Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

Abstract

Background: Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions.

Results: [(125)I]Insulin binding (K(d) = 54 ± 6 nM) at the oocyte plasma membrane activates membrane serine protease(s), followed by the loss of low affinity ouabain binding sites, with a concomitant 3-4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na(+)/Ca2(+) exchange, increased endocytosis, decreased Na(+) conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi). Hyperpolarization is largely due to Na(+)-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na(+)/glucose cotransport. The Na(+) sym- and antiporter systems are driven by the Na(+) free energy gradient generated by Na(+)/K(+)-ATPase. Shifts in α and/or β Na(+)-pump subunits to caveolar (lipid raft) membrane regions may activate Na/K-ATPase and contribute to the Na(+) free energy gradient and the increase in both Na(+)/glucose co-transport and pHi.

Conclusions: Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na(+) channels and mobilizes fully functional Na(+)-pumps, generating a Na(+) free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

Figure 1

Comparison of mammalian and Xenopus laevis: insulin and insulin receptor structures. The upper panel compares the amino acid sequences of Porcine (Accession #P01315) and X. laevis (Accession #P12706) insulin-1. The lower panel compares the alignment of the α-subunits of the X. laevis insulin receptor (Accession #Q9PVZ4; sequence 38–754) and human insulin receptor (Accession #P06213; sequence 28–758). Data are from the unprocessed sequences as listed in the Swiss Protein Knowledgebase (http://www.uniprot.org).

Figure 2

Scatchard type plot of [¹²⁵I]insulin binding to the plasma-vitelline membrane complex of denuded R. pipiens oocytes. Intact, denuded prophase oocytes were incubated in Ringer’s solution containing [¹²⁵I]insulin for 15 min at 20–22°C, the oocytes removed, rinsed with Ringer’s solution and the membranes removed and counted as described in Methods. The [¹²⁵I]insulin binding data were corrected by subtracting the constant binding fraction.

Figure 3

A comparison of the time course of insulin effects on ⁴⁵Ca²⁺/Na⁺ exchange and fluid phase uptake ([¹⁴C]insulin) by isolated denuded R. pipiens oocytes at 20–22°C, plotted as rate of change per unit time (dy/dt). The values are in pmols/oocyte for ⁴⁵Ca²⁺ and [¹⁴C]inulin, respectively. Each point presents a pooled sample of 5–10 sibling oocytes and the values shown are typical for 4 experiments in late winter-early spring. For details see Methods.

Figure 4

A comparison of the time course of insulin effects on membrane hyperpolarization (E_mV), intracellular pH (pH_i) and [¹⁴C]2-deoxy-D-glucose uptake by isolated denuded R. pipiens oocytes at 20–22°C. The graphs represent changes in various parameters relative to untreated oocytes. Each point presents a pooled sample of 5–10 sibling oocytes and is typical for 4 experiments in late winter-early spring Rana. For details see Table 2 and Methods.

Figure 5

Effect of 5 μM insulin on serine protease activity in isolated, intact plasma-vitelline membranes from prophase-arrested Rana pipiens oocytes (● - ●). p-Tosyl-l-arginine methyl ester (TAME) was used as a serine protease-specific substrate and the serine protease inhibitor phenylmethyl-sulfonyl-flouride (PMSF) was added 15 min prior to insulin (▲ - ▲). The control is represented by (○ - ○). Values shown are from a typical experiment using sibling oocytes. The mean ± SD values for serine protease activity for oocytes from four R. pipiens females at 30 min was 26.4 ± 2.57 absorbancy units for 5 μM insulin, 8.23 ± 1.27 absorbancy units for 0 insulin (controls) and 0 absorbancy units for oocytes treated with both 5 μM insulin and PMSF. For details see methods.

Figure 6

A comparison of the topology of four plasma membrane proteins associated with the initial insulin response. TMHMM projections were generated using the server at the Center for Bological Sequence Analysis, Technical University of Denmark DTU. From top to bottom the proteins are: the β-subunit of the insulin receptor (Accession #Q9PVZ4), the membrane serine protease 8 (Prostasin, Accession #Q16651), the protease-activated receptor (PAR2, Accession #Q5U791) and the amiloride-sensitive Na⁺ channel subunit β (Accession #P51169). The amino acid sequences are those published in the Swiss Protein Knowledgebase (http://www.uniprot.org).

Figure 7

A comparison of the topology of four putative plasma membrane enzymes that respond to exogenous insulin. TMHMM projections were generated as described in Figure 6. From top to bottom the enzymes are: the catalytic α-subunit of Na⁺/K⁺-ATPase (Accession #Q92123), Na⁺/H⁺-exchanger (Accession #P70009), the Na⁺/Ca²⁺-exchanger (Accession #Q91849) and Na⁺ -glucose cotransporter ((SGLT2, Accession #P31639). The amino acid sequences are those published in the Swiss Protein Knowledgebase (http://www.uniprot.org).