Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts

Area of Science:

• Reproductive Biology
• Developmental Biology
• Genetics

Background:

• Successful production of embryos via nuclear transfer (NT) using cultured somatic cells is multifactorial.
• Understanding gene expression differences in NT embryos compared to in vitro (IVP) and in vivo embryos is crucial for improving cloning efficiency.

Purpose of the Study:

• To investigate the effects of activation protocols, donor cell cycle status (G(0) or G(1)), and donor cell passage number on mRNA abundance in NT blastocysts.
• To compare the relative abundance (RA) of eight specific mRNAs in NT embryos with IVP and in vivo-derived embryos.

Main Methods:

• Nuclear transfer (NT) was performed using cultured somatic cells.
• Two activation protocols (FBA and AFS) were tested.
• Donor cells at G(0) or G(1) cell cycle stages and different passage numbers (P5/6, P8) were used.
• Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was employed to measure the relative abundance (RA) of eight target mRNAs (DNMT, Mash2, Glut-1, Hsp, Dc II, E-cad, IF, Igf2r).
• Results were compared to in vitro-produced (IVP) and in vivo-derived embryos.

Main Results:

• Heat shock protein 70.1 (Hsp) mRNA was not detected in NT embryos but was present in IVP embryos.
• Interferon tau (IF) transcript levels were significantly increased in NT embryos produced with simultaneous fusion and activation (AFS) compared to fusion before activation (FBA).
• Use of G(0) or G(1) donor cells for NT significantly reduced DNA methyltransferase (DNMT) transcripts and increased mammalian achaete-scute homologue (Mash2) transcripts compared to IVP embryos.
• IF transcript levels were elevated in NT blastocysts derived from G(1) cells compared to IVP and G(0) NT embryos.
• Donor cell passage number affected gene expression, with higher passage numbers (P8) showing increased IF mRNA compared to P5/6 and IVP embryos.
• In vivo-derived embryos showed decreased DNMT and increased Mash2 transcripts compared to in vitro-produced embryos.
• Culture conditions for IVP embryos also influenced gene expression (e.g., Hsp, Mash2, IF).

Conclusions:

• Nuclear transfer (NT) derived embryos exhibit distinct gene expression patterns compared to in vitro-produced (IVP) and in vivo-derived embryos, depending on specific cloning procedures.
• Aberrant expression of genes involved in stress adaptation, trophoblastic function, and DNA methylation was observed in NT embryos during preimplantation development.
• Optimization of activation protocols, donor cell status, and culture conditions is critical for improving the developmental competence of cloned embryos.