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.

Keywords: Granulosa cells; Microarray; Ovarian follicle; Pig; Primary culture.

Fig. 1

Heat map representation of differentially expressed genes belonging to the “cell cycle checkpoint”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition”, “cell cycle phase transition”, “cell cycle process”, “cell cycle” and “cell division” GO BP terms. Arbitrary signal intensity acquired from microarray analysis is represented by colors (green, higher; red, lower expression). log2 signal intensity values for any single gene were resized to Row z-score scale (from − 2, the lowest expression to + 2, the highest expression for single gene)

Fig. 2

The circular visualization of the results of gene-annotation enrichment analysis. The outer circle shows a scatter plot for each term of the logFC of the assigned genes. Green circles display up-regulation and red ones down-regulation. The inner circle is the representation of z-score. The size and the color of the bar correspond to the value of z-score

Fig. 3

The representation of the mutual relationship between 20 chosen genes that belongs to “cell cycle checkpoint”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition”, “cell cycle phase transition”, “cell cycle process”, “cell cycle” and “cell division” GO BP terms. The ribbons indicate which gene belongs to which categories. The colors of 3 inner bars near each gene corresponds to logFC after 48 h, 96 h and 144 h, respectively. The genes were sorted by logFC

Fig. 4

Heatmap showing the gene occurrence between 20 chosen genes that belongs “cell cycle checkpoint”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition”, “cell cycle phase transition”, “cell cycle process”, “cell cycle” and “cell division” GO BP terms. Yellow color indicates the gene occurrence in indicated GO BP term. The intensity of colour correlates with number of GO BP Terms that selected gene belongs to

Fig. 5

STRING-generated interaction network among 20 chosen genes belonging to the “cell cycle checkpoint”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition”, “cell cycle phase transition”, “cell cycle process”, “cell cycle” and “cell division” GO BP terms. The intensity of the edges reflects the strength of interaction score

Fig. 6

Functional interaction (FI) between 20 chosen genes that belongs to “cell cycle checkpoint”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition”, “cell cycle phase transition”, “cell cycle process”, “cell cycle” and “cell division” GO BP terms. In the following figure “− >“ stands for activating/catalyzing, “− |” for inhibition, “–” for FIs extracted from complexes or inputs, and “—” for predicted FIs

Fig. 7

The results of RT-qPCR validation of microarray results presented in the form of a bar graph

Fig. 8

Mid-part histological sections of crossbred Landrace gilts ovaries, stained with H&E, representing their structure and follicles in all stages of development. a, c, e, g Whole ovaries (scale bars: 5000 µm), b, d, f, h selected areas of a, c, e, g observed in higher magnification (scale bars: B, D-50 µm, F-100 µm, H-500 µm). Arrows: 1—primordial follicles, 2—oocyte, 3—follicular cells, 4—tunica albuginea, 5—germinal epithelium, 6—primary follicle, 7—granulosa cells, 8—secondary follicle, 9—antrum, 10—zona pellucida, 11—theca interna and theca externa, 12—mature follicle, 13—corona radiata, 14—cumulus oophorus

Fig. 9

Microphotograph representing separated mature follicles classified into 3 groups according to their size (H&E staining). a Small follicle (< 3 mm; scale bar—500 µm), b medium follicle (3–5 mm; scale bar—1000 µm), c large follicles (> 5 mm; scale bar—1000 µm). Arrows: 1—antrum, 2—granulosa cells, 3—theca interna and theca externa

Fig. 10

A diagram of a cross-section of the ovarian follicle, outlining its layout and features enabling functions of the distinct granulosa types