Project description:Background Correct achievement of early ovarian folliculogenesis is a crucial phase for further ovarian function. This process is closely regulated by cell-cell interactions and coordinated expression of genes from oocyte and granulosa cells. But, despite of the large number of studies, little is known about the precise gene expression patterns driving early folliculogenesis. The experimental limitations concerned the very small size of these follicles and the mixture of the different developmental stages within an ovary that make the study of isolated follicular components much more difficult. The recently developed laser capture microdissection (LCM) technique coupled with microarrays experiments is promising in addressing the molecular specificity of each follicular compartment. Nevertheless, the isolation of unique cells or group of cells is still challenging to maintain RNA quality during this process and to obtain sufficient amount of RNA. In this study, we described a method allowing the analysis of oocyte and granulosa cells gene expression during the first stages of sheep early folliculogenesis. Results First we developed a new fixation protocol using a frizzed 70% ethanol fixation solution that ensures correct single cell capture and RNA integrity during microdissection time. After LCM capture of the compartments and follicular stages, RNA extraction and amplification, the expression of 6 oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and 3 granulosa cell-specific genes (KITLG, GATA4, AMH) confirmed the purity of the samples and documented their ovine expression profiles. Then, using bovine Affymetrix chip, we identified for the first time, a global gene expression for each follicular compartment during early developmental stages. Particularly the granulosa cell data set is quite unique. 1050 granulosa cell specific transcripts compared to oocyte and 759 oocyte specific transcripts were detected. The analysis of the expression of 2 genes (SIRT7, FST) confirmed this specificity of expression. Finally, the integration of the data stated the 3 main physiological events involved in early folliculogenesis and provided descriptive elements that confirmed the relevance and the potential of the LCM-derived RNAs. Conclusions This method should contribute through an additional genome wide expression profiling to give insights on molecular mechanisms involved in stage transitions and cell type interplays. The 2 ovine follicular compartments (i.e. granulosa cells (G) and oocytes (O) were captured using LCM technology for each early stage (primordial (Pd), primary (Pm), secondary (Sec) follicles. The RNA of each group was extracted using Picopure RNA Isolation kit (Arcturus) and subjected to 2 round T7 amplification (RiboAmp®HS PLUS kit, Arcturus). Ovine microarray experiments were performed using the Affymetrix Bovine Expression Array. First the quality of the cross-species hybridizations was checked by comparison of hybridization data of ovine fetal ovary RNA with bovine fetal ovary ones, generated with the Affymetrix standard protocol (protocole 1). Then, three biotin-labeling protocols were compared from ovine fetal ovary total RNA: protocol 1; protocol 2 (Biotin-labeled cRNAs were synthesized following Affymetrix protocol using the second-round cDNAs from RiboAmp®HS kit as templates); protocol 3 (Arcturus biotin turboTM labeling kit from aRNA after the 2 round amplification using RiboAmp®HS kit). Last , one LCM-derived aRNA sample of each group was labeled using the Arcturus biotin turboTM labeling kit (protocol 3) and hybridized to Affymetrix Bovine Expression arrays. Images were interpreted using Microarray Suite version 5.0 (MAS 5.0) in GCOS with scaling (100) and without normalization.

Project description:The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. Experiment Overall Design: We generated a database of the human oocyte transcriptome by comparing the transcripts in the oocyte with the reference samples, which contain mRNA from several somatic tissues. Experiment Overall Design: Human oocytes were obtained from three patients undergoing an assisted reproductive treatment (ART) at the Unit of Reproductive Medicine of Clinica Las Condes, Santiago, Chile. It is important to emphasize that the routine in vitro fertilization protocol at Clinica Las Condes calls for fertilizing only those oocytes that will be transferred into the uterus of the patient. Therefore, there is always a surplus of oocytes. We then had the opportunity to use specific criteria to select donors as follows: (i) <35 years old, (ii) reproductively healthy with regular ovulatory cycles, (iii) male factor as the only cause of infertility, and (iv) considerable number of developing follicles that assured spared oocytes. The experimental protocol was reviewed and approved by a local independent Ethics Review Board. All donors signed informed consent. At the time this manuscript was submitted, all three donors had already conceived; two of them got pregnant during the ART cycle in which our samples were collected, and the third one got pregnant after a spontaneous cycle with artificial insemination using donated sperm. Ovarian stimulation, oocyte retrieval, and cell lysis were performed as described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Experiment Overall Design: Three groups of 10 oocytes each were used. Reference RNA (100 Î¼g) was prepared by mixing 10 Î¼g of total RNA from each of 10 different normal human tissues, including skeletal muscle, kidney, lung, colon, liver, spleen, breast, brain, heart, and stomach (Ambion). Experiment Overall Design: Transcriptional profile of each sample was probed by using Affymetrix Human Genome U133 Plus 2.0 GeneChips.

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Global gene analysis of oocytes from early stages in human folliculogenesis

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