One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG‐LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96‐well cell culture plate coated with or without XG‐LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG‐LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG‐LBG gels for 24 hr had high expression levels of F‐actin and a highly even distribution of E‐cadherin. In addition, embryos developed on XG‐LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP‐CTGF signalling.
Embryo-maternal reproductive tract interactions are pivotal for successful pregnancy. The present study predicted the molecules modulating embryo-uterine communication by comparing two sets
of differentially expressed genes (DEGs): DEGs in uterine epithelial cells (UECs) collected from the uterus with and without blastocysts and DEGs between blastocysts developed
in
vivo
and
in vitro
. Cows were subjected to super ovulation (SOV), followed by insemination or non-insemination at estrus (SOV + AI and SOV cows). Seven days after
estrus, the uterus was flushed to collect UECs, and the presence of blastocysts in the uterus was confirmed. UECs were subjected to RNA-Sequencing (RNA-Seq) to identify DEGs. Publicly
available RNA-Seq data of
in vivo
and
in vitro
developed bovine blastocysts were used to determine DEGs. Then, using ingenuity pathway analysis, activated-
and inhibited-upstream regulators (USRs) for UECs in blastocysts were compared with those for blastocysts developed
in vivo
. RNA-Seq of UECs revealed that the DEGs were
associated with immune response and cell adhesion pathways. The activated and inhibited USRs of UECs derived from SOV+ AI cows overlapped with the activated and inhibited USRs of blastocysts
developed
in vivo
. Overlapping activated USRs include leukemia inhibitory factor, interleukin 6, fibroblast growth factor-2, transforming growth factor beta-1, and epidermal
growth factor. In conclusion, the present study predicted the molecules that potentially mediate communication between the developing embryo and the uterus
in vivo
and
prepare the uterus for pregnancy.
The present study examined the effect of polysaccharides gels made of xanthan gum and locust bean gum (gel culture system) on oocyte maturation and explored the molecules causing the beneficial effect of the gel culture system. Oocytes and cumulus cells complexes were collected from slaughterhouse-derived ovaries and cultured on a plastic plate or gel. The gel culture system improved the rate of development to the blastocyst stage. The oocytes that matured on the gel contained high lipid contents and F-actin formation, and the resultant 8-cell stage embryos had low DNA methylation levels compared to their plate counterparts. RNA sequencing of the oocytes and embryos revealed the differentially expressed genes between the gel and plate culture systems, and upstream regulator analysis revealed estradiol and TGFB1 as top activated upstream molecules. The medium of the gel culture system contained higher concentrations of estradiol and TGFB1 than that of the plate cultures system. Supplementation of the maturation medium with either estradiol or TGFB1 resulted in high lipid content in oocytes. In addition, TGFB1 improved the developmental ability of the oocytes and increased F-actin content while reducing DNA methylation levels in the 8-cell stage embryos. In conclusion, the gel culture system is useful for embryo production, potentially through the upregulation of TGFB1.
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