Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation

Abstract

Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 μM) on the percentage of cultured cumulus-oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥ 8-cell embryos (1 μM, P < 0.05; 10 μM, P < 0.01; and 0.1 μM, 10 μM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 μM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 μM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.

AMPK: a master energy regulator for gonadal function

“…In addition, AICAR inhibited cumulus expansion, which normally occurs in response to FSH and/or epidermal growth factor (EGF) during IVM (Harper and Brackett, 1993 ; Lonergan et al, 1996 ; Ritter et al, 2015 ). The results in porcine are supported by those in bovine where supplementation of metformin during embryo in vitro production resulted in AMPK mediated activation of TSC2 (Pikiou et al, 2015 ) and probably a reduction in TOR complex signaling and protein synthesis inhibition. In bovine COCs, metformin blocks meiotic progression at the germinal vesicle stage, activates AMPK, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation.…”

Metformin in Reproductive Biology

“…The cell-to cell contact with trophectoderm was also altered because of an increase in tight junction permeability ( 55 ). In vivo , metformin reduced apoptosis in blastocysts of obese mice ( 56 ) possibly through an increase in NAMPT expression, but induced early bovine embryo arrest ( 57 ). Taken together, these studies demonstrate metformin's important contribution in the cross-talk between somatic cells and oocytes for the normal development of high-quality female germ cells and embryo developmental competence.…”

The effect of nutrition and metabolic status on the development of follicles, oocytes and embryos in ruminants

“…Although there are some differences among species, AMPK appears to be important for the breakdown of the germinal vesicle during nuclear maturation of the oocyte. Recently, Pikiou et al (2013) suggested that activation of AMPK in response to metformin could decrease the ability of bovine oocytes to cleave following in vitro fertilization (Pikiou et al, 2013). Although the role of AMPK in nuclear maturation could be to promote early embryonic development, it is unclear whether AMPK is involved in cytoplasmic maturation of oocytes.…”