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Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

ABSTRACT: BACKGROUND: Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model. METHODS: We determined the expression of 128 miRNAs by real time PCR in early-gestational (gestational day (GD) 42) and mid-gestational (GD75) sheep ovaries and testes. Expression data were further examined and validated by bioinformatic analysis. RESULTS: Expression analysis revealed significant differences between ovaries and testes among 24 miRNAs at GD42, and 43 miRNAs at GD75. Bioinformatic analysis revealed that a number of differentially expressed miRNAs are predicted to target genes known to be important in mammalian gonadal development, including ESR1, CYP19A1, and SOX9. In situ hybridization revealed miR-22 localization within fetal testicular cords. As estrogen signaling is important in human and sheep ovarian development, these data indicate that miR-22 is involved in repressing estrogen signaling within fetal testes. CONCLUSIONS: Based on our results we postulate that gene expression networks underlying fetal gonadal development are regulated by miRNAs.

SUBMITTER: Torley KJ

PROVIDER: S-EPMC3027096 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

Torley Katie J KJ da Silveira Juliano C JC Smith Peter P Anthony Russell V RV Veeramachaneni D N Rao DN Winger Quinton A QA Bouma Gerrit J GJ

Reproductive biology and endocrinology : RB&E 20110111

<h4>Background</h4>Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.<h4>Methods</h4>We determine ...[more]

PMID: 21223560

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Project description:Reptiles constitute a highly diverse group of vertebrates, with their evolutionary lineages having diverged relatively early. The types of sex determination exemplify the diversity of reptiles; however, there are limited data regarding the gonadal development in squamate reptiles. Geckos constitute a group that is increasingly used in research and that serves as a potential reptilian model organism. The aim of this study was to trace the changes in the structure of developing gonads in the embryos of three gecko species: the crested gecko, leopard gecko, and mourning gecko. These species represent different families of the Gekkota infraorder and exhibit different types of sex determination. Gonadal development was examined from the formation of the earliest gonadal ridges through the development of undifferentiated gonadal structures, sex differentiation of gonads, and the formation of testicular and ovarian structures. The study showed that the gonadal primordia of these three gecko species formed on the most dorsally located surface of the dorsal mesentery, and both the coelomic epithelium and the nephric mesenchyme contributed to their development. As in other reptile species, primordial germ cells settled in the gonadal ridges, and the undifferentiated gonad was composed of a cortex and a medulla. Ovarian differentiation started with the thickening of the gonadal cortex and proliferation of germ cells in this region. A characteristic feature of the developing gecko ovaries was the thickened crescent-shaped cortex on the medial and ventral surfaces of the ovaries. The ovarian medulla also grew and exhibited diverse tendencies to form cords. In the leopard gecko, advanced cord-like structures with lumens were observed in the ovaries, which were not seen in the crested gecko. Testicular differentiation was characterized by cortical thinning and the disappearance of germ cells in this region. In the medulla, the development of distinct cords with early lumen formation was noted. A characteristic feature of embryonic gonads was their growth in a horizontal plane. In this study, gonadal development was characterized by several features that are shared by geckos and other reptiles, along with features that are specific only to geckos.

Project description:BACKGROUND:Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes. METHODS:Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher's exact test were used to determine statistical significance. RESULTS:Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes. CONCLUSIONS:Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.

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Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

Publications

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

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