The improvement of porcine oocyte in vitro maturation systems through antioxidant and lipid metabolism upregulation

Abstract

In vitro maturation (IVM) is one of the routinely used systems for the in vitro production of embryos in pigs. It offers mature oocytes from immature status and then develops to blastocyst stage under in vitro condition. However, the maturation rate and the developmental competence of embryos derived from IVM oocytes are significantly lower than those of oocytes matured in vivo. In this study, I attempted to investigate the oocyte maturation and their subsequent embryonic development were influenced by antioxidative and lipid metabolic functions. Firstly, to determine the antioxidant property of spermine on IVM of porcine oocytes and their embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). As a result, treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. Nevertheless, spermine treatment groups showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control. Furthermore, 10 µM of spermine supported significantly higher blastocyst formation rates after PA and SCNT than the control group. Therefore, it can be concluded that 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression. Knockout serum replacement (KSR) is composed of small organic molecules, trace elements, and three proteins, namely insulin, transferrin, and lipid-rich albumin, without any undefined growth factors or differentiation-promoting factors. This study was conducted to examine the effects of KSR on oocyte maturation and embryonic development after PA in pigs. Although the cumulus cell expansion index was significant lower in 5% and 10% KSR compare to the control and 10% porcine follicular fluid (pFF), the 5% KSR supplementation was significantly increased the mRNA expression of Ptgs1, Has2 and Tnfaip6 than control group. Moreover, the mRNA expression of GDF9, BMP15 and Cdc2 were increased in 5% KSR, in line with these results, the protein levels of GDF9 and BMP15 were also upregulated in 5% KSR and 10% pFF. In case of the Bax/Bcl2 ratio, it was decreased in 5% KSR and 10% pFF both of oocytes and cumulus cells, in line with ROS, and GSH results in oocytes. In addition, rates of oocyte maturation and blastocyst formation after PA were significantly higher in the 5% KSR supplemented group than control group and more similar to those of the 10% pFF supplemented group. Finally, I conformed that 5% KSR has antioxidant property which played a crucial role in the acquisition of oocyte development and subsequent embryonic development. Secondly, I analyzed the effect of lipid metabolism during porcine oocyte maturation by KSR and melatonin treatment. To investigate the mechanism of KSR during IVM, I focused on lipid metabolism and carried out experiments. In this study, I evaluated (i) the expression of lipid metabolism genes in cumulus cells and oocytes, (ii) measure the fluorescence staining intensity of BODIPY-LD, BODIPY-FA and BODIPY-ATP in oocytes and (iii) determine the influence of 5% KSR and 10% pFF on embryo development after SCNT and in vitro fertilization (IVF). Rates of blastocyst formation after SCNT and IVF were significantly higher in the 5% KSR supplemented group than in the control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of BODIPY-LD, BODIPY-FA and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which were significantly higher than the control. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In summary, 5% KSR increased blastocyst formation rate after SCNT and IVF with providing an essential energy by upregulation of lipid metabolism. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM. The results of melatonin treatment as another supplementation showed that it significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI-58, HSL and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid β-oxidation (CPT1a, CPT1b, CPT2 and ACADS) and mitochondrial biogenesis (PGC-1α, TFAM and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria and ATP. Moreover, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, β-oxidation and mitochondrial biogenesis-related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development. In addition, melatonin treatment increased SCNT efficiency for in vitro development, and one of 3 recipients was pregnant by melatonin treatment. In conclusion, I demonstrated that supplementation with exogenous antioxidants such as spermine and KSR, which effectively contributed to oocyte maturation and embryonic development by increasing the intracellular GSH levels, and decreasing the ROS levels. In addition, KSR and melatonin upregulated lipid metabolism in porcine oocytes thus provided an essential energy source to promote and improve oocyte quality and subsequent embryo development. It is feasible that upregulation of lipid metabolism and antioxidation are indispensable mechanisms for improving porcine oocyte in vitro maturation systems.