Éditeur(s) : HAL CCSD
Résumé : Spermatogenesis in mammals is a complex biological function including cellular processes such as proliferation, meiosis and differentiation, aiming to the production of male gametes in the testis. If the seminiferous epithelium is well described in terms of organization and cellular morphology of cells that compose it, the processes by which undifferentiated diploid germ cells enter meiosis and give haploid cells that undergo many morphological transformations, are not fully decrypted. These processes rely on the coordinated and sequential expression of genes, including specific products for each stage of germ cell development These gene products are essential at each key stage of spermatogenesis. Transcriptomics since the 1990s, and proteomics since the 2000s have contributed to the improved. understanding of these mechanisms. A long term proteomic study aiming at characterizing the proteomes of Sertoli cells and germ cells, and a recent study that characterized and quantified the transcriptome of isolated rat testicular cells at high resolution using de novo sequencing of transcripts (RNA-Seq), have been the basis of my thesis work. The latter study showed the accumulation of long non-Coding RNAs (lncRNAs) and testicular unannotated transcripts (TUTs) at meiotic and post-Meiotic stages of spermatogenesis in the rat. In this context, my thesis work aimed at validating the coding potential of many genes expressed in germ cells using RNA-Seq combined with shotgun proteomics, a so-Called PIT (Proteomics Informed by transcriptomics) approach. In this approach, the protein sequences translated from the transcripts assembled by RNA-Seq in the different testicular cell types are integrated into a custom database of protein sequences used to query mass spectrometry data obtained from proteins of meiotic and post-Meiotic cells. The PIT approach showed that 69 TUTs or lncRNA (corresponding to 44 loci) code for proteins in meiotic cells and post meiotic cells, and we confirmed experimentally the meiotic and post-Meiotic expression for two new transcripts encoding for VAMP9, a protein of the SNARE family, and a new testicular enolase T-ENOL. The post-Meiotic expression of T-ENOL protein was confirmed by immunohistochemistry using a polyclonal antibody raised against the recombinant protein. This approach also allowed us to identify new isoforms of known proteins, specific to each stage of spermatogenesis. Germ cells and Sertoli cells maintain a dialogue which is necessary to the success of spermatogenesis and spermiogenesis. Another part of my work aimed at identifying membrane proteins, in germ cells and residual bodies, that may be involved in the dialogue between Sertoli cells and germ cells, using a ICPL relative quantification proteomic approach. The ICPL analysis enabled us to establish a list of 166 proteins whose expression is differential between pachytene spermatocytes, round spermatids and residual bodies. Their differential expression suggests that these proteins may play a role in spermiogenesis. Thanks to the Gene Ontology annotations, a list of 8 proteins with a putative role in signal transduction, cell recognition or differentiation, thus potentially involved in the dialogue between Sertoli and germ cells was drawn. In addition, I provided a first proteome of rat Sertoli cells, germ cells and residual bodies obtained by shotgun proteomics.
La spermatogenèse chez les mammifères est une fonction biologique complexe incluant des processus de prolifération cellulaire, de méiose et de différenciation uniques visant à la production des gamètes mâles au sein du testicule. Si l’épithélium séminifère est bien décrit sur le plan de son organisation et de la morphologie des cellules qui le composent, les processus par lesquels les cellules germinales diploïdes indifférenciées entrent en méiose pour donner ensuite des cellules haploïdes subissant par la suite de nombreuses transformations morphologiques, ne sont pas totalement décryptés. Ils reposent sur l’expression coordonnée et séquentielle de gènes dont les produits spécifiques de chaque stade de développement des cellules germinales sont essentiels aux étapes clés de la spermatogenèse. La transcriptomique depuis les années 1990 et la protéomique depuis les années 2000 ont contribué à l’amélioration de la connaissance de ces mécanismes. Une étude protéomique visant à caractériser par des approches systématiques et différentielles les protéomes des cellules de Sertoli et de la lignée germinale, et d’autre part une étude récente, réalisée dans notre unité, qui a permis de caractériser et de quantifier le transcriptome des cellules testiculaires isolées de rat en utilisant le séquençage de novo des transcrits (RNA-Seq), ont été à la base de mes travaux de thèse. Cette dernière étude a mis en évidence l’accumulation de longs ARNs non codants (lncRNAs) et de transcrits testiculaires non annotés (TUTs) aux stades méiotique et post- méiotique de la spermatogenèse chez le rat. Dans ce contexte, mon travail a consisté à valider le potentiel codant de nombreux gènes exprimés dans les cellules germinales par une approche dite PIT (Proteomics Informed by Transcriptomics) couplant protéomique Shotgun et RNA-Seq. Dans ce type d’approche, les séquences protéiques déduites des transcrits des différents types cellulaires, assemblés par RNA-Seq, sont intégrées dans une base personnalisée de séquences protéiques utilisée pour interroger les données de spectrométrie de masse obtenues à partir de protéines de cellules méiotiques et post-Méiotiques. L’approche PIT a permis de montrer que 69 TUTs ou lncRNA (correspondant à 44 loci) codent pour des protéines dans les cellules méiotiques et post méiotiques. L’expression post-Méiotique de deux nouveaux transcrits, l’un codant pour la protéine VAMP9, une protéine de la famille SNARE, et l’autre pour une nouvelle énolase T-ENOL a pu être confirmée. L’expression post-Méiotique de T-ENOL a été confirmée par immunohistochimie à l’aide d’un anticorps polyclonal produit contre la protéine recombinante. Cette approche nous a également permis d’identifier de nouvelles isoformes de protéines connues spécifiques de chaque stade de la spermatogenèse. Les cellules germinales et les cellules de Sertoli entretiennent le dialogue nécessaire au bon déroulement de la spermatogenèse. Une autre partie de mon travail a consisté à identifier des protéines membranaires des cellules germinales et des corps résiduels, susceptibles d’intervenir dans le dialogue entre les cellules de Sertoli et les cellules germinales, par une approche protéomique de quantification relative ICPL. Cette approche a permis d’établir une liste de 166 protéines différentiellement exprimées entre les spermatocytes pachytène, les spermatides rondes et les corps résiduels, qui sont susceptibles de jouer un rôle dans la spermiogénèse. Grâce aux annotations de le Gene Ontology, j’ai pu établir une liste de 8 protéines ayant un rôle supposé dans la transduction du signal, la reconnaissance cellulaire ou bien la différenciation. Par ailleurs, j’ai pu établir par protéomique Shotgun un premier protéome des cellules de Sertoli, des cellules germinales et des corps résiduels chez le rat.
https://tel.archives-ouvertes.fr/tel-01127389

Droits : info:eu-repo/semantics/OpenAccess
NNT : 2014REN1S064
tel-01127389
https://tel.archives-ouvertes.fr/tel-01127389
https://tel.archives-ouvertes.fr/tel-01127389/document
https://tel.archives-ouvertes.fr/tel-01127389/file/2014REN1S064.pdf

Éditeur(s) : HAL CCSD Federation of American Society of Experimental Biology
Résumé : International audience
Testes of most male mammals present the particularity of being externalized from the body and are consequently slightly cooler than core body temperature (4-8 degrees C below). Although, hypothermia of the testis is known to increase germ cells apoptosis, little is known about the underlying molecular mechanisms, including cold sensors, transduction pathways, and apoptosis triggers. In this study, using a functional knockout mouse model of the cold and menthol receptors, dubbed transient receptor potential melastatine 8 (TRPM8) channels, we found that TRPM8 initiated the cold-shock response by differentially modulating cold- and heat-shock proteins. Besides, apoptosis of germ cells increased in proportion to the cooling level in control mice but was independent of temperature in knockout mice. We also observed that the rate of germ cell death correlated positively with the reactive oxygen species level and negatively with the expression of the detoxifying enzymes. This result suggests that the TRPM8 sensor is a key determinant of germ cell fate under hypothermic stimulation.Borowiec, A.-S., Sion, B., Chalmel, F., Rolland, A. D., Lemonnier, L., De Clerck, T., Bokhobza, A., Derouiche, S., Dewailly, E., Slomianny, C., Mauduit, C., Benahmed, M., Roudbaraki, M., Jegou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.
ISSN: 0892-6638

hal-01390973
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390973
DOI : 10.1096/fj.201600257R
PUBMED : 27317670

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
EXOSC10 is a catalytic subunit of the exosome that processes biologically active transcripts, degrades aberrant mRNAs and targets certain long non-coding RNAs (lncRNAs). The yeast orthologue Rrp6 is required for efficient growth and gametogenesis, and becomes unstable during meiosis. However, nothing is known about the localization, stability and function of EXOSC10 in the rodent male germline. We detect the protein in nucleoli and the cytoplasm of mitotic and meiotic germ cells, and find that it transiently associates with the XY body, a structure targeted by meiotic sex chromosome inactivation (MSCI). Finally, EXOSC10 becomes unstable at later stages of gamete development. To determine Exosc10's meiotic function, we inactivated the gene specifically in male germ cells using cre recombinase controlled by Stra8 or Ddx4/Vasa promoters. Mutant mice have small testes, show impaired germ cell differentiation and are subfertile. Our results demonstrate that EXOSC10 is post-translationally regulated in germ cells, associate the protein with epigenetic chromosome silencing, and reveal its essential role in germ cell growth and development.
ISSN: 2045-2322

hal-01647082
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647082
DOI : 10.1038/s41598-017-14643-y
PUBMED : 29118343

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
BACKGROUND: Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS: We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS: Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS: Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.
ISSN: 0268-1161

hal-00877243
https://hal.archives-ouvertes.fr/hal-00877243
DOI : 10.1093/humrep/des301
PUBMED : 22926843

Éditeur(s) : HAL CCSD
Résumé : International audience
STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase₉) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.
Molecular Human Reproduction

hal-01647081
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647081
DOI : 10.1093/molehr/gax054
PUBMED : 29040674

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.
ISSN: 1355-8382

hal-00985894
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00985894
DOI : 10.1261/rna.043729.113
PUBMED : 24554440

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10(-9)-10(-5) M for 24 or 48 h. Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10(-9) M; P < 0.05), BPB (10(-9) M; P < 0.05), BPS (10(-9) and 10(-8) M; P < 0.05) and BADGE (10(-5) M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. N/A. Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis.
ISSN: 0268-1161

hal-01578582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582/file/Parallel%20assessment%20of%20the%20effects%20of%20bisphenol%20A_accepted.pdf
DOI : 10.1093/humrep/dex093

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm (''preformation'' or ``maternal inheritance'') or by inductive signaling (classically termed ``epigenesis'' or ``zygotic induction''). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent ``interstitial'' cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.
ISSN: 0012-1606

hal-01544487
https://hal.archives-ouvertes.fr/hal-01544487
DOI : 10.1016/j.ydbio.2012.01.018

Éditeur(s) : HAL CCSD
Résumé : International audience
Studies report that foetal exposure to paracetamol by maternal consumption can interfere with male reproductive development. Moreover, recent biomonitoring data report widespread presence of paracetamol in German and Danish populations, suggesting exposure via secondary (non-pharmaceutical) sources such as metabolic conversion from the ubiquitous industrial compound aniline. In this study we investigated the extent to which paracetamol and aniline can interfere with female reproductive development. Intrauterine exposure to paracetamol by gavage of pregnant dams resulted in shortening of the anogenital distance in adult offspring, suggesting that foetal hormone signalling had been disturbed. Female offspring of paracetamol-exposed mothers had ovaries with diminished follicle reserve and reduced fertility. Foetal gonads of exposed animals had also reduced gonocyte numbers, suggesting that the reduced follicle count in adults could be due to early disruption of germ cell development. However, ex vivo cultures of ovaries from 12.5 days post coitum foetuses showed no decrease in proliferation or expression following exposure to paracetamol. This suggests that the effect of paracetamol occurs prior to this developmental stage. Accordingly, using embryonic stem cells as a proxy for primordial germ cells we show that paracetamol is an inhibitor of cellular proliferation, but without cytotoxic effects. Collectively, our data show that intrauterine exposure to paracetamol at levels commonly observed in pregnant women, as well as its precursor aniline, may block primordial germ cell proliferation, ultimately leading to reduced follicle reserves and compromised reproductive capacity later in life
Toxicological Sciences

hal-01259211
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01259211
DOI : 10.1093/toxsci/kfv332
PUBMED : 26732887

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture.
ISSN: 1932-6203

hal-01129957
https://hal.archives-ouvertes.fr/hal-01129957
https://hal.archives-ouvertes.fr/hal-01129957/document
https://hal.archives-ouvertes.fr/hal-01129957/file/2014_FChalmel_PlosOne_%7B1236BFCE-4830-400D-B816-4B6651311900%7D.pdf
DOI : 10.1371/journal.pone.0104418

Éditeur(s) : Springer
Résumé : The transforming growth factor (TGF)-beta superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-beta gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-beta appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-beta family members. The function of og TGF-beta protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-beta protein, and we demonstrate that og TGF-beta is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-beta in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-beta function tends to reduce gonadic area. We conclude that mature og TGF-beta probably functions as an activator of germ cells development in oyster.
Marine Biotechnology (1436-2228) (Springer), 2011-10 , Vol. 13 , N. 5 , P. 971-980

Droits : Springer Science+Business Media, LLC 2011
http://archimer.ifremer.fr/doc/00042/15371/13438.pdf
DOI:10.1007/s10126-010-9361-4
http://archimer.ifremer.fr/doc/00042/15371/

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture.
PLoS ONE

hal-01063901
https://hal.archives-ouvertes.fr/hal-01063901
DOI : 10.1371/journal.pone.0104418
PUBMED : 25111155

Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
Spermatogenesis is a complex process, dependent upon the successive activation and/or repression of thousands of gene products, and ends with the production of haploid male gametes. RNA sequencing of male germ cells in the rat identified thousands of novel testicular unannotated transcripts (TUTs). Although such RNAs are usually annotated as long noncoding RNAs (lncRNAs), it is possible that some of these TUTs code for protein. To test this possibility, we used a "proteomics informed by transcriptomics" (PIT) strategy combining RNA sequencing data with shotgun proteomics analyses of spermatocytes and spermatids in the rat. Among 3559 TUTs and 506 lncRNAs found in meiotic and postmeiotic germ cells, 44 encoded at least one peptide. We showed that these novel high-confidence protein-coding loci exhibit several genomic features intermediate between those of lncRNAs and mRNAs. We experimentally validated the testicular expression pattern of two of these novel protein-coding gene candidates, both highly conserved in mammals: one for a vesicle-associated membrane protein we named VAMP-9, and the other for an enolase domain-containing protein. This study confirms the potential of PIT approaches for the discovery of protein-coding transcripts initially thought to be untranslated or unknown transcripts. Our results contribute to the understanding of spermatogenesis by characterizing two novel proteins, implicated by their strong expression in germ cells. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD000872.
ISSN: 0006-3363

hal-01121802
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01121802
PUBMED : 25210130
DOI : 10.1095/biolreprod.114.122416

Éditeur(s) : HAL CCSD
Résumé : International audience
The SOX8 and SOX9 transcription factors are involved in, among others, sex differentiation, male gonad development and adult maintenance of spermatogenesis. Sox8(-/-) mice lacking Sox9 in Sertoli cells fail to form testis cords and cannot establish spermatogenesis. Although genetic and histological data show an important role for these transcription factors in regulating spermatogenesis, it is not clear which genes depend upon them at a genome-wide level. To identify transcripts that respond to the absence of Sox8 in all cells and Sox9 in Sertoli cells we measured mRNA concentrations in testicular samples from mice at 0, 6 and 18 days post-partum. In total, 621 and 629 transcripts were found at decreased or increased levels, respectively, at different time points in the mutant as compared to the control samples. These mRNAs were categorized as preferentially expressed in Sertoli cells or germ cells using data obtained with male and female gonad samples and enriched testicular cell populations. Five candidate genes were validated at the protein level. Furthermore, we identified putative direct SOX8 and SOX9 target genes by integrating predicted SOX-binding sites present in potential regulatory regions upstream of the transcription start site. Finally, we used protein network data to gain insight into the effects on regulatory interactions that occur when Sox8 and Sox9 are absent in developing Sertoli cells. The integration of testicular samples with enriched Sertoli cells, germ cells and female gonads enabled us to broadly distinguish transcripts directly affected in Sertoli cells from others that respond to secondary events in testicular cell types. Thus, combined RNA profiling signals, motif predictions and network data identified putative SOX8/SOX9 target genes in Sertoli cells and yielded insight into regulatory interactions that depend upon these transcription factors. In addition, our results will facilitate the interpretation of genome-wide in vivo SOX8 and SOX9 DNA binding data.
Andrology

hal-00877013
https://hal.archives-ouvertes.fr/hal-00877013
DOI : 10.1111/j.2047-2927.2012.00049.x
PUBMED : 23315995

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
STUDY QUESTION: Can protein biomarkers of the male genital tract be identified in human seminal plasma? SUMMARY ANSWER: We identified potential biomarkers for each of the organs participating in the secretions of the human seminal plasma. WHAT IS KNOWN ALREADY: The seminal plasma fulfills critical functions for fertility by providing spermatozoa with a protective milieu, promoting their final maturation and modulating the immune responsiveness of the female reproductive tract. It is also considered to be a promising source of biomarkers of male infertility and/or pathologies of the male genital tract. STUDY DESIGN, SIZE, DURATION: This study combines proteomic analyses of normal seminal plasma together with transcriptomic gene expression profiling of human healthy tissues. MATERIALS, SETTING, METHODS: Non-liquefied seminal plasma proteins from a healthy donor were prefractionated using two sequential Proteominer™ libraries. Eight subproteome fractions were collected, trypsin digested and subjected to three successive mass spectrometry analyses for peptide characterization. The list of identified proteins was compared with and merged with other available data sets of the human seminal plasma proteome. The expression of corresponding genes was then investigated using tissue transcriptome profiles to determine where, along the male reproductive tract, these proteins were produced. Finally, tissue specificity of a selected subset of biomarker candidates was validated on human tissues. MAIN RESULTS AND THE ROLE OF CHANCE: We first performed a proteomic analysis of the human seminal plasma and identified 699 proteins. By comparing our protein list with other previous proteomic data sets, we found that 2545 unique proteins have been described so far in the human seminal plasma. We then profiled their expression at the gene level and identified 83 testis, 42 epididymis, 7 seminal vesicle and 17 prostate candidate protein markers. For a subset of testis-specific candidates, i.e. TKTL1, LDHC and PGK2, we further validated their germ cell expression and demonstrated that such markers could distinguish between semen from fertile and infertile men. LIMITATIONS, REASONS FOR CAUTION: While some of the markers we identified are well-known tissue-specific products, further dedicated studies to validate the biomarker status of new candidates will be required. Additionally, whether or not the abundance of these proteins is indeed decreased in some specific pathological situations remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Using an integrative genomics approach, we identified biomarker candidates for each of the organs participating in the seminal plasma production. In this study, we essentially focused on germ cell markers and their potential application for the diagnosis of male infertility. Other types of markers also deserve a focused attention given their potential predictive value for various reproductive disorders, notably for prostate cancers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Proteomics Core Facility at Biogenouest and was funded by Conseil Régional de Bretagne, IBiSA and Agence de la Biomédecine grants. The authors declare that there exists a competing financial interest in this work that is related to a patent application on the use of identified germ cell-specific proteins in an antibody-based assay (Fertichip™) to predict the successful testicular biopsy outcomes in human non-obstructive azoospermia.
ISSN: 0268-1161

inserm-00814146
http://www.hal.inserm.fr/inserm-00814146
DOI : 10.1093/humrep/des360
PUBMED : 23024119

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.
ISSN: 1932-6203

inserm-00825763
http://www.hal.inserm.fr/inserm-00825763
http://www.hal.inserm.fr/inserm-00825763/document
http://www.hal.inserm.fr/inserm-00825763/file/Rolland_et_al.pdf
DOI : 10.1371/journal.pone.0053302

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
BOI1 and BOI2 are paralogs important for the actin cytoskeleton andpolar growth. BOI1 encodes a meiotic transcript isoform with an extended5-untranslated region predicted to impair protein translation. It is,however, unknown how the isoform is repressed during mitosis, and ifBoi1 is present during sporulation. By interpreting microarray data fromMATa cells, MATa/ cells, a starving MAT/ control, and a meiosis-impairedrrp6 mutant, we classified BOI1's extended isoform as earlymeiosis-specific. These results were confirmed by RNA-Sequencing, andextended by a 5-RACE assay and Northern blotting, showing that meioticcells induce the long isoform while the mitotic isoform remainsdetectable during meiosis. We provide evidence via motif predictions, anin vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6histone deacetylase complex, which represses meiotic genes duringmitosis, also prevents the induction of BOI1's 5-extended isoform inmitosis by direct binding of Ume6 to its URS1 target. Finally, we findthat Boi1 protein levels decline when cells switch from fermentation torespiration and sporulation. The histone deacetylase Rpd3 is conserved,and eukaryotic genes frequently encode transcripts with variable 5-UTRs.Our findings are therefore relevant for regulatory mechanisms involvedin the control of transcript isoforms in multi-cellular organisms.
ISSN: 0950-382X

hal-01122120
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01122120
DOI : 10.1111/mmi.12976
PUBMED : 25708805

Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
The Chromosome-Centric Human Proteome Project (C-HPP) aims at cataloguing the proteins as gene products encoded by the human genome in a chromosome-centric manner. The existence of products of about 82% of the genes has been confirmed at the protein level. However, the number of so-called "missing proteins" remains significant. It was recently suggested that the expression of proteins that have been systematically missed might be restricted to particular organs or cell types, for example, the testis. Testicular function, and spermatogenesis in particular, is conditioned by the successive activation or repression of thousands of genes and proteins including numerous germ cell- and testis-specific products. Both the testis and postmeiotic germ cells are thus promising sites at which to search for missing proteins, and ejaculated spermatozoa are a potential source of proteins whose expression is restricted to the germ cell lineage. A trans-chromosome-based data analysis was performed to catalog missing proteins in total protein extracts from isolated human spermatozoa. We have identified and manually validated peptide matches to 89 missing proteins in human spermatozoa. In addition, we carefully validated three proteins that were scored as uncertain in the latest neXtProt release (09.19.2014). A focus was then given to the 12 missing proteins encoded on chromosomes 2 and 14, some of which may putatively play roles in ciliation and flagellum mechanistics. The expression pattern of C2orf57 and TEX37 was confirmed in the adult testis by immunohistochemistry. On the basis of transcript expression during human spermatogenesis, we further consider the potential for discovering additional missing proteins in the testicular postmeiotic germ cell lineage and in ejaculated spermatozoa. This project was conducted as part of the C-HPP initiatives on chromosomes 14 (France) and 2 (Switzerland). The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD002367
ISSN: 1535-3893

hal-01187320
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01187320
DOI : 10.1021/acs.jproteome.5b00170
PUBMED : 26168773

Éditeur(s) : HAL CCSD
Résumé : International audience
Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.
Systems Biology in Reproductive Medicine

hal-00877749
https://hal.archives-ouvertes.fr/hal-00877749
DOI : 10.3109/19396368.2012.691943
PUBMED : 22788530

Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
The endonuclease DICER that processes micro-RNAs and small interfering RNAs is essential for normal spermatogenesis and male fertility. We previously showed that the deletion of Dicer1 gene in postnatal spermatogonia in mice using Ngn3 promoter-driven Cre expression caused severe defects in the morphogenesis of haploid spermatid to mature spermatozoon, including problems in cell polarization and nuclear elongation. In this study, we further analyzed the same mouse model and revealed that absence of functional DICER in differentiating male germ cells induces disorganization of the cell-cell junctions in the seminiferous epithelium. We detected discontinuous and irregular apical ectoplasmic specializations between elongating spermatids and Sertoli cells. The defective anchoring of spermatids to Sertoli cells caused a premature release of spermatids into the lumen. Our findings may help also explain the abnormal elongation process of remaining spermatids because these junctions and the correct positioning of germ cells in the epithelium are critically important for the progression of spermiogenesis. Interestingly, cell adhesion-related genes were generally upregulated in Dicer1 knockout germ cells. Claudin 5 ( Cldn5 ) was among the most upregulated genes and we show that the polarized localization of CLAUDIN5 in the apical ectoplasmic specializations was lost in Dicer1 knockout spermatids. Our results suggest that DICER-dependent pathways control the formation and organization of cell-cell junctions in the seminiferous epithelium via the regulation of cell adhesion-related genes
ISSN: 0006-3363

hal-01274239
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01274239
DOI : 10.1095/biolreprod.115.131938
PUBMED : 26510868