Au cours de la spermiogenèse, une réorganisation très importante de la chromatine se produit : la quasi-totalité des histones sont progressivement enlevées et remplacées par des protéines de transition puis par des protamines. Mon travail de thèse a alors consister à caractériser l'organisation finale de cette chromatine dans les spermatozoïdes ainsi que les mécanismes mis en jeu. Nos résultats montrent pour la première fois l'existence, dans les spermatides condensées de souris, de structures nucléoprotéiques contenant l'histone testiculaire TH2B ainsi que de nouveaux variants d'histones identifiés au laboratoire. De plus, ces structures organisent de manière préférentielle l'hétérochromatine péricentromérique de ces cellules. Cette réorganisation impliquerait la protéine chaperonne NAP1L4, qui pourrait être ciblée sur la chromatine en interagissant avec les queues N-terminales des histones H3. D'autres régions particulières du génome pourraient être la cible de la réorganisation différentielle de la chromatine par ces structures, permettant de les différencier du reste du génome et constituant ainsi le support d'une information épigénétique mâle transmise lors de la fécondation. L'analyse des protéines présentes dans les cellules germinales nous a également permis d'identifier deux autres protéines, HMGB4 et FYTTD1, qui ne semblent pas impliqués dans le processus de réorganisation de la chromatine dans les stades tardifs de la spermiogénèse. Néanmoins, le rôle potentiel de FYTTD1 dans le phénomène de maturation des ARN, ces derniers pouvant être un autre support de l'information épigénétique, pourrait s'avérer être un nouveau champ d'investigations très intéressant.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Univ., Diss., 2006--Giessen

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

In cultured medaka testis fragments, cells remained viable for the entire culture period (17h), and spermatids that developed from spermatocytes were viable and motile. Primary cultures were characterized over a period of two days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained in dynamic equilibrium in vitro for several days. Proliferating cells were predominant among clusters of suspended cells, as determined by BrdU labeling, and CFSE and propidium iodide PI labeling. Based on cytological criteria, the proliferating cells were mostly spermatogonia and possibly also preleptotene spermatocytes. Differentiation of spermatocytes into spermatids or spermatozoa was also observed, mainly among the suspended cells. These results suggest that the organ and primary culture systems are suitable systems for studying the effects of substances that interfere with spermatogenesis in the medaka, a model vertebrate. The organ and primary culture systems were used to analyze the effects of a synthetic estrogen, EE2, on cell proliferation in medaka testis. Both organ and primary culture were suitable for this purpose consistently small concentrations (0.01 and 1 nM) of EE2 stimulated cell proliferation slightly, while higher concentrations (100 nM) had an inhibitory effect. To investigate the effect of phytoestrogens on cell proliferation in spermatogenesis, selected flavonoids [genistein (1, 10, 100 µg/ml), quercetin (0.01, 1, 100 µM), and 8-prenylnarigenin (0.001, 0.1, 1, 10 µM)] were added to medaka testis primary cultures. Genistein and quercetin inhibited cell proliferation in the cultures while 8-prenylnarigenin had no effect. In a second series of experiments the addition of genistein (10 µg/ml) to primary cultures significantly inhibited both cell proliferation and cell differentiation as determined by flow cytometry using CFSE/PI labeling.

In cultured medaka testis fragments, cells remained viable for the entire culture period (17h), and spermatids that developed from spermatocytes were viable and motile. Primary cultures were characterized over a period of two days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained in dynamic equilibrium in vitro for several days. Proliferating cells were predominant among clusters of suspended cells, as determined by BrdU labeling, and CFSE and propidium iodide PI labeling. Based on cytological criteria, the proliferating cells were mostly spermatogonia and possibly also preleptotene spermatocytes. Differentiation of spermatocytes into spermatids or spermatozoa was also observed, mainly among the suspended cells. These results suggest that the organ and primary culture systems are suitable systems for studying the effects of substances that interfere with spermatogenesis in the medaka, a model vertebrate. The organ and primary culture systems were used to analyze the effects of a synthetic estrogen, EE2, on cell proliferation in medaka testis. Both organ and primary culture were suitable for this purpose consistently small concentrations (0.01 and 1 nM) of EE2 stimulated cell proliferation slightly, while higher concentrations (100 nM) had an inhibitory effect. To investigate the effect of phytoestrogens on cell proliferation in spermatogenesis, selected flavonoids [genistein (1, 10, 100 µg/ml), quercetin (0.01, 1, 100 µM), and 8-prenylnarigenin (0.001, 0.1, 1, 10 µM)] were added to medaka testis primary cultures. Genistein and quercetin inhibited cell proliferation in the cultures while 8-prenylnarigenin had no effect. In a second series of experiments the addition of genistein (10 µg/ml) to primary cultures significantly inhibited both cell proliferation and cell differentiation as determined by flow cytometry using CFSE/PI labeling.

Au cours de la maturation des gamètes mâles a lieu un événement unique : les structures classiques qui ordonnent l'ADN sont complètement bouleversées et remplacées par une nouvelle, de composition et d'agencement différents, qui permet au noyau du spermatozoïde final une compaction qui n'est retrouvée nulle part ailleurs. Les signaux épigénétiques ainsi que les événements qui se déroulent alors au niveau moléculaire ne sont pas encore bien élucidés. C'est dans l'optique d'une meilleure compréhension de ce phénomène que la protéine Atad2 a été identifiée et caractérisée. Ce facteur, qui possède un bromodomaine ainsi qu'un domaine AAA, est retrouvé sous deux formes chez la souris. La plus courte, strictement testiculaire, est capable de se lier à la chromatine acétylée. De plus, comme les autres membres de la famille des AAAATPases, Atad2 est capable de se multimériser. Cette multimérisation régule son affinité à la chromatine. Des études plus poussées ont montré un rôle dans l'activité transcriptionnelle ainsi que dans la stabilité des structures basales d'ordonnancement de l'information génétique, les nucléosomes. De plus, chez l'humain, Atad2 a été retrouvé exprimé de façon aberrante dans certaines tumeurs. L'élucidation du rôle de cette protéine dans les cellules germinales et somatiques apportera ainsi des informations sur le changement complet de la nature chromatinienne au cours de la spermatogenèse, et peut-être permettra peut-être une meilleure appréhension de l'activité génomique ayant lieux dans les cellules tumorales.

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

No description available.