Ultrastructure of the reactivated corpus luteum after embryonic diapause in the marsupial,Macropus rufogriseus banksianus

Summary During embryonic diapause in the red-necked wallaby, M. r. banksianus, both the corpus luteum and uterine blastocyst remain dormant, and are reactivated following removal of the suckling pouch young (RPY). The morphology of dormant and reactivated corpora lutea has been studied throughout the 26.5 days of delayed gestation. Corpora lutea at 0, 2 1/2, 4, 9, 14, 21 and 25 days after RPY were fixed by perfusion. From day 4 to day 14 after RPY there was a progressive increase in the amount of smooth endoplasmic reticulum and the numbers of mitochondria. However there was a decrease in mitochondrial size from 1–2 m in diameter (0 days after RPY) to 0.5–1 m (14 days after RPY). Densely-staining granules (approximately 0.2 m in diameter) were first observed in the luteal cells at 4 days after RPY. The maximum density of granules was observed at 21 days after RPY. Shortly before birth (25 days after RPY) the number of secretory granules had significantly decreased and the features of cellular regression were evident. As with the eutherian mammals, the wallaby luteal cells have all the structural organelles associated with steroid hormone production. The numbers of densely-staining granules are greatest at 21 days after RPY and may reflect the luteal progesterone content since similar granules in the sheep and cow have been shown to be associated with elevated levels of progesterone.     

Development of a sediment bioassay to determine bioavailability of PAHs to fish

Recent work has shown that MFO induction and loss of control of steroid hormone production occurs in fish after exposure to pulp mill effluents, PCBs, PAHs, and some pesticides. We had recently developed laboratory assays to evaluate the effluents on these responses, but were lacking a protocol for a sediment assay. This paper describes the development of a sediment test capable of demonstrating MFO induction in fish. MFO responses were evident in rainbow trout within 4 days of exposure to contaminated sediments. Further testing showed that fish were responding to chemicals from the sediments, but not from bottom water, and a survey of sediment from thirteen contaminated areas showed that MFO induction more closely paralleled PAH levels in the sediments than the observed PCB concentrations. The sites showing MFO induction were also the sites where sediment toxicity was demonstrated with laboratory bioassays using Daphnia magna and Hyalella azteca. The protocol has been further refined to describe the quantity of sediment required and duration of testing. This test will enable us to study the biochemical effects of exposure to contaminated sediments. The protocol could also be used to prioritize areas of contamination and to evaluate dredging impacts and remediation success.     

Eggshell spot scoring methods cannot be used as a reliable proxy to determine pigment quantity

Eggshell maculation of most passerines is due to the deposition of the pigment protoporphyrin which is produced during biosynthesis of blood haem. Its functional significance has only received empirical attention in recent years. This interest has generated a number of hypotheses of which some remain untested partly because the quantification of protoporphyrin is analytically challenging and can be prohibitively expensive. Many studies have therefore used the extent of eggshell spotting as a proxy for total eggshell protoporphyrin concentration, although this has not been formally tested. Pigment scoring involves recording visible eggshell pigment attributes, such as spot intensity, distribution and size. Since even immaculate eggs can contain some protoporphyrin, there remains doubt over the degree to which visible pigment correlates with total pigment content of the shell. In this study, we test whether visible pigment scoring can be used as a proxy for protoporphyrin concentration of an eggshell. We use pigmented eggshells of two common British passerine species to compare eggshell spot intensity, distribution and spot size (as used by the visual pigment scoring method) with direct measures of eggshell protoporphyrin concentration. In addition, we compared an alternative method of pigment scoring, the pixel pigment scoring method, using a computer programme to quantify the number of pixels exceeding a specified colour threshold. We demonstrate that although results from both scoring methods were positively correlated with eggshell protoporphyrin concentrations, the correlations were not sufficiently strong to be used as surrogates in studies where actual pigment concentrations are required.     

Use of a multiple addition bioassay to determine limiting nutrients in eagle lake,California

This investigation uses anin vitro enrichment bioassay (in which all essential nutrients except one are added to each culture) to determine which nutrients are important to algal growth in Eagle Lake. The technique was developed when it was discovered that addition of individual nutrients produced little if any growth response. Laboratory bioassays correlated well with comparative studies in the lake. A great deal of variation was found throughout the year but P, N, Fe, and S were found to be limiting at one time or another. The north and south basins of the lake, which differ in morphometry, were also found to differ in the intensity spectrum of limitation. While P was the most important nutrient in both basins, the other nutrients were more limiting in the north basin than the south, and Fe, which was least limiting in the south, was very important in the north. The multiple enrichment bioassay has several advantages over other bioassays. Supported in part by Research Corporation     

Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis

Whereas most mammalian cells require extracellular signals to suppress apoptosis, preimplantation embryos can survive and develop to the blastocyst stage in defined medium without added serum or growth factors. Since cells of these embryos are capable of undergoing apoptosis, it has been suggested that their lack of dependence upon exogenous growth factors results from the production of endogenous growth factors that suppress apoptosis by an autocrine signaling mechanism. In the present study, we have examined the growth factor requirements and intracellular signaling pathways that suppress apoptosis in both mouse preimplantation embryos and embryonic stem (ES) cells, which are derived from the blastocyst inner cell mass. Cultured ES cells, in contrast to intact embryos, required serum growth factors to prevent apoptosis. Suppression of ES cell apoptosis by serum growth factors required the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, since apoptosis was rapidly induced by inhibition of PI 3-kinase with LY294002. In contrast, inhibition of MEK/ERK signaling with U0126 or of mTOR with rapamycin had no detectable effect on ES cell survival. Thus, like most mammalian cells, the survival of ES cells is mediated by growth factor stimulation of PI 3-kinase signaling. Treatment with LY294002 (but not with U0126 or rapamycin) similarly induced apoptosis of mouse blastocysts in serum-free medium, indicating that intact preimplantation embryos are also dependent upon PI 3-kinase signaling for survival. These results demonstrate that PI 3-kinase signaling is required to suppress apoptosis of both ES cells and intact preimplantation embryos, consistent with the hypothesis that survival of preimplantation embryos is maintained by endogenous growth factors that stimulate the PI 3-kinase pathway.     

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

Evaluation of three techniques used to determine surfactant phytotoxicity

Three techniques were used to measure and compare the phytotoxicities of Triton X-45 and Agral 90 (two organic surfactants) with two organosilicone surfactants (Silwet L-77 and L-7607), and to compare these four non-ionic compounds with a cationic tallow amine surfactant (Hyspray). Total ion efflux and ethylene response methods were used in vivo and in vitro, while a betacyanin efflux method was an in vitro system only. The first two methods, using intact leaves, were considered to be more closely related to normal spraying conditions than the betacyanin efflux test which used explant material. However, the use of intact leaves was thought to bias the results in favour of the leaf penetration properties of the surfactants rather than their phytotoxicities. The in vitro pigment efflux method provided a simple way of ranking the surfactants in order of their potential phytotoxicities, especially with regard to effects on membrane permeability. This ranking, in increasing order of toxicity, was: Silwet L-7607, Silwet L-77, Agral 90, Triton X-45 and Hyspray.     

Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila

A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. Previous work has shown that (1) the two strains age in the same manner, (2) the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and (3) the extended longevity phenotype is significantly modulated by the larval environment. In this report, we investigate the resistance of the L and R strains to the lethal effects of dietary paraquat. We show that, within the limitations of our described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing us to identify long-lived animals at a young age. Finally, we show that the age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements which we have previously noted in this system.     

Maternal control of cold and desiccation tolerance in eggs of the band-legged ground cricket Dianemobius nigrofasciatus in relation to embryonic diapause

Cold and desiccation tolerance was investigated in the eggs of the band‐legged ground cricket Dianemobius nigrofasciatus in relation to embryonic diapause. Diapause eggs were more tolerant to both desiccation and cold than non‐diapause eggs. In addition, diapause‐destined eggs on day zero (0–12 h after being laid) already showed high tolerance to these stresses before entering diapause. This clearly indicates that stress tolerance, like diapause, is controlled by photoperiod, but is not directly associated with diapause itself. Because the acquisition of stress tolerance predates the onset of diapause, it is plausible that diapause programming during some period before the onset of diapause is involved in the acquisition of stress tolerance. Weights and sizes were nearly identical in short‐day and long‐day eggs until day five. Sorbitol, a major sugar alcohol in eggs of D. nigrofasciatus, was accumulated at the same level in short‐day and long‐day eggs on days zero and five. These results indicate that the surface‐to‐volume ratio as well as the accumulation of sugar alcohol is not involved in the acquisition of stress tolerance. Maternal factors are clearly involved in the acquisition of stress tolerance in D. nigrofasciatus eggs, but the physiological mechanisms underlying the tolerance are still unclear.     

Geographic variation in embryonic development time and stage of diapause in a grasshopper

Embryonic development times and the stage at which embryonic diapause occurs varied dramatically among 23 populations of the Melanoplus sanguinipes/ devastator species complex in California, USA. Grasshoppers were collected from a wide range of latitudes (32°57N to 41°20N) and altitudes (10m to 3031 m), spanning much of the variation in climatic conditions experienced by these insects in California. When reared in a common garden in the laboratory, total embryonic development times were positively correlated to the mean annual temperature of the habitat from which the grasshoppers were collected (varying from about 19 days to 32 days when reared at 27°C). These grasshoppers overwinter as diapausing eggs and the proportion of embryonic development completed prior to diapause was significantly higher in populations collected from cool habitats (>70%) than in populations collected from warm environments (<26%). The length of pre-diapause development time is determined by the stage of embryonic development at which diapause occurs, and varies considerably among populations of these grasshoppers; grasshoppers from warmer environments tend to diapause at very early stages of embryogenesis, while grasshoppers from cooler environments diapause at very late stages. The combined effect of variation in embryonic development times and variation in the stage at which diapause occurs results in a dramatic reduction in the time needed to hatch in the spring; populations from warm environments required up to 20 days (at 27°C) to hatch while populations from cool environments required as few as 5 days to complete embryonic development prior to hatching. Egg size also varied significantly among populations, but tended to be larger in populations with shorter embryonic development times. Significant family effects were observed for development time and stage of diapause, suggesting significant heritabilities for these traits, although maternal effects may also contribute to family level variation. We interpret these findings to support the hypothesis that embryonic development time and the stage of embryonic diapause have evolved as adaptations to prevailing season lengths in the study populations.     

Summer diapause in Daphnia as a reaction to the presence of fish

A chemical signal, released by a fish predator under summer-likehigh water temperature and long-day photoperiod, caused theformation of resting eggs in a clone of Daphnia magna. No ephippialfemales were recorded and no ephippia were released in the controltreatment during 45 days of the experiment. When exposed tofish water, the fraction of ephippial females reached a maximumof 3.7%, a value comparable to that registered in summer inthe GroBer Binnensee (Northern Germany), a hypertrophic lakeinhabited by fish, which was the source lake for our experimentalclone. The number of ephippia released within 45 days was onaverage 34 ± 22. Ephippia formation could not resultfrom the between-treatment differences in population density,and related patterns of food depletion, since no substantialdifference between control and fish treatment was observed.Instead, specific information on the presence of a predatorprovided a cue which induced the formation of resting eggs inDaphnia. Under heavy predation and very low survival probabilityof parthenogenetic females, ephippia formation in summer canbe adaptive, i.e. higher fitness can be achieved through survivalin the diapausing state than through the immediate reproductivegain via parthenogenesis.     

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.     

Esterases in relation to embryonic diapause in the field cricket,Teleogryllus commodus

Fourteen esterase isozymes were detected by polyacrylamide gel electrophoresis in homogenates of eggs of the field cricket Teleogryllus commodus, at various stages of development. One esterase isozyme appeared during embryonic diapause termination effected by prolonged chilling of the eggs.Esterase activity towards methyl butyrate in T. commodus egg homogenates increased with time. Sonication generated the maximum levels of esterase activity observed. Methyl butyrate esterase activity in T. commodus eggs was high during the first few days of embryogenesis; fell markedly during the period of rapid water uptake, and then remained fairly constant during diapause, diapause development, and during resumed embryogenesis.     

The cockroach heart as a bioassay organ

Detailed procedures are presented for denervation of the American cockroach heart, Periplaneta americana L., by removal of the lateral cardiac nerve cords. Results of bioassay with the innervated heart are also presented and compared to responses obtained from identical assay conditions with the denervated heart.The response of the innervated heart to 10^-3 M sodium azide, slight changes in the concentration of sodium ion, reduced glutathione, saline dilutions, and low amounts of ethanol and acetone was characterized by the immediate appearance of irregularities in the heartbeat. In contrast, the responses of the denervated heart to the first three of these compounds were always characterized by a smooth even heartbeat changing gradually in amplitude and/or rate and/or contractile state of the alary muscles. All assay conditions examined caused qualitatively less response in the beat of the denervated heart.Irregularities in the beat of the innervated heart which were induced in bioassay were found to be due to extreme sensitivity of the spontaneously active cardiac neurons.

---

Zusammenfassung Das isolierte abdominale Herz von Periplaneta americana L. reagiert auf Durchspülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung mit einem unmittelbaren Anstieg des Tonus bis zu fast systolischer Hemmung. Danach verringert das Herz den Tonus allmählich, schlägt unregelmäßig und tritt in eine kurze Periode diastolischer Pause ein, bevor es in Diastole stehen bleibt. Die Herzschlagaktivität wurde wieder aufgenommen, wenn das Natriumazid durch Spülung mit physiologischer Kochsalzlösung ersetzt wird.Nach Entfernung beider Paare der Herzseitennerven gab das Herz von P. americana auf Bespülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung keine Initialreaktion. Statt dessen stieg der Herzschlag nach 2 min allmählich leicht an und die Herzschlagamplitude nahm im Verlaufe von 6 min sehr allmählich ab. Nach 6 min blieb das Herz in Diastole stehen.Die Herzreaktionen der amerikanischen Küchenschabe waren bei Auftropf-Tests mit anormaler Natriumchloridkonzentration, herabgesetztem Glutathion und Lösungsmitteln ähnlich den für Natriumazid beschriebenen. Die Reaktion des denervierten Herzens ist durch das Fehlen von Unregelmäßigkeiten im Herzschlagmuster gekennzeichnet.Sowohl das denervierte wie das innervierte Herz reagieren auf Testtropfen von nur etwas anormalen Calciumchloridkonzentrationen.

---

---

Supported by USPHS Training grant PHS GM 1076.     

Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila.

A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. Previous work has shown that 1) the two strains age in the same manner, 2) the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and 3) the extended longevity phenotype is significantly modulated by the larval environment. In this report, we investigate the resistance of the L and R strains to the lethal effects of dietary paraquat. We show that, within the limitations of our described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing us to identify long-lived animals at a young age. Finally, we show that the age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements which we have previously noted in this system.     

Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust.