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New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte—granulosa cells—are characte...

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Autores principales: Kulus, Magdalena, Sujka-Kordowska, Patrycja, Konwerska, Aneta, Celichowski, Piotr, Kranc, Wiesława, Kulus, Jakub, Piotrowska-Kempisty, Hanna, Antosik, Paweł, Bukowska, Dorota, Iżycki, Dariusz, Bruska, Małgorzata, Zabel, Maciej, Nowicki, Michał, Kempisty, Bartosz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721001/
https://www.ncbi.nlm.nih.gov/pubmed/31443152
http://dx.doi.org/10.3390/ijms20163966
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author Kulus, Magdalena
Sujka-Kordowska, Patrycja
Konwerska, Aneta
Celichowski, Piotr
Kranc, Wiesława
Kulus, Jakub
Piotrowska-Kempisty, Hanna
Antosik, Paweł
Bukowska, Dorota
Iżycki, Dariusz
Bruska, Małgorzata
Zabel, Maciej
Nowicki, Michał
Kempisty, Bartosz
author_facet Kulus, Magdalena
Sujka-Kordowska, Patrycja
Konwerska, Aneta
Celichowski, Piotr
Kranc, Wiesława
Kulus, Jakub
Piotrowska-Kempisty, Hanna
Antosik, Paweł
Bukowska, Dorota
Iżycki, Dariusz
Bruska, Małgorzata
Zabel, Maciej
Nowicki, Michał
Kempisty, Bartosz
author_sort Kulus, Magdalena
collection PubMed
description Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte—granulosa cells—are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h—before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: “Cell morphogenesis”, “cell differentiation” and “cell development” were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.
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spelling pubmed-67210012019-09-10 New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles Kulus, Magdalena Sujka-Kordowska, Patrycja Konwerska, Aneta Celichowski, Piotr Kranc, Wiesława Kulus, Jakub Piotrowska-Kempisty, Hanna Antosik, Paweł Bukowska, Dorota Iżycki, Dariusz Bruska, Małgorzata Zabel, Maciej Nowicki, Michał Kempisty, Bartosz Int J Mol Sci Article Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte—granulosa cells—are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h—before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: “Cell morphogenesis”, “cell differentiation” and “cell development” were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits. MDPI 2019-08-15 /pmc/articles/PMC6721001/ /pubmed/31443152 http://dx.doi.org/10.3390/ijms20163966 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kulus, Magdalena
Sujka-Kordowska, Patrycja
Konwerska, Aneta
Celichowski, Piotr
Kranc, Wiesława
Kulus, Jakub
Piotrowska-Kempisty, Hanna
Antosik, Paweł
Bukowska, Dorota
Iżycki, Dariusz
Bruska, Małgorzata
Zabel, Maciej
Nowicki, Michał
Kempisty, Bartosz
New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title_full New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title_fullStr New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title_full_unstemmed New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title_short New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles
title_sort new molecular markers involved in regulation of ovarian granulosa cell morphogenesis, development and differentiation during short-term primary in vitro culture—transcriptomic and histochemical study based on ovaries and individual separated follicles
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721001/
https://www.ncbi.nlm.nih.gov/pubmed/31443152
http://dx.doi.org/10.3390/ijms20163966
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