C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation

Abstract

Background Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. Methods The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28–32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. Results c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. Conclusions The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Effects of stem cell factor in follicular fluid and granulosa cells on oocyte maturity and clinical pregnancy

“…[25] They observed a positive correlation between SCF expression levels in FF and serum and MII oocyte proportion, which was consistent with the results of our study. We also observed that the number of high-quality embryos, proportion of highquality embryos, and SCF mRNA concentration were significantly higher in the clinical pregnancy group than in the nonclinical pregnancy group, which was consistent with the findings of Tan et al [10] In IF analysis, SCF protein expression levels were higher in the clinical pregnancy group than in the Based on the results of qPCR and IF, it can be concluded that SCF concentration may be an influencing factor in the clinical pregnancy group and pregnancy outcome. We constructed a ROC curve of SCF in GCs combined with serum AMH and found that SCF alone had a good effect on predicting oocyte maturity and clinical pregnancy, although serum AMH is the currently recognized evaluation index.…”

“…Ligand-receptor interactions establish communication between oocytes and GCs, participate in the development and growth of primordial follicles and the appearance of preovulatory dominant follicles, [1,9] and play an important role in early embryonic development and blastocyst formation. [10,11] Mouse and rat ovaries have been used to detect the expression of phosphoinositide 3 kinase/serine-threonine protein XW, LZ, DN, and, AX contributed equally to this work.…”

“…Ligand–receptor interactions establish communication between oocytes and GCs, participate in the development and growth of primordial follicles and the appearance of preovulatory dominant follicles, [ 1 , 9 ] and play an important role in early embryonic development and blastocyst formation. [ 10 , 11 ]…”

GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression

“…The embryo assessment criteria were executed as the previously described [ 18 ]. A good quality embryo should consist of 7–9 blastomeres with a uniform size, and the fragment proportion should be less than 20% at day 3 for human 2PN embryos after fertilization.…”

“…Activated c-kit receptor was associated with multiple kinds of biological events, including proliferation, differentiation, migration and apoptosis, in many types of cells [ 16 , 17 ]. Our previous study reported the expression of c-kit receptor on human embryo and demonstrated that added exogenous SCF to activate c-kit receptor significantly promoted embryo development [ 18 ]. Recently, we found the expression of c-kit receptor on human endometrial stromal cells (ESCs).…”