Bradley J. Stith

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate.

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis.

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.

Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes.

An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mares serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis.

Stimulation of the intracellular portion of the human insulin receptor by the antidiabetic drug metformin

Our prior work suggested that the antidiabetic metformin must enter the cell to act and that the drug stimulates tyrosine kinase activity. We now report that therapeutic concentrations (approximately 1 microg/mL) of metformin stimulated the tyrosine kinase activity of the intracellular portion of the beta-subunit of the human insulin receptor (IPbetaIRK), the intracellular portion of the epidermal growth factor receptor and pp60-src, but not cAMP-dependent protein kinase. A derivative of metformin unable to lower glucose was ineffective in stimulating IPbetaIRK. Two derivatives more effective than metformin in patients were also more effective than metformin in stimulating IPbetaIRK. Higher levels (10-100 microg/mL) of metformin or methylglyoxyl bis(guanylhydrazone) inhibited the tyrosine kinases, and this inhibition may be responsible for the ability of these two drugs to block cell proliferation.

Nongenomic action of progesterone: activation of Xenopus oocyte phospholipase C through a plasma membrane-associated tyrosine kinase.

Using a plasma membrane-cortex preparation (wherein the nucleus and> 90% of the total cell protein are removed), progesterone stimulated tyrosine kinase activity that stimulated phospholipase C. Although it has been known for over 20 yr that progesterone acts at the plasma membrane of Xenopus oocytes to induce oocyte maturation, this is the first report that progesterone stimulates this tyrosine kinase activity that is associated with the oocyte plasma membrane and cortex. A tyrosine kinase inhibitor (tyrphostin B46) inhibited steroid stimulation of tyrosine kinase and phospholipase C (PLC) activities, but did not block lipase C stimulation by G protein activators. A fusion protein that contains tandem N- and C-terminal SH2 domains of PLCγ also blocked progesterone stimulation of PLC (a fusion protein with the SH2 domain from Shc was ineffective). Lowering the Ca2+ concentration in the medium inhibited progesterone, but not guanosine 5′-O-(3-thiotriphosphate), stimulation of PLC, and the effects of proges...

Insulin and progesterone increase 32PO4-labeling of phospholipids and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

After a 4-6 h induction period, insulin or progesterone induces Xenopus oocytes to enter prophase of meiosis. During the period of induction, both insulin and progesterone induced an increase in 32PO4 labeling of phosphatidylcholine and phosphatidylinositol. Through a mass assay, we found that insulin and progesterone increase inositol 1,4,5-trisphosphate (IP3) at about 15-30 s, 15 min and at about 2-3 h (0.5 GVBD50) after hormone addition. Since IP3 increases were small (from a basal of 66 to 104 nM), the results agree with prior conclusions that progesterone does not induce a large, cytosolic calcium elevation. Insulin is probably acting through the insulin-like growth factor-1 receptor as insulin concentrations greater than about 50 nM are required to increase IP3.

Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG.

Reduced Chrna7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABAA receptor subunits

Decreased expression of CHRNA7, the gene encoding the α7(∗) subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7(∗) receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in the hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout (KO) mice using quantitative Western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in the postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia.

Protein kinase C initially inhibits the induction of meiotic cell division in Xenopus oocytes.

We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and in vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.