Effects of a gel culture system made of polysaccharides (xanthan gum and locust bean gum) on in vitro bovine oocyte development and gene expression of the granulosa cells

Abstract

Xanthan gum (XG) and locust bean gum (LBG) are nontoxic polysaccharides that produce culture substrates. The present study examined the effect of XG-LBG gel on in vitro bovine oocyte growth and gene expression in granulosa cells. Oocytes and granulosa cell complexes (OGCs) were cultured in vitro on plastic culture plate (Plate) or XG-LBG gel for 16 days. OGCs formed a dome-like cavity surrounding the oocytes on plate but formed a spherical follicle structure on XG-LBG gel. The total granulosa cell numbers of the OGCs and their survival rate was greater for OGCs cultured on XG-LBG gel than for those cultured on plate. Oocytes grown on XG-LBG gels had higher lipid and mitochondrial content, as well as a larger diameter, than their plate counterparts. When oocytes grown in vitro were subjected to in vitro maturation and fertilization, the normal fertilization rate was significantly higher for oocytes developed on XG-LBG gel than that of oocytes cultured on the plate counterpart. RNAseq of the granulosa cells revealed that genes associated with focal adhesion, phosphatidylinositol 3'-kinase-Akt and Hippo signaling, and regulation of actin cytoskeleton were upregulated in granulosa cells of OGCs cultured on XG-LBG gel compared with those cultured on plate. K E Y W O R D S bovine, early antral follicle, locust bean gum, oocyte, xanthan gum 1 | INTRODUCTION Bovine embryos are produced from oocytes collected from antral follicles (AFs; 3-5 mm in diameter), but the number of large AFs is limited. Establishing methods for in vitro oocyte growth from small AFs has a great impact on expanding oocyte resources for domestic animals, humans, and endangered wild animals; however, the efficiency of in vitro oocyte development is still low, and oocytes grown in vitro generally have poor developmental competence compared with oocytes collected from large AFs. The major differences between plastic-based in vitro culture conditions versus intra-follicular environments include the presence of focal adhesions, interactions with the extracellular matrix (ECM), and stiffness of the substrate. Although the medium compositions and hormones in in vitro oocyte culture conditions have been studied, the stiffness of the culture substrate has not been extensively studied. Regarding simple modifications to the cell culture conditions, Munakata et al. (2017) grew porcine oocyte granulosa cell complexes

Beneficial Effect of Polysaccharide Gel Made of Xanthan Gum and Locust Bean Gum on Bovine Oocytes

“…The XG-LGB gel had abundant hydroxyl oxide-bound residues and was negatively charged, with soft and low adhesive character to the cellular membrane; consecutively, granulosa cells cultured on the gel did not attach to the gel substrate and formed a spheroid cell mass [ 6 ]. In general, cells proliferate on a stiff substrate through focal adhesion activation and ECM–cell interactions, and this conventional notion cannot explain our findings that cumulus cells and oocytes cultured on soft and less adhesive XG-LGB gel promoted focal adhesion and ECM receptor interactions in both cells and oocytes.…”

“…In line with this, PI3K signaling was a top enriched pathway associated with the DEGs in cumulus cells; however, whether the same mechanism works in gel–cell interaction remains unclear. Our previous report showed that embryos cultured on the XG-LGB gel upregulated actin formation and cadherin localization [ 5 ], and oocyte granulosa cell complexes derived from early antral follicles cultured on the XG-LGB gel upregulated genes associated with focal adhesion and ECM interactions [ 6 ]. Gel substrate then transfers certain signals to activate focal adhesion and ECM interaction-related molecules on cumulus cells and oocytes.…”

“…For the IVM of oocytes, 100 μL of IVM medium was placed on gel substrate or on plastic plate without gel (10 COCs/well). Concentration of the gel was determined based on the previous reports [ 5 , 6 ].…”

“…We developed a soft gel culture substrate made of polysaccharides, xanthan gum (XG), and locust bean gum (LBG), and reported that the gel culture system improved the in vitro growth of oocytes derived from immature small follicles (early antral follicles) and the in vitro development of early developmental stage bovine embryos [ 5 , 6 ]. Herein, the gel culture system activated genes associated with the focal adhesion and actine-mediated signal transduction; however, the key factors causing these changes are yet to be defined.…”

In Vitro Growth of Mammalian Follicles and Oocytes

“…There are several aspects of the efforts employed to improve culture systems, and these include the preparation of ovarian tissues [18], isolation of follicles and oocytes [19,20], design of culture plates [21][22][23], creation of a matrix for media [24][25][26], and inclusion of supplements into media [27]. Additionally, the study of in vivo follicular development [28] and related signaling pathways [29,30] can be the best means of achieving reconstitution of the follicular environment in living animals.…”