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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

1
Veterinary Center, Nicolaus Copernicus University in Torun, 87-100 Toruń, Poland
2
Department of Histology and Embryology, Poznan University of Medical Sciences, 61-701 Poznan, Poland
3
Department of Anatomy, Poznan University of Medical Sciences, 61-701 Poznan, Poland
4
Department of Toxicology, Poznan University of Medical Sciences, 61-701 Poznan, Poland
5
Chair of Biotechnology, Department of Cancer Immunology, Poznan University of Medical Sciences, 61-701 Poznan, Poland
6
Department of Histology and Embryology, Wroclaw Medical University, 50-367 Wroclaw, Poland
7
Division of Anatomy and Histology, University of Zielona Gora, 65-417 Zielona Góra, Poland
8
Department of Obstetrics and Gynecology, University Hospital and Masaryk University, 601 77 Brno, Czech Republic
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(16), 3966; https://doi.org/10.3390/ijms20163966
Received: 18 July 2019 / Revised: 9 August 2019 / Accepted: 12 August 2019 / Published: 15 August 2019
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

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. View Full-Text
Keywords: pig; granulosa; cells morphogenesis pig; granulosa; cells morphogenesis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Kulus, M.; Sujka-Kordowska, P.; Konwerska, A.; Celichowski, P.; Kranc, W.; Kulus, J.; Piotrowska-Kempisty, H.; Antosik, P.; Bukowska, D.; Iżycki, D.; Bruska, M.; Zabel, M.; Nowicki, M.; Kempisty, B. 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. Int. J. Mol. Sci. 2019, 20, 3966.

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