Abstract
In vitro culture of ovarian granulosa cells and theca cells has been very important for our understanding of their function and regulation. One of the most eagerly sought attributes of cell culture is the use of chemically-defined conditions. However, even under such in vitro conditions cell behaviour could differ from the in vivo situation because of differences in oxygen tension, nutrients, adhesion matrix and other factors. To examine this further we compared the transcriptomes of both granulosa cells and cells from the theca interna that were cultured in what are arguably the best in vitro conditions for maintaining the ‘follicular’ phenotypes of both tissue types, as displayed by their respective freshly-isolated counterparts. The array data analysed are from recently published data and use the same sizes of bovine follicles (small antral 3–6 mm) and the same Affymetrix arrays. We conducted analysis using Partek, Ingenuity Pathway Analysis and GOEAST. Principal Component Analysis (PCA) and hierarchical clustering clearly separated the in vivo from the in vitro groups for both cells types and transcriptomes were more homogeneous upon culture. In both cell cultures behaviours associated with cell adhesion, migration and interaction with matrix or substrate were more abundant. However, the pathways involved generally differed between the two cell types. With the thecal cultures a gene expression signature of an immune response was more abundant, probably by leukocytes amongst the cells cultured from the theca interna. These results indicate differences between in vivo and in vitro that should be considered when interpreting in vitro data.
Highlights
In ovaries oocytes develop within follicles which at the initial primordial stage are composed of an inactive oocyte surrounded by a quiescent population of epithelial granulosa cells
RNA was extracted from granulosa cells and theca interna of each follicle preparation and two μg of RNA per sample was processed for hybridisation to a Bovine Affymetrix Genome Array (Australian Genomics Research Facility, Parkville and ACRF Cancer Genomics facility, Adelaide) whereby ten arrays each of either granulosa cells or theca interna were available for analysis as was previously published by our groups [11,12]
The theca cells were subjected to four treatments: luteinising hormone (LH) only (160 pg/ml), bone morphogenic protein-6 (BMP-6) only (10 ng/ml), LH plus BMP-6 or control [14], but only control and LH treated cells were analysed here
Summary
In ovaries oocytes develop within follicles which at the initial primordial stage are composed of an inactive oocyte surrounded by a quiescent population of epithelial granulosa cells. The steroidogenic cells of the theca, on the other hand, express LH receptors from an early stage and respond to LH by synthesising androgens Both the granulosa cells and the theca interna cells are key somatic cell types whose function and regulation are pivotal to follicle development, steroidogenesis and female fertility. Anchorage-independent culture was used to examine the stem cell population of granulosa cells [3,4] and this was followed by a serum free system [5], in which granulosa cells retained the ability to produce oestradiol In vivo, this feature of granulosa cells is seen during the final stages of antral follicle maturation and in large follicles approaching ovulatory size [6]. Steroid hormone production was monitored and confirmed that both the granulosa and thecal cells maintained their follicular phenotypes [13,14]
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