5 The activity of cyclin-dependent protein kinase p34cdc2 is regulated by phosphorylation. In this study, we show that the presence of phosphatase inhibitors can have major effects on the levels of phosphorylation and the activities of the kinase extracted from cells. The combination of okadaic acid and sodium vanadate was most effective in protecting p34cdc2 against cellular phosphatases. In the absence of these inhibitors, p34cdc2 was dephosphorylated with an altered activity, indicating that phosphatase activities remained high during extractions. In contrast to when both inhibitors were used, lower activity of the kinase was found when only sodium vanadate was used, whereas higher activity was found in the presence of okadaic acid. Other conventional phosphatase inhibitors such as NaP, NaRS03 and glycero12-phosphate, were not effective in preventing dephosphorylation from p34cdc2 in whole cell lysates.

The Cell Cycle

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLCζ as the molecular trigger for development of a fertilised egg into an embryo.

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.

The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.

/pdf/the-endoplasmic-reticulum-structure-function-and-response-to-46s4vmf7yp.pdf

The endoplasmic reticulum: structure, function and response to cellular signaling

To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

https://www.physiology.org/doi/pdf/10.1152/physrev.1999.79.4.1089

Measurement of Intracellular Calcium

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

Fertilization of the immature, prophase I-arrested mouse oocyte produces multiple Ca2+ transients similar to those of the mature, metaphase II egg; however, the first Ca2+ transient is much lower in amplitude and shorter in duration. In contrast to prophase I-arrested oocytes, maturing oocytes fertilized after germinal vesicle breakdown have first Ca2+ transients similar to those of mature fertilized eggs. Immature, prophase-arrested oocytes release less Ca2+ in response to injection of inositol 1,4,5-trisphosphate (IP3) than eggs. At high concentrations, the sulfhydryl reagent, thimerosal (200 microM), causes Ca2+ oscillations in eggs and produces similar oscillations in oocytes. A lower concentration of thimerosal (25 microM) does not cause Ca2+ oscillations, but does sensitize IP3-induced Ca2+ release in both eggs and oocytes, since IP3-induced Ca2+ release is enhanced in the presence of 25 microM thimerosal. Incubation of oocytes in 25 microM thimerosal before injection of 2.2 microM IP3 causes oocytes to release as much Ca2+ as is released in eggs injected with 2.2 microM IP3. These results indicate that immature mouse oocytes possess intracellular stores of releasable Ca2+ similar in size to Ca2+ stores in eggs; however, these stores are less sensitive to IP3. Development of the IP3-induced Ca2+ release mechanism may be an important component of maturation; at fertilization of the egg, Ca2+ must be elevated to levels sufficient to activate further development and establish a block to polyspermy. Mouse oocytes appear to develop an increased sensitivity to IP3 during the course of oocyte maturation.

/pdf/regulation-of-intracellular-calcium-in-the-mouse-egg-calcium-365mn08gjp.pdf

Regulation of intracellular calcium in the mouse egg: calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation.

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei.

http://www.jbc.org/content/267/25/17624.full.pdf

Douglas Kline

Papers

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

Regulation of intracellular calcium in the mouse egg: calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation.

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei.

Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg.