67 SUPPLEMENTATION WITH EPIDERMAL GROWTH FACTOR AND GLIAL CELL-DERIVED NEUROTROPIC FACTOR DURING PORCINE OOCYTE MATURATION STIMULATES BLASTOCYST HATCHING RATE AND QUALITY

Abstract

The growth factors glial cell-derived neurotropic factor (GDNF) and epidermal growth factor (EGF) have previously been shown to elicit a functional effect on oocyte competence and embryo development; however, their effects on embryo quality remain elusive. The objective of the present study was to investigate whether supplementation with EGF, GDNF, or a combination of the 2 growth factors during in vitro porcine oocyte maturation has a beneficial effect on the rate of embryonic development, cell number and relative expression of genes related to apoptosis (anti-apoptotic: BCLX; pro-apoptotic: BAK, CASPASE), cellular stress (HSP70), mitochondrial function (TFAM) and pluripotency (TERT). Cumulus-oocyte complexes (COC) aspirated from slaughterhouse ovaries were matured for 42 h in maturation medium supplemented with either 50 μg mL^–1 of EGF (n = 3398), 50 μg mL^–1 of GDNF (n = 1870), or a combination of both growth factors [EGF (50 μg mL^–1) + GDNF (50 μg mL^–1); EGF+GDNF-group; n = 1993). Subsequently, the oocytes were inseminated, the cleavage rate scored and the resultant embryos cultured in vitro for 7 days. The experiments were replicated a total of 7 times and zona-enclosed blastocysts were collected for Hoechst staining (cell counting) and for gene expression analyses. For gene expression analyses, total RNA was isolated from pools of embryos (n = 10), reverse transcribed into cDNA and subjected to comparative real-time PCR using the delta-delta Ct method (2^–ΔΔCT) and statistical method with a significance level of P < 0.05. The blastocyst rate was significantly lowest in the GDNF group (P < 0.05), whereas the hatched blastocyst rate (P < 0.05) was highest in the EGF+GDNF-group (Table 1). The GDNF-group displayed 2-fold up-regulation of BAK compared with the EGF group (P < 0.05), 1.75-fold up-regulation of TFAM compared with the EGF+GDNF group (P < 0.05) and 2.4-fold up-regulation of CASPASE compared with the EGF+GDNF group (P < 0.05). In contrast, an 11-fold decrease of TERT compared with the EGF+GDNF and EGF group was observed (P < 0.05). The EGF+GDNF-group displayed 1.16 and 1.56-fold up-regulation of BCLX compared with the EGF and GDNF (P < 0.05) groups, respectively and a 1.1-fold up-regulation of TERT compared with the EGF group; in contrast, there was a 1.35 and 1.25 fold down-regulation of CASPASE and TFAM compared with the EGF group, respectively. The EGF-group displayed the lowest (P < 0.05) expression level of BAK. In conclusion, compared among treatments, supplementation with a combination of EGF and GDNF during porcine in vitro oocyte maturation resulted in an increased rate of hatched blastocysts and increased expression of anti-apoptotic (BCLX) and pluripotency related (TERT) genes, whereas it resulted in decreased expression of the pro-apoptotic gene (CASPASE). The present study may help to improve the culture conditions, which are important for optimizing blastocyst rates and quality.

Table 1.  Effect of using growth factor in in vitro maturation on the cleavage rate and blastocyst yield (mean ± standard error of the means) in vitro