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

Investigation of orthohantavirus seroprevalence in northern rural areas of Denizli province, Turkey.

Orthohantaviruses infect humans via inhalation of the viral particles in the excreta of infected rodents or direct contact with infected rodents. The infections caused by Puumala orthohantavirus (PUUV) and Dobrava-Belgrade orthohantavirus (DOBV) have been reported in Turkey. Serum samples of 346 healthy volunteers who are in the high-risk group of Orthohantavirus infections among the residents of Çal, Baklan, Çivril, and Bekilli counties, located in the northeast part of Denizli province, were used in this study. The samples were screened and confirmed using commercial ELISA and immunoblot tests, which detect IgG antibodies against DOBV, PUUV, and Hantaan orthohantavirus. IgG antibodies against PUUV were detected in the samples of 2 volunteers (2/346, 0.6%). One was a veterinarian and the other a farmer and they live in the Baklan and Çal counties, respectively. Both of them have a high probability of exposure to the virus, based on their occupation and living conditions. However, no symptoms were found in the clinical findings of both cases. This study is the first publication of reported PUUV seropositivities from the southwestern part of Turkey