and CA12 expression with corresponding increase in the bicarbonate-dependent Isc, suggesting a novel role of estrogen in regulating uterine bicarbonate secretion.
He, Qiong.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3247.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 163-176).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

環氧合酶-2(COX-2)是在花生四烯酸(AA)轉化為前列腺素H₂(PGH₂)的過程中最重要的限速酶，PGH2再進一步被合成為各種前列腺素，包括前列腺素E₂(PGE₂), 因此，COX-2在前列腺素的合成中起著舉足輕重的作用。COX-2在受到例如感染和炎症等刺激的情況下被誘導，迅速大量地產生。越來越多的證據證明瞭COX-2在許多細胞反應和病理生理過程中起重要作用, 其中, 對COX-2在炎症中的作用研究最深入。
囊性纖維化病(CF)是一種由於編碼囊性纖維化跨膜轉導調節器(CFTR)基因的突變所引起的常染色體隱性遺傳疾病。CFTR是在上皮細胞中廣泛表達的環磷酸腺苷(cAMP)依賴的陰離子通道。愈來愈多的證據顯示, CF的呼吸道上皮處於過量炎症因子和前列腺素的微環境中, 最終導致了在CF肺部病變中觀察到的超炎症反應. 但其中的機制仍未闡明. 本研究觀察到, 相對於野生型人類支氣管上皮細胞系(16HBE14o-), CF的人類支氣管上皮細胞系(CFBE41o-)中NFκB的活化, COX-2的表達和PGE₂的產量增加. 此外, CFTR基因敲除小鼠顯示出升高的NFκB活性和COX-2表達水準, 提示CFTR基因的缺失介導了超炎症反應的信號. 我們還驗證了一條PKA和CREB參與介導的PGE₂產生的正回饋通路. 更重要的是, 在CFBE41o-細胞中過表達CFTR顯著地抑制了COX-2的表達. 用LPS或者PGE₂處理16HBE14o-細胞導致了野生型CFTR表達的顯著升高. 這些實驗結果提示了CFTR可能參與對COX-2/PGE₂的負調節. 因此, CFTR負調節PGE₂介導的炎症反應. 這個調節機制的缺陷可能導致在CF炎症反應的組織中觀察到的過量的NFκB活化和過量PGE₂產生.
我們證實了睾丸中也存在這條CFTR負調節COX-2/PGE₂的通路. 由於隱睾處於比陰囊溫度高的腹腔中, 在隱睾中, 我們觀察到了高溫導致的CFTR下調,伴隨著COX-2的上調以及緊密連接蛋白(ZO-1, occludin)的下調. 這種CFTR和COX-2的負相關在小鼠睾丸高熱動物模型以及CFTR基因敲除小鼠模型中也被證實. 為了模擬隱睾的病理狀況, 我們提高原代睾丸支援細胞的培養溫度至37°C. 與在32°C培養條件下的對照細胞相比, 37C培養的支持細胞中CFTR表達顯著下調, 而COX-2表達顯著上調. 用CFTR的抑制劑CFTRinh-172處理支持細胞48小時後, COX-2的表達也上升了. 抑制或者敲除支持細胞中的CFTR都引起了ZO-1和occludin表達水準的下降, 從而損傷了支持細胞間的緊密連接. NFκB或者PGE₂的抑制劑都能逆轉ZO-1和occludin表達水準的下降. PGE₂同樣導致了支援細胞間緊密連接的損傷. 以上結果提示CFTR對緊密連接的調節作用是通過NFκB/COX-2/PGE₂通路實現的. 本研究闡明了在支持細胞中, CFTR通過負調節NFκB/COX-2/PGE₂通路調節緊密連接, 從而參與了隱睾導致的生精障礙的病理過程.
總之, 本研究論證了CFTR/COX-2/PGE₂通路在CF呼吸道的超炎症反應以及隱睾導致的生精障礙兩個病理過程中的作用, 說明了CFTR在呼吸系統和男性生殖系統中維持細胞因子穩態的重要作用. CF肺中CFTR的缺失或者隱睾病中CFTR表達水準的下降可能導致了呼吸道中過剩炎症反應和生精障礙.
Cyclooxygenase-2 (COX-2) is a pivotal rate-limiting enzyme responsible for the production of prostaglandins by converting arachidonic acid (AA) to prostaglandin H₂ (PGH₂), which is further metabolized to various prostaglandins, including PGE₂. COX-2 is inducible and increases dramatically upon stimulation, such as infection and inflammation. Accumulating evidences have demonstrated the important role of COX-2 in many cellular responses and pathophysiological processes, especially inflammation.
Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-dependent anion channel expressed in many epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased COX-2 expression and over-production of prostaglandin E₂ (PGE₂) in human CF bronchial epithelial cell line (CFBE41o-) with elevated NFκB activity compared to a wild-type bronchial epithelial cell line (16HBE14o-). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NFκB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE₂ involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o- cells were challenged with LPS as well as PGE₂, indicating possible involvement of CFTR in the negative regulation of COX-2/PGE₂. These results suggest that CFTR is a negative regulator of PGE₂-mediated inflammatory response, defect of which may result in excessive activation of NFκB, leading to over production of PGE2 as seen in inflammatory CF tissues.
This negative regulation of COX-2/PGE₂ pathway by CFTR was also identified in the testis in the present study. Downregulation of CFTR accompanied by upregulation of COX-2/PGE₂ and downregulation of tight junction proteins, including ZO-1 and occludin, were observed in a cryptorchidism mouse model with elevated testis in the abdomen, at which the temperature is several degrees higher than that in the scrotum. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and in CF mice. Culturing primary Sertoli cells at a temperature of 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression compared to the physiological condition of 32°C. Increase of COX-2 expression was also detected 48 hours after administrating CFTRinh-172 to the cells. Inhibition or knockdown of CFTR led to decreased ZO-1 and occludin expression and impaired tight junction in Sertoli cells, which could be mimicked by PGE₂, but reversed by NFκB and COX-2 inhibitors, suggesting that regulation of tight junction by CFTR is mediated by NFκB /COX-2/PGE₂ pathway. This study illustrates that CFTR may be involved in regulating testicular tight junctions through its negative regulation of NFκB/COX-2/PGE₂ pathway in Sertoli cells, defect of which may result in spermatogenesis defect in cryptorchidism.
Taken together, the present study has demonstrated the role of CFTR/ NFκB /COX-2/PGE₂ pathway in two pathological processes, exaggerated inflammation in CF airway and defective spermatogenesis in cryptorchidism, indicating that CFTR is critical for maintaining cytokine homeostasis in respiratory system and male reproductive system. Defect of CFTR in CF lung and downregulation of CFTR in cryptorchidism may contribute to the excessive lung inflammation and impaired spermatogenesis respectively.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chen, Jing.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 109-121).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
ABSTRACT --- p.i
摘要 --- p.iv
ACKNOWLEDGEMENT --- p.vi
LIST OF PUBLICATIONS --- p.vii
ABBREVIATIONS --- p.xii
LIST OF FIGURES AND TABLES --- p.xvi
Chapter 1 --- Chpter 1: Overview --- p.1
Chapter 1.1 --- CFTR and Cystic Fibrosis --- p.1
Chapter 1.1.1 --- Cystic Fibrosis --- p.1
Chapter 1.1.2 --- Structure of CFTR --- p.2
Chapter 1.1.3 --- Mutations of CFTR --- p.2
Chapter 1.1.4 --- Channel and signal transduction function of CFTR --- p.3
Chapter 1.1.5 --- Interaction of CFTR with other proteins --- p.4
Chapter 1.1.6 --- Regulation of CFTR --- p.5
Chapter 1.2 --- COX-2 and PGE₂ --- p.6
Chapter 1.2.1 --- Biosynthesis of PGE₂ --- p.6
Chapter 1.2.2 --- Pathophysiologic roles of COX-2 and PGE₂ --- p.7
Chapter 1.2.3 --- Role of COX-2/PGE₂ in inflammation --- p.7
Chapter 1.2.4 --- Regulation of COX-2 --- p.8
Chapter 1.2.4.1 --- Regulation of COX-2 by NF-κB --- p.9
Chapter 1.2.4.2 --- Regulation of COX-2 by CREB --- p.10
Chapter 1.3 --- Link between CFTR and NF-κB --- p.11
Chapter 1.4 --- General hypothesis and aims of study --- p.12
Chapter 2 --- Chapter 2: CFTR negatively regulates COX-2/PGE₂ positive loop in feedback loop in inflammation --- p.13
Chapter 2.1 --- Introduction --- p.13
Chapter 2.1.1 --- Airway inflammation in Cystic Fibrosis --- p.13
Chapter 2.1.2 --- Current theories on the causes of pulmonary inflammation in CF --- p.13
Chapter 2.1.2.1 --- Theory one --- p.14
Chapter 2.1.2.2 --- Theory two --- p.16
Chapter 2.1.3 --- Role of airway epithelia in CF airway inflammation --- p.16
Chapter 2.1.4 --- Link between CFTR and NF-κB in pulmonary inflammation --- p.17
Chapter 2.1.5 --- Link between CFTR and COX-2/PGE₂ in pulmonary inflammation --- p.18
Chapter 2.1.6 --- Hypothesis and aims of study --- p.18
Chapter 2.2 --- Materials and methods --- p.20
Chapter 2.2.1 --- Cell culture materials --- p.20
Chapter 2.2.2 --- Animals --- p.20
Chapter 2.2.3 --- Chemicals, drugs and assay kits --- p.20
Chapter 2.2.4 --- Antibodies --- p.22
Chapter 2.2.5 --- Cell culture. --- p.22
Chapter 2.2.6 --- Animal models and procedures --- p.23
Chapter 2.2.7 --- Manipulation of RNA and QRT-PCR --- p.23
Chapter 2.2.8 --- Manipulation of protein and Western blot --- p.25
Chapter 2.2.9 --- Histological and morphological --- p.27
Chapter 2.2.9.1 --- Tissue section. --- p.28
Chapter 2.2.9.2 --- Hematoxylin and eosin staining --- p.28
Chapter 2.2.9.3 --- Immunohistochemistry --- p.28
Chapter 2.2.10 --- PGE₂ EIA --- p.29
Chapter 2.2.11 --- Statistical analysis --- p.30
Chapter 2.3 --- Results --- p.30
Chapter 2.3.1 --- Increased expression of NF-κB and COX-2 in the lung of CF mice --- p.31
Chapter 2.3.2 --- Defect of CFTR leads to increased COX-2 expression in CF cell line --- p.31
Chapter 2.3.3 --- Increased expression of COX-2 in CF cells is attributed to NF-κB activation --- p.33
Chapter 2.3.4 --- A positive feedback loop from PGE₂ to COX-2 is mediated by PGE₂/cAMP/PKA/p-CREB pathway --- p.34
Chapter 2.3.5 --- PGE₂ increase the expression of CFTR protein in 16HBE14o- but not in CFBE41o- cells --- p.35
Chapter 2.4 --- Discussion --- p.47
Chapter 2.5 --- Conclusion --- p.51
Chapter 3 --- Chapter 3: Role of CFTR/COX-2/PGE₂ Pathway in the Regulation of Junctional Complex Proteins in Sertoli Cells and its Implication in Spermatogenesis Defect in Cryptorchidism --- p.53
Chapter 3.1 --- Introduction --- p.53
Chapter 3.1.1 --- Spermatogenesis.p53
Chapter 3.1.1.1 --- Structure of the seminiferous tubules --- p.53
Chapter 3.1.1.2 --- Role of Sertoli cells in spermatogenesis --- p.55
Chapter 3.1.1.3 --- Role of junctional complexes in spermatogenesis --- p.55
Chapter 3.1.2 --- Junctional complexes in the testis --- p.59
Chapter 3.1.2.1 --- Tight Junction --- p.59
Chapter 3.1.2.2 --- Anchoring Junction. --- p.60
Chapter 3.1.2.3 --- Cross talk between TJs and AJs --- p.60
Chapter 3.1.3 --- Cryptorchidism --- p.61
Chapter 3.1.3.1 --- Causes and consequences of Cryptorchidism --- p.61
Chapter 3.1.3.2 --- Elevated temperature caused by cryptorchidism greatly contributes to defective spermatogenesis --- p.62
Chapter 3.1.3.3 --- Changes of Sertoli cells in cryptorchidim contributing to defective spermatogenesis. --- p.62
Chapter 3.1.3.4 --- Disruption of junctional complexes in heat shock and cryptorchidism. --- p.65
Chapter 3.1.4 --- CFTR and spermatogenesis --- p.66
Chapter 3.1.4.1 --- Expression of CFTR in Sertoli cells in testis --- p.66
Chapter 3.1.4.2 --- Temperature sensitive processing of CFTR protein --- p.66
Chapter 3.1.4.3 --- CFTR and junctional complex --- p.67
Chapter 3.1.4.4 --- CFTR and male reproduction --- p.68
Chapter 3.1.4.5 --- Role of CFTR in spermatogenesis --- p.68
Chapter 3.1.5 --- Prostaglandins and male fertility --- p.69
Chapter 3.1.5.1 --- Expression of COX-2 in testis. --- p.69
Chapter 3.1.5.2 --- Role of prostaglandins in spermatogenesis --- p.70
Chapter 3.1.5.3 --- Regulation of junctional complexes by PGE₂ --- p.70
Chapter 3.1.5.4 --- Prostaglandins in cryptorchidism --- p.72
Chapter 3.1.6 --- Hypothesis and aims of study --- p.73
Chapter 3.2 --- Materials and Methods --- p.74
Chapter 3.2.1 --- Cell culture materials --- p.74
Chapter 3.2.2 --- Drugs and Reagents --- p.74
Chapter 3.2.3 --- Antibodies --- p.74
Chapter 3.2.4 --- Animals --- p.75
Chapter 3.2.4.1 --- Mice artificial cryptorchidism model --- p.75
Chapter 3.2.4.2 --- Mice testes hyperthermia model --- p.75
Chapter 3.2.5 --- Sertoli cell primary culture --- p.76
Chapter 3.2.6 --- siRNA against CFTR and transfection --- p.76
Chapter 3.2.7 --- Examination of assembly and destruction of assembly of inter-Sertoli TJs --- p.77
Chapter 3.2.8 --- Manipulation of RNA and Real-Time Quantitative RT-PCR (QRT-PCR) --- p.77
Chapter 3.2.9 --- Manipulation of protein and western blot --- p.77
Chapter 3.2.10 --- Histological and morphological studies --- p.78
Chapter 3.2.10.1 --- Immunofluorescence of ZO-1 Staining in Sertoli cells --- p.78
Chapter 3.2.10.2 --- Immunofluorescent staining of ZO-1, Occludin and β-Catenin in testes --- p.78
Chapter 3.2.11 --- PGE₂ EIA --- p.79
Chapter 3.2.12 --- Statistical Analysis --- p.79
Chapter 3.3 --- Results --- p.79
Chapter 3.3.1 --- Downregulation of CFTR is associated with upregulation of COX-2 in mice cryptorchidism model, mice testes hyperthermia model, and CF mice testes --- p.79
Chapter 3.3.2 --- Negative regulation of COX-2 by CFTR is mediated by NF-κB --- p.81
Chapter 3.3.3 --- Decreased tight junction proteins expression and increased anchoring junction proteins expression in cryptorchid testes. --- p.81
Chapter 3.3.4 --- Elevation of culture temperature results in downregulation of CFTR and upregulation of COX-2 in primary cultured rat sertoli cells --- p.82
Chapter 3.3.5 --- Defect of functional CFTR leads to increased COX-2 expression. --- p.83
Chapter 3.3.6 --- CFTR regulates TJ protein expression and TJ formation through NF-κB/COX-2/PGE₂. --- p.83
Chapter 3.4 --- Discussion --- p.100
Chapter 3.5 --- Conclusion --- p.104
Chapter 4 --- Chapter 4: General Discussion --- p.105
Chapter 4.1 --- The immunosuppressive function of PGE₂ in CF lung disease and cryptorchidism-induced infertility. --- p.105
Chapter 4.2 --- Importance of CFTR/ NF-κB /COX-2/PGE₂ pathway in inflammation-based diseases. --- p.106
Chapter 4.3 --- Possible implications of CFTR/NF-κB /COX-2/PGE₂ pathway in cancer --- p.107
Chapter 4.4 --- Concluding remarks --- p.108

Cheung King Ho.
"August 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 140-158).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

Infertility is a widespread health problem, caused by the male factor in about half of all cases, and in about a half of the infertile men the cause is unknown. In a significant number of these men, genetic etiology is assumed. Current routine methods of laboratory diagnostics, which include karyotype examination, exclusion of mutations in the CFTR gene, and Y chromosome microdeletions, do not usually reveal the cause of infertility. That is why researchers' efforts aim at detecting mutations in other genes that are causing male infertility. In recent years, animal models have been used to identify many genes necessary for fertility. Based on these findings, 12 candidate genes have been selected (CAPZA3, CDC14B, CDC42, CNTROB, CSNK2A2, GOPC, HOOK1, HRB, OAZ3, ODF1, RIMBP3, SPATA16) that are essential for spermatogenesis. Mouse or rat mutants in these genes are primarily associated with oligoasthenoteratozoospermia, since they are involved in sperm morphogenesis. However, the phenotype spectrum may comprise also azoospermia. The purpose of the thesis was to determine the sequence of the afore mentioned genes in infertile men with impaired spermatogenesis and to reveal presence or absence of pathogenic mutations in these genes, using cDNA and genomic DNA from peripheral blood. The candidate genes were...