RESUMEN
The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.
Asunto(s)
Células Epiteliales , Transcriptoma , Animales , Femenino , Porcinos , Células Epiteliales/metabolismo , Células Epiteliales/citología , Perfilación de la Expresión Génica , Células Cultivadas , Oviductos/metabolismo , Oviductos/citología , Técnicas de Cultivo de Célula/métodos , Regulación de la Expresión Génica , Trompas Uterinas/metabolismo , Trompas Uterinas/citologíaRESUMEN
In vitro meat production presents a potential viable alternative for meat consumption, which could provide the consumer with a product indistinguishable from the original, with very similar nutritional and culinary values. Indeed, the alternative products currently accessible often lack comparable nutritional value or culinary attributes to their animal-derived counterparts. This creates challenges for their global acceptance, particularly in countries where meat consumption holds cultural significance. However, while cultured meat research has been progressing rapidly in recent years, some significant obstacles still need to be overcome before its possible commercialization. Hence, this review summarizes the most current knowledge regarding the history of cultured meat, the currently used cell sources and methods used for the purpose of in vitro meat production, with particular focus on the role of bioreactors, scaffolds and microcarriers in overcoming the current obstacles. The authors put the potential microcarrier and scaffold-based solutions in a context, discussing the ways in which they can impact the way forward for the technology, including the use of considering the potential practical and societal barriers to implementing it as a viable food source worldwide.
RESUMEN
BACKGROUND: Cell cycle regulation influences the proliferation of granulosa cells and affects many processes related to ovarian folliclular growth and ovulation. Abnormal regulation of the cell cycle can lead to many diseases within the ovary. The aim of this study was to describe the expression profile of genes within granulosa cells, which are related to the formation of the cytoskeleton, organization of cell organelles inside the cell, and regulation of cell division. Established in vitro primary cultures from porcine ovarian follicle granulosa cells were maintained for 48, 96, 144 h and evaluated via microarray expression analysis. RESULTS: Analyzed genes were assigned to 12 gene ontology groups "actin cytoskeleton organization", "actin filament organization", "actin filament-based process", "cell-matrix adhesion", "cell-substrate adhesion", "chromosome segregation", "chromosome separation", "cytoskeleton organization", "DNA integrity checkpoint", "DNA replication initiation", "organelle fision", "organelle organization". Among the genes with significantly changed expression, those whose role in processes within the ovary are selected for consideration. Genes with increased expression include (ITGA11, CNN1, CCl2, TPM2, ACTN1, VCAM-1, COL3A1, GSN, FRMD6, PLK2). Genes with reduced expression inlcude (KIF14, TACC3, ESPL1, CDC45, TTK, CDC20, CDK1, FBXO5, NEK2-NIMA, CCNE2). For the results obtained by microarray expressions, quantitative validation by RT-qPCR was performed. CONCLUSIONS: The results indicated expression profile of genes, which can be considered as new molecular markers of cellular processes involved in signaling, cell structure organization. The expression profile of selected genes brings new insight into regulation of physiological processes in porcine follicular granulosa cells during primary in vitro culture.
RESUMEN
Exosomal regulation is intimately involved in key cellular processes, such as migration, proliferation, and adhesion. By participating in the regulation of basic mechanisms, extracellular vesicles are important in intercellular signaling and the functioning of the mammalian reproductive system. The complexity of intercellular interactions in the ovarian follicle is also based on multilevel intercellular signaling, including the mechanisms involving cadherins, integrins, and the extracellular matrix. The processes in the ovary leading to the formation of a fertilization-ready oocyte are extremely complex at the molecular level and depend on the oocyte's ongoing relationship with granulosa cells. An analysis of gene expression from material obtained from a primary in vitro culture of porcine granulosa cells was employed using microarray technology. Genes with the highest expression (LIPG, HSD3B1, CLIP4, LOX, ANKRD1, FMOD, SHAS2, TAGLN, ITGA8, MXRA5, and NEXN) and the lowest expression levels (DAPL1, HSD17B1, SNX31, FST, NEBL, CXCL10, RGS2, MAL2, IHH, and TRIB2) were selected for further analysis. The gene expression results obtained from the microarrays were validated using quantitative RT-qPCR. Exosomes may play important roles regarding intercellular signaling between granulosa cells. Therefore, exosomes may have significant applications in regenerative medicine, targeted therapy, and assisted reproduction technologies.
Asunto(s)
Células de la Granulosa , Folículo Ovárico , Femenino , Porcinos , Animales , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Oocitos/metabolismo , Ovario/metabolismo , Proliferación Celular/genética , MamíferosRESUMEN
The extracellular matrix (ECM) in granulosa cells is functionally very important, and it is involved in many processes related to ovarian follicle growth and ovulation. The aim of this study was to describe the expression profile of genes within granulosa cells that are associated with extracellular matrix formation, intercellular signaling, and cell-cell fusion. The material for this study was ovaries of sexually mature pigs obtained from a commercial slaughterhouse. Laboratory-derived granulosa cells (GCs) from ovarian follicles were cultured in a primary in vitro culture model. The extracted genetic material (0, 48, 96, and 144 h) were subjected to microarray expression analysis. Among 81 genes, 66 showed increased expression and only 15 showed decreased expression were assigned to 7 gene ontology groups "extracellular matrix binding", "extracellular matrix structural constituent", "binding, bridging", "cadherin binding", "cell adhesion molecule binding", "collagen binding" and "cadherin binding involved in cell-cell adhesion". The 10 genes with the highest expression (POSTN, ITGA2, FN1, LAMB1, ITGB3, CHI3L1, PCOLCE2, CAV1, DCN, COL14A1) and 10 of the most down-regulated (SPP1, IRS1, CNTLN, TMPO, PAICS, ANK2, ADAM23, ABI3BP, DNAJB1, IGF1) were selected for further analysis. The results were validated by RT-qPCR. The current results may serve as preliminary data for further analyses using in vitro granulosa cell cultures in assisted reproduction technologies, studies of pathological processes in the ovary as well as in the use of the stemness potential of GCs.
RESUMEN
The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus-oocyte complex (COC) show evolving gene expression profiles throughout the oocytes' lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs' gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the "defense response to other organism", "defense response", "defense response to virus", "cytokine secretion", "cytokine production" and "cytokine-mediated signaling pathway" GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells.
Asunto(s)
Células del Cúmulo/inmunología , Citocinas/metabolismo , Inmunidad/genética , Oocitos/inmunología , Ovulación/inmunología , Adulto , Proliferación Celular/genética , Células Cultivadas , Células del Cúmulo/metabolismo , Femenino , Fertilización In Vitro , Perfilación de la Expresión Génica , Humanos , Oocitos/metabolismo , Ovulación/genética , Inducción de la Ovulación , Cultivo Primario de Células , Transducción de Señal/genética , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The methylated resveratrol analogue 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) has been revealed to exert the anti-cancer activity by a block of the cell cycle at the G2/M phase, apoptosis induction, and metastasis inhibition. These biological events may be involved in crosstalk with the epidermal growth factor receptor (EGFR), which belongs to the ErbB family of receptor tyrosine kinases. Several cancer therapeutic approaches employ small molecules capable of inhibiting tyrosine kinases (e.g., gefitinib). According to more recent reports, combining gefitinib with chemotherapeutics, such as cisplatin, seems to be more effective than monotherapy. The present study aimed to assess the molecular mechanism of the potential anti-proliferative activity of individual and combined treatments with DMU-214 and gefitinib in SCC-25 and CAL-27 human tongue cancer cell lines. We showed for the first time the anti-cancer effects of DMU-214, gefitinib, and their combination in tongue cancer cells triggered via cell cycle arrest, apoptosis induction, and inhibition of the EGFR signaling pathway. The anti-proliferative effects of DMU-214 and gefitinib are also suggested to be related to the EGFR and EGFRP (phosphorylated epidermal growth factor receptor) expression status since we found significantly weaker cytotoxic activity of the compounds tested in SCC-25 cells, which overexpressed EGFR and EGFRP proteins.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Lengua/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ciclo Celular , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Gefitinib/administración & dosificación , Humanos , Resveratrol/administración & dosificación , Resveratrol/análogos & derivados , Neoplasias de la Lengua/tratamiento farmacológico , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/metabolismo , Células Tumorales CultivadasRESUMEN
Following the publication of this paper, the authors have requested that, on p. 4412 of the above article in the Funding section of the Declarations, the acknowledgement to one of the funding sources should be removed from the paper; essentially, the reference to grant no. 2018/31/B/NZ5/02475, formulated by the Polish National Science Centre (grant providing institution), should be removed from the paper. Therefore, the revised version of the Funding section paragraph should read as follows: Funding: The present study was supported by a grant from Poznan University of Medical Sciences (grant no. 502140222736710694). The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 4403-4414, 2019, DOI: 10.3892/mmr.2019.10709].
RESUMEN
The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.
Asunto(s)
Células del Cúmulo/metabolismo , Neovascularización Fisiológica , Células Madre/metabolismo , Femenino , Humanos , Síndrome del Ovario Poliquístico/metabolismo , Insuficiencia Ovárica Primaria/metabolismoRESUMEN
Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.
Asunto(s)
Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Oocitos/metabolismo , Oviductos/citología , Animales , Diferenciación Celular/genética , Forma de la Célula/genética , Células Cultivadas , Regulación hacia Abajo/genética , Femenino , Ontología de Genes , Redes Reguladoras de Genes , Marcadores Genéticos , Transducción de Señal/genética , Porcinos , Transcriptoma , Regulación hacia Arriba/genéticaRESUMEN
In the growing ovarian follicle, the maturing oocyte is accompanied by cumulus (CCs) and granulosa (GCs) cells. Currently, there remain many unanswered questions about the epithelial origin of these cells. Global and targeted gene transcript levels were assessed on 1, 7, 15, 30 days of culture for CCs and GCs. Detailed analysis of the genes belonging to epithelial cell-associated ontological groups allowed us to assess a total of 168 genes expressed in CCs (97 genes) and GCs (71 genes) during long-term in vitro culture. Expression changes of the analyzed genes allowed the identification of the group of genes: TGFBR3, PTGS2, PRKX, AHI1, and IL11, whose expression decreased the most and the group of ANXA3, DKK1, CCND1, STC1, CAV1, and SFRP4 genes, whose expression significantly increased. These genes' expression indicates CCs and GCs epithelialization processes and their epithelial origin. Expression change analysis of genes involved in epithelization processes in GCs and CCs during their in vitro culture made it possible to describe the most significantly altered of the 11 genes. Detailed analysis of gene expression in these two cell populations at different time intervals confirms their ovarian surface epithelial origin. Furthermore, some gene expression profiles appear to have tumorigenic properties, suggesting that granulosa cells may play a role in cancerogenesis.
RESUMEN
Aquaporins constitute a group of water channel proteins located in numerous cell types. These are pore-forming transmembrane proteins, which mediate the specific passage of water molecules through membranes. It is well-known that water homeostasis plays a crucial role in different reproductive processes, e.g., oocyte transport, hormonal secretion, completion of successful fertilization, blastocyst formation, pregnancy, and birth. Further, aquaporins are involved in the process of spermatogenesis, and they have been reported to be involved during the storage of spermatozoa. It is noteworthy that aquaporins are relevant for the physiological function of specific parts in the female reproductive system, which will be presented in detail in the first section of this review. Moreover, they are relevant in different pathologies in the female reproductive system. The contribution of aquaporins in selected reproductive disorders and aging will be summarized in the second section of this review, followed by a section dedicated to aquaporin-related proteins. Since the relevance of aquaporins for the male reproductive system has been reviewed several times in the recent past, this review aims to provide an update on the distribution and impact of aquaporins only in the female reproductive system. Therefore, this paper seeks to determine the physiological and patho-physiological relevance of aquaporins on female reproduction, and female reproductive aging.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/fisiología , Acuaporinas/metabolismo , Genitales Femeninos/metabolismo , Genitales Femeninos/fisiología , Mamíferos/metabolismo , Mamíferos/fisiología , Animales , Femenino , Humanos , Masculino , Espermatozoides/metabolismo , Espermatozoides/fisiologíaRESUMEN
Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, "muscle cell development", "muscle cell differentiation", "muscle contraction", "muscle organ development", "muscle organ morphogenesis", "muscle structure development", "muscle system process" and "muscle tissue development". The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.
RESUMEN
Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.
Asunto(s)
Diferenciación Celular , Gránulos Citoplasmáticos/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/metabolismo , Transcriptoma , Animales , Células Cultivadas , Femenino , Oocitos/citología , PorcinosRESUMEN
In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Senescencia Celular/genética , Células del Cúmulo/citología , Células del Cúmulo/metabolismo , Perfilación de la Expresión Génica , Adulto , Biomarcadores/metabolismo , Muerte Celular/genética , Forma de la Célula/genética , Regulación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Humanos , Análisis de Componente Principal , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The process of neural tissue formation is associated primarily with the course of neurogenesis during embryonic life. The source of neurallike cells is stem cells, which, under the influence of appropriate differentiating factors, may differentiate/transdifferentiate towards a neurallike lineage. The present study suggested that, under longterm in vitro culture conditions, human ovarian granulosa cells (GCs), obtained from granulosarich follicular fluid, acquired new properties and expressed genes characteristic of the ontological groups 'neurogenesis' (GO:0022008), 'neuronal precursor cell proliferation' (GO:0061351) and 'nervous system development' (GO:0007399), which are closely related to the formation of neurons. The present study collected GCs from 20 women referred for the procedure of in vitro fertilization. Cells were maintained in longterm in vitro culture for 30 days, and RNA was isolated after 1, 7, 15 and 30 days of culture. The expression profile of individual genes was determined using the Affymetrix microarray method. The 131 genes with the highest expression change in relation to day 1 of culture were then selected; the 10 most affected genes found to be primarily involved in nerve cell formation processes were chosen for consideration in this study: CLDN11, OXTR, DFNA5, ATP8B1, ITGA3, CD9, FRY, NANOS1, CRIM1 and NTN4. The results of the present study revealed that these genes may be considered potential markers of the uninduced differentiation potential of GCs. In addition, it was suggested that GCs may be used to develop a cell line showing neuronal characteristics after 30 days of cultivation. In addition, due to their potential, these cells could possibly be used in the treatment of neurodegenerative diseases, not only in the form of 'cultured neurons' but also as producers of factors involved in the regeneration of the nervous system.
Asunto(s)
Transdiferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Células-Madre Neurales/citología , Neurogénesis/genética , Neuronas/citología , Adolescente , Adulto , Proliferación Celular/genética , Forma de la Célula/genética , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Adulto JovenRESUMEN
Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.
Asunto(s)
Hormonas/metabolismo , Técnicas de Maduración In Vitro de los Oocitos , Lípidos/análisis , Oocitos/metabolismo , Animales , Células Cultivadas , Eosina Amarillenta-(YS)/química , Femenino , Hematoxilina/química , Hormonas/genética , Oocitos/crecimiento & desarrollo , Oxazinas/química , Transducción de Señal , PorcinosRESUMEN
The oogenesis and folliculogenesis are closely linked and occur simultaneously in the growing ovarian follicles. Biochemical and morphological changes in oocytes (OC) and surrounding granulosa cells (GCs) are highly complex and depend on many factors, including intercellular communication. GCs are cells with many functions, often crucial for the proper viability of the oocyte and subsequent positive fertilization. The purpose of this study was to analyze gene expression in porcine GCs, to define differentially expressed genes belongs to the "cell growth", "aging", "positive regulation of cell death", "apoptotic process", "regulation of cell death", "cell death" and "negative regulation of cell death" ontology groups during the short - term primary in vitro culture. Microarrays were employed to study the transcriptome contained in the total RNA of the cultured GCs. Ovaries were obtained after slaughter, from 40 gilts of swine aged 170 days. The cells were obtained through puncture of the ovaries, collection of follicular fluid, removal of the cumulus - oocyte complexes and centrifugation. The cells were then cultured in vitro. The RNA material was obtained before the culture was established (0h) and then after 48h, 96h and 144h of its course. From 182 differently expressed genes belonging to the these ontology groups, we have selected POSTN, FN1, FMOD, ITGB3, DCN, SERPINB2, SFRP2, IGFBP5, EMP1, and CCL2 which were upregulated, as well as DAPL1, ESR1, IHH, TGFBR3, PPARD, PDK4, TXNIP, IFIT3, CSRNP3, and TNFSF10 genes whose expression was downregulated during the time of in vitro culture of the GCs. The significance of the differential gene expression is to provide new information on the molecular aspects of in vitro granulosa cell culture.
Asunto(s)
Apoptosis/fisiología , Supervivencia Celular/fisiología , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/fisiología , Animales , Células Cultivadas , Regulación hacia Abajo , Femenino , Análisis por Matrices de Proteínas , Porcinos , Transcriptoma , Regulación hacia ArribaRESUMEN
The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.
Asunto(s)
Ciclo Celular/genética , Células de la Granulosa/citología , Folículo Ovárico/citología , Transcriptoma , Animales , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , PorcinosRESUMEN
Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively wellknown, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RTqPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNAdependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.