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Coenzyme and Cofactor Metabolism Belongs to Biochemical Processes

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Coenzyme and Cofactor Metabolism Belongs to Biochemical Processes Nawrocki et al. Medical Journal of Cell Biology 2019 DOI: 10.2478/acb-2019-0021 Received: 22.10.2019 Accepted: 26.11.2019 Coenzyme and cofactor metabolism belongs to biochemical processes significantly regulated in human granulosa cells collected after IVF during long-term primary in vitro culture Mariusz J. Nawrocki1 2 3, Piotr Celichowski4, Leszek Pawelczyk3 3, Claudia Dompe5, Bartosz Kempisty1,4,6, Paul Mozdziak7 , Rafał Sibiak , Maciej Brązert , Błażej Chermuła Abstract Granulosa cells (GCs) provide the microenvironment necessary for the development of the follicle and the maturation of the oocyte. GCs are associated with reproductive system function and the maintenance of pregnancy by participating in the synthesis of steroid hormones. Many authors point to new ways of using GCs in regenerative medicine and indicate the significant plasticity of this cell population, suggesting that GCs can undergo a transdifferentiation process. Employing primary in vitro cell cultures and high-thro- ughput transcriptome analysis via Affymetrix microarrays, this study describes groups of genes associated with enzymatic reactions. 52 genes were identified belonging to four gene ontology biological process terms (GO BP): “coenzyme biosynthetic process”, “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process”. All identified genes showed reduction in the level of mRNA expression during long-term in vitro cultivation. Significanthe transcriptomic profile variability was exhi- bited for the genes (ELOVL5, ELOVL6 and GPAM) involved in enzymatic regulation of fatty acid metabolism. Running title: Enzymatic regulation in granulosa cells Keywords: granulosa cells, in vitro culture, coenzyme, cofactor 1Department of Anatomy, Poznan University of Medical Sciences, Poznan, Poland 2Division of Reproduction, Department of Obstetrics, Gynecology and Gynecologic Oncology, Poznan University of Medical Sciences, Poznan, Poland 3Department of Infertility and Reproductive Endocrinology, Poznan University of Medical Sciences, Poznan, Poland 4Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland 5The School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, UK 6Department of Obstetrics and Gynecology, University Hospital and Masaryk University, Brno, Czech Republic 7Physiology Graduate Program, North Carolina State University, Raleigh, North Carolina * Correspondence: [email protected] Full list of author information is available at the end of article 153 Nawrocki et al. Medical Journal of Cell Biology (2019) Introduction in this study. All animals were housed under identi- Ovarian follicles are formed during the process of cal conditions. folliculogenesis and oogenesis. The follicle consists of the oocyte, the follicular fluid and the oocyte-as- Collection of porcine ovaries and in vitro culture sociated cells: granulosa cells (GCs). GCs provide of granulosa cells the microenvironment necessary for the develop- The ovaries (n = 80) and the reproductive ment of the follicle and the maturation of the oo- tracts were recovered at slaughter and transport- cyte [1]. GCs also influence the development of the ed to the laboratory at 38°C in 0.9% NaCl within female gamete in a paracrine and autocrine manner 30 min. The ovaries of each animal were placed [2]. Paracrine communication leads to the estab- in phosphate-buffered saline supplemented with lishment of bidirectional cooperation affecting the fetal bovine serum (Sigma-Aldrich Co., St. Louis, hormonal production and the expression of genes MO). Thereafter, single preovulatory large follicles associated with follicular development [3]. GCs are with an estimated diameter >5 mm (n = 300) were associated with the functioning of the reproductive opened into a sterile Petri dish by puncturing them system and the maintenance of pregnancy by par- with a 5 mL syringe and 20-G needle, recovering ticipating in the synthesis of steroid hormones. The the cumulus–oocyte complexes (COCs) and follicu- physiological and morphological changes occurring lar fluid (FF). The GCs were extracted from the FF, in GCs during the formation of the ovarian follicle whereas the COCs were discarded. Culture medium have been well characterized. In contrasts, the deep consisted of Dulbecco’s modified Eagle’s medium molecular mechanisms and the gene interactions of (Sigma-Aldrich) supplemented with 2% fetal calf GCs are still insufficiently described. serum (PAA, Linz, Austria) and l-glutamine. The With a large probability for application as the final concentration of l-glutamine in the medium grafts in regenerative medicine, the establishment was 4 mM (diluted from the 200 mM stock solu- and characterization of in vitro cell cultures (IVC) tion; Invitrogen). Moreover, 10 mg/mL gentamicin may be the most prospective methods of tissue and (Invitrogen), 10,000 U/mL penicillin, and 10,000 organ regeneration,. Some studies point to future mg/mL streptomycin (Invitrogen) were added to potential of GCs in regenerative medicine and in the culture medium. Cells were cultivated at 38.5°C transplantology. GCs containing follicular fluid af- under aerobic conditions (5% CO2). Once the adher- ter being transferred for utilization during routine ent cells were >80% confluent, they were detached in vitro fertilization procedures may be the source by treating them with 0.05% trypsin– ethylenedi- of stem cells. Several authors clearly indicate these aminetetraacetic acid (Invitrogen) for 3 min and previously unidentified properties of GCs. Oppo- they were then counted using a Z2 counter and cell sitely, many articles suggest that GCs are subject to viability analyzer (ViCell XR 2.03; both Beckman a transdifferentiation process [6,7]. During this pro- Coulter). For our experiments, 3 106 cells per dish cess, under the influence of the appropriate factors, were used for culture. the mature cell is reprogrammed and it changes into another cell type [4,5]. Kossowska-Tomaszczuk et al. RNA extraction from granulosa cells [6,7] indicated that GCs have stem-like properties. Oviductal epithelial cells were pooled and har- The aim of study is to furtherly characterize GCs vested at 24h, 7 days, 15 days and 30 days after the at the molecular level and obtain more insight in the beginning of the in vitro culture. The total RNA was variability of the transcriptomic profile during long- extracted from samples using TRI Reagent (Sigma, St term cell culture. Affymetrix microarray assays Louis, MO, USA) according to the method described revealed the regulatory peptides and enzymes in- volved in the functioning of the mechanism of enzy- quantified by measuring the optical density (OD) at matic reactions. The focus of the current study was 260by Chomczyński nm (NanoDrop and spectrophotometer; Sacchi [8]. Then the Thermo RNA wasSci- to examine transcript variability of genes belong- entific, Waltham, MA, USA). Afterwards, 100 ng of an ing to “coenzyme biosynthetic process”, “coenzyme RNA sample were used for downstream analysis. metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process” gene ontology bi- Microarray expression analysis and statistics ological process terms (GO BP) during OECs long- In the first step, the whole RNA (100 ng) from term primary culture in vitro. It will delineate the each sample was subjected to two rounds of sense transcriptomic profile variability for the genes cDNA amplification (Ambion® WT Expression Kit, (ELOVL5, ELOVL6 and GPAM) involved in enzymatic provided by Ambion, Austin, TX, USA). The synthe- regulation of fatty acid metabolism. sis of cRNA was performed by in vitro transcription (16 h, 40 °C). Subsequently, cRNA was purified and Materials and methods retranscribed into cDNA. The cDNA samples were Animals then used for biotin labeling and fragmentation us- A total of 40 crossbred Landrace gilts with a me- ing an Affymetrix GeneChip® WT Terminal Labeling dian age of 170 days and weight of 98 kg were used and Hybridization kit (Affymetrix). These biotin-la- 154 Nawrocki et al. Medical Journal of Cell Biology (2019) beled samples were loaded onto and hybridized to covery rate. The selection of significantly altered the Affymetrix® Porcine Gene 1.1 ST Array Strip genes was based on a p-value beneath 0.05 and ex- (48°C/20 h). Hybridization was conducted at 48 °C pression higher than two-fold. for 20 h, employing an AccuBlock™ Digital Dry Bath Differentially expressed genes were subjected to (Labnet International, Inc., Edison, NJ, USA) hybrid- selection by examination of genes involved in cell mi- ization oven. Subsequently, according to technical gration regulation. The differentially expressed gene protocol, microarrays were washed and stained list (separated for up- and down-regulated genes) using an Affymetrix GeneAtlas™ Fluidics Station was uploaded to the DAVID software (Database for (Affymetrix, Santa Clara, CA, USA). The strips were Annotation, Visualization and Integrated Discov- scanned using an Affymetrix GeneAtlas™ Imaging ery) [9], where genes belonging to the terms of all Station (Affymetrix, Santa Clara, CA, USA) and the three Gene Ontology (GO) domains were extracted. scans of the microarrays were saved on hard drives Expression data of these genes were also subjected as *.CEL files for downstream data analysis. to a hierarchical clusterization procedure, and their All of the presented analyses and graphs were expression values were presented as a heat map. performed using Bioconductor and R program-
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