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Influence of Gross Ovarian Stage on Transcript Profiles in Fathead Minnow (Pimephales promelas) Ovary Tissue

ABSTRACT: Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species are complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative PCR and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. Multiple lines of evidence supported the conclusion that variation in the transcriptional profile was primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary tissue. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few putative molecular markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the study, targets including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous spawning fish species.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE18254 | GEO | 2010/05/01

SECONDARY ACCESSION(S): PRJNA119595

REPOSITORIES: GEO

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Project description:Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species are complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative PCR and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. Multiple lines of evidence supported the conclusion that variation in the transcriptional profile was primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary tissue. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few putative molecular markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the study, targets including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous spawning fish species. Relative abundance of approximately 15,000 RNA transcripts in 26 ovary samples and 4 expelled oocyte (egg) samples, representing the five different histoclasses defined for the present study, was evaluated using fathead minnow oligonucleotide microarrays. Fathead minnow 15,000 gene arrays were purchased from Agilent (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturerM-bM-^@M-^Ys protocol and recommendations. One ug of total RNA was used for all hybridizations. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA). An Axon GenePixM-BM-. 4000B Microarray Scanner (Molecular Devices Inc., city, state) was used to scan microarray images at 5 M-NM-<m resolution.

Project description:Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in primordial follicle development. Over 1500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a subnetwork associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e. Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction. For GSM509254-9: We used microarrays to determine genes expressed differentially between control and CTGF (connective tissue growth factor) treated P4 ovary. For GSM510028-65: We used microarrays to determine genes expressed differentially between control and growth factors treated P4 ovary and co-expressed in the same manner for P4 ovaries treated with 8 growth factors AMH, BMP4, GDNF, FGF2, FGF7, KITLG, LIF, PDGFab. For GSM509254-9: RNA samples from 3 control groups are compared to 3 CTGF treated ovary groups. For GSM510028-65: RNA samples from 14 control samples are compared to 3 growth factor treated ovary samples for each AMH, BMP4, GDNF, FGF2, FGF7, KITLG, LIF, PDGFab treatment.

Project description:Primordial follicle assembly is a process that occurs in the embryonic or early post natal ovary in which oocyte nests break down to form individual primordial follicles. The size of this initial pool of primordial follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFbeta-1) on follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system for two days to measure the effect of these factors on follicle assembly. In addition, ovaries were cultured and treated for ten days to determine the potential of CTGF and TGFbeta-1 to manipulate the primordial follicle pool size over a longer developmental time period. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled follicles. TGFbeta-1 had no effect on primordial follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFbeta-1 was found to cause an increase in the proportion of assembled follicles. Interestingly, treatment with TGFbeta-1 alone resulted in fewer total oocytes in the ovary and decreased the primordial follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFbeta-1 stimulates primordial follicle assembly and TGFbeta-1 can decrease the primordial follicle pool size. CTGF was found to regulate the ovarian transcriptome during primordial follicle assembly and an integrative network of genes was identified. CTGF is one of the first growth factors shown to promote primordial follicle assembly, while TGFbeta-1 is one of the first factors shown to decrease the primordial follicle pool size. These observations suggest the possibility of manipulating primordial follicle pool size and influencing female reproductive lifespan. We used microarrays to determine genes expressed differentially between control and CTGF (connective tissue growth factor) treated P0 ovary RNA samples from 3 control groups are compared to 3 CTGF treated ovary groups

Project description:Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells invaginate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 53) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 25 markers for GREL and other cells we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are separated from the stroma by a basal lamina. Around 130 days of gestation as the stroma expands laterally below the GREL cells on the surface thus establishing a sub-epithelial basal lamina and an epithelial-stromal interface, and it is at this stage that a mature surface epithelium develops from the GREL cells. The stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not invaginate into the ovary to form the granulosa cells of follicles. Microarray analysis of gene expression was conducted on different cell types cultured from fetal ovaries to identify possible markers of somatic cells during development. Two different cell types: Gonadal Ridge Epithelial-Like cells (n=2, 130 days gestation) and adult fibroblasts (n=1) were cultured from digested bovine fetal ovaries and gene expression compared with each other by Bovine Genome Affy array analysis in Partek Genomics Suite software, to identify possible markers of somatic cells during development.

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Influence of Gross Ovarian Stage on Transcript Profiles in Fathead Minnow (Pimephales promelas) Ovary Tissue

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