Osmolarity at early culture stage affects development and expression of apoptosis related genes (bax‐α and bcl‐xl) in pre‐implantation porcine NT embryos

Abstract

This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.

Oil-free culture system for in vitro bovine embryo production

“…The evaporation increased osmolality and salt concentration, which is harmful to oocyte and embryo viability. Regardless of the evidence that high osmolality during early embryonic stages decreases the number of apoptotic cells and improves blastocyst formation, the increase in osmolality is highly detrimental for later stages of embryo development (Nguyen et al, 2003;Hwang et al, 2008). Bovine embryos require a low constant osmolality, near 270 mOsm kg -1 (reviewed by Wright and Bondioli, 1981).…”

“…There is some evidence that osmolality of culture medium must remain stable during all the IVP process (Kruger et al, 1985). However, recent data indicates that increased osmolality in the first two days of culture improved in vitro development and reduced apoptosis through regulation of Bax-alpha/Bclxl gene expression in porcine embryos (Hwang et al, 2008). Osmotic stresses can also damage DNA, affect DNA replication, DNA transcription and mRNA translation, leading to cellular damage (Burg et al, 2007).…”

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

“…These results demonstrate that the optimal osmolarities required before and after early cleavage for the parthenogenetic development of pig oocytes are different. Recent reports have indicated that there is a preference for physiological osmolarities during the first 2 to 3 days of culturing pig in vitro -fertilized embryos [13], activated oocytes [20,21,22] and SCNT embryos [14, 21, 22], although the oocytes/embryos have to be transferred into hypotonic media subsequently in order to maintain in vitro development. The results of the present study are consistent with these reports.…”

“…When cultured in isotonic media during the first 2 days, the development of pig electrically activated oocytes and SCNT embryos improved because of the reduction in their fragmentation rates [21]. The same culture conditions also reduced apoptosis in pig SCNT embryos, resulting in improved development [14]. These results suggest that culture in hypotonic media induces excessive apoptosis and fragmentation in pig oocytes and embryos compared with isotonic media because of unphysiological conditions.…”

“…Osmolarities greater than 300 mOsm are detrimental to mammalian embryos [2, 6, 7, 10, 11]. Porcine zygote medium-3 (PZM-3) [12] at 260–280 mOsm [13, 14] is the most common culture medium used in pigs for 1-cell embryos produced in vitro and in vivo , artificially activated oocytes and cloned embryos produced by nuclear transfer. The osmolarity of PZM-3 is much lower than the physiological osmolarity of 318 to 321 mOsm for pig oviductal fluid [13].…”