A tridimensional atlas of the developing human head.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell-death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a "pre-granulosa-like" identity, while other revert to a "supporting progenitor-like" cell identity, most of them being "intersex" because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.

Uterine defects and estradiol-dependent development of oviductal diverticula in mice lacking the SMAD4 C-terminal Mad homology 2 domain.

In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.

Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43 mM ethanol and 25 nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.

Transcriptional profiling of the developing rat ovary following intrauterine exposure to the endocrine disruptors diethylstilbestrol and ketoconazole.

Exposure to endocrine-disrupting chemicals (EDCs) during development may cause reproductive disorders in women. Although female reproductive endpoints are assessed in rodent toxicity studies, a concern is that typical endpoints are not sensitive enough to detect chemicals of concern to human health. If so, measured endpoints must be improved or new biomarkers of effects included. Herein, we have characterized the dynamic transcriptional landscape of developing rat ovaries exposed to two well-known EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), by 3' RNA sequencing. Rats were orally exposed from day 7 of gestation until birth, and from postnatal day 1 until days 6, 14 or 22. Three exposure doses for each chemical were used: 3, 6 and 12 µg/kg bw/day of DES; 3, 6, 12 mg/kg bw/day of KTZ. The transcriptome changed dynamically during perinatal development in control ovaries, with 1137 differentially expressed genes (DEGs) partitioned into 3 broad expression patterns. A cross-species deconvolution strategy based on a mouse ovary developmental cell atlas was used to map any changes to ovarian cellularity across the perinatal period to allow for characterization of actual changes to gene transcript levels. A total of 184 DEGs were observed across dose groups and developmental stages in DES-exposed ovaries, and 111 DEGs in KTZ-exposed ovaries across dose groups and developmental stages. Based on our analyses, we have identified new candidate biomarkers for female reproductive toxicity induced by EDC, including Kcne2, Calb2 and Insl3.

Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue.

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.

Cytosine modifications modulate the chromatin architecture of transcriptional enhancers.

Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.

The ReproGenomics Viewer: a multi-omics and cross-species resource compatible with single-cell studies for the reproductive science community.

MOTIVATION: Recent advances in transcriptomics have enabled unprecedented insight into gene expression analysis at a single-cell resolution. While it is anticipated that the number of publications based on such technologies will increase in the next decade, there is currently no public resource to centralize and enable scientists to explore single-cell datasets published in the field of reproductive biology. RESULTS: Here, we present a major update of the ReproGenomics Viewer, a cross-species and cross-technology web-based resource of manually-curated sequencing datasets related to reproduction. The redesign of the ReproGenomics Viewer's architecture is accompanied by significant growth of the database content including several landmark single-cell RNA-sequencing datasets. The implementation of additional tools enables users to visualize and browse the complex, high-dimensional data now being generated in the reproductive field. AVAILABILITY AND IMPLEMENTATION: The ReproGenomics Viewer resource is freely accessible at http://rgv.genouest.org. The website is implemented in Python, JavaScript and MongoDB, and is compatible with all major browsers. Source codes can be downloaded from https://github.com/fchalmel/RGV. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Male Fertility Gene Atlas: a web tool for collecting and integrating OMICS data in the context of male infertility.

STUDY QUESTION: How can one design and implement a system that provides a comprehensive overview of research results in the field of epi-/genetics of male infertility and germ cells? SUMMARY ANSWER: Working at the interface of literature search engines and raw data repositories, the newly developed Male Fertility Gene Atlas (MFGA) provides a system that can represent aggregated results from scientific publications in a standardized way and perform advanced searches, for example based on the conditions (phenotypes) and genes related to male infertility. WHAT IS KNOWN ALREADY: PubMed and Google Scholar are established search engines for research literature. Additionally, repositories like Gene Expression Omnibus and Sequence Read Archive provide access to raw data. Selected processed data can be accessed by visualization tools like the ReproGenomics Viewer. STUDY DESIGN, SIZE, DURATION: The MFGA was developed in a time frame of 18 months under a rapid prototyping approach. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the context of the Clinical Research Unit 'Male Germ Cells' (CRU326), a group of around 50 domain experts in the fields of male infertility and germ cells helped to develop the requirements engineering and feedback loops. They provided a set of 39 representative and heterogeneous publications to establish a basis for the system requirements. MAIN RESULTS AND THE ROLE OF CHANCE: The MFGA is freely available online at https://mfga.uni-muenster.de. To date, it contains 115 data sets corresponding to 54 manually curated publications and provides an advanced search function based on study conditions, meta-information and genes, whereby it returns the publications' exact tables and figures that fit the search request as well as a list of the most frequently investigated genes in the result set. Currently, study data for 31 different tissue types, 32 different cell types and 20 conditions are available. Also, ∼8000 and ∼1000 distinct genes have been found to be mentioned in at least 10 and 15 of the publications, respectively. LARGE SCALE DATA: Not applicable because no novel data were produced. LIMITATIONS, REASONS FOR CAUTION: For the most part, the content of the system currently includes the selected publications from the development process. However, a structured process for the prospective literature search and inclusion into the MFGA has been defined and is currently implemented. WIDER IMPLICATIONS OF THE FINDINGS: The technical implementation of the MFGA allows for accommodating a wide range of heterogeneous data from aggregated research results. This implementation can be transferred to other diseases to establish comparable systems and generally support research in the medical field. STUDY FUNDING/COMPETING INTEREST(S): This work was carried out within the frame of the German Research Foundation (DFG) Clinical Research Unit 'Male Germ Cells: from Genes to Function' (CRU326). The authors declare no conflicts of interest.

Dynamics of the transcriptional landscape during human fetal testis and ovary development.

STUDY QUESTION: Which transcriptional program triggers sex differentiation in bipotential gonads and downstream cellular events governing fetal testis and ovary development in humans? SUMMARY ANSWER: The characterization of a dynamically regulated protein-coding and non-coding transcriptional landscape in developing human gonads of both sexes highlights a large number of potential key regulators that show an early sexually dimorphic expression pattern. WHAT IS KNOWN ALREADY: Gonadal sex differentiation is orchestrated by a sexually dimorphic gene expression program in XX and XY developing fetal gonads. A comprehensive characterization of its non-coding counterpart offers promising perspectives for deciphering the molecular events underpinning gonad development and for a complete understanding of the etiology of disorders of sex development in humans. STUDY DESIGN, SIZE, DURATION: To further investigate the protein-coding and non-coding transcriptional landscape during gonad differentiation, we used RNA-sequencing (RNA-seq) and characterized the RNA content of human fetal testis (N = 24) and ovaries (N = 24) from 6 to 17 postconceptional week (PCW), a key period in sex determination and gonad development. PARTICIPANTS/MATERIALS, SETTING, METHODS: First trimester fetuses (6-12 PCW) and second trimester fetuses (13-14 and 17 PCW) were obtained from legally induced normally progressing terminations of pregnancy. Total RNA was extracted from whole human fetal gonads and sequenced as paired-end 2 × 50 base reads. Resulting sequences were mapped to the human genome, allowing for the assembly and quantification of corresponding transcripts. MAIN RESULTS AND THE ROLE OF CHANCE: This RNA-seq analysis of human fetal testes and ovaries at seven key developmental stages led to the reconstruction of 22 080 transcripts differentially expressed during testicular and/or ovarian development. In addition to 8935 transcripts displaying sex-independent differential expression during gonad development, the comparison of testes and ovaries enabled the discrimination of 13 145 transcripts that show a sexually dimorphic expression profile. The latter include 1479 transcripts differentially expressed as early as 6 PCW, including 39 transcription factors, 40 long non-coding RNAs and 20 novel genes. Despite the use of stringent filtration criteria (expression cut-off of at least 1 fragment per kilobase of exon model per million reads mapped, fold change of at least 2 and false discovery rate adjusted P values of less than <1%), the possibility of assembly artifacts and of false-positive differentially expressed transcripts cannot be fully ruled out. LARGE-SCALE DATA: Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI GEO under accession number GSE116278. LIMITATIONS, REASONS FOR CAUTION: The intrinsic nature of this bulk analysis, i.e. the sequencing of transcripts from whole gonads, does not allow direct identification of the cellular origin(s) of the transcripts characterized. Potential cellular dilution effects (e.g. as a result of distinct proliferation rates in XX and XY gonads) may account for a few of the expression profiles identified as being sexually dimorphic. Finally, transcriptome alterations that would result from exposure to pre-abortive drugs cannot be completely excluded. Although we demonstrated the high quality of the sorted cell populations used for experimental validations using quantitative RT-PCR, it cannot be totally excluded that some germline expression may correspond to cell contamination by, for example, macrophages. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study has led to the identification of 1000 protein-coding and non-coding candidate genes showing an early, sexually dimorphic, expression pattern that have not previously been associated with sex differentiation. Collectively, these results increase our understanding of gonad development in humans, and contribute significantly to the identification of new candidate genes involved in fetal gonad differentiation. The results also provide a unique resource that may improve our understanding of the fetal origin of testicular and ovarian dysgenesis syndromes, including cryptorchidism and testicular cancers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the French National Institute of Health and Medical Research (Inserm), the University of Rennes 1, the French School of Public Health (EHESP), the Swiss National Science Foundation [SNF n° CRS115_171007 to B.J.], the French National Research Agency [ANR n° 16-CE14-0017-02 and n° 18-CE14-0038-02 to F.C.], the Medical Research Council [MR/L010011/1 to P.A.F.] and the European Community's Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to P.A.F.] and from the European Union's Horizon 2020 Research and Innovation Programme [under grant agreement no 825100 to P.A.F. and S.M.G.]. There are no competing interests related to this study.

Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.

Characterization of Glyceollins as Novel Aryl Hydrocarbon Receptor Ligands and Their Role in Cell Migration.

Recent studies strongly support the use of the aryl hydrocarbon receptor (AhR) as a therapeutic target in breast cancer. Glyceollins, a group of soybean phytoalexins, are known to exert therapeutic effects in chronic human diseases and also in cancer. To investigate the interaction between glyceollin I (GI), glyceollin II (GII) and AhR, a computational docking analysis, luciferase assays, immunofluorescence and transcriptome analyses were performed with different cancer cell lines. The docking experiments predicted that GI and GII can enter into the AhR binding pocket, but their interactions with the amino acids of the binding site differ, in part, from those interacting with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Both GI and GII were able to weakly and partially activate AhR, with GII being more potent. The results from the transcriptome assays showed that approximately 10% of the genes regulated by TCDD were also modified by both GI and GII, which could have either antagonistic or synergistic effects upon TCDD activation. In addition, we report here, on the basis of phenotype, that GI and GII inhibit the migration of triple-negative (ER-, PgR-, HER2NEU-) MDA-MB-231 breast cancer cells, and that they inhibit the expression of genes which code for important regulators of cell migration and invasion in cancer tissues. In conclusion, GI and GII are AhR ligands that should be further investigated to determine their usefulness in cancer treatments.

TOXsIgN: a cross-species repository for toxicogenomic signatures.

Motivation: At the same time that toxicologists express increasing concern about reproducibility in this field, the development of dedicated databases has already smoothed the path toward improving the storage and exchange of raw toxicogenomic data. Nevertheless, none provides access to analyzed and interpreted data as originally reported in scientific publications. Given the increasing demand for access to this information, we developed TOXsIgN, a repository for TOXicogenomic sIgNatures. Results: The TOXsIgN repository provides a flexible environment that facilitates online submission, storage and retrieval of toxicogenomic signatures by the scientific community. It currently hosts 754 projects that describe more than 450 distinct chemicals and their 8491 associated signatures. It also provides users with a working environment containing a powerful search engine as well as bioinformatics/biostatistics modules that enable signature comparisons or enrichment analyses. Availability and implementation: The TOXsIgN repository is freely accessible at http://toxsign.genouest.org. Website implemented in Python, JavaScript and MongoDB, with all major browsers supported. Supplementary information: Supplementary data are available at Bioinformatics online.

Genome-Wide Transcriptional and Functional Analysis of Human T Lymphocytes Treated with Benzo[α]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, known to affect T lymphocytes. However, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, benzo[α]pyrene (B[α]P), using a microarray-based transcriptome analysis. After a 48 h exposure to B[α]P, we identified 158 genes differentially expressed in T lymphocytes, including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, but also others not previously shown to be targeted by B[α]P such as genes encoding the gap junction beta (GJB)-2 and 6 proteins. Functional enrichment analysis revealed that these candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signaling pathways; a marked alteration in T lymphocyte recruitment was also observed. Using functional tests in transwell migration experiments, B[α]P was then shown to significantly decrease the chemokine (C-X-C motif) ligand 12-induced chemotaxis and transendothelial migration of T lymphocytes. In total, this study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, thus providing a more thorough understanding of the molecular basis of the immunotoxicity of PAHs.

Defining the human sperm microtubulome: an integrated genomics approach.

Sperm motility notably depends on the structural integrity of the flagellum and the regulation of microtubule dynamics. Although researchers have started to use "omics" techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined. In this study, we defined a core set of 116 gene products associated with the human sperm microtubulome (including products potentially involved in abnormal ciliary phenotypes and male infertility disorders). To this end, we designed and applied an integrated genomics workflow and combined relevant proteomics, transcriptomics, and interactomics datasets to reconstruct a dynamic interactome map. By further integrating phenotypic information, we defined a disease-interaction network; this enabled us to highlight a number of novel factors potentially associated with altered sperm motility and male fertility. Lastly, we experimentally validated the expression pattern of two candidate genes (CUL3 and DCDC2C) that had never previously been associated with male germline differentiation. Our analysis suggested that CUL3 and DCDC2C's products have important roles in the sperm flagellum. Taken as a whole, our results demonstrate that an integrated genomics strategy can highlight relevant molecular factors in specific sperm components. This approach could be easily extended by including other "omics" data (from asthenozoospermic men, for example) and identifying other critical proteins from the human sperm microtubulome.

Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

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°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., Jégou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Glyceollins trigger anti-proliferative effects through estradiol-dependent and independent pathways in breast cancer cells.

BACKGROUND: Estrogen receptors (ER) α and ß are found in both women and men in many tissues, where they have different functions, including having roles in cell proliferation and differentiation of the reproductive tract. In addition to estradiol (E2), a natural hormone, numerous compounds are able to bind ERs and modulate their activities. Among these compounds, phytoestrogens such as isoflavones, which are found in plants, are promising therapeutics for several pathologies. Glyceollins are second metabolites of isoflavones that are mainly produced in soybean in response to an elicitor. They have potentially therapeutic actions in breast cancer by reducing the proliferation of cancer cells. However, the molecular mechanisms driving these effects remain elusive. METHODS: First, to determine the proliferative or anti-proliferative effects of glyceollins, in vivo and in vitro approaches were used. The length of epithelial duct in mammary gland as well as uterotrophy after treatment by E2 and glyceollins and their effect on proliferation of different breast cell line were assessed. Secondly, the ability of glyceollin to activate ER was assessed by luciferase assay. Finally, to unravel molecular mechanisms involved by glyceollins, transcriptomic analysis was performed on MCF-7 breast cancer cells. RESULTS: In this study, we show that synthetic versions of glyceollin I and II exert anti-proliferative effects in vivo in mouse mammary glands and in vitro in different ER-positive and ER-negative breast cell lines. Using transcriptomic analysis, we produce for the first time an integrated view of gene regulation in response to glyceollins and reveal that these phytochemicals act through at least two major pathways. One pathway involving FOXM1 and ERα is directly linked to proliferation. The other involves the HIF family and reveals that stress is a potential factor in the anti-proliferative effects of glyceollins due to its role in increasing the expression of REDD1, an mTORC1 inhibitor. CONCLUSION: Overall, our study clearly shows that glyceollins exert anti-proliferative effects by reducing the expression of genes encoding cell cycle and mitosis-associated factors and biomarkers overexpressed in cancers and by increasing the expression of growth arrest-related genes. These results reinforce the therapeutic potential of glyceollins for breast cancer.