New Gene Markers of Angiogenesis and Blood Vessels Development in Porcine Ovarian Granulosa Cells during Short-Term Primary Culture In Vitro

Abstract

The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of “angiogenesis,” “blood vessel development,” “blood vessel morphogenesis,” “cardiovascular system development,” and “vasculature development” for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC’s in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro

“…Fibronectin is a major component of the extracellular matrix, also involved in the migration of cells associated with the basement membrane from connective tissue [59]. The expression of this gene was also recorded in the aforementioned studies on bovine GCs and theca cells [42] and on swine GCs [25,60], where an increase in expression during primary in vitro culture was observed. Significant expression of this gene in reproductive system tissues may provide insight into its role in ovarian follicle and oocyte morphogenesis.…”

“…Downregulation of IHH, ITGB1, and CD9 connected with folliculogenesis or oogenesis was revealed after IVM. IHH (Indian hedgehog signaling molecule), whose expression in the mucous membrane of the murine uterus was caused by the addition of progesterone [93], and its expression was documented in swine GCs [25,60], also the ITGB1 (integrin subunit beta 1) gene associated with the embryogenesis processes [94], and the CD9 gene, whose contribution to fertility is not yet clear, although it is known to be important in gametes [95]. CD9 is an important factor associated with oocyte membrane and microvilli, playing an active part in the fusion of oocyte and sperm [96].…”

Coenzyme and cofactor metabolism belongs to biochemical processes significantly regulated in human granulosa cells collected after IVF during long-term primary in vitro culture

“…However, the molecular mechanisms and gene interactions of GCs are still insufficiently described, Previous studies are an important introduction to a better understanding of all key mechanisms in this cell population [13]. In addition to indicating numerous molecular markers that are key to many physiological and biochemical processes occurring in these cells [13][14][15], the very high plasticity of GCs and their potential to differentiate into other cell populations were demonstrated [16][17][18]. In the present study, by employing primary in vitro long-term culture and microarray approach, groups of genes associated with enzymatic reactions were described.…”

Involvement of GJA1 and Gap Junctional Intercellular Communication between Cumulus Cells and Oocytes from Women with PCOS

“…During folliculogenesis, oocytes receive important signaling and nutrition only through communication with the cumulus cells around them. Through this communication, oocytes and cumulus cells complete folliculogenesis, oocyte maturation, and ovulation [9][10][11][12]. Therefore, microcommunication plays a critical role in oocyte maturation.…”