Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes

Abstract

Purpose: stem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development.

Methods: using exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos.

Results: addition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing.

Conclusion: SCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Fig. 1

Effect of siRNA-mediated c-Kit downregulation on the development of mouse one-cell embryos. After microinjection, the embryos were cultured in serum-free KSOM medium containing 100 ng/ml SCF. The data are presented as the mean±SEM. ^a,b Values within the same column with different superscripts are significantly different (P < 0.05). Note: Control (116 zygotes, uninjected group), Sham (112 zygotes, vehicle-injected group), c-Kit siRNA (161 zygotes, CGA CCT TTT ATA GGC ACG T target siRNA-injected group). Early Bla: early blastocyst (less than 50% cavity), Bla: blastocyst (greater than 50% cavity), Expanded Bla: expanded blastocyst, Hatching Bla: hatching blastocyst

Fig. 2

Specific reduction of c-Kit mRNA and protein expression by siRNA injection. Quantification of c-Kit mRNA in mouse blastocysts by RT-PCR (a) and real-time RT-PCR (b). c-Kit siRNA or sham (vehicle) was injected into one-cell mouse embryos. Injected and control embryos were cultured in serum-free KSOM medium containing 100 ng/ml SCF. Total RNA isolated from blastocyst stage embryos was reverse transcribed and amplified. c Western blot of c-Kit in extracts from 50 blastocysts in the sham- and c-Kit siRNA-injected groups. d Embryos at each stage were fixed and stained with an anti-c-Kit antibody. Right, lower panel depicts a blastocyst stained with secondary antibody only as a negative control. Sham and c-Kit siRNA groups were analyzed at the same exposure time and more than 10 embryos were used for the fluorescence quantitation

Fig. 3

Effect of c-Kit siRNA on proliferation and apoptosis of blastomeres. a Proliferating blastomeres were detected by an anti-PCNA antibody (green). b Apoptosis of blastomeres was detected by TUNEL assay (TMR-red staining, arrows indicate apoptotic cells). Graphical representation of the results in panels A and B show the percentage of proliferating cells (c; PCNA) and the percentage of apoptotic cells (c; TUNEL) that developed into blastocysts following injection of c-Kit siRNA. The data are presented as the mean±SEM. ^a,b Values within the same column with different superscripts are significantly different (P < 0.05). Sham and c-Kit siRNA groups were analyzed at the same exposure time and more than 10 embryos (Sham; n = 33 and c-Kit siRNA: n = 33) were used for the cell number count

Fig. 4

a Specific reduction of total Akt and p-Akt protein expression by c-Kit siRNA. Blastocyst stage embryos were fixed and stained with an anti-p-Akt and anti-total Akt antibody. Changes in total Akt and p-Akt protein levels were evaluated following injection of sham and c-Kit siRNA into one-cell mouse embryos. Sham and siRNA groups at the same exposure length. b Western blot of p-Akt in extracts from 50 blastocysts in the sham- and c-Kit siRNA-injected groups. c PCR band shows semi-quantitative RT-PCR results performed prior to real-time RT-PCR (Negative: no reverse transcriptase). d Quantification of mRNA for the downstream targets of Akt (mTOR, tuberin, IKK and Bad) in mouse blastocysts by real-time RT-PCR. The data are presented as the mean±SEM. ^a,b Values within the same column with different superscripts are significantly different (P < 0.05)