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.

Keywords: C-kit; ETV5; Embryonic development; IVF; In vitro culture.

Fig. 1

The expression of c-kit in human immature oocytes, 3PN embryos at eight-cell stage (day 3) and blastocysts developed from 3PN embryos (day 6). Immunofluorescence staining showed that c-kit protein was also expressed on the surface of human immature oocytes, the surface of blastomeres of human 3PN embryos (day 3) and the surface of human 3PN blastocysts (day 6). Negative control was stained with second antibody alone

Fig. 2

Comparison of the ETV5 expression in human 3PN embryos between SCF group, SCF + imatinib group and control group. Western Blot (a) and RT-PCR (b) showed that the expression of ETV5 was significantly higher in blastocysts than that in non-blastocysts in all groups. Furthermore, the results showed that the level of ETV5 was significantly higher in SCF group than that in other groups (*P < 0.05, ** P < 0.01, *** P < 0.001)

Fig. 3

Comparison of the ETV5 expression and ERK phosphorylation in mouse 2PN embryos between SCF group, SCF + imatinib group and control group. Western Blot (a) showed an increased ETV5 expression and a higher phosphorylation level of MEK/ERK signal molecule in mouse embryos from SCF group than those from other groups. RT-PCR (b) showed that the expression of ETV5 was significantly higher in blastocysts than that in non-blastocysts in all groups (*P < 0.05, ** P < 0.01)

Fig. 4

Comparison of the ETV5 expression and ERK phosphorylation in mouse 2PN embryos between SCF group, SCF + U0126 group. Western Blot (a) and RT-PCT (b) showed that after inhibiting MEK/ERK signaling activation by U0126, the expression of ETV5 was significantly decreased (*P < 0.05, ** P < 0.01, *** P < 0.001)