Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism

Abstract

A follicular spike in cyclic AMP (cAMP) and its subsequent degradation to AMP promotes oocyte maturation and ovulation. In vitro matured (IVM) oocytes do not receive the cAMP increase that occurs in vivo, and artificial elevation of cAMP in IVM cumulus-oocyte complexes improves oocyte developmental potential. This study examined whether mouse oocytes can use the cAMP degradation product AMP to generate ATP via the adenosine salvage pathway, and examined whether pharmacological elevation of cAMP in IVM cumulus-oocyte complexes alters ATP levels. Oocytes cultured with isotopic ¹³C₅-AMP dose-dependently produced ¹³C₅-ATP, however total cellular ATP remained constant. Pharmacological elevation of cAMP using forskolin and IBMX prior to IVM decreased oocyte ATP and ATP:ADP ratio, and promoted activity of the energy regulator AMPK. Conversely, cumulus cells exhibited higher ATP and no change in AMPK. Culture of oocytes without their cumulus cells or inhibition of their gap-junctional communication yielded lower oocyte ¹³C₅-ATP, indicating that cumulus cells facilitate ATP production via the adenosine salvage pathway. In conclusion, this study demonstrates that mouse oocytes can generate ATP from AMP via the adenosine salvage pathway, and cAMP elevation alters adenine nucleotide metabolism and may provide AMP for energy production via the adenosine salvage pathway during the energetically demanding process of meiotic maturation.

Figure 1

Cellular adenosine metabolism in relation to cAMP-elevating pre-IVM treatment. COC cAMP increases during the peri-ovular period and through pharmacological elevation during pre-IVM. Cyclic AMP is generated by adenylate cyclase (AC) from its substrate ATP and is hydrolysed to AMP by phosphodiesterases (PDE). AMP can be recycled to ATP via the adenosine salvage pathway. The energy sensing enzyme AMP-activated protein kinase (AMPK) is activated by shifts in ATP:AMP and ATP:ADP ratios. CK, creatine kinase; AK, adenylate kinase; Cr, creatine; PCr, phosphocreatine; IBMX, 3-isobutyl-1-methylxanthine; IVM, oocyte in vitro maturation.

Figure 2

Oocytes utilise AMP for ATP production via the adenosine salvage pathway. Iodoacetamide (IAC) was used to inhibit creatine kinase activity in order to perturb the interconversion of ADP and ATP. COCs (A) or denuded oocytes (B) were cultured for 3 h ± ¹³C₅-AMP and then intra-oocyte ¹³C₅-AMP, ¹³C₅-ADP and ¹³C₅-ATP were measured by LC-MS/MS (n = 4 biological replicates). COCs were cultured with ¹³C₅-AMP ± the gap-junction uncoupler carbenoxolone (CBX) for 3 h and intra-oocyte ¹³C₅-ATP was measured (n = 4 biological replicates) (C). COCs were cultured for 3 h with increasing concentrations of ¹³C₅-AMP and the peak areas of intra-oocyte ¹³C₅-ATP and endogenous ATP were measured (n = 1 biological replicate) (D). COCs were cultured for 3 h ± ¹³C₅-AMP (1 mM) and intra-oocyte ¹³C₅-ATP was measured (n = 4 biological replicates) (E). ns, not significantly different (P ˃ 0.05, t-test), ^#Not detected; data are mean ± SEM.

Figure 3

Elevated COC cAMP alters intra-oocyte adenine nucleotide levels throughout oocyte maturation. COCs were either not exposed (control, 0 h pre-IVM) or were exposed to 2 h of pre-IVM with FSK + IBMX prior to IVM culture in the presence of FSH for up to 16 h. Cumulus cells were then removed and intra-oocyte ATP (A), ADP (B), and the ATP:ADP ratio (C) were measured (mean ± SEM). T, treatment; D, oocyte culture duration; *Significantly different (P < 0.05, two-way ANOVA); ns, not significantly different. Oocyte meiotic maturation was assessed after 2, 3, 4 and 16 hours of culture (D). *Significantly different to control (P < 0.05, χ²). N = 3 biological replicates.

Figure 4

Elevated COC cAMP alters intra-oocyte AMPK production. COCs were either untreated (control) or pre-treated for 2 h (pre-IVM) with FSK + IBMX, then cultured without treatments in the presence of FSH for 2 h or 16 h. Cumulus cells were then removed and intra-oocyte phosphorylated (pAMPK) and total (tAMPK) AMPK were measured at 2 h (A,C) or 16 h (B,D). N = 3 biological replicates, data are mean ± SEM. *Significantly different (P < 0.05, t-test).

Figure 5

Elevated COC cAMP alters adenine nucleotide levels. COCs were either untreated (control) or pre-treated for 2 h (pre-IVM) with FSK + IBMX, then cultured without treatments in the presence of FSH for 3 h. COC ATP (A), ADP (B), AMP (C) were measured. COC ATP:ADP (D), ATP:AMP (E) ratios and energy charge (F) were calculated. N = 4 biological replicates, data are mean ± SEM. T, treatment; D, oocyte culture duration; *Significantly different (P < 0.05, two-way ANOVA); ns, not significantly different.

Figure 6

Elevated COC cAMP does not impact COC AMPK production and activity. COCs were either untreated (control) or pre-treated for 2 h (pre-IVM) with FSK + IBMX, then cultured without treatments in the presence of FSH for 2 h or 16 h. COC phosphorylated (pAMPK) and total (tAMPK) AMPK levels were measured at 2 h (A,C) or 16 h (B,D). N = 3 biological replicates, data are mean ± SEM. Data within graphs were not significantly different (P ˃0.05, t-test).

Figure 7

Hypothesised model of the impact of cAMP modulation in the cumulus-oocyte complex on oocyte energy production. Oocyte meiotic resumption and pharmacological cAMP upregulation via pre-IVM generate AMP from cAMP in cumulus cells which can be used via the adenosine salvage pathway to regenerate ATP in the oocyte. Upregulation of cAMP in the cumulus-oocyte complex leads to a decrease in intra-oocyte ATP, decreasing the ATP:ADP ratio and upregulating AMPK protein expression to restrain energy depletion. Pre-IVM also increases COC ATP by stimulating cumulus cell glycolysis. TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AMPK, AMP-activated protein kinase.