Sertoli cells (SCs) are somatic cells located in the testes. They are the only cells in direct contact with germ cells and play a key role in process of spermatogenesis. New insights in the biology of SCs are highlighting the immunological function of these cells: germ cells protection by maintaining the immunoprotective niche, creating the blood- testis barrier and local modulation of the immune response to spermatic cells. Immunomodulatory activity of SCs is preserved after their allo- and xenogeneic transplantation, and thus SCs prolongs survival not only of themselves but also of cells transplanted with them. The aim of this thesis was to study the survival and migration of SCs precursors (TSC) in mice recipients. The project is employing the neonatal tolerance phenomenon and evolutionary distinct donor organism, Xenopus tropicalis, to monitor conserved mechanisms of immune system (IS) modulation using SCs. SCs were detectable in the lungs and thymus 7 days after transplantation. The phenotype of immune cells was not altered 30 days after transplantation, however we detected changes in cytokine environment, namely increased levels of cytokines typical for Th2 and Treg immune responses. In vitro experiments further confirmed IS modulation by SCs - changing the phenotype of macrophages to alternatively...

La stérilité affecte à l’heure actuelle près de 15-20 % des couples et sa prévalence est en progression depuis quatre à cinq décennies. Cette progression évolue parallèlement à la prévalence de l’épidémie d’obésité et de syndrome métabolique dans le monde. De multiples arguments physiologiques et épidémiologiques chez l’Homme soutiennent l’hypothèse de l’influence de l’homéostasie des lipides sur la fonction gonadique. En particulier, le cholestérol est un facteur clef dans la régulation de la stéroïdogenèse et de la gamétogenèse. Bien que les atteintes gonadiques semblent multifactorielles, les mécanismes moléculaires restent méconnus dans la majorité des cas. Les Liver X receptors (LXRα et β) sont des récepteurs nucléaires activés par les oxystérols. Ce sont classiquement des régulateurs du métabolisme lipidique. Plusieurs études ont démontré l’importance de ces récepteurs dans la physiologie des gonades. Ce travail identifie les rôles multiples des LXRs dans le maintien de la fertilité masculine et féminine, et décrit l’effet de l’homéostasie du cholestérol sur la maturation des cellules germinales dans le testicule et l’ovaire. Ce travail se concentre sur l’analyse comparative d’une lignée de souris ré-exprimant LXRβ (Lxrαϐ-/-:AMHLxrϐ) sous contrôle de promoteur d’AMH humain (expression spécifique dans les cellules de Sertoli dans le testicule et les cellules de granulosa dans l’ovaire) sur un fond génétique de souris Lxrαϐ-/-. L’absence d’un isoforme d LXR aboutit à des défauts spécifiques dans un type cellulaire du testicule. Néanmoins, le dysfonctionnement d’un type cellulaire est compensé. En effet, de multiples défauts sont nécessaires pour aboutir à la stérilité. LXR dans les cellules de granulosa est critique pour la maturation et la survie des ovocytes, l’ovulation, et par conséquence pour la fertilité. Ainsi, LXRβ est une cible potentielle pour réguler la fertilité féminine et la prévention de syndrome d’hyperstimulation ovarienne. Nos résultats ouvrent des perspectives pour des nouvelles cibles diagnostiques et pronostiques dans la fertilité. / Sterility affects 15 % of French population and its prevalence is propagating since four or five decades. Many human physiological and epidemiological arguments support the impact of lipid homeostasis on the gonads; indeed, cholesterol is a key regulator of steroidogenesis and gametogenesis. Nevertheless, the molecular mechanisms remain hidden. Liver X receptors alpha and beta (LXRα and β) belong to the superfamily of nuclear receptors and are activated upon binding to oxysterols. LXRs are mainly implicated in cholesterol homeostasis. Increasing bulk of literature identified these non-steroid nuclear receptors as major regulators of the gonad physiology. This work uncovers previously unidentified putative roles of LXRs and ability of cholecterol excess to alter male and female germ cell maturation. Herein, we analyse a new mouse strain (Lxrαϐ-/-:AMHLxrϐ) re-expressing LXRβ under control of AMH promoter (specific to Sertoli in testis and granulosa cells in ovary) in a background of Lxrαϐ-/- mouse. Our results identify LXRs as primordial to maintain male and female fertility. They have pleotropic « non-classical » roles ranging from lipid homeostasis to the regulation of germ cell maturation and bi-directional control of steroid synthesis. If the cellular defects in the absence of LXRs within the testis are significant, they are generally compensated and consequently, single cell compartment is tolerated. Unlike the testis, LXRβ in the granulosa cells is « the regulator » of multiple mechanisms essential for follicle maturation, ovocyte survival and for controlled ovulation. LXRβ is therefore a potnetial target to regulate female fertility and to prevent ovarian hyperstimulation syndrome. Our results open the perspectives for the identification of new diagnostic and/or prognostic markers in both male and female fertility.

Made available in DSpace on 2015-10-06T13:03:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-25. Added 1 bitstream(s) on 2015-10-06T13:19:00Z : No. of bitstreams: 1 000847999.pdf: 2110688 bytes, checksum: 3cfbc2629db1b4c973c6917a12690b53 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As células de Sertoli, encontradas no epitélio seminífero, formam a barreira hematotesticular através de suas junções comunicantes e de oclusão, responsável por proporcionar um ambiente protegido e coordenar o processo espermatogênico, fundamental para a manutenção da fertilidade masculina. A dinâmica de formação da barreira hematotesticular está diretamente relacionada com o equilíbrio entre síntese e degradação de moléculas especializadas, e sofre influência através de regulação androgênica e de fatores de crescimento específicos como o TGF-β2, sendo particularmente sensível aos fatores ambientais. A exposição à desreguladores endócrinos (DEs), capazes de interferir na modulação da barreira, pode levar a infertilidade. Atualmente o fator masculino pode ser responsável por 30-50% dos casos de infertilidade, sendo de grande importância o estabelecimento da relação entre a exposição aos DEs e o processo de formação dos gametas. Este estudo objetivou correlacionar a ação de dois desreguladores endócrinos, o Monobutil ftalato (MBP) e o Bisfenol A (BPA), em diferentes períodos de exposição, a partir de uma dose de baixa toxicidade, sobre a linhagem HSec de células de Sertoli humanas (in vitro) e sua possível ação sobre proteínas da barreira hematotesticular e proteínas regulatórias envolvidas na modulação da degradação e síntese dessa barreira. Para tal, as células da linhagem HSec foram cultivadas em meio específico para a expansão e submetidas à exposição ao MBP e ao BPA em baixas doses por 6 e 48 horas. Em seguida, as células foram isoladas e submetidas à extração de proteínas. Foram analisadas as proteínas: Ocludina, Ncaderina, β-catenina e Receptor de Andrógeno (AR) por Western Blot. Os resultados foram comparados para os dois agentes nos diferentes períodos de exposição em relação às células não expostas, mostrando que os dois agentes foram capazes de reduzir a... / Sertoli cells, found in the seminiferous epithelium, form the blood-testis barrier via their gap and tight junctions, responsible for providing a protected environment and coordinate the spermatogenic process, essential for male fertility maintenance. The dynamics of the blood-testis barrier formation is directly related to the balance between synthesis and degradation of specialized molecules, and is influenced by androgen regulation and specific growth factors such as TGF-β2, being particularly sensitive to environmental factors. Exposure to endocrine disruptors (EDs), which interfere in the barrier modulation, can lead to infertility. Currently the male factor may be responsible for 30-50% of infertility cases, being the establishment of the relationship between exposure to EDs and the process of gametes formation of great importance. This study aimed to correlate the action of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, from a dose of low toxicity on the HSec lineage of human Sertoli cells (in vitro) and its possible action on proteins of blood-testis barrier and regulatory proteins involved in modulation of degradation and synthesis of this barrier. To this end, cells were cultured in HSec lineage specific medium and exposed to MBP and BPA in low doses for 6 and 48 hours. Then, cells were isolated and subjected to protein extraction. Proteins analyzed were: occludin, Ncadherin, β-catenin and Androgen Receptor (AR) by Western blot. The results were compared for the two agents in different exposure times to non-treated cells, showing that both agents were able to reduce the expression of important BTB proteins, presenting potential for disrupting the structure and maintenance of the barrier and, consequently, sperm production and male fertility

Analisou-se por métodos quantitativos e qualitativos o testículo de roedores histricomorfos nas fases, pré-púbere, púbere e adulto da Paca (Cuniculus paca), Cutia (Dasyprocta leporina) e Porquinho-da-índia (Cavia porcellus). Mediante as técnicas de microscopia de luz, microscopia eletrônica de varredura, microscopia eletrônica de transmissão e método estereológico. Verificou-se a influência do Letrozol um inibidor da enzima aromatase nas dosagens de 0,15mg\\kg e 1,0 mg\\kg, sobre a morfologia testicular de pacas macho. Este modelo animal apresenta importância, comercial e científica sendo excelente alternativa para a pesquisa, colaborando com o desenvolvimento de investigações vitais ao homem e aos próprios animais. Os resultados desta investigação demonstraram, que os aspectos qualitativos da morfologia testicular do Porquinho-da-índia, Cutia e de Paca foram semelhante nas fases pré-púbere, púbere e adulta. As avaliações quantitativas do Porquinho-da-índia e Cutia realizados mediante o método estereológico permitiram quantificar parâmetros, sem hipóteses de forma, tamanho, orientação ou distribuição das partículas contadas, demonstrando ser um método imparcial e confiável para ser aplicado em estudos de reprodução. Considerando o estrógeno ser essencial para a função da espermatogênese, o efeito do Letrozol no testículo da paca (Cuniculus paca), sugeriu que a inibição da enzima aromatase mediante a utilização de Letrozol na dosagem de 0,15mg \\kg, sugeriu em um aumento no número das células de Sertoli, refletindo sobre as demais células testiculares, comparando com o testículo basal (controle). O testículo tratado (Letrozol) demonstrou epitélio germinativo íntegro, sendo este dado importante para a reprodução. No animal púbere houve um aumento no número de células de- Sertoli, semelhante ao descrito anteriormente. O epitélio seminífero mostrou com alterações morfológicas, tais como proliferação celular, presença de células germinativas com características apoptóticas, e vacúolos e núcleos em picnose. Estes dados poderão ser úteis para o estudo da Síndrome da Disgenesia testicular / Analysis by quantitative and qualitative methods testicular histricomorfs rodents in phases, pre-pubertal, pubertal and adult paca (Cuniculus paca), agouti (Dasyprocta leporina) and guinea-pig (Cavia porcellus). By means of light microscopy techniques, scanning electron microscopy, transmission electron microscopy, stereological method and the influence of an aromatase inhibitor, the Letrozole in dosages of 0.15 mg\\kg and 1.0 mg\\kg on testicular male morphology paca. This animal model has importance, excellent commercial and scientific alternative to research, contributing to the development of vital research to humans and from animals themselves. The results of this research have shown that the qualitative aspects of testicular morphology guinea-pig, Agouti and paca were similar in pre-pubertal, pubertal and adult stages. Quantitative assessments of the guinea-pig and agouti performed by the stereological method allowed quantify parameters, no change of shape, size, orientation or distribution of particles counted, it is proving to be a partial and reliable method to be applied to reproduction studies. Considering the estrogen is essential for the function of spermatogenesis, the effect of Letrozole in the testis of paca (Cuniculus paca), suggested that inhibition of aromatase enzyme through the use of Letrozole in a dosage of 0.15 mg\\kg suggested in an increase in number of Sertoli cells, reflecting on the other testicular cells, comparing the baseline testis (control). The treaty testis (Letrozole) showed intact germinal epithelium, which is important data for reproduction. In pubertal animals there was an increase in the number of de Sertoli cells, similar to that described above. The seminiferous epithelium showed to morphological changes such as cell proliferation, the presence of germ cells with apoptotic features and vacuoles and nuclei picnosis. These data may be useful for the study of testicular dysgenesis syndrome

Magister Scientiae (Medical Bioscience) - MSc(MBS) / There is increasing concern worldwide by researchers with regards to the assessing of safety and therapeutic consumption of the plants used in traditional medicine. Date palm pollen (DPP) and flax seed have been used traditionally to improve fertility in Libya. DPP extracts have shown several reproductive beneficial effects. In vivo, studies have revealed the ability of DPP to increase sperm concentrations, ameliorate the testicular toxicity induced by cadmium and lead, raise testosterone, as well as LH and FSH hormone levels. Flax seed phytochemical analysis showed lots of valuable components such as lignans and α linolenic acid to which were attributed its positive health effects like antitumor, antioxidant and protective effects against coronary heart diseases. Moreover, flax lignans have both estrogenic and antiestrogenic potential. This study was aimed at testing the effects of Libyan DPP and flax seed on the Sertoli (TM4) cell line and human breast adenocarcinoma (MCF - 7) cell line. Different concentrations (0.01, 0.1, 1, 10, 100 and 1000 μg/ml) of ethanolic extracts of DPP and flax seed, respectively, were used to assess the morphology of TM4 and MCF - 7 cells after 24 and 72 hours exposure. Mitochondrial dehydrogenase activity as a marker of cell viability was measured by MTT assay after 24 and 72 hours exposure. Apoptotic effects were assessed by flow cytometeric APO percentage assay. TM4 cell production of Inhibin - B hormone and GGT enzyme activity under the effects of DPP or flax seed was determined by use of ELISA kits. Transepithelial electrical resistance (TEER) assay were used to detect the effect of DPP or flax seed on TM4 cell monolayer integrity. Finally the plants potential phytoestrogenic activity was determined by use of E - SCREEN assay in MCF – 7 breast cancer cells. Higher concentrations of DPP significantly increased the activity of mitochondrial dehydrogenase enzyme of TM4 cells after 24 hours associated with increasing cell number as detected in a microphotograph. Flax seed concentrations less than 100 μg/ml reduced TM4 cell viability but there were no morphological changes visible after 24 hours. MCF - 7 cells viability was reduced after 24 and 72 hours treatment with DPP and flax seed. DPP concentrations beyond 1 μg/ml significantly raised the TEER of TM4 monolayer over 72 hours while flax seed treatments caused a significant increase only after 72 hours of exposure. TM4 cells GGT activity increased significantly after exposure to higher concentrations of DPP and all flax seed concentrations. Significant stimulatory effects of all the concentrations of DPP or flax seed on TM4 inhibin - B hormone productions have been detected. Apoptotic studies showed no significant changes. E - SCREEN assay resulted in significant reduction in MCF - 7 proliferation rate under the effect of low concentrations of DPP or flax seed. Higher concentrations of the plant extracts, however, stated to increase MCF – 7 cell proliferation, this exerts weak estrogenic activities. In conclusion, the main finding of this study is that DPP and flax seed showed stimulatory effects on TM4 cells proliferation. The resistivity of TM4 cells monolayer which reflect the integrity of blood – testis barrier (BTB) was also significantly increased as well as inhibin - B production and GGT enzyme activity. In addition DPP and flax seed respectively showed inhibitory effects on MCF - 7 cells viability. This study indicated that DPP or flax seed may enhance spermatogenesis through their stimulatory action on Sertoli cells. Moreover, both plants could reduce breast cancer cells viability. However, further investigations are required to elucidate the exact mechanisms behind these obtained findings.

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Objetivo: Avaliar a função das células testiculares de Sertoli em homens com lúpus eritematoso sistêmico (LES). Métodos: Trinta e quatro pacientes consecutivos foram prospectivamente selecionados para avaliar a função das células testiculares de Sertoli pelos níveis séricos da inibina B. Características clínicas, tratamento, análises dos espermatozóides, avaliação urológica, ultrasonografia testicular, hormônios e anticorpos anti-espermatozóides foram avaliados. Resultados: Os pacientes foram subdivididos em dois grupos: níveis séricos reduzidos (Grupo 1, n=8) e níveis séricos normais (Grupo 2, n=26) de inibina B. As medianas da concentração média de espermatozóides (p=0,024), da contagem total de espermatozóides (p=0,023) e da contagem total de espermatozóides móveis (p=0,025) foram menores no Grupo 1. Os níveis de inibina B foram positivamente correlacionados com a concentração de espermatozóides (r=0,343) e contagem total de espermatozóides móveis (r=0,357), e negativamente correlacionados com FSH (r=0,699) e LH (r=0,397). A mediana de inibina B sérica foi menor nos pacientes com LES tratados com pulsoterapia com ciclofosfamida endovenosa (CICIV) comparada aos não tratados com este medicamento (p=0,031). Avaliação dos 26 pacientes com LES e níveis normais de inibina B e FSH revelou que a mediana da relação inibina B/FSH foi menor nos pacientes lúpicos com oligozoospermia comparada aos pacientes com normozoospermia (p=0,004). Esta relação também foi menor nos pacientes com LES tratados com CICIV do que naqueles sem esta terapia (p=0,04). Conclusão: Este é o primeiro estudo que identificou uma alta freqüência de disfunção das células testiculares de Sertoli em homens com LES associada a anormalidades dos espermatozóides. Outros estudos prospectivos são necessários para determinar se os níveis séricos de inibina e a relação inibina B/FSH serão um marcador útil e precoce de toxicidade pela CICIV neste pacientes / Objective: To assess the testicular Sertoli cell function in male SLE patients. Methods: 34 consecutive patients were prospectively selected to evaluate the testicular Sertoli cell function by serum inhibin B levels. Clinical features, treatment, semen analysis, urologic evaluation, testicular ultrasound, hormones, and antisperm antibodies were determined. Results: Patients were subdivided into two Groups: low serum inhibin B (Group 1, n=8) and normal levels (Group 2, n=26). The medians of sperm concentration (p=0.024), total sperm count (p=0.023) and total motile sperm count (p=0.025) were lower in Group 1. Inhibin B levels were positively correlated with sperm concentration (r=0.343), total motile sperm count (r=0.357), and negatively correlated with FSH (r=0.699) and LH (r=0.397). The median serum inhibin B was lower in SLE patients treated with intravenous cyclophosphamide pulsetherapy (IVCYC) compared to those without this therapy (p=0.031). Further evaluation of the 26 SLE patients with normal inhibin B and FSH levels revealed that medians of inhibin B/FSH ratio were lower in SLE patients with oligozoospermia compared to normozoospermia (p=0.004). This ratio was also lower in SLE patients treated with IVCYC than those without this therapy (p=0.04). Conclusions: This is the first study to identify a high frequency of testicular Sertoli cell dysfunction in male SLE associated with sperm abnormalities. Further prospective studies are necessary to determine if inhibin levels and inhibin B/FSH ratio will be an earlier and useful marker of IVCYC toxicity in these patients

Objetivo: Avaliar a função das células testiculares de Sertoli em homens com lúpus eritematoso sistêmico (LES). Métodos: Trinta e quatro pacientes consecutivos foram prospectivamente selecionados para avaliar a função das células testiculares de Sertoli pelos níveis séricos da inibina B. Características clínicas, tratamento, análises dos espermatozóides, avaliação urológica, ultrasonografia testicular, hormônios e anticorpos anti-espermatozóides foram avaliados. Resultados: Os pacientes foram subdivididos em dois grupos: níveis séricos reduzidos (Grupo 1, n=8) e níveis séricos normais (Grupo 2, n=26) de inibina B. As medianas da concentração média de espermatozóides (p=0,024), da contagem total de espermatozóides (p=0,023) e da contagem total de espermatozóides móveis (p=0,025) foram menores no Grupo 1. Os níveis de inibina B foram positivamente correlacionados com a concentração de espermatozóides (r=0,343) e contagem total de espermatozóides móveis (r=0,357), e negativamente correlacionados com FSH (r=0,699) e LH (r=0,397). A mediana de inibina B sérica foi menor nos pacientes com LES tratados com pulsoterapia com ciclofosfamida endovenosa (CICIV) comparada aos não tratados com este medicamento (p=0,031). Avaliação dos 26 pacientes com LES e níveis normais de inibina B e FSH revelou que a mediana da relação inibina B/FSH foi menor nos pacientes lúpicos com oligozoospermia comparada aos pacientes com normozoospermia (p=0,004). Esta relação também foi menor nos pacientes com LES tratados com CICIV do que naqueles sem esta terapia (p=0,04). Conclusão: Este é o primeiro estudo que identificou uma alta freqüência de disfunção das células testiculares de Sertoli em homens com LES associada a anormalidades dos espermatozóides. Outros estudos prospectivos são necessários para determinar se os níveis séricos de inibina e a relação inibina B/FSH serão um marcador útil e precoce de toxicidade pela CICIV neste pacientes / Objective: To assess the testicular Sertoli cell function in male SLE patients. Methods: 34 consecutive patients were prospectively selected to evaluate the testicular Sertoli cell function by serum inhibin B levels. Clinical features, treatment, semen analysis, urologic evaluation, testicular ultrasound, hormones, and antisperm antibodies were determined. Results: Patients were subdivided into two Groups: low serum inhibin B (Group 1, n=8) and normal levels (Group 2, n=26). The medians of sperm concentration (p=0.024), total sperm count (p=0.023) and total motile sperm count (p=0.025) were lower in Group 1. Inhibin B levels were positively correlated with sperm concentration (r=0.343), total motile sperm count (r=0.357), and negatively correlated with FSH (r=0.699) and LH (r=0.397). The median serum inhibin B was lower in SLE patients treated with intravenous cyclophosphamide pulsetherapy (IVCYC) compared to those without this therapy (p=0.031). Further evaluation of the 26 SLE patients with normal inhibin B and FSH levels revealed that medians of inhibin B/FSH ratio were lower in SLE patients with oligozoospermia compared to normozoospermia (p=0.004). This ratio was also lower in SLE patients treated with IVCYC than those without this therapy (p=0.04). Conclusions: This is the first study to identify a high frequency of testicular Sertoli cell dysfunction in male SLE associated with sperm abnormalities. Further prospective studies are necessary to determine if inhibin levels and inhibin B/FSH ratio will be an earlier and useful marker of IVCYC toxicity in these patients

<p>The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies.</p> <p>Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male&nbsp / reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P &le / 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum.</p> <p>It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies.</p> <p>&nbsp / </p>