Early antral follicle (EAF)-derived porcine oocytes develop more readily on polyacrylamide-gel (PAG) than on plastic plates. Xanthan gum (XG) and locust bean gum (LBG) are edible polysaccharides. We investigated XG-LBG gel supports in the development of EAF-derived porcine oocytes. XG and LBG were mixed in a 1:1 ratio to form a substrate. We cultured oocyte granulosa cell complexes (OGCs) from the EAFs on XG-LBG gels of various concentrations. The oocyte diameters were comparable among the 0.3, 0.5, and 1.0% gels; granulosa cell proliferation was greater on the 1.0% gel. The proliferation and survival rates of the granulosa cells, and the histone H4 at lysine 12 acetylation levels were higher in OGCs cultured on 1.0% XG-LBG than those grown on 0.3% PAG. Development to the blastocyst stage was 13.8% for the XG-LBG gels and 9.4% for PAG. In conclusion, XG-LBG are safe and efficient substrates for in vitro culture of oocytes.
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
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