Mammalian Eggs Research Articles

Development of new immunocontraceptives-industrial perspective.

Contraception has been practiced for thousands of years. Nevertheless, it took until the late 1950s and early 1960s before a major breakthrough in contraceptive technology was achieved by the introduction of oral hormonal contraceptives. However, we have not succeeded in the development of non-hormonal contraceptive that is comparable to the pill regarding its efficacy, safety, and acceptance. The immunological interference with the complex fertilization process is a very attractive target in this respect, whereby the zona pellucida, a non-cellular surrounding of all mammalian eggs, represents a potentially ideal target. Another interesting target are sperm: either for the development of a female contraceptive or for a male contraceptive, although the latter approach does not look very promising so far. In conclusion, given the enormous impact on mankind of a growing world population and given the very individual needs for contraceptive methods of different women in one and the same country and in different cultures we should make widely available a whole set of suitable, adjusted methods of fertility control and this includes the search for an effective method of male fertility control.

Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice.

Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that plays important roles during early development. The mouse egg zona pellucida is constructed of three glycoproteins, called mZP1, mZP2, and mZP3. The gene encoding mZP3 is expressed only by growing oocytes during a 2- to 3-week period of oogenesis. Here, the mZP3 gene was disrupted by targeted mutagenesis using homologous recombination in mouse embryonic stem cells. Viable female mice homozygous for the mutated mZP3 allele (mZP3-/-) were obtained. These mice are indistinguishable in appearance from wild-type (mZP3+/+) and heterozygous (mZP3+/-) littermates. However, although ovaries of juvenile and adult mZP3-/- females possess growing and fully grown oocytes, the oocytes completely lack a zona pellucida. Consistent with this observation, eggs recovered from oviducts of superovulated, adult mZP3-/- females also lack a zona pellucida. Thus far, mZP3-/- females mated with wild-type males have failed to become pregnant.

Birth of normal mice after removal of the supernumerary male pronucleus from polyspermic zygotes

Each year, world wide, tens of thousands of zygotes derived from the in-vitro insemination of human oocytes undergo polyspermic fertilization. These embryos must presently be discarded because it has never been demonstrated in any mammal that polyspermic zygotes can develop normally to term after removal of the supernumerary male pronucleus. Our study was undertaken to test the developmental potential of polyspermic zygotes corrected by micromanipulation. Mouse oocytes were inseminated zona-free, and polyspermic zygotes were manipulated so as to remove one of the two male pronuclei. Surviving embryos were then observed for further development in vitro and after transfer into pseudopregnant females. Of 58 zygotes manipulated, 18 developed to the blastocyst stage and were transferred. Five animals (two male and three females) were born. The agouti coat colour marker confirmed the genotypes of the gametes. All five animals developed to normal-appearing adults, and all five produced at least 10 normal offspring. One adult founder animal was karyotyped and found to have a normal chromosome complement. These results demonstrate for the first time that a mammalian egg that has undergone polyspermic fertilization can develop normally after restoration of the diploid state by micromanipulation. Accordingly, the results provide impetus for attempting to rescue polyspermic human embryos.

A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes.

Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins (1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca(2+)-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.

EARLY STEPS OF SPERM–EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95 kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, in in vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.

Calcium oscillations in mammalian eggs triggered by a soluble sperm protein.

At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo. It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated. Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion. Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs. Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head. Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes. This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

Soluble sperm factors and Ca2+ release in eggs at fertilization.

All eggs are activated at fertilization by an increase in intracellular free Ca2+. How the spermatozoa triggers the release of intracellular Ca2+ has not been established in any species. One hypothesis is that spermatozoa introduce a Ca(2+)-releasing factor into the cytoplasm after gamete membrane fusion. It has been suggested that spermatozoa may introduce Ca2+ itself, or a small molecular weight factor that triggers the release of Ca2+ from intracellular stores. However, these suggestions are not consistent with a number of experiments in vertebrate eggs. Here, I present the argument that spermatozoa cause intracellular Ca2+ oscillations in mammalian eggs by introducing a specific protein called an oscillogen. It is suggested that it does not mediate its effects by causing increased inositol, 1,4,5-trisphosphate (InsP3) production, but instead directly affects the ability of the stores to undergo Ca(2+)-induced Ca2+ release. The sperm oscillogen hypothesis may help explain the patterns of intracellular Ca2+ waves that occur during fertilization in hamster eggs, as well as the ability of an injection of live spermatozoa to activate development in human eggs.

11 Cytoskeleton in Teleost Eggs and Early Embryos: Contributions to Cytoarchitecture and Motile Events

Successful interaction between gametes initiates a cascade of pathways that lead to cleavage, cellular differentiation, morphogenesis, and eventual development of an adult organism. Shortly after fertilization of the teleost egg, the egg cortex undergoes a predictable sequence of structural changes in response to intracellular signals. Early transformation of the cortex includes formation of the fertilization cone and second polar body, sperm entry into the egg, cortical granule exocytosis, and surface membrane retrieval by endocytosis. Subsequently, cytoplasm moves toward the animal pole where it accumulates in preparation for cell division and cellular rearrangement. By analogy to other cell systems that show secretion, mitosis, intracellular cytoplasmic flow, and cell migration, it is predicted that similar events in eggs are mediated by action of the cytoskeleton. Most of the knowledge about the egg cytoskeleton has been obtained from analyses of sea urchin, amphibian, and mammalian eggs. By comparison, the composition, organization, and function of the egg cytoskeleton in teleost fishes have received considerably less attention. The eggs of teleost fish have long provided favorable material for the study of vertebrate development. Unlike mammalian and amphibian eggs, teleost eggs can be obtained in large numbers, are optically transparent, and are easily accessible for experimental manipulation. Furthermore, teleosts now also offer the possibility of employing genetic and molecular approaches to investigate the role of the cytoskeleton in early development. This chapter surveys the teleost egg cytoskeleton and evaluates its proposed role(s) in events of fertilization, ooplasmic segregation, and gastrulation.

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida binding characteristics.

AQN-1 and AQN-3 form part of the complement of surface-associated lectins which coat the plasma membrane overlying the acrosomal cap of in vitro capacitated boar spermatozoa. They belong to the spermadhesin protein family and have binding affinity for glycoconjugates of the zona pellucida, the extracellular investment surrounding mammalian eggs. The oligosaccharide and zona pellucida binding characteristics of spermadhesins AQN-1 and AQN-3 were investigated using a solid-phase assay and biotinylated glycoprotein ligands. Both sperm proteins bind glycoproteins containing Gal beta (1-4)-GlcNAc and Gal beta-(1-3)-GalNAc oligosaccharide sequences with dissociation constants (Kd) of 0.08 to 0.8 microM, and to zona pellucida glycoproteins with Kd = 0.15-0.25 microM. However, 5-N-acetyl neuraminic acid alpha (2-3/6)-linked to the galactose residue decreases the affinity of glycosylated ligands to AQN-1 three-fold, although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glycoproteins with either a linear or tri- and tetraantennary carbohydrates than to those containing diantennary N-acetyllactosamine structures. The similar but distinct oligosaccharide recognition capabilities of spermadhesins AQN-1 and AQN-3 (this work) and AWN-1 (Dostálová, Z, Calvete, J.J., Sanz, L., and Töpfer-Petersen, E. (1995) Eur. J. Biochem. 230, 329-336) suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying similar although distinct carbohydrate-recognition abilities.

The cortical reaction and development of activation competence in mammalian oocytes.

Blocks to polyspermic fertilization are necessary to prevent the incorporation of two sperm nuclei into a zygote's genome, which would result in abnormal development. Many mammalian eggs utilize both an extracellular zona pellucida block to polyspermy and a plasma membrane block. Although little is known about the plasma membrane block in mammals, fertilization results in zona glycoprotein modifications caused by enzymes released by the egg and its cortical granules (CG). This article reviews other recent investigations demonstrating that the oocyte's ability to cause CG release and the block to polyspermy develops near the time of ovulation. The development of normal "activation competence' is likely to involve preovulatory changes in the oocyte's ability to signal the release of intracellular calcium as well as to respond to this calcium increase, resulting in CG exocytosis. Because normal activation competence appears to have a brief temporal window after oocyte meiotic maturation is resumed and since the oocytes are collected at various stages in assisted reproductive procedures, these studies are relevant to optimizing clinical success.

A role for intermediate filaments in the establishment of the primitive epithelia during mammalian embryogenesis

Investigations of the cytoskeleton in mammalian eggs and embryos have revealed the existence of an unusual array of crosslinked intermediate filaments composed of cytokeratins 5, 6, 16, and ‘Z’ that are referred to as cytoskeletal sheets. We have been investigating the function of these cytoskeletal sheets during embryogenesis. In this investigation we report the rapid appearance of extensive arrays of tonofilaments extending across blastomeres and in association with intercellular desmosomal junctions appearing at the time the embryo hatches from its zona pellucida, through the time of implantation of the embryo into the uterine wall. Just prior to the time of gastrulation these tonofilaments disappear. Electron microscopy and immunoconfocal microscopy demonstrate that the tonofilaments are composed of cytokeratins characteristic of the type found earlier in development, that is types 5 and 6; whereas, cytokeratin type 8 which has been shown to be synthesized in blastocysts is localized primarily at perinuclear regions. Cytokeratins 8 and 18 are synthesized to about the same extent as actin at the time the tonofilaments appear whereas the synthesis of cytokeratins 5 an 6 is greatly reduced. Our results suggest that cytokeratins 5 and 6 in the tonofilaments may arise from the stored form of cytokeratins in the cytoskeletal sheets. Consequently, our results suggest that the sheets may serve as a maternal reserve of cytokeratin employed by the embryo at the time of implantation to form extensive arrays of tonofilaments in the embryo that likely provide structural integrity to the embryo as it is subjected to mechanical stress during invasion and implantation into the uterine wall.

Absence of an intracellular pH change following fertilisation of the mouse egg.

The intracellular pH of the mouse egg was measured during fertilisation to determine whether an increase in pH accompanies activation of this mammalian egg. The pH-sensitive dye BCECF [2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein] was introduced into the mouse egg by incubation in BCECF-AM or by microinjection of dextran-conjugated BCECF. The cells were also loaded with the DNA-specific fluorochrome Hoechst 33342 to confirm fertilisation by observation of Hoechst-stained, decondensing sperm heads in the cytoplasm. The ratio of emission intensities for the dye (494/440 nm excitation wavelengths) was monitored continuously with a photon-counting photomultiplier tube. There was no change in pH during or after fertilisation. Control eggs displayed the expected increase in pH when exposed to NH4Cl. In other experiments, intracellular pH and intracellular Ca2+ were monitored simultaneously during fertilisation. The eggs were injected with BCECF dextran and Fura dextran. Fluorescence emission was recorded at excitation wavelengths of 495 nm (BCECF, pH-sensitive wavelength) and 385 nm (Fura, Ca(2+)-sensitive wavelength). A decrease in emission intensity at 385 nm excitation clearly marked the repetitive Ca2+ transients at egg activation. There was no change in the fluorescence emitted at 495 nm excitation, indicating an absence of any change in intracellular pH. These results indicate that intracellular alkalinisation of the cytoplasm does not accompany activation of this vertebrate egg.

Low temperature and fertilization‐induced Ca2+ changes in rat eggs

In mammalian eggs, activation by sperm that leads to resumption of meiosis is characterized by an explosive transient increase in intracellular calcium ion concentration ([Ca2+]i), followed by [Ca2+]i oscillations. In addition to the spermatozoon, various treatments can induce parthenogenetic activation, accompanied by an elevation of [Ca2+]i. It has been reported that cooling can induce egg activation, yet the mechanism of this phenomenon has not been elucidated. In the present study we followed changes in egg [Ca2+]i (measured by Fura-2 fluorescence ratio imaging) during activation by cooling, using conditions that ensure a low rate of spontaneous activation. Our present findings demonstrate that cooling induces egg activation as manifested by [Ca2+]i transient(s) and second polar body extrusion. Seventy-eight of 104 eggs responded to cooling with increased [Ca2+]i. Thirty-five percent of the responding eggs displayed a single [Ca2+]i transient, while 65% exhibited at least two [Ca2+]i transients within the time window of the experiment (30-40 min). Twenty-two percent of these eggs displayed high-frequency oscillations (intervals of 3.5-5.9 min). In these eggs, the overall pattern of calcium dynamics was similar to that observed in eggs activated by sperm, as judged by the transient's intervals, duration, and a gradual increase in the amplitude of successive transients. The amplitudes of [Ca2+]i transients, however, were 2-3 times lower. We propose that cooling affects [Ca2+]i homeostasis to produce fertilization-like changes in [Ca2+]i, possibly associated with parthenogenetic activation. Moreover, great care should be exercised to prevent temperature changes during egg handling.

The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes.

Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 microM ryanodine and 4-8 microM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 microM. Prior injection of either 50 microM ruthenium red, 50 microM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10-20 mM caffeine or 200 microM ryanodine, but prior injection of 50 microM ruthenium red or procaine completely inhibited the effect of 10-20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 microM concentration of ruthenium red or procaine only partially blocked the effect of 200 microM ryanodine, but 200 microM ruthenium red or procaine completely blocked the effect of 200 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

Stimulation of repetitive calcium transients in mouse eggs.

1. We have combined cell membrane electroporation by electrical field (EF) stimulation with a rapid perfusion system in order to stimulate repetitive increases in cytoplasmic free [Ca2+] ([Ca2+]i) in mouse eggs. [Ca2+]i was monitored by ratio fluorescent measurements of intracellular indo-1 on individual eggs. The conditions required to cause different types of [Ca2+]i increases were established and the effects of these [Ca2+]i changes upon egg activation examined. 2. The rapid perfusion of non-ionic medium caused a single [Ca2+]i increase. However, to generate repetitive [Ca2+]i increases, eggs were exposed to EF pulses in the presence of Ca2+ and then washed rapidly with culture medium. Sequential EF pulse application led to prolonged elevation of [Ca2+]i levels and eventual cell lysis unless rapid reperfusion with culture medium was achieved. Transient increases in [Ca2+]i in eggs could also be generated by EF pulses in the presence of inositol 1,4,5-trisphosphate (InsP3). 3. In response to EF stimulation fertilized eggs showed [Ca2+]i increases that were enhanced relative to unfertilized eggs. The responses in these fertilized eggs were often followed by repetitive [Ca2+]i oscillations, despite the fact that the [Ca2+]i oscillations associated with sperm penetration had ceased by this stage. 4. In unfertilized mouse eggs the [Ca2+]i increases appeared to be due to direct cation influx since repeated EF pulses caused repeated influx of Mn2+ as monitored by quenching of fluorescence of fura-2 loaded eggs. 5. Under conditions that stimulated reproducible patterns of [Ca2+]i transients we found that a single large [Ca2+]i transient did not cause significant egg activation, but that inducing repetitive [Ca2+]i transients was effective in activating eggs. The speed of activation as judged by the rate of pronuclear formation was also dependent upon the frequency of pulse application. 6. These data show that combining EF pulses with a rapid and precise sequential perfusion system can be used to manipulate [Ca2+]i levels in mammalian eggs. This provides a means of artificial mimicry of the [Ca2+]i transients seen after fertilization. It appears that Ca2+ influx during EF pulses does not cause significant Ca2+ release from internal stores in unfertilized eggs, but after fertilization Ca2+ influx does induce Ca2+ release. It is also apparent that mouse eggs are more successfully activated by repetitive [Ca2+]i increases than by single large [Ca2+]i rises. We suggest that our data provide direct evidence for the hypothesis that a cellular response to oscillations of intracellular [Ca2+]i can be distinct from that to monotonic rises in [Ca2+]i.

Protein Kinase M, the Cytosolic Counterpart of Protein Kinase C, Remodels the Internal Cytoskeleton of the Mammalian Egg during Activation

We have investigated mechanisms by which intracellular signals act to restructure the spatial organization of the cytoskeleton as the mammalian egg is converted into the zygote. Four distinct approaches (one cytological, two biochemical, and one pharmacological) demonstrate protein kinase C (PKC) and its cytosolic active counterpart, PKM, act in succession at the time of egg activation. PKM serves to remodel the internal cytoskeleton. The cytological approach mapped the distribution of kinase over time using the PKC reporter dye, Rim-1, which demonstrated a temporal shift in kinase distribution from its initial site of activation at the plasma membrane to its subsequent association with a cross-linked network of intermediate filaments referred to as sheets. The first of two biochemical analyses, Western blot analysis, demonstrated that eggs activated with calcium ionophore contained PKC in the detergent-soluble fraction and PKM in the sheet enriched fraction. Prior to egg activation, PKM is not detected in the sheet-enriched fraction; only PKC is detected in the detergent-soluble fraction of these eggs. The second biochemical analysis demonstrated, via [32P]ATP labeling of a known PKC substrate, that the PKM generated from activation of PKC in response to activation of eggs with calcium ionophore is an active kinase and is located in the sheet-enriched fraction. In addition, purified forms of PKM, but not PKC, could be shown to act on the internal cytoskeleton when perfused into a permeabilized egg system. Pharmacological treatments demonstrate that elevation of [Ca2+]i does not act directly to alter the internal cytoskeleton of the egg. Our results suggest that this kinase is employed at the time of fertilization to provide an internal chronometer acting first at the cell periphery as PKC and subsequently in the cell interior as PKM.

5 Nuclear Transplantation in Mammalian Eggs and Embryos

Publisher Summary This chapter discusses the progress of nuclear transplantation studies in mammals, the important biological factors influencing the development of eggs and embryos reconstituted by nuclear transplantation, and the prospects for future studies in this field. The transplantation of a nucleus to an appropriate cytoplast and the subsequent analysis of its embryonic development provide the most vigorous and direct tests of genomic totipotency of a cell nucleus. The most advanced studies are those carried out in amphibia. Totipotency, the capacity to direct the formation of fertile frogs, has been demonstrated for the nuclei of many kinds of embryonic cells; pluripotency, as judged by the development of heart-beating larvae, has been proved for all the cell nuclei tested to date.

3 Mechanisms of Calcium Regulation in Sea Urchin Eggs and their Activities during Fertilization

Publisher Summary This chapter discusses the current evidence of Ca 2+ regulation in sea-urchin eggs during fertilization. Studies on calcium regulation during the fertilization of mammalian eggs and comparative changes in deuterostome eggs during fertilization are also discussed in the chapter, along with other events associated with egg membranes during fertilization. The regulation of intracellular Ca 2+ activity ([Ca 2+ ] i ) in a wide variety of cells is well established as an important event during signal transduction of numerous extrinsic stimuli. Different sources, including influx of external Ca 2+ and release from the intracellular stores, contribute to the rise in [Ca 2+ ] i . Irrespective of the cell type, resting [Ca 2+ ] is generally in the range of 50–200 n M and accounts for only a minute fraction of the total cellular Ca 2+ content, therefore the bulk of the cellular Ca 2+ is bound and may reside within the membrane-bound organelles.