Abstract
The aims of this study were to demonstrate the stress-associated developmental events and
the effects ofstress-modulating agents on in vitro development and embryo quality in cattle.
First, to investigate whether the regulation of oxidative stress by supplementing sericin, a
potential antioxidant during in vitro culture (IVC) can contribute to the production of
bovine embryos with high viability in terms of thermotolerance, in vitro fertilized-embryos
were cultured in IVC medium supplemented with 0.1% sericin until Day 7, and then exposed
to heat stress (HS) at 40.5°C for 6 h in the sericin-free medium. Following HS, subsequently
embryos were cultured in sericin-free medium until Day 8. The results show that the
expression of genes for HSPA1A and BAX in blastocysts obtained from culture without
sericin significantly increased (P < 0.05) by HS (at 40.5°C for 6 h on Day 7) treatment
compared to the non-HS control group; whereas blastocysts obtained from culture with
sericin showed significantly decreased (P < 0.05) these gene expression to a level
comparable to that in the non-HS control group. In addition, expression of IFN-tau, a
maternal pregnancy recognition gene was increased in blastocysts produced by sericin
culture. Moreover, TUNEL-positive cells number was significantly lower in blastocysts
produced by sericin culture than in the HS control group. These findings indicate that
sericin supplementation during IVC is useful for production of embryo with high tolerance
in cattle.
Next, to examine the effects of endoplasmic reticulum (ER) stress during IVC on
developmental kinetics and cryo-tolerance in embryos, tauroursodeoxycholic acid
(TUDCA) and/ tunicamycin (TM), an ER stress inhibitor and inducer, respectively, were
supplemented during IVC. As a result, treatment of TUDCA (10 µM) restored the detrimental effects of TM-induced ER stress impairments in embryos development and
quality. In addition, TUDCA (10 µM) significantly suppressed reactive oxygen species
(ROS) generation, apoptosis and expression of ER stress markers (GRP78, ATF4, ATF6,
IER1, sXBP1, CHOP and BAX); while it increased anti-apoptotic BCL2 gene and
glutathione levels compared to the control. Moreover, ER stress inhibition via TUDCА (10
µM) enhances embryo cryo-tolerance after vitrification. Based on these findings, a followup study conducted to examine whether ER stress attenuation via TUDCA during IVM
influences oocyte developmental competences. The results show that addition of TUDCA
(100 µM) during IVM significantly decreased ROS, apoptosis and ER stress-induced
protein/gene levels in matured COCs; thereby increases the maturation rate, and
subsequent embryos development. Collectively, these findings suggest that controlling ER
stress during IVM or IVC improves in vitro embryo development with high cryo-tolerance.
Finally, to explore whether the regulation of embryo autophagic activity during culture
influences preimplantation embryo development and quality at the viewpoint of gene
expression, rapamycin was used to induce, while wortmannin was used to suppress embryo
autophagic activity during IVC. Surprisingly, autophagy is highly activated in embryos at
4-cell stage by rapamycin treatment (100 nM), as evidenced by significant upregulation of
autophagy triggering molecules (LC3, ATG5, ATG7) and suppression of mTOR expression,
and rapid maternal mRNA degradation. Further, induction of autophagy influences the
blastocyst outcomes and activates many developmentally related genes (BCL2, MnSOD,
SOX2, POUSF1, NANOG, PLAC8, IFN-tau, and GLUT5) in blastocysts. For the first, this
research focus on the extent of embryo autophagic activity via rapamycin during culture
involves with activation of many genes that are essential for blastocyst outcomes, quality
and viability. In conclusion, these findings suggest that an appropriate balance between oxidative and ER stress along with regulation of autophagy during culture influences in
vitro early embryogenesis and may contribute to the development of strategies for the
production of bovine blastocysts with high developmental competence.