Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

Abstract

BackgroundSuccessful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.ResultsWe developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.ConclusionsThe ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

Genome-wide transcriptome analysis between Small-tail Han sheep and the Surabaya fur sheep using high-throughput RNA sequencing

“…The real-time RT-PCR results showed that TGFb-associated genes were upregulated in BB and CC genotype, which implied that TGFb played important roles in high prolificacy of Small-tail Han sheep. BMPs belong to TGFb superfamily, and Bonnet et al (2011) have proved that BMP15 was expressed in sheep granulosa cells and oocytes during early follicular development by laser capture microdissection and which played important role in follicular development and ovarian function (Otsuka et al 2011, Paulini & Melo 2011. In our study, latent TGFb-binding protein 1 was highly upregulated in Small-tail Han sheep, especially in BB genotype, implying that it might play critical roles for high prolificacy.…”

Estradiol supports in vitro development of bovine early antral follicles

“…With continued in vitro culture, the expression of many parts of these genes is enhanced in the AF group. In addition, ITGA6, THBS1, THBS2, VEGF, AMH, FST and INHBA were reported to express high in growing follicles (Le Bellego et al 2002, Nilsson et al 2003, Greenaway et al 2005, Tesfaye et al 2009, Rosales-Torres et al 2010, Bonnet et al 2011 and Table 5 Expression of marker genes in granulosa cells.…”

Characterization and Comparative Analyses of Muscle Transcriptomes in Dorper and Small-Tailed Han Sheep Using RNA-Seq Technique

“…Over the past decade, microarray hybridization has been commonly used to measure gene expression [6–10]. Using the first specialized transcriptome-wide sheep DNA oligonucleotide microarray, the gene expression profiles of the developing fetal ovine longissimus dorsa of the Texel sheep and Ujumqin sheep were investigated [11].…”