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The rates of water loss of domestic chicken eggs were varied during incubation to measure the osmoregulatory ability of the avian embryo. Egg water loss was increased by drilling holes in the eggshell over the airspace on day 13 (I = 21 days) and then placing these eggs in a low relative humidity (r.h.: 0-10%) incubator until hatch. Egg water loss was decreased by placing other eggs in a high-r.h. (85-90%) incubator on day 0. Eggs with low water loss (approximately 6% of initial fresh mass [IFM]) produced embryos and yolks that were not different in wet or dry mass when compared to control eggs that lost approximately 12% of IFM. However, 1-4 gm of excess albumen were left in low-water-loss eggs on day 21. Hatching success was 71% and 89% for low and control eggs, respectively. Low egg water loss did not appear to disturb embryonic growth. The allantoic fluid volume and millimolar allantoic Na+ and Cl- ions declined faster with high and slower with low rates of water loss. Thus, excess water was lost as a result of increased movement of water out of allantoic fluid, which was due to increased active transport of Na+ ions by the chorioallantoic membrane (CAM). Eggs with high water loss had elevated Cl- levels after day 17 in plasma and amniotic fluid, which indicated a period of osmotic stress after depletion of allantoic fluid between day 18 and hatch. The decrease in wet embryo mass measured in embryos from high-water-loss eggs was due principally to dehydration of skin. Embryonic skin may serve as an emergency water reservoir during osmotic stress. Dehydrated chicks produced from high-water-loss eggs were 6 gm less in wet mass at hatch compared to controls. However, these chicks regained the water deficit 7 days after hatch and grew at a rate not different from control chicks through 6 weeks of age. Total egg water loss of 12% of IFM results in highest hatching success. However, water losses between 6% and 20% of IFM do not appear to affect adversely the growth or water content of the chick. Water losses above 20% of IFM cause early depletion of allantoic fluid, prolong the period of osmotic stress, and result in subsequent dehydration of blood, amniotic fluid, and embryonic skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

The production of inositol 1,4,5-trisphosphate (InsP3) has been reported to mediate the transient rise in intracellular Ca2+ activity ([Ca2+]i) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP3 during fertilization is still lacking. We investigated this question by blocking the InsP3 synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca2+ release in a dose-dependent manner, although above 15 microM U73122 eggs showed an elevated resting [Ca2+]i and a lower fertilization rate. The inhibition of Ca2+ transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca2+ response. U73122 also prevented the accompanying rise in intracellular pH (pHi), which is mediated by the activation of the Na+-H+ antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol, which is the other hydrolytic product of phosphatidylinositol 4,5-bisphosphate by PLC. Further evidence of the specificity of U73122 action was inhibition of the increase in InsP3 mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTPgammaS-induced Ca2+ release and pHi rise in unfertilized eggs. These results suggested that the transient rise in Ca2+ in sea urchin during fertilization requires the production of InsP3.

Several lines of evidence suggest that ionic messengers are primary agents in the metabolic derepression which occurs at fertilisation. The derepression at fertilisation or parthenogenetic activation of the sea urchin egg occurs in two main phases. The first phase, which triggers the early events of fertilisation, is mediated by transitory increase of intracellular free calcium. The second, which triggers the late events of fertilisation, is mediated by a rise in the intracellular pH (refs 4-6). The transition from the early events of fertilisation of sea urchin eggs to the late events requires a minimal concentration of sodium in the external medium. External Na+ is required for the acid effux which follows fertilisation. Na+ requirement and the acid effux have been correlated in a hypothesis which proposes that internal protons are exchanged for external Na+ (refs 8, 9). By using pH-sensitive microelectrodes, we have examined the relationship between external Na+ and internal pH more closely. We demonstrate here that the increase of the intracellular pH following egg activation does require external Na+. However, the relative insensitivity of the alkalisation of the egg cytoplasm to large reductions of external Na+ is evidence against the Na-H exchange hypothesis.

Fertilization initiates a transient increase in intracellular Ca21 principally by Ca2' release from intracellular stores. Possible multiple Ca21 stores and multiple receptor regulation of the same store have been reported. Here we report the presence of at least two independent intracellular Ca2+ stores in the sea urchin egg, which are released during fertilization. Ca2' release from one store is mediated by inositol 1,4,5-trisphosphate (IP3) and is sensitive to low molecular weight heparin. The other store is heparin insensitive and independent of IP3 regulation, but the regulatory factor remains unidentified. A transient increase in Ca2+ in heparinloaded eggs is observed during fertilization, which suggests that IP3-independent Ca2' release mediates the production of IP3