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.
Highlights
Owing to their genetic, anatomic, and physiologic similarities with humans [1], porcine animal models are commonly used for studying human diseases, developing novel medical treatment therapies, and xenotransplantation
Effects of human recombinant G-CSF (hrG-CSF) treatment during in vitro culture on porcine pre-implantation embryos almost no expression of CSF3 and its receptor (CSF3R) was detected in cumulus cells and parthenogenetic blastocysts expressed neither CSF3 nor its receptor (Fig 1)
To identify the optimal concentration of hrG-CSF for the embryonic development of porcine parthenotes and in vitro fertilization (IVF)-derived embryos, embryos were cultured in in vitro culture (IVC) medium containing 0, 10, 50, and 100 ng/mL hrG-CSF as previously described [38] for 168 h (7 days)
Summary
Anatomic, and physiologic similarities with humans [1], porcine animal models are commonly used for studying human diseases, developing novel medical treatment therapies, and xenotransplantation. Despite extensive research in previous decades, conditions for the production of in vitro porcine embryos remain suboptimal [3]. The investigation of maternally-derived factors could aid in vitro production methods. Pre-implantation embryos developed from the zygote to blastocyst stage must traverse the maternal oviduct to the uterus. During this process, numerous maternal growth factors, cytokines, and nutrients provide essential conditions for embryonic development [4,5,6,7]. In previous studies, these maternal factors have been shown to affect embryonic development, blastocyst formation rates, blastocyst cell number, metabolism, and apoptosis [4,5,6,7]
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