Estrogen related receptor β (
Mouse oocytes and embryos were obtained from superovulated mice. The expression of
Expression of
Treatment of CPP-ESRRB during cultivation could increase embryos' expression of
Various techniques have been introduced to satisfy the demand for suitable mammalian oocyte and embryo culture systems. For example, a co-culture system with somatic cells such as cumulus cells could increase the fertilization and blastocyst formation rate [
In a previous study, recombinant protein conjugated with cell penetrating peptide (CPP) using 7X-arginine (R7) was translocated into human mesenchymal stem cells or ES cells efficiently with biological activity [
Four- to 5-week-old female and 8- to 10-week-old male B6D2F1 mice (Samtako, Seoul, Korea) were kept under controlled light and temperature conditions with free access to water and food under 12 hours light and 12 hours dark. All animal experiments were approved by the Animal Care and Use Committee of CHA University. The female mice were superovulated via intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG, Sigma-Aldrich, St. Louis, MO, USA), followed by injection with 5 IU hCG after 48 hours (Sigma-Aldrich). The female mice were then mated with the male mice and their vaginal plugs were checked at 12 hours after mating.
Two-cell embryos were collected from the oviduct at 46 hours after hCG injection and cultured in potassium simple optimization medium amino acid (KSOM, Millipore, Billerica, MA, USA) under mineral oil (Sage, Trumbull, CT, USA) in an incubator at 37℃ and 5% CO2 in air. Then, 8-cell embryos derived
Germinal vesicle-stage (GV) oocytes were collected from the ovaries at 48 hours after PMSG injection. Metaphase II (MII)-stage oocytes were collected after
In order to link
The number of ICM and trophectoderm (TE) cells in the blastocysts derived from embryo experiments were counted by differential staining using two chromatin-specific fluorochromes with different fluorescent spectra: propidium iodide (Sigma-Aldrich), which enters only cells with damaged membranes, and bisbenzimide (Hoechst 33342, Sigma-Aldrich), which passes through both damaged and intact membranes. At day 5, the zona pellucida (ZP) of the collected blastocysts was removed by brief exposure to acid Tyrode's solution (Sigma-Aldrich). The ZP-free embryos were exposed to a 1:5 dilution of whole rabbit anti-mouse serum (Sigma-Aldrich) for 1 hour and washed three times with DPBS (Hyclone) containing 0.1% BSA for 5 minutes each. Then, the embryos were placed into a 1:10 dilution of guinea pig complement (Sigma-Aldrich) for 1 hour. Propidium iodide and bisbenzimide were added to the complement solution to a final concentration of 10 µg/mL and 10 µg/mL, respectively. Then, they were briefly washed in DPBS (Hyclone) containing 0.1% BSA (Sigma-Aldrich) and were mounted between the slide and coverslip and examined under ultraviolet light using an Axio Imager A2 microscope (Carl Zeiss, Jena, Germany) fitted for epifluorescence. The nuclei of the ICM were labeled with bisbenzimide and appeared blue, while the nuclei of the TE cells appeared pink.
Total cellular RNA was extracted from 15 GV oocytes and embryos using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The first strand of cDNA was synthesized using a PrimeScript 1st strand cDNA Synthesis kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. Reverse transcription polymerase chain reaction (PCR) was conducted using G-Tag (Cosmogene Tech, Seoul, Korea) and specific primer sets (
Western blot analysis was performed to characterize the CPP-ESRRB and confirm delivery of CPP-ESRRB protein into the embryos. Each sample containing 60 embryos, which were separated by 12% SDS-PAGE; after that, they were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) stored for 90 minutes. Blocking was performed in DPBS containing 3% BSA and 0.1% Tween-20 (Sigma-Aldrich) for 2 hours at room temperature. The membranes were incubated in blocking solution with anti-ESRRB (Santa Cruz Biotech-nology, Santa Cruz, CA, USA, 1:200) or anti-His-tag (Sigma-Aldrich, 1:700) overnight, and then each sample was evaluated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted to 1:2,000 with DPBS containing 0.1% Tween-20 (Sigma-Aldrich) for 1 hour. Immunoreactivity was detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and developed on Hyperfilm ECL X-ray film (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Embryos cultured with or without CPP-ESRRB for 24 hours and 48 hours were fixed in 4% paraformaldehyde with 0.1% BSA (Sigma-Aldrich) at 4℃ for 30 minutes, and washed twice with DPBS (Hyclone) containing 0.1% BSA. After that, the embryos were permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 20 minutes and blocked in DPBS (Hyclone) with 3% BSA and 0.05% Tween-20 (Sigma-Aldrich) for 2 hours at 4℃. They were then stained overnight at 4℃ with primary antibodies anti-His-taq (1:100, mouse polyclonal, Sigma-Aldrich) or anti-ESRRB (1:100, rabbit polyclonal, H70, Santa Cruz), followed by 2 hours incubation at 4℃ with Alexa Fluor 555-labeled goat anti-rabbit, 488-labeled goat anti-mouse secondary antibodies (Molecular Probes, Eugene, OR, USA) diluted to 1:200 with 3% BSA in DPBS (Hyclone). All of the samples were counterstained with 1 µg/mL 4, 6-diamidino 2-phenyindiol (DAPI, Sigma Aldrich) diluted to 1:1000 with DPBS for 15 minutes at room temperature. All sample images were captured with an LSM 510 META confocal laser-scanning microscope (Carl Zeiss). The stained cell images were reconstructed using LSM 510 META software.
Unless otherwise specified, data are expressed as means±SE. The data were analyzed for statistical significance with the Student's
To verify the cell numbers of blastocysts derived from the groups cultured
mRNA of
The expression of CPP-ESRRB protein was induced by IPTG and was purified using His-tag at the C-terminal. The purified CPP-ESRRB protein was characterized through electrophoresis using SDS-PAGE gel and staining with Coomassie Brilliant blue R250 (
To observe the delivery of CPP-ESRRB recombinant protein into embryos during
As shown in the
After the embryos were cultured in KSOM with or without 2 µg/mL of CPP-ESRRB for 48 hours,
According to recent reports, there are three mRNA alternative splicing isoforms of human
In the present study, we used a CPP protein delivery system known as protein transduction domains or membrane transduction peptides. They have been thought to be a useful tool due to their ability to translocate across cellular membranes [
In conclusion, recombinant CPP-ESRRB could be easily delivered into embryos by CPP and may increase the level of
This research was partly supported by a grant from the Priority Research Centers Program (2009-0093821) and the BK21 PLUS Program (22A20130012640) through the NRF funded by the Ministry of Education, Korea.
No potential conflict of interest relevant to this article was reported.
Comparison of cell numbers and expression of
Expression patterns of estrogen related receptor β (
Characterization of recombinant cell-penetrating peptide-conjugated estrogen-related receptor β (CPP-ESRRB). (A) Confirmation of pure isolation of CPP-ESRRB using staining with Coomassie Brilliant blue. (B) Confirmation of pure isolation of CPP-ESRRB by Western blot analysis using specific antibody against ESRRB.
Localization of recombinant cell-penetrating peptide-conjugated estrogen-related receptor β (CPP-ESRRB) in mouse embryos according to treatment time. (A) Protein transduction of 7R-tagged ESRRB protein into embryos was detected by immunocytochemistry. CPP-ESRRB protein (2 µg/mL) was added to the culture medium for the embryos. The embryos were fixed at 24 hours (morulas) and 48 hours (blastocysts) after treatment and then analyzed by immunostaining using 6× His-taq and ESRRB antibodies. The nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI), and the images were merged. (B) The relative intensity was increased in the CPP-ESRRB-treated groups compared to the non-treated group. (C) Delivery of CPP-ESRRB protein into embryos was confirmed and analyzed by western blot analysis. The scale bars are 5 µm. *Significantly different (
Comparison of cell numbers and expression of Oct4 mRNA between blastocysts treated with/without cell-penetrating peptide-conjugated estrogen-related receptor β (CPP-ESRRB). (A) Blastulation rates in the CPP-ESRRB-treated group were compared with those from the non-treated one at different times after hCG injection. (B) The numbers of inner cell mass (ICM), trophectoderm (TE), and total cells in the blastocysts obtained from the CPP-ESRRB-treated group were compared with those from the non-treated one. (C, D) The expression levels of
Primer sequences for real-time polymerase chain reaction
F, forward; R, reverse.