Cargando…

Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density

BACKGROUND: 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, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC tra...

Descripción completa

Detalles Bibliográficos
Autores principales: Baufeld, Anja, Koczan, Dirk, Vanselow, Jens
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217602/
https://www.ncbi.nlm.nih.gov/pubmed/28056989
http://dx.doi.org/10.1186/s12958-016-0221-6
_version_ 1782492139701141504
author Baufeld, Anja
Koczan, Dirk
Vanselow, Jens
author_facet Baufeld, Anja
Koczan, Dirk
Vanselow, Jens
author_sort Baufeld, Anja
collection PubMed
description BACKGROUND: 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, 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. METHODS: GC 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. RESULTS: The 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. CONCLUSIONS: From 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12958-016-0221-6) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5217602
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-52176022017-01-09 Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density Baufeld, Anja Koczan, Dirk Vanselow, Jens Reprod Biol Endocrinol Research BACKGROUND: 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, 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. METHODS: GC 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. RESULTS: The 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. CONCLUSIONS: From 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12958-016-0221-6) contains supplementary material, which is available to authorized users. BioMed Central 2017-01-05 /pmc/articles/PMC5217602/ /pubmed/28056989 http://dx.doi.org/10.1186/s12958-016-0221-6 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Baufeld, Anja
Koczan, Dirk
Vanselow, Jens
Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title_full Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title_fullStr Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title_full_unstemmed Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title_short Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
title_sort induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217602/
https://www.ncbi.nlm.nih.gov/pubmed/28056989
http://dx.doi.org/10.1186/s12958-016-0221-6
work_keys_str_mv AT baufeldanja inductionofalteredgeneexpressionprofilesinculturedbovinegranulosacellsathighcelldensity
AT koczandirk inductionofalteredgeneexpressionprofilesinculturedbovinegranulosacellsathighcelldensity
AT vanselowjens inductionofalteredgeneexpressionprofilesinculturedbovinegranulosacellsathighcelldensity