Abstract
BackgroundIn previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.MethodsGC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.ResultsThe data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified “AMPK signaling” as well as “cAMP-mediated signaling” as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.ConclusionsFrom these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.
Highlights
In previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, resembling an early stage of luteinization
Expression profiling of GC cultured at different cell densities As a first approach the mRNA microarray data were subjected to principal component analysis (PCA) to reduce the multidimensionality of datasets and to identify principal components with the highest variation
The gene expression levels were tightly clustered in GC cultured at normal density, but to a much lesser degree at high cell density
Summary
In previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, resembling an early stage of luteinization. VNN2, RGS2 and PTGS2, encoding vanin-2 (vascular noninflammatory molecule 2), the regulator of G protein signaling and the key enzyme for prostaglandin synthesis cyclooxygenase-2 showed an up-regulation as observed in vivo after LH stimulation [4,5,6,7,8] Besides these drastic changes in the gene expression profiles, the follicle cell layers convert into the physiologically and morphologically different corpus luteum (CL) after ovulation. For a proper function of the CL a highly developed vascular system is essential, highlighting the importance of angiogenesis, which is involved in follicular and CL development [12, 14,15,16] From this point of view a profound change of angiogenic factors should be visible in the altered gene expression profile of cultured GC, suggestively mimicking the process of early luteinization. In order to validate the used in vitro model, the data were compared with a previous in vivo transcriptome analysis studying effects of the pre-ovulatory LH surge on the transcriptome of theca and granulosa cells [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
