Die vorliegende Arbeit untersucht die Steroidogenese und die Expression Leydig-Zellspezifischer Gene in Kulturen postnataler fetaler Leydig-Zellen (PFLC). Die Stimulation von PFLC mit hCG und (Bu)2cAMP bewirkt eine Steigerung der Testosteronproduktion in vitro. Es wurde eine zeitabhängige Abschwächung der Testosteronproduktion durch (Bu)2cAMPstimulierte PFLC beobachtet. Diese war begleitet von einer Akkumulation von Progesteron im Kulturmedium und einer Suppression der Expression von P450c17 auf dem translatorischen Level. Während der Kultivierung verloren PFLC ihre Fähigkeit der Expression Leydig-Zell-spezifischer Gene (z.B. 3βHSD, P450c17, Insl3). Dieses Phänomen konnte durch Stimulation mit (Bu)2cAMP rückgängig gemacht werden. Außerdem zeigte sich, dass PDGFα allein und in Kombination mit (Bu)2cAMP signifikant die Proliferation der PFLC in vitro stimulierte. Die vorliegende Arbeit deutet darauf hin, dass cAMP-aktivierte Signalkaskaden eine wichtige Rolle in der Regulation von Differenzierung und Funktion von PFLC spielen.

The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture. / Ph. D.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Le virus de l’Artérite Virale Équine (EAV) est un virus à ARN simple brin positif, appartenant à la famille des Arteriviridae, dans l’ordre des Nidovirales. C’est un virus spécifique des équidés qui peut être transmis par voies respiratoire et vénérienne. Lors de la primo-infection, l’EAV peut entraîner des signes cliniques grippaux, mais de façon plus grave, il peut aussi provoquer l’avortement des juments gestantes ainsi que la mort des nouveau-nés. L’EAV représente donc un enjeu économique majeur pour la filière équine. Suite à la primo-infection, ce virus peut persister dans de l’appareil reproducteur de certains étalons. Les mécanismes de cette persistance ne sont pas connus.Au cours de cette thèse, le premier modèle in vitro d’infection non lytique d’une lignée issue de l’appareil reproducteur mâle par l’EAV a été développé. L’infection de ces cellules de Leydig a montré une induction de l’expression de nombreux gènes de l’immunité innée dont ceux codant pour des cytokines pro-inflammatoires et des chimiokines qui permettraient le recrutement de cellules de l’immunité innée au niveau des testicules, et qui pourraient expliquer l’orchite observée chez certains étalons lors de la phase aiguë de l’infection. Pour les étalons infectés de façon persistante, la castration et les traitements anti-GnRH peuvent permettre la suppression de la persistance du virus, suggérant ainsi une implication de la testostérone dans la persistance du virus. Les cellules TM3 exprimant le récepteur aux androgènes, des essais de traitements ont été réalisés. Les premiers résultats préliminaires semblent indiquer que les cellules TM3 ne répondent pas ou peu au stimulus hormonal. Cependant, des tests de prétraitement par la testostérone seraient à envisager afin d’en étudier les conséquences sur le cycle viral. Ce modèle d’infection non lytique reste cependant un modèle intéressant pouvant être utilisé afin d’étudier les relations hôte-pathogène et pouvant aider à comprendre les mécanismes impliqués dans la persistance de l’EAV. / Equine Arteritis Virus (EAV) is a positive-strand RNA virus, which belongs to the Arteriviridae familly, in the Nidovirales order. It is an equid specific virus that can be transmitted by respiratory and venereal routes. During primary infection, EAV can induce flu-like clinical signs, but worse, it may also cause the abortion of pregnant mares and newborn foal death. EAV is therefore a main economic challenge for the horse industry. Following primary infection, this virus is able to persist in the reproductive tract of some stallions. The mechanisms of this persistence remain unknown.During this thesis, the first in vitro model of an EAV non-lytic infection of a male reproductive tract cell line has been developed. EAV infection of these Leydig cells induced the expression of numerous innate immune genes including those coding for pro-inflammatory cytokines and chemokines, which could recruit innate immune cells to testicles and which could explain the orchitis observed in some stallions during primary infection.For persistently infected stallions, castration and anti-GnRH treatments can suppress EAV persistence, suggesting an involvement of testosterone in the virus persistence. Since TM3 cells express the androgen receptor, treatment trials have been performed. The first preliminary results suggest TM3 cells do not respond to the hormonal stimulus, or only a little. However, pretreatment trials should be realized to study the consequences on the viral cycle.Nevertheless, this non-lytic infection model is still an interesting model that can be used to study the host-pathogen relationship and that could help understanding the mechanisms involved in EAV persistence.

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Submitted by (lucia.rodrigues@ufrpe.br) on 2016-05-31T15:10:49Z No. of bitstreams: 1 Bruno Mendes Tenorio.pdf: 4101398 bytes, checksum: d81bf09fb306302eb9abbcd6d926a6a7 (MD5) / Made available in DSpace on 2016-05-31T15:10:49Z (GMT). No. of bitstreams: 1 Bruno Mendes Tenorio.pdf: 4101398 bytes, checksum: d81bf09fb306302eb9abbcd6d926a6a7 (MD5) Previous issue date: 2011-12-12 / In recent decades, humans have been exposed to various exogenous physical agents, such as high temperatures emitted by industrial devices and electromagnetic fields emitted by the electricity distribution networks and electronic devices. Researches demonstrated that these agents can cause reproductive disorders. The chapter 1 of this study aimed to investigate the possible effects of low frequency EMF exposure of 60 Hz and 1 mT from day 13 of gestation up to puberty at 21 postnatal days. The results showed that exposure to EMF reduced parameters related to the lumen, epithelium and seminiferous tubules. It was also observed an increase in the volume of blood vessels and connective tissue cells in animals exposed to EMF. The plasma testosterone did not change due to EMF exposure. The exposure to EMF of 60 Hz and 1 mT from gestation to puberty can induce a delay in testicular development. In chapter 2, the aim was to evaluate the effects of exposure to low frequency EMF (60 Hz and 1 mT) from the 13th day of gestation up to 90 postnatal days (adult). The plasma testosterone concentration was not changed by EMF exposure. However, the histopathological and histomorphometrical analysis showed testicular degeneration in a subset of animals exposed to EMF. The degenerative process severity varied among affected animals, indicating an individual sensitivity to EMF. The electron microscopy analysis also showed testicular degeneration, the main change was high electrodense mitochondria with loss of normal organization and cristae. The exposure to EMF of 60 Hz and 1 mT seems to alter spermatogenesis and reduce the fertility. Chapter 3 aimed to analyze the effects of heat shock (43 °C / 15 min.) and Hsp90 inhibition on CaV3 T-type calcium channels and calciumactivated potassium channels (BKCa) in Leydig cells. The results showed that exposure to heat shock induced a reduction in BKCa channels steady-state current (49.8%), maximum conductance (68.9%) and activation time constant (31.9%). Heat shock slowed the BKCa channels activation and reduced its voltage dependence. Hsp90 inhibition did not change BKCa channels. The CaV3 channels current was not affected by heat shock or Hsp90 inhibition. The heat shock can inhibit BKCa channels, the Hsp90 does not seem to be involved in this process. / Nas últimas décadas, os seres humanos vêm sendo expostos a vários agentes físicos exógenos, como temperaturas elevadas induzidas por equipamentos industriais e a exposição aos campos eletromagnéticos (CEM) emitidos por aparelhos eletrônicos. Pesquisas vêm demonstrando que estes agentes podem gerar distúrbios reprodutivos. O objetivo do capítulo 1 do presente trabalho foi investigar o possível efeito da exposição ao CEM de baixa frequência de 60 Hz e 1 mT desde o 13º dia de gestação até a puberdade aos 21 dias pós-natal. Os resultados demonstraram que a exposição ao CEM reduziu os parâmetros relacionados ao lúmen, epitélio e túbulos seminíferos. Também foi observado o aumento no volume dos vasos sanguíneos e das células do tecido conjuntivo nos animais expostos ao CEM. A testosterona plasmática não se alterou devido à exposição ao CEM. A exposição ao CEM de 60 Hz e 1 mT desde a gestação até a puberdade pode induzir o retardo no desenvolvimento testicular. No capítulo 2, o objetivo foi avaliar o efeito da exposição ao CEM de baixa frequência (60 Hz e 1 mT) desde o 13º dia de gestação até os 90 dias pós-natal (adulto). A concentração da testosterona plasmática não foi alterada devido à exposição ao CEM. Entretanto, as análises histopatológicas e histomorfométricas demonstraram degeneração testicular em um sub-grupo dos animais expostos ao CEM. A gravidade do processo degenerativo variou entre os indivíduos afetados, indicando uma sensibilidade individual ao CEM. A análise de microscopia eletrônica também demonstrou degeneração testicular, sendo a principal alteração observada a presença de mitocôndrias eletrodensas e com perda da sua organização e cristas. A exposição ao CEM de 60 Hz e 1 mT parece alterar a espermatogênese e reduzir a fertilidade. No capítulo 3, o presente trabalho objetivou analisar os efeitos do choque térmico (43ºC / 15 min.) e da inibição da Hsp90 nos canais de cálcio CaV3 tipo-T e potássio ativado por cálcio (BKCa) em células de Leydig. Os resultados obtidos revelaram que nos canais BKCa o choque térmico reduziu a corrente do estado estacionário em 49,8%, a condutância máxima em 68,9% e a constante de tempo de ativação em 31,9%. O choque térmico tornou mais lenta a ativação dos canais BKCa e reduziu sua dependência de voltagem. A inibição da Hsp90 não alterou os canais BKCa. A corrente dos canais CaV3 tipo-T não foi afetada pelo estresse térmico ou pela inibição da Hsp90. A exposição das células de Leydig à temperatura elevada pode inibir os canais BKCa, a Hsp90 parece não estar envolvida neste processo.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-20T20:06:17Z No. of bitstreams: 1 leonardotoshiooshio.pdf: 6184732 bytes, checksum: 2ae5dded31a5fbae3336c3f4555afe20 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T15:26:54Z (GMT) No. of bitstreams: 1 leonardotoshiooshio.pdf: 6184732 bytes, checksum: 2ae5dded31a5fbae3336c3f4555afe20 (MD5) / Made available in DSpace on 2016-07-13T15:26:54Z (GMT). No. of bitstreams: 1 leonardotoshiooshio.pdf: 6184732 bytes, checksum: 2ae5dded31a5fbae3336c3f4555afe20 (MD5) Previous issue date: 2012-08-31 / O Extrato de Ginkgo biloba (EGb) é um dos fitoterápicos mais consumidos no mundo e tem sido utilizado no tratamento da disfunção erétil e como afrodisíaco. O presente trabalho teve como objetivo avaliar a toxicidade sistêmica do EGb e o efeito sobre o sistema reprodutor masculino de ratos Wistar. Oitenta animais de três meses de idade foram tratados com água destilada (Grupo Controle) e extrato aquoso de Ginkgo biloba nas seguintes doses: 3,5 (EGb 3,5); 7,0 (EGb 7,0) e 14,0 mg/kg (EGb 14,0) uma vez ao dia, por 56 dias consecutivos. Foram avaliados o peso corporal, estimativa de consumo diário de ração, indícios de sinais clínicos de toxicidade, peso de órgãos e glândulas acessórias do sistema reprodutor masculino e dados histométricos testiculares. Espermatozoides foram coletados da cauda do epidídimo e foram submetidos à contagem e avaliados quanto à vitalidade e morfologia. Foram realizados hemograma completo, dosagem bioquímica sérica de ureia, creatinina e alanina aminotransferase (ALT) e concentração de testosterona total sérica. Não foram observados nos animais sinais clínicos de toxicidade sistêmica e mortes. Apesar de ter ocorrido diferenças estatísticas significativas na estimativa de consumo de ração, hemoglobinometria, concentração de hemoglobina globular média e volume das células de Leydig, concluise que o EGb no presente trabalho, e com as doses utilizadas, não causou toxicidade sistêmica nem promoveu alteração na morfometria testicular e qualidade espermática de ratos Wistar. / Ginkgo biloba Extract (GBE) is one of the most consumed herbal medicines in the world and it has been used in the treatment of erectile dysfunction and as an aphrodisiac. The present study had as objective to evaluate the GBE systemic toxicity and its effect on male reproductive system of Wistar rats. Eighty animals of threemonth- old age were treated with distilled water (Control Group) and aqueous extract of Ginkgo biloba in the following doses: 3.5 (GBE 3.5); 7.0 (GBE 7.0) and 14.0 mg/kg (GBE 14.0) once per day, for 56 consecutive days. Body weight, daily food consumption estimation, toxicity clinical signs evidences, male reproductive system organs and accessory glands weights and testis histometric analysis data were evaluated. Spermatozoa were collected from the cauda epididymis and were subjected to counting and evaluations of vitality and morphology. Complete blood count test, serum biochemistry test of urea, creatinine and alanine aminotransferase (ALT) and total serum testosterone levels were realized. Clinical signs of systemic toxicity and deaths were not seen. In spite of it had had significant statistical differences on daily food consumption estimation, hemoglobinometry, mean corpuscular hemoglobin concentration and Leydig cell volume, it was concluded that GBE in the present study and with the doses used, did not cause systemic toxicity nor promoted alterations on testicular morphometry and spermatic quality of Wistar rats.

L’impact de l’utilisation des agropesticides sur la fonction de reproduction mâle est évalué chez des agriculteurs de Djutitsa (Ouest Cameroun). Le pesticide le plus utilisé par ces agriculteurs est testé in vivo sur la fonction de reproduction du rat mâle, ex vivo et/ou in vitro sur la stéroïdogenèse Leydigienne afin d’évaluer son implication dans les déséquilibres observés chez les agriculteurs. Ensuite, l’effet protecteur des extraits de Carpolobia alba et Basella alba contre les effets délétères dudit pesticide est évalué in vivo chez des rats mâles, ainsi que leur influence sur la production de la protéine de transport des stéroïdes (SHBG) par les cellules HepG2. Les résultats montrent que les agriculteurs utilisent 25 agropesticides et présentent des symptômes liés à une protection inadéquate. De plus, ils présentent une diminution de la testostérone avec une élévation de l’androstènedione sériques comparé au groupe témoin non exposé (P < 0,05) et ces déséquilibres androgéniques sont accompagnés de difficultés de reproduction. Le manèbe est l’agropesticide le plus utilisé et perturbe chez le rat mâle la stéroïdogenèse Leydigienne (à travers l’inhibition de la CYP11A1) et la fertilité. Cette fertilité est améliorée/rétablie lorsque le pesticide est concomitamment administré avec l’un des extraits, suggérant ainsi l’effet protecteur desdits extraits qui est imputable à leur activité androgénique également démontré chez les rats. Toutefois, ces extraits et le manèbe n’affectent pas la production de la SHBG in vitro. Ces résultats soulignent l’altération de la fonction de reproduction mâle par les agropesticides et l’effet bénéfique des plantes médicinales / The effect of agropesticides use on male reproductive function was evaluated in male farmers in Djutitsa (West Cameroon). The most frequently used agropesticide by farmers was selected and tested in vivo on the reproductive function of male rats, ex vivo and/or in vitro on Leydig cells steroidogenesis, in order to evaluate its implication in disorders observed in farmers. The ability of 2 Cameroonian medicinal plants (Carpolobia alba and Basella alba) to prevent pesticide-induced toxicity was also investigated in vivo in male rats. Furthermore, both extracts and selected pesticide were tested on the release of a steroid transporter protein (SHBG) by HepG2 cells. The results showed that farmers used 25 agropesticides and presented symptoms related to inadequate protection. Moreover, they had lower serum testosterone and higher androstenedione levels compared to a control group with no history of agropesticide exposure (P < 0.05), and these androgenic imbalances were accompanied by difficulties of reproduction. Maneb was the most common ingredient, and its administration to male rats resulted in decrease/alteration of Leydig cells steroidogenesis (through inhibition of CYP11A1) as well as fertility. The latter fertility was improved/restored when maneb was coadministrated to rats with any of the plant extracts, suggesting their protective effect that may be attributed to their proven androgenic activity. However maneb and the plant extracts did not affect SHBG release by HepG2 cells. These results highlight agropesticides deleterious effect on male reproductive function, which may be prevented by the investigated plant extracts

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.