While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-09Bitstream added on 2014-06-13T18:39:54Z : No. of bitstreams: 1 maia_fa_me_botfm.pdf: 761192 bytes, checksum: af7cd1654f8ff045bdb288b848b037dc (MD5) / A infertilidade é definida pela inabilidade de engravidar após 12 meses ou mais de coito regular não protegido. O uso de polivitamínico e polimineral parece influenciar na qualidade seminal. Dados da literatura sobre o uso oral isolado ou combinado desses micronutrientes na melhoria dos parâmetros seminais e eventual fertilidade são controversos e escassos. Analisar os parâmetros seminais de indivíduos inférteis em uso de polivitamínico e polimineral e compará-los com indivíduos normais, comprovadamente férteis sem uso destas substâncias. Foram analisados os parâmetros Seminais de 57 casais inférteis acompanhados no ambulatório de esterilidade do Hospital das Clínicas da Faculdade de Medicina de Botucatu, no período de 2003 a 2007. Nos indivíduos inférteis a análise seminal foi realizada antes e com 90 dias de micronutrientes por via oral os quais foram comparados com 50 indivíduos saudáveis comprovadamente férteis sem uso destas substâncias. A avaliação do sêmen foi feita de acordo com os critérios da Organização Mundial de Saúde - OMS (1999) e morfologia de Kruger et al. (1986). A análise estatística foi feita utilizando os testes t de Student, Mann-Whitney e Wilcoxon, considerando um nível de significância de 5%. Os indivíduos inférteis e os comprovadamente férteis apresentaram similaridade quanto a idade (31,0±5,6 versus 30,3±6,5) (p=0,55) e ao tabagismo (29,8% versus 22,0) (p=0,36). Nos indivíduos inférteis, o uso desses micronutrientes aumentou significativamente a morfologia tanto pelos critérios estabelecidos pela OMS (18,3±9,6 para 22,6±11,8) (p=0,006) e por Kruger (6,9±4,1 para 9,1±5,2) (p=0,002). Verificou-se que os homens inférteis antes do uso de micronutrientes quando comparados aos férteis apresentavam significantemente uma menor concentração de espermatozóides/ml (68,0[37,8;101,2]... / Infertility is defined as the inability to become pregnant after 12 months or more of regular unprotected intercourse. The use of multivitamin/multimineral supplements seems to influence semen quality. Data on the isolated or combined use of these micronutrients to improve semen parameters and eventual fertility are controversial and scarce. To assess semen parameters in infertile individuals using multivitamin and multimineral supplements in comparison with healthy proven fertile individuals not using these substances. Semen parameters were evaluated in 57 infertile couples followed up in the Sterility Outpatient Clinic of Botucatu Medical School between 2003 and 2007. Semen analysis was performed before and after 90 days of oral micronutrient use in infertile individuals that were compared with 50 healthy proven fertile individuals not using these substances. Semen was evaluated according to the recommendations of the World Health Organization- WHO (1999) and the criteria described by Kruger et al. (1986). Statistical analysis was carried out using Student’s t test and the tests of Mann-Whitney and Wilcoxon with significance set at 5%. Infertile and proven fertile individuals showed similar age (31.0±5.6 versus 30.3±6.5) (p=0.55) and smoking status (29.8% versus 22.0) (p=0.36). In infertile individuals, the use of micronutrients significantly improved morphology according to the criteria of WHO (18.3±9.6 to 22.6±11.8) (p=0.006) and Kruger (6.9±4.1 to 9.1±5.2) (p=0.002). Before micronutrient use, infertile individuals compared with fertile males showed lower spermatozoa/ml concentration (68.0[37.8;101.2] versus 96.5[49.0;144.2]) and vitality (85,4±9,2 versus 89.6±6.9) and higher leukocyte count (600.0 [300.0;121.5] versus 350.0[100.0;675.0]). In infertile individuals using multivitamin and multimineral supplements, semen parameters... (Complete abstract click electronic access below)

Magister Scientiae - MSc / Male infertility in humans has increased in the last few decades and could be as high as 40%, while up to 50% of these men have ''unexplained'' (idiopathic) infertility. Although newly developed molecular techniques have great value in detecting subtle causes of male infertility, more detailed sperm functional tests are required to identify compromised fertility, especially in a clinical set-up. Since ethical constraints often preclude the pursuit of many basic research questions in humans, non-human primates (NHPs) have been identified as key models in human-related studies. NHPs are often used in studies on male fertility/infertility, IVF or assisted reproductive technology (ART) procedures, male contraception and reproductive toxicology. However, comparing results of NHP and human studies require that techniques used for assessment must be objective, standardized and sensitive to recognize compromised sperm function. The aim of this study was to evaluate standard sperm functional tests and develop new functional tests using NHP sperm, specifically from vervet monkeys (Chlorocebus aethiops), chacma baboons (Papio ursinus) and rhesus monkeys (Macaca mulatta), for application in human and NHP studies and to ultimately develop a basic primate model. The sperm functions investigated included sperm motility, longevity, vitality, DNA integrity, acrosome reaction, and hyperactivation. The sperm functional tests evaluated were: CASA motility analysis; Sperm Longevity test; Eosin-Nigrosin and Hoechst and Propidium Iodide staining, as well as the use of WST-1 cytotoxicity assay for vitality; the TUNEL assay for DNA integrity; Acrosome Intactness Test; and induction of hyperactivation via stimulants. The validity of each test was investigated by inhibiting sperm function through the use of copper sulphate and cadmium chloride. All functional tests were successfully performed across all three species, except the TUNEL assay for DNA integrity, and was further used for validation testing. Validation testing proved that all sperm functional parameters were significantly affected by the highest concentrations of the chemicals (250 µg/ml CuSO4 and 500 µg/ml CdCl2) and if not significant, trends of reduction were seen. The tests employed were therefore sensitive to the inhibitory effect of the metals. By evaluating these established sperm functional tests we found that primates would serve as good models for research study. Furthermore, we optimized and modified techniques for sperm and functional analysis in these three primate species and this study will standardize protocols for use in future studies on male infertility. Additionally, comparing human and NHP sperm function can possibly reveal or explain the high infertility rates in humans.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Passage of spermatozoa through the female reproductive tract is essential for the regulation of fertilization, ensuring that healthy sperm reach the oocyte. Previous studies were devoted to morphological selection of sperm cells by the cervical mucus. However, research prove that the loss of integrity of the sperm plasma membrane is associated with infertile men, irrespective of their normal semen parameters. This indicates that the sperm plasma membrane plays an important role in fertilization. Further studies indicated that sperm surface proteins assist penetration through the female reproductive tract and would therefore provide useful insight in understanding other factors associated with male infertility. The aim of this project was to determine if there are any differences between sperm surface proteins of fertile donor samples in relation to infertile patient samples using different separation techniques and different detergents. Three different sperm separation techniques were employed, including wash, swim-up (SU) and Percoll density gradient centrifugation (DGC).Parallel to this, the deoxy-ribose nucleic acid (DNA) fragmentation of these cells were analysed for comparison of the extent of DNA damage induced due to different separation techniques used. This provided evidence that the best separation technique is the DGC as it minimises the amount of DNA fragmentation caused. Four different detergents were used in the process of extracting the membrane proteins from spermatozoa, namely sodium dodecyl sulphate (SDS), saponin,cetyl-trimethyl-ammonium bromide (CTAB), and TWEEN-20. The membrane proteins were then separated on a12% SDS poly-acrylamide gel electrophoresis (PAGE), and analysed by Coomassie blue and silver staining techniques as well as densitometry. Due to the different chemical nature of the detergents that extracted different surface proteins, CTAB (cationic) and SDS (anionic) extracted the most because of its strong solubilising abilities as non-ionic detergents. Common proteins that were extracted in donor samples included; 115, 92.5, 89, 61, 55.5, 51.5, 47, 44.5, 43, 38.5, 34 and 28 kDa proteins. In patients, commonly occurring proteins included; 92.5, 74.5, 70, 60.5, 51.5, 50, 44.5, 43, 36, 29.5, and 25.5 kDa proteins. Marked differences were found between membrane proteins extracted from donor samples in comparison to patient samples. Identification of these proteins was done using the SwissProt database and a literature search. Mostly non-genomic progesterone receptors were identified; others included oestrogen receptor, a phosphotyrosyl protein, P34H, equatorial segment protein, mannose lectin receptor, human guanylylcyclase receptor, epididymal protease inhibitor receptor, PH30 and estradiol binding protein. The function of the membrane surface proteins identified in this study plays a vital role in fertilization. A few of these functions include sperm attachment and binding to the oocyte as well as penetration thereof. Others play a role in signalling events such as capacitation, hyperactivation and acrosome reaction. The absence of these proteins in patient sperm possibly accounts for the functional inability to successfully achieve fertilization suggesting that this provides molecular insight to reasons for infertility amongst men. In addition to this, proteins presented by patient samples that were absent in healthy donors may too account for their infertility status. Estradiol binding protein and PH30 are two proteins presented only in patient samples. Their function plays a role in the inhibition of the acrosome reaction and sperm-egg fusion, respectively. In conclusion, these differences in protein expression between fertile donors and patients may form the molecular basis of infertility amongst men and indicates possibilities for novel proteonomic approaches to improve andrological diagnosis in future.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

The full text of the thesis is currently restricted. / The full text will be available at the end of the embargo period: 1st Nov 2021

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: CHAPTER 1 A review of the application of traditional semen parameters for the investigation and diagnosis of male infertility and the role of predictive values in assisted reproductive techniques (ART) is presented. The importance of sperm morphology, with special emphasis on sperm morphology evaluation, is discussed. Also presented is an overview of the physiology of sperm DNA, the process of spermatogenesis, as well as the contribution of the spermatozoon to the embryo. The different causes of sperm DNA damage and techniques to determine DNA damage in spermatozoa are described. A survey is presented of the correlation of sperm DNA with sperm morphology. CHAPTER 2 All the materials and methods applicable to this study are described. Sperm morphology assessment and two different sperm DNA tests, the chromomycin A3 (CMA3) staining test and the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay, are discussed in detail. CHAPTER 3 Results obtained in this study are presented. Results include the prevalence of abnormal sperm DNA and association with sperm morphology, specifically in the p-pattern and g-pattern morphology groups. Further results include the correlation of sperm morphology and sperm DNA with fertilization in vitro, embryo quality and pregnancy outcome. The percentage CMA3 positive spermatozoa (abnormal DNA) and percentage TUNEL positive spermatozoa (abnormal DNA) had a significant negative association with normal sperm morphology. P-pattern and g-pattern morphology groups differed significantly from each other for both CMA3 and TUNEL. A significant positive association between CMA3 and TUNEL was observed. No association between the percentage normal sperm morphology, percentage CMA3 positive spermatozoa and percentage TUNEL positive spermatozoa and IUI pregnancy outcome was observed. A significant negative association between the percentage TUNEL positive spermatozoa and IVF/ICSI pregnancy outcome was established. The percentage CMA3 positive spermatozoa had a significant positive (unexpected) association with IVF/ICSI pregnancy outcome. There was no association between the three variables and IVF/ICSI fertilization rates. A significant positive association between the percentage normal sperm morphology and IVF/ICSI embryo quality was found. There was a significant positive association between the percentage CMA3 positive spermatozoa and IVF/ICSI embryo quality (unexpected). The percentage TUNEL positive spermatozoa and IVF/ICSI embryo quality was negatively associated. CHAPTER 4 Interpretation of the results and future perspectives are discussed. The CMA3 staining test and TUNEL assay has a limited ability to distinguish between the p-pattern and g-pattern morphology groups. P-pattern spermatozoa are more likely to possess poor chromatin packaging and show increased levels of DNA fragmentation, but some p-pattern patients also may have normal DNA and g-pattern patients abnormal DNA. It is recommended that a sperm DNA test should be implemented routinely in andrology laboratories for the clinical diagnosis of sperm DNA damage in patients. / AFRIKAANSE OPSOMMING: HOOFSTUK 1 'n Samevatting wat handel oor die toepassing van tradisionele semen parameters vir die evaluasie en diagnose van manlike infertiliteit, asook die rol van voorspellingswaardes in kunsmatige voortplantingstegnieke word voorgelê. Die belangrikheid van sperm morfologie, met die klem op sperm morfologie evaluering, word ook bespreek. 'n Oorsig van sperm DNS fisiologie, die proses van spermatogenese, sowel as die sperm se bydrae tot die embrio word hier aangebied. Die verskillende oorsake van sperm DNS skade en die tegnieke om sperm DNS skade vas te stel, asook die die korrelasie tussen sperm DNS en sperm morfologie word ook bespreek. HOOFSTUK 2 Alle materiale en metodes wat van toepassing is op hierdie studie word beskryf. Sperm morfologie evaluering en twee verskillende sperm DNS toetse, die chromomycin A3 (CMA3) kleuringstoets en die “terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL)" toets, word ook in meer besonderhede aangebied. HOOFSTUK 3 Resultate wat verkry is tydens hierdie studie word hier uiteengesit. Resultate behels die voorkomsyfer van abnormale DNS en die assosiasie met sperm morfologie, spesifiek in die p-patroon en g-patroon. Verdere resultate sluit die korrelasie van sperm morfologie en sperm DNS met bevrugting in vitro, embriokwaliteit en swangerskap uitkomste in. Die persentasie CMA3 positiewe sperme (abnormale DNS) en persentasie TUNEL positiewe sperme (abnormale DNS) het 'n betekenisvolle negatiewe assosiasie met normale sperm morfologie getoon. P-patroon en g-patroon morfologie groepe het betekenisvol van mekaar verskil vir beide CMA3 en TUNEL. 'n Betekenisvolle positiewe assosiasie is tussen CMA3 en TUNEL waargeneem. Geen assosiasie is tussen die persentasie normale sperm morfologie, persentasie CMA3 positiewe sperme en persentasie TUNEL positiewe sperme en IUI swangerskap uitkomste waargeneem nie. 'n Betekenisvolle negatiewe assosiasie is tussen die persentasie TUNEL positiewe sperme en IVB/ICSI swangerskap uitkomste vasgestel. Die persentasie CMA3 positiewe sperme het 'n betekenisvolle positiewe (onverwags) assosiasie met IVB/ICSI swangeskap uitkomste opgewys. Daar was geen assosiasie tussen die drie veranderlikes en IVB/ICSI bevrugting nie. 'n Betekenisvolle positiewe assosiasie is tussen die persentasie normale sperm morfologie en IVB/ICSI embryo kwaliteit waargeneem. Daar was 'n betekenisvolle positiewe assosiasie tussen die persentasie CMA3 positiewe sperme en IVB/ICSI embrio kwaliteit (onverwags). Die persentasie TUNEL positiewe sperme het 'n negatiewe assosiasie met IVB/ICSI embrio kwaliteit getoon. HOOFSTUK 4 Interpretasie van die resultate en toekomstige vooruitsigte is bespreek. Die CMA3 kleuringstoets en TUNEL toets het 'n beperkte vermoë om tussen die p-patroon en g-patroon morfologie groepe te onderskei. P-patroon spermatozoa sal heel waarskynlik oor swakker chromatien verpakking en meer DNS fragmentasie beskik. Sommige p-patroon pasiënte mag egter normale DNS toon, terwyl g-patroon pasiënte abnormale DNS het. Die implementering van 'n sperm DNS toets in andrologie laboratoriums, vir die kliniese diagnose van sperm DNS skade in pasiënte, word aanbeveel.

Abstrakt (svenska) Titel: Infertilitet – vems rätt att bestämma? En kvalitativ intervjustudie kring barnmorskors erfarenheter av att arbeta med infertilitet. Författare: Rebecka Kristensen Berlin, Umeå Universitet -Institutionen för Omvårdnad   Syfte: Att undersöka hur barnmorskans erfarenheter av parens reaktioner uppfattas i samband med infertilitetsproblematik. Studiedesign: En kvalitativ intervjustudie innefattande fyra barnmorskor har genomförts på en barnmorskemottagning. Intervjumaterialet analyserades utifrån kvalitativ innehållsanalys, vilket i korthet innebar följande steg: Materialet transkriberades ordagrant och bröts därefter ned i meningsbärande enheter, för att kunna sortera data utifrån innehåll. Kondensering utfördes i syfte att lyfta fram kärninnehållet. Abstraktion utfördes för att få fram koder, i syfte att reducera mängden text men även för att därefter kunna sammanfoga snarlika ämnen till underkategorier, vilka sedan grupperades i huvudkategorier tillhörande ett gemensamt tema. Resultat: De fyra huvudkategorier som uppkom var känslor, kommunikation, rättighet och prevention. Känslor: Oro, stress samt nedstämdhet var vanligt förekommande. Mäns känslor var svårare att identifiera då kvinnor ibland kom ensamma till besöken, samt män ibland dolde känslor bakom aggressivitet. Kommunikation: God information skapade förtroende. Råd om livsstilsförändringar samt vidareremittering var en viktig form av stöd. Rättighet: Ingen har rätt till barn, men alla ansågs ha rätt att försöka få barn. Privatekonomi samt samhällets krav påverkar dock i stor utsträckning paren vid misslyckade försök eller önskan om ytterligare barn. Prevention: Vikten av information kring infertilitet vid undervisning i skolan, vid preventivmedelsamtal och vid cellprovtagning poängterades. Slutsats: Att vara väl införstådd med de känslor som kan uppstå i samband med infertilitet möjliggör att ge tillfredställande stöd. Särskilda ansträngningar behöver göras för att nå män, såväl emotionellt som preventivt. Stort utrymme för förbättring finns gällande preventiva åtgärder, i syfte att fördjupa allmänhetens kunskaper kring infertilitet. Nyckelord: kvinnlig infertilitet; manlig infertilitet; känslor; barnmorska; kvalitativ forskning / Abstract (English)     Title: Infertility. Who´s right to decide? A qualitative interview study regarding midwives experiences to work with infertility. Author: Rebecka Kristensen Berlin, Umeå University- Department of nursing   Objective: To investigate midwives thoughts and experiences regarding couples reactions in connection with infertility.  Study design: A qualitative interview study comprising four midwives have been conducted. The resulting data was analyzed using qualitative content analysis, consisting of the following steps: The material was transcribed verbatim and then broken down into sentences, to aid in sorting the data based on content. Condensation was carried out in order to highlight the core content. Abstraction was performed to obtain codes, in order to reduce the amount of text , but also to subsequently merge similar content into subcategories , which were then grouped into categories. Results: Four main categories emerged. Feelings: anxiety, stress and depression were common. Men’s feelings were more difficult to identify since women sometimes came alone to appointments, and men sometimes concealed their feelings behind aggression. Communication: Good information created trust. Advice on lifestyle changes and referrals were important forms of support. Empowerment: No-one has the right to a child, but the right to try to have a child. Personal and societal demands, however, affect the couples at failure to conceive or desire for additional children. Prevention: The importance of information about fertility in school, at contraception counselling and routine smear exams was emphasized. Conclusion: Awareness of the feelings that infertility may give rise to, enables providing satisfactory support. Further efforts need to be made to reach males emotionally as well as preventatively. There is great possibility for improvement in regards to public awareness about infertility. Keywords: female infertility; male infertility; emotions; midwife; qualitative research

Les récepteurs couplés aux protéines G (RCPG) sont impliqués dans la majeure partie des communications intercellulaires. Un grand nombre de RCPG ont été découverts en comparant la séquence des récepteurs connus avec les données fournies par le séquençage du génome humain. Pour plus d'une centaine de ces récepteurs, le ligand activateur ou agoniste est inconnu. Ces récepteurs sont dès lors qualifiés d'orphelins. Les LGR forment une sous-famille de RCPG structurellement proches de la rhodopsine qui comprend les récepteurs aux hormones glycoprotéiques (TSH, LH, hCG, FSH) et à la relaxine. LGR4 est un membre de cette famille dont ni la fonction précise, ni l'agoniste ne sont connus. Dans un premier temps, une cartographie détaillée de l'expression de Lgr4 chez la souris a été obtenue. Nous avons tiré parti de l'existence d'une lignée de souris transgéniques dont le gène Lgr4 a été interrompu par l'introduction d'une cassette comportant deux marqueurs histologiques. L'activité beta-galactosidase d'un de ces marqueurs a été analysée chez les souris hétérozygotes. Ces dernières ne présentent pas de phénotype particulier, ce qui permet d'estimer que l'expression des marqueurs rend effectivement compte de l'expression normale du gène Lgr4. Lgr4 est exprimé dans un grand nombre de structures, notamment dans le cartilage, le rein, les appareils reproducteurs mâle et femelle et certaines cellules du système nerveux. Ensuite, le phénotype des souris homozygotes pour l'inactivation de Lgr4 (LGR4KO) a été exploré. Ces souris présentent à la naissance un poids inférieur à leurs congénères des autres phénotypes. Les mâles sont stériles à cause d'une malformation des tubules efférents et de l'épididyme. Un blocage au niveau des tubules efférents reliant le testicule à l'épididyme contraint les spermatozoïdes à s'accumuler à la sortie du testicule, dans la région du rete testis. De plus, les tubes de l'épididyme, pourtant normaux à la naissance, ne s'allongent pas pour former la structure convolutée habituelle. L'épithélium de ces tubes est aplati et est entouré d'une quantité anormalement élevée de mésenchyme. Dans un troisième temps, des outils nécessaires aux futures tentatives d'identification de l'agoniste naturel de LGR4 ont été réalisés. Il s'agit : (1) d'anticorps monoclonaux dirigés contre la partie extracellulaire du récepteur humain. (2) d'un appât moléculaire pour la ‘pêche au ligand’. Cet appât est constitué du domaine extracellulaire du récepteur humain couplé à un marqueur histologique. (3) d'une construction peptidique constituée du domaine extracellulaire du récepteur humain couplé à une queue poly-histidine. Cette construction est destinée à servir de greffon lors de chromatographies d'affinités devant permettre de purifier le ligand. (4) de lignées cellulaires exprimant le récepteur LGR4 humain ainsi que le système æquorine devant permettre de détecter l'activation de ce récepteur. Les données apportées par ce travail montrent un rôle important du récepteur LGR4 au cours du développement et permettent de circonscrire le champ des recherches futures. Ceci, ainsi que les outils moléculaires développés, constitue une base pour l'identification future de l'agoniste et la détermination précise de la fonction de LGR4.

<p>The chromatin integrity in sperm cells is vital for successful pregnancy. In this</p><p>study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization</p><p>(IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were</p><p>purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test</p><p>Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation</p><p>Index (DFI) of 30 % and the results correlated with fertilization, cleavage and</p><p>pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower</p><p>cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro</p><p>may be independent of the degree of DNA-damage, the embryonic development could be</p><p>seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a</p><p>DFI threshold of 30 %.</p>