Sains Malaysiana 45(4)(2016): 575–580

Developmental Potential of Mouse Single Blastomere Derived from Isolated 2-, 4- and 8- Cell Embryos into Blastocyst and Inner Cell Mass (ICM) Outgrowths

(Potensi Perkembangan Sel Mencit Blastomer Tunggal daripada Pencilan Sel Embrio 2-, 4- dan 8- kepada Blastosista dan Pertumbuhan Luar Jisim Sel Dalaman (ICM))

ASDIANA AMRI, RAMLI BIN ABDULLAH & WAN KHADIJAH WAN EMBONG*

Animal Biotechnology-Embryo Laboratory (ABEL), Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

Received: 21 January 2015/Accepted: 6 October 2015

ABSTRACT

The application of blastomere separation (BS) technique to produce embryonic stem cell (ESC) from inner cell mass (ICM) is not yet perfected due to low cleavage efficiency of single blastomere. Thefore, the aims of this study were to evaluate the developmental potential of single blastomere (SB) derived from embryos at different stages to blastocyst and the potential of ICM outgrowth. The female mice of ICR strain (age: 8-12 weeks; n=47) were superovulated using gonadotrophins and mated with ICR strain males (age: 10-14 weeks; n=20). The 2-cell embryos (n=366) were flushed from the oviduct of the treated females and cultured in vitro before assigned into the following groups: a) 2-cell, b) 4-cell, c) 8-cell and d) control prior blastomere separation. The SBs were cultured in vitro and daily observations were conducted to record the cleavage rates up to Day 5. Developmental rate of 2-cell derived SB (77.28±6.77) was greater than 4-cell (63.70±5.35) and 8-cell (55.73±3.35), corresponding to the results of ICM outgrowth at 2-cell (69.29±4.13), gave higher rate followed by 4-cell (55.73±7.81) and 8-cell (41.85±3.58). Diameter of blastocyst decreased as the SB parent embryo stage increased, with the respective ratio of 5 (diameter of SB blastocyst): 3 (diameter of SB) (2-cell: 92.55±1.59 vs. 56.48±0.40; 4-cell: 78.71±1.37 vs. 44.02±0.49 and 8-cell: 64.13±2.20 vs. 35.68±0.34) as well as total cell number in blastocyst (2-cell: 43.00±1.48; 4-cell: 28.33±1.15; 8-cell: 8.80±0.58). In conclusion, SB at different stages of mouse embryos successfully develop to blastocysts in vitro that can be used as ICM source, which is a prerequisite for establishment of ESC outgrowth.

Keywords: Diameter of blastocyst; embryonic stem cell; inner cell mass isolated single blastomere; mouse embryos

ABSTRAK

Penggunaan aplikasi teknik pemisahan blastomer (BS) untuk menghasilkan sel stem embrio (ESC) daripada jisim sel dalaman (ICM) masih belum sempurna kerana kecekapan belahan blastomer tunggal masih rendah. Tujuan kajian ini adalah untuk menilai potensi perkembangan blastomer tunggal (SB) yang dihasilkan daripada embrio pada pelbagai peringkat blastosista dan potensi pertumbuhan luar ICM. Strain ICR mencit betina (umur: 8-12 minggu; n=47) telah disuperovulasi menggunakan gonadotrofin dan disenyawakan dengan strain ICR jantan (umur:10-14 minggu; n=20). Embrio 2-sel (n=366) dikeluarkan daripada oviduktus betina yang dirawat dan dikultur secara in vitro sebelum diumpukkan ke dalam kumpulan berikut: 2-sel, 4-sel, 8-sel dan kawalan sebelum pengasingan blastomer. SB telah dikultur secara in vitro dan pemerhatian harian telah dijalankan untuk merekod kadar belahan sehingga hari ke-5. Kadar perkembangan diperoleh daripada 2-sel SB (77.28±6.77) adalah lebih besar daripada 4-sel (63.70±5.35) dan 8-sel (55.73±3.35), sama seperti keputusan hasil pertumbuhan luar ICM pada 2-sel (69.29±4.13), memberikan kadar yang lebih tinggi diikuti dengan 4-sel (55.73± 7.81) dan 8-sel (41.85± 3.58). Diameter blastosista menurun apabila peringkat SB induk embrio meningkat dengan nisbah 5 (diameter blastosista SB): 3 (diameter SB) (2-sel: 92.55±1.59 lawan 56.48±0.40; 4-sel: 78.71±1.37 lawan 44,02±0.49 dan 8-sel: 64.13±2.20 lawan 35.68±0.34) dan juga jumlah sel dalam blastosista (2-sel: 43.00±1.48; 4-sel: 28.33±1.15; 8-sel: 8.80±0.58). Kesimpulannya, SB pada pelbagai peringkat embrio mencit berjaya dibangunkan ke peringkat in vitro blastosista yang boleh digunakan sebagai sumber ICM, yang merupakan pra-syarat bagi penubuhan pertumbuhan luar ESC.

Kata kunci: Diameter blastosista; jisim sel dalaman; pencilan blastomer tunggal; sel stem embrionik; embrio mencit

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*Corresponding author; email: wkhadi@um.edu.my