A possible bio-assay for chromosome movement in isolated mitotic apparatus

We isolated mitotic apparatus (MA) from sea urchin zygotes using several methods. The isolated MA were injected into nucleated frog eggs or into enucleated frog eggs, and 24 h later we determined whether or not the eggs had undergone normal cleavage. Normal cleavage occurred in about 50% of the cases when MA isolated in glycerol-dimethyl sulphoxide were injected into either nucleated or enucleated frog eggs. Likewise for MA isolated initially in hexylene glycol and transferred immediately into glycerol-dimethyl sulphoxide. No cleavage occurred when MA isolated in hexylene glycol (and stored in hexylene glycol) were injected into frog eggs. We discuss two possible interpretations of the results. In one interpretation cleavage of the frog eggs is a bioassay, measuring the ability of the isolated MA to support chromosome movement. In the other interpretation the isolated MA contribute only nuclear material and cleavage initiation factors, and there is no chromosome movement in the isolated MA per se.     

Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.     

Sex-Dependent Changes in Striatal Dopamine Transport in Preadolescent Rats Exposed Prenatally and/or Postnatally to Methamphetamine

Methamphetamine (MA) is the most commonly used psychostimulant drug, the chronic abuse of which leads to neurodegenerative changes in the brain. The global use of MA is increasing, including in pregnant women. Since MA can cross both placental and haematoencephalic barriers and is also present in maternal milk, children of chronically abused mothers are exposed prenatally as well as postnatally. Women seem to be more vulnerable to some aspects of MA abuse than men. MA is thought to exert its effects among others via direct interactions with dopamine transporters (DATs) in the brain tissue. Sexual dimorphism of the DAT system could be a base of sex-dependent actions of MA observed in behavioural and neurochemical studies. Possible sex differences in the DATs of preadolescent offspring exposed to MA prenatally and/or postnatally have not yet been evaluated. We examined the striatal synaptosomal DATs (the activity and density of surface expressed DATs and total DAT expression) in preadolescent male and female Wistar rats (31–35-day old animals) exposed prenatally and/or postnatally to MA (daily 5 mg/kg, s.c. to mothers during pregnancy and lactation). To distinguish between specific and nonspecific effects of MA on DATs, we also evaluated the in vitro effects of lipophilic MA on the fluidity of striatal membranes isolated from preadolescent and young adult rats of both sexes. We observed similar changes in the DATs of preadolescent rats exposed prenatally or postnatally (MA-mediated drop in the reserve pool but no alterations in surface-expressed DATs). However, prenatal exposure evoked significant changes in males and postnatal exposure in females. A significant decrease in the activity of surface-expressed DATs was found only in postnatally exposed females sensitized to MA via prenatal exposure. MA applied in vitro increased the fluidity of striatal membranes of preadolescent female but not male rats. In summary, DATs of preadolescent males are more sensitive to prenatal MA exposure via changes in the reserve pool and those of preadolescent females to postnatal MA exposure via the same mechanism. The combination of prenatal and postnatal MA exposure increases the risk of dopaminergic deficits via alterations in the activity of surface-expressed DATs especially in preadolescent females. MA-mediated changes in DATs of preadolescent females could be still enhanced via nonspecific disordering actions of MA on striatal membranes.     

Calcium, Sulfhydryls and the Mitotic Apparatus

The in vivo mitotic apparatus (MA) of clam, worm and sea urchineggs may be augmented or dispersed by application of specificantimitotic agents. Glycols, which are antimitotic at concentrationsof 1–3% in sea water, cause a rapid massive increase inMA volume and retardation as seen in the polarizing microscope.Caffeine and dinitrophenol (DNP) cause a rapid disappearanceof the MA by shrinkage. Glycol effects can be balanced by DNPor caffeine if the agents are applied at the proper time andconcentration although normal cleavage does not ensue. Analysisof DNP and caffeine shrinkage suggests that they act indirectlyby causing release of calcium from intracellular stores, calciumcausing inactivation of polymerizable tubulin. DNP could causerelease of calcium either from mitochondria (if egg mitochondriahave a calcium uptake system) or by causing a decrease in ATPlevels which would inactivate calcium uptake systems such asthe Petzelt Ca⁺² ATP-ase or Kinoshita Ca⁺² uptake vesicles. Caffeine, while an inhibitor of the phosphodiesterase for cyclicAMP does not cause inhibition of the MA through the cyclic nucleotide.It is found that caffeine (and other methyl xanthines) causean inhibition of glutathione reductase activity in treated eggs.It is postulated that the calcium ATP-ases in the egg may becontrolled by reversible oxidation and reduction of their sulfhydrylsthus regulating calcium concentration in the cytosol. The possiblemodes of action of this system are discussed.     

DNA barcoding reveals the temporal community composition of drifting fish eggs in the lower Hongshui River,China

Determining the temporal community composition of fish eggs in particular regions and understanding the reproductive times of regional fish taxa are key aspects of the management and regulation of regional fish stocks. However, it is extremely difficult to accurately identify fish eggs due to the absence of diagnostic morphological characters. We sampled fish eggs in the lower Hongshuihe River (an upper mainstem of the Pearl River) between May and September 2020. We then used DNA barcoding to determine the species composition of the egg pool and to predict the spawning periods of the identified species. A total of 641 eggs and 17 larvae were chosen for molecular identification; 397 eggs and 17 larvae yielded high‐quality barcoding sequences. The high failure rate (~38%) was most likely due to long‐term storage in low concentrations of ethanol prior to molecular analysis. We successfully classified 392 eggs into 10 species and 13 larvae into four species using public databases. Most of the species identified in the egg pool were small and/or benthic, and migratory species were rare. This may partially reflect the adverse effects of hydropower cascade development in this river section. We also found that spawning periods tended to be species‐specific. Our study provides a reference for the conservation and management of regional fishery stocks.     

Developmental defects of female-sterile mutants of Drosophila melanogaster

Gans et al. (1975) isolated female-sterile mutants, viable and normal in the homozygous state, producing apparently normal eggs which were unable to develop or developed into defective embryos or adults, even when fertilized with wild-type sperm. Developmental abnormalities of these mutants were surveyed by observing living and fixed material. The following types of mutants were distinguished according to the predominant developmental defect: (i) Eggs not developing at all, mostly remaining unfertilized. (ii) Eggs stopping development after a few cleavages, some with polyploid nuclei. (iii) Eggs stopping development at various embryonic stages, with haploid nuclei. (iv) Eggs with abnormal blastoderm, not developing further or giving abnormal embryos. (v) Eggs with abnormal gastrula, in one case with excessive invaginations, in another case with germ band failing to elongate. (vi) Eggs with embryos dying at different stages, without easily visible effects. Several of the mutants were temperature sensitive. In all the mutants there were eggs that died without developing. Most of the developmental defects appear to be due to general metabolic disturbances of the egg, not directly related to morphogenesis. There were no mutants affecting determination of a particular adult organ. The closest to a morphological type of mutation were those with abnormal blastoderm having successive bands of nuclei of different sizes. Those mutants were thermosensitive; at permissive termperatures they developed into agametic adults or adults with various defects of abdomens, wings or eyes. The nature of maternal genetic control of early morphogensis was discussed.     

Interpretation of Tracer Data: Significance of the Number of Terms in Specific Activity Functions

It has already been shown that the number of pools in an open system in the steady state cannot be determined from the number of exponential terms in the specific activity function of a pool, even if the data were free from experimental error. However, some information is conveyed by the number of exponential terms. The information is different depending upon whether the data are obtained from the pool into which the tracer is introduced or from another pool. In the latter case, the number of exponential terms is shown to indicate the maximum number of intermediate pools involved in the shortest path of transfer of material from the injected pool to the pool in question. With regard to the former case, this paper is restricted to functions with two exponential terms and shows which systems of n pools (n >/= 2) are consistent with such data. Consequently, biexponential experimental curves can be interpreted in terms of models consisting of an unrestricted number of pools in which each pool is defined in terms of fast mixing. The generalization to cases of functions with more than two exponential terms can be carried out in a similar manner.     

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.     

Fertilization of newt coelomic eggs in the absence of jelly envelope material

Summary Fertilization ofCynops pyrrhogaster (Japanese newt) coelomic eggs was studied in the absence of jelly envelope material or synthetic high polymer. An undiluted sperm fluid from the vas deferens fertilized coelomic eggs in the absence of the jelly envelope material or synthetic high polymer. The fertilized eggs developed beyond gastrulae and formed tail bud embryos. These results indicate that the fertilization process does not depend upon the presence of jelly envelope material or synthetic high polymer and that the sperms within the vas deferens are already capable of fertilizing the eggs inC. pyrrhogaster. The sperm suspension in Holtfreter's balanced salt solution (H-sperm) fertilized the coelomic eggs without the jelly envelope material or synthetic high polymer. These eggs had been suspended in Holtfreter's balanced salt solution (H) or in 1/20 strength H (1/20 H) prior to insemination (H-eggs or 1/20 H-eggs). In contrast, the sperm suspension in 1/20 H (1/20 H-sperm) did not fertilize 1/20 H-eggs, but dit H-eggs. In the latter case, H surrounding the eggs may affect sperms, allowing them to be fertilized. The 1/20 H-sperms regained their ability to fertilize 1/20 H-eggs on re-exposure to H. The 1/20 H-sperm also fertilized jelly eggs. The results of the dejellied egg experiment showed the same pattern. These results indicate that the sperms within the vas deferens lose their capacity to fertilize 1/20 H-eggs on exposure to low ionic strength solution (1/20 H); this capacity is restored by exposure to high ionic strength solution (H) or to jelly envelope.     

ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.     

鼎湖山森林土壤活性碳及惰性碳沿海拔梯度的变化

对鼎湖山3个不同海拔高度下的沟谷雨林(LA)、低地常绿阔叶林(MA)和山地常绿阔叶林(UA)的土壤活性碳库和惰性碳库进行了研究。结果表明:(1)土壤总碳库仅在30—45 cm土层中存在显著差异且碳库大小随着海拔的增加而增加。(2)土壤微生物生物量碳(MBC)碳库在0—15 cm是LA和MA显著大于UA,在30—45 cm是MA和UA显著高于LA,在45—60 cm土层中MA最大。水溶性碳(WSOC)和颗粒碳(POC)碳库均不随海拔高度而改变。WSOC碳库占总碳库的百分比仅在30—45cm土层中存在差异且大小顺序为:LAUAMA,POC碳库占总碳库的百分比仅在土层15—30 cm上存在显著差异且MA比值最大。易氧化性碳(ROC)碳库及占总碳库百分比都是在表层土壤(0—15 cm)中产生显著变化,且UA极显著地大于LA和MA。(3)惰性碳(RC)碳库仅在深层土壤中存在显著差异且MA中RC碳库最大,UA次之,LA最小。RC碳库占总碳库比值仅在表层土壤0—15 cm存在显著差异且UA最大。表层土壤中ROC碳库和RC碳库占总碳库百分比的增加是导致中高海拔森林土壤总碳库最大的主要原因。(4)不同海拔高度上森林土壤理化性质与土壤碳库组成存在显著相关,土壤理化性质的改变是引起不同海拔高度森林土壤碳库组成变化的重要原因。     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.     

Magnetic alignment in mammals and other animals

Magnetic alignment (MA) constitutes the simplest directional response to the geomagnetic field. In contrast to magnetic compass orientation, MA is not goal directed and represents a spontaneous, fixed directional response. Because animals tend to align their bodies along or perpendicular to the magnetic field lines, MA typically leads to bimodal or quadrimodal orientation, although there is also growing evidence for a fixed unimodal orientation not necessarily coinciding with the magnetic cardinal directions. MA has been demonstrated in diverse animals including insects, amphibians, fish, and mammals. Alignment can be expressed by animals during resting as well as on the move (e.g. while grazing, hunting, feeding, etc.). Here, we briefly survey characteristic features and classical examples of MA and review the current knowledge about the occurrence of MA in mammals. In addition, we summarize what is known about mechanisms underlying MA and discuss its prospective biological functions. Finally, we highlight some physiological effects of alignment along the magnetic field axes reported in humans. We argue that the phenomenon of MA adds a new paradigm that can be exploited for investigation of magnetoreception in mammals.     

Aster formation in vitro is nucleated by granules isolated from the mitotic apparatus

Mitotic apparatuses (MAs) isolated from sea urchin eggs contained clusters of granular material in their centrospheres. After cold treatment and mild agitation, the MA fraction formed asters when combined with tubulin. Many microtubules grew from isolated centrospheres most of which were covered with astral residues. Homogenization of the isolated MA fraction dispersed the centrospheres which broke into fragments or into aggregates of small granules that formed small asters when tubulin was added. Electron microscopy showed that more than ten microtubules were nucleated from a granular aggregate composed of several approximately 90-nm granules. The aster-forming activity was lost with time when the MAs were kept at 0 degree C. Only glycerol stabilized this activity. The aster-forming activity also was heat labile and trypsin sensitive, but it was resistant to RNase treatment. When the dispersed MAs were extracted with a buffer solution of high ionic strength, aster-forming activity was recovered only in the extract; that is, when the extract had been dialyzed against a solution of low ionic strength, the fine granules self assembled and retained their aster-forming ability.     

Ultrastructural studies on the intracellular fate of 125I-nerve growth factor in cultured rat sympathetic neurons

Primary cell cultures of sympathetic neurons from rat were exposed to 125I-nerve growth factor (NGF) and the fate of the NGF in the cell was followed using electron microscopic autoradiography. The intracellular localization of NGF was determined in the cell bodies and in the proximal neurites of neurons that had been grown in three-chamber dishes, following 5 or 24 hr of retrograde transport of NGF from the distal portions of the neurites. Label in the proximal neurites was predominantly associated with lysosomes and multivesicular bodies (MVBs), and at 5 hr elongated tubular elements were especially heavily labeled. Most of the label in the cell bodies was concentrated in lysosomes and MVBs. Lysosomes accounted for the largest fraction (45-60%) of the grains in the cell body, with a labeling density (LD = % grains/% area) of 3-5, while MVBs accounted for 5-10% of the grains with an LD of 5-20. We observed no evidence of nuclear labeling after 5 or 24 hr of retrograde transport. Mass cultures of neurons were incubated for 22 hr with NGF in the presence of the lysosomal inhibitors chloroquine (CQ, 0.05 mM) or methylamine (MA, 10 mM). In both agents the lysosomes were swollen with membranous material but still sequestered NGF, especially in CQ where the lysosomes were associated with almost 65% of the grains and had an LD of 6. CQ and MA had different effects on the morphology of the MVBs: in CQ they were few in number and compact while in MA they were numerous and appeared swollen and vacuolated. We observed no evidence for the nuclear accumulation of NGF even in the presence of the lysosomotropic agents.     

Studies on the role of intestinal bacteria in metabolism of synthetic and natural steroid hormones

Administration of antimicrobial agents to subjects taking oral contraceptives has been reported to lead to contraceptive failure and subsequent pregnancy. In women taking oral contraceptives antimicrobial agents could have an effect on both endogenous hormone levels and on the metabolism of the exogenously administered steroids. To investigate these possibilities, antimicrobial agents were administered for short periods to normal women taking various steroid drugs: Megestrol acetate (MA), medroxyprogesterone acetate (MPA), norethisterone (NET), a combination of NET and ethinylestradiol (EE) or a combination of lynestrenol and EE. During ampicillin administration the 24-h morning plasma concentrations of MA, MPA and NET were increased compared to the control values. In the MA and MPA experiments the afternoon values were determined and also found to be increased. In the subjects taking oral contraceptives plasma EE concentration showed a tendency to decrease during ampicillin administration on the third, fourth or fifth morning of ampicillin administration, but was never lower than the pretreatment values. In other experiments plasma estrone (E1) and estradiol (E2), urinary total E1, E2 and estriol (E3) and fecal unconjugated and conjugated E1, E2 or E3 were determined by RIA before, during and after administration of oxytetracycline (2 X 500 mg/day for 5 days) to 5 young male subjects. Furthermore urinary and fecal estrogens were determined in 1 male subject after administration of erythromycin for 6 days and in 2 normally menstruating women after tetracycline and trimethoprim administration, respectively. During treatment with antimicrobial drugs an increase in the excretion of fecal conjugated and, with the exception of the oxytetracycline experiments, also of unconjugated estrogens paralleled a decrease in urinary estrogen excretion, especially for E2 and E3. In both urine and feces the E1/E2 and E1 + E2/E3 ratios increased due to diminished reductive metabolism of estrogens in the gut. No significant effects on plasma unconjugated estrogen concentrations were observed. The results suggest that the intestinal bacterial flora plays a significant role in estrogen metabolism. However, further studies are necessary, because our results do not explain why administration of antibiotics may cause contraceptive failure.     

Final oocyte maturation in vivo and in vitro in marine fishes with pelagic eggs; yolk protein hydrolysis and free amino acid content

The role of free amino acids (FAA) in oocyte hydration during final maturation has been studied in plaice Pleuronectes platessa and lemon sole Microstomus kitt by in vivo and in vitro measurements. In vitro final maturation was initiated by the administration of human chorionic gonadotropin on large vitellogenic oocytes. The eggs produced in vitro had the same fraction of their total amino acid pool present in the free form as the in vivo hydrated eggs, regardless of whether FAA had been present in the incubation medium or not. The FAA pool in the mature egg was increased 10–15 times that of the oocyte, and the two FAA pool profiles differed strongly. The FAA profiles of the egg groups (intra- as well as interspecific) were almost identical except that the taurine content was lower in eggs in vitro . A major protein band of about 100 kDa was present on SDS electrophoretic gels of oocytes but missing on gels of hydrated eggs. This protein, presumably a lipovitellin, is the most likely origin of the egg FAA pool. We suggest that marine fishes with pelagic eggs share a common mechanism for oocyte hydration whereby partial hydrolysis of specific yolk proteins to FAA creates a major part of the osmotic potential needed for the water influx.     

Effect of ethylenediaminetetraacetic acid-Tris(hydroxymethyl)aminomethane on release of the acid-soluble nucleotide pool and on breakdown of ribosomal ribonucleic acid in Escherichia coli

Brief treatment of Escherichia coli with 2 x 10(-4)m ethylenediaminetetraacetic acid (EDTA)-0.12 m tris(hydroxymethyl)aminomethane (Tris), pH 8.0, or 0.12 m Tris alone resulted in the release of the acid-soluble nucleotide pool at 3 or 23 C. Exposure to EDTA-Tris for up to 90 min at 3 C did not result in the release of increasing amounts of 260-mmu-absorbing material. At 23 and 37 C, EDTA-Tris resulted in a steady increase in acid-soluble 260-mmu-absorbing material. Previous growth environment did not alter the release. There appeared to be degradation of 23S ribonucleic acid (RNA) after 10 min of exposure at 23 C. In addition, there was degradation of nucleotides to nucleosides and bases. This occured either within the cells with altered permeability or in the periplasmic space. This occurred in the presence of EDTA and Tris but was not seen with EDTA-phosphate. The mechanism of this degradation is unclear, since it occurs in ribonuclease I-deficient strains. Exposure to Tris buffer for long periods of time at 23 C resulted in release of the nucleotide pool and in degradation of RNA and nucleotides. These studies point out that the EDTA-Tris effect on E. coli must be divided into two parts, an early (4 to 5 min) change in permeability and a later phase of actual RNA breakdown and nucleotide degradation. Studies utilizing EDTA and Tris as agents altering permeability must thus be viewed with caution. Although the cells are viable, they have lost their acid-soluble nucleotide pool and have undergone degradation of some ribosomal RNA.     

Developmental and biochemical alterations caused by tunicamycin inBufo arenarum embryos

Summary Bufo arenarum eggs at late blastula and gastrula were treated with tunicamycin, an inhibitor of glycoprotein glycosylation, to investigate its effects on morphogenesis and neural induction. Because of the low permeability of the amphibian egg to a number of drugs, the blastocoel was opened surgically prior to treatment. Almost all of the eggs treated with the antimetabolite, at a concentration of 10 g/ml, from late blastula stage for 24h exhibited exogastrulation. The effect is dose- and stage-dependent as shown by the lower proportion of exogastrulae obtained when eggs are treated at a lower concentration (5 g/ml) or after the onset of gastrulation. Treatment with the antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. The most striking biochemical effect was an enhanced uptake of glucose, mannose and leucine. The incorporation of mannose into acid-insoluble material was severely inhibited by tunicamycin, with a concomitant decrease of leucine incorporation into the acid-soluble pool.