Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture

Abstract

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.

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes

“…Ożegowska et al [34] determined the gene expression profile of cultured porcine oocytes, in which estrogen stimulation caused significant downregulation of TXNIP expression during in vitro maturation. A recently described study also showed down-regulation of this gene during primary culture of porcine granulosa cells [22,35]. In agreement with these previous studies, the present study demonstrated that the TXNIP gene was downregulated in cultured porcine OECs in vitro, which may suggest that TXNIP downregulation could serve as a marker for epithelial cell growth.…”

Expression of genes regulating cell division in porcine follicular granulosa cells

“…All of the down-regulated expression genes are cell cycle related and have been described to be involved in cell division. Interestingly, a similar direction of expression of genes responsible for cell division was obtained in in vitro experiments on human [ 88 ] and porcine granulosa cells [ 89 ]. These genes affecting cell proliferation within the ovary affect granulosa cell function [ 90 , 91 ], while at the same time may cause many diseases within the ovary [ 86 , 87 ].…”

Exploration of the Antioxidant Effect of Spermidine on the Ovary and Screening and Identification of Differentially Expressed Proteins

“…This gene is also important in the maturation of mouse oocytes [ 71 ]. These results provide a potential reference for the study of mammalian follicular mechanisms and provide a possible new genetic marker for the study of the pig granulosa cell cycle [ 72 ]. These data suggest that future research directions will involve the exploration of the specific functions of TNXIP in the ovary.…”