Identification of transcripts involved in meiosis and follicle formation during ovine ovary development

<p>Abstract</p> <p>Background</p> <p>The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep.</p> <p>Results</p> <p>In order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep.</p> <p>Conclusion</p> <p>We showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.</p

TOPAZ1, a Novel Germ Cell-Specific Expressed Gene Conserved during Evolution across Vertebrates

BACKGROUND: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene. PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis. CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ lin

First Evidence of Bone Morphogenetic Protein 1 Expression and Activity in Sheep Ovarian Follicles1

Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1′s dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth

Structural and Functional Characterization of a Testicular Long Non-coding RNA (4930463O16Rik) Identified in the Meiotic Arrest of the Mouse Topaz1–/– Testes

International audienceSpermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reported Topaz1 as a germ cell-specific gene highly conserved in vertebrates. Topaz1 knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterize Topaz1 –/– testes, we used RNA-sequencing analyses at two different developmental stages (P16 and P18). The absence of TOPAZ1 disturbed the expression of genes involved in microtubule and/or cilium mobility, biological processes required for spermatogenesis. Moreover, a quarter of P18 dysregulated genes are long non-coding RNAs (lncRNAs), and three of them are testis-specific and located in spermatocytes, their expression starting between P11 and P15. The suppression of one of them, 4939463O16Rik , did not alter fertility although sperm parameters were disturbed and sperm concentration fell. The transcriptome of P18- 4939463O16Rik –/– testes was altered and the molecular pathways affected included microtubule-based processes, the regulation of cilium movement and spermatogenesis. The absence of TOPAZ1 protein or 4930463O16Rik produced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, although Topaz1 is essential for the meiosis in male germ cells and regulate the expression of numerous lncRNAs, these studies have identified a Topaz1 regulated lncRNA ( 4930463O16Rik ) that is key for both sperm production and motility

Integration of multi-tissues data. An example from bovine embryos

International audienceThe increasing availability of large multi-tissue data sets which contain gene expression measurements across different tissues and individuals provided unprecedented opportunities to investigate transcriptome variation across tissues and individuals, and may reveal interactions between genes and tissues. The corresponding data set is a three-dimensional array: genes, individuals and tissues (or recording times). We present here the so-called “Partial Triadic Analysis”,(PTA), a well suited statistical tool to get a clear representation of a spatial series of matrices, one for each tissue. PTA is an extension of PCA and allows one to find a structure common to every matrix and to study its stability across tissues. PTA consists in three steps: i) the interstructure step, where are compared and analyzed the relationships between the different datasets, ii) the compromise step, where all datasets are integrated into an optimum weighted average, the compromise (or consensus) table, and iii) the intrastructure step, where the single-transcriptome are compared to the compromise in order to analyze commonalities and discrepancies.PTA was applied to transcriptomic data from the ANR (Agence Nationale de la Recherche) funded BoSexDim project, consisting in 19 embryo transcriptomes recorded at D40 (40 days after fertilization) and structured by sex (Male / Female) and type (in vivo / in vitro). These transcriptomes were recorded for four tissues (brain, liver, gonad and placenta). PTA shows a compromise structured by sex (first axis) and type (second axis). The same set of genes contribute the most to the sex structuration whatever the tissue. However, the differentiation of in vivo vs in vitro embryos was not made by the same genes according to tissues. Some genes showed an inconsistent, even contradictory behaviour, with an overexpression in one tissue and an underexpression in another one. This example highlights the power of the Partial Triadic Analysis for depicting the variability of the transcriptome structure across various tissues. Acknowledgement: This research was funded by the ANR French organization (BoSexDim project). We are grateful to the Bosexdim consortium members for producing the biological material

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility

International audienceDMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1 −/− rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1 −/− ovaries did not undergo meiosis and folliculogenesis. XX DMRT1 −/− adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility

Early mammalian embryo response to environmental changes: should we take the sex into account?

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