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

<p>Abstract</p> <p>Background</p> <p>Successful 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.</p> <p>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.</p> <p>Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.</p> <p>Results</p> <p>We 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 (<it>SOHLH2</it>, <it>MAEL</it>, <it>MATER</it>, <it>VASA</it>, <it>GDF9</it>, <it>BMP15</it>) and three granulosa cell-specific genes (<it>KL</it>, <it>GATA4</it>, <it>AMH</it>).</p> <p>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.</p> <p>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.</p> <p>Conclusions</p> <p>The 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.</p

Increased free fetal DNA levels in early pregnancy plasma of women who subsequently develop preeclampsia and intrauterine growth restriction

Artículo de publicación ISI.Objective To determine if maternal plasma ffDNA is increased early in pregnancies which subsequently develop preeclampsia (PE) and intrauterine growth restriction (IUGR). Methods Blood was obtained at 11–14 weeks and plasma stored. Among those who delivered a male infant and had a birth weight under the tenth centile and/or PE, we divided them into those who delivered before 35 weeks (9) and those who delivered after this gestation (15). A third group with uncomplicated pregnancies was used as controls (24). Real time-polymerase chain reaction (RT-PCR) was carried out to detect the multi-copy Y chromosome associated DSY14 gene. Results There were no differences between the ffDNA levels in the group delivered after 35 weeks and the control group (2.23ge/mL–1.61ge/mL p = 0.39). However, the levels of ffDNA at 11–14 weeks were statistically, significantly higher in patients that delivered before 35 weeks (4.34ge/mL–1.61ge/mL p = 0.0018). A logistic regression analysis shows that for every unit (1ge/mL) in which ffDNA increases, the likelihood of having PE or a fetus growing under the tenth centile delivered before 35 weeks increases by 1.67 times (CI 1.13–2.47). Conclusion The concentration of ffDNA is significantly higher even during early pregnancy, in patients who subsequently develop PE and/or IUGR and are delivered before 35 weeks. Copyright 2009 John Wiley & Sons, Ltd. KEY WORDS: preeclampsia; maternal disease; placental disease; DNA; fetal Cells; nucleic acids and proteins; maternal serum Screening; fetal and placental pathology