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Systematic discovery and classification of ovarian fertility factors.

ABSTRACT: Female infertility syndromes are among the most prevalent chronic health disorders in women, but their molecular basis remains unknown because of the complexity of oogenesis and uncertainty regarding the number and identity of ovarian factors controlling the assembly, preservation, and maturation of ovarian follicles. To systematically discover such ovarian fertility factors en masse, we employed a mouse model (Foxo3), where follicles are assembled normally but are then synchronously activated. Gene expression profiling of mutant and normal ovaries led to the identification a surprisingly large set of ovarian factors. The set included the vast majority of known ovarian factors, many of which when mutated produce female sterility phenotypes, but most were novel. Subsequent analyses revealed novel classes of ovarian factors and significant overrpresentation on the X chromosome, among other insights into the general properties of oogenesis genes and their patterns of expression. Keywords: time course, ovarian fertility factors, Foxo3 mutant

ORGANISM(S): Mus musculus

PROVIDER: GSE8249 | GEO | 2007/08/02

SECONDARY ACCESSION(S): PRJNA101193

REPOSITORIES: GEO

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Project description:Female infertility syndromes are among the most prevalent chronic health disorders in women, but their molecular basis remains unknown because of the complexity of oogenesis and uncertainty regarding the number and identity of ovarian factors controlling the assembly, preservation, and maturation of ovarian follicles. To systematically discover such ovarian fertility factors en masse, we employed a mouse model (Foxo3), where follicles are assembled normally but are then synchronously activated. Gene expression profiling of mutant and normal ovaries led to the identification a surprisingly large set of ovarian factors. The set included the vast majority of known ovarian factors, many of which when mutated produce female sterility phenotypes, but most were novel. Subsequent analyses revealed novel classes of ovarian factors and significant overrpresentation on the X chromosome, among other insights into the general properties of oogenesis genes and their patterns of expression. Experiment Overall Design: Total ovarian RNA from +/+ and -/- ovaries at PND1, 3, 7, and 14 (n=3 replicates per timepoint and genotype, a total of 24 microarrays) was subjected to linear RNA amplification and hybridized to Affymetrix 430 2.0 mouse whole-genome microarrays, which interrogate >39K transcripts including the vast majority of protein-coding genes. We also profiled 14 somatic tissues. Additionally, to provide more refined views of gene expression, we profiled adult ovaries, adult testis, KitlSl/KitlSl-d testis (devoid of germ cells except for rare spermatogonia) (Shinohara et al., 2000), ES cells, laser-capture microdissected (LCM) primary oocytes, LCM somatic cells (granulosa cells + surrounding stroma), superovulated unfertilized eggs, cumulus granulosa cells, and E11 Foxo3 +/+ and -/- embryos. Each array data set was independently normalized by global median scaling, and the signal strengths were averaged for those samples for which replicates were available (PND1-14).

Project description:Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra- and extra-cellular ice formation, which is a major driver of cell cryoinjury. Using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study. Multilayered secondary follicles with a diameter of 130 -160 µm were mechanically isolated from 16-day-old CD-1 female mice and vitrified using the closed vitrification method. After a 2-week storage in liquid nitrogen, vitrified follicles were warmed, and together with freshly-harvested follicles, cultured for 8 days using the eIVFG method. Next, mature antral follicles from day 8 were collected for single-follicle RNA sequencing using the SMART-seq RNA-seq method. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Using the criterion of fold change >2 or <0.5 and a false discovery rate (FDR) adjusted p-value < 0.05, there were 35 differentially expressed genes out of a total of 11,806 detected genes between vitrified and fresh follicles, among which 17 were up-regulated and 18 were down-regulated in vitrified follicles. To the best of our knowledge, none of those differentially expressed genes identified between vitrified and fresh follicles have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis over these 35 genes using the functional annotation tool DAVID revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of several genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles, including granulosa cell-specific genes (Cyp19a1, Amh, Inha, Inhba, Inhbb, and Fshr), theca cell-specific genes (Cyp17a1, Col1a2, and Col3a1), and oocyte-specific genes (Gdf9, Bmp15, Zp1, Zp2, and Zp3). Equally important, Lhcgr, the gene that is primarily expressed in theca cells of preantral follicles and then greatly induced in mural granulosa cells in antral follicles to prepare for the luteinizing hormone (LH) surge and ovulation, also had comparable mRNA expression levels between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves the follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system. In conclusion, our study re-evaluates individual follicle cryopreservation by using the closed vitrification method in a more in-depth perspective and proves that vitrification preserves the follicular cell transcriptome in the eIVFG system, thus providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.

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Systematic discovery and classification of ovarian fertility factors.

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