Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos.

Abstract

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0-3, Days 4-7, and Days 0-7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0-7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.