Amount of maternal body fat significantly affected the quality of isolated mouse preimplantation embryos and slowed down their development

The aim of our study was to investigate the effect of maternal obesity on the quality and developmental capabilities of in vivo-derived preimplantation embryos. A two-generation dietary model, based on mice overfeeding during intrauterine and early postnatal development, was used to produce four types of female animals: with physiological (7%–8%), slightly elevated (8%–11%), highly elevated (>11%), and low (<7%) amounts of body fat. Spontaneously ovulating females (5–6 weeks old) were mated with male animals and subjected to embryo isolation at Day 4. Stereomicroscopical evaluation of collected embryos showed that the amount of maternal body fat did not affect the average number of collected embryos per dam. However, significant differences were found in the stage-distribution of isolated embryos: dams with highly elevated body fat and dams with low fat delivered decreased numbers of blastocysts and increased numbers of lower-stage or degenerated embryos compared with dams with physiological or slightly elevated fat value. Fluorescence staining showed that blastocysts isolated from dams with high and low percentage of body fat contained significantly higher numbers of dead cells. Most of such dead cells were of apoptotic origin. In contrast, the amount of maternal body fat did not affect blastocyst growth—the average numbers of cells per blastocyst were comparable in all groups. In conclusion, highly elevated or decreased amount of maternal body fat slowed down the development and negatively affected the quality of naturally in vivo-derived preimplantation embryos. No negative effect of slightly elevated fat was observed.     

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.     

Sexual dimorphism and female reproductive cycle in the scincid lizard Trachylepis vittata (Olivier, 1804) in Lebanon (Reptilia: Scincidae)

Sexual dimorphism and the female reproductive cycle were studied in a population of the viviparous lizard Trachylepis vittata at 2000 m a.s.l. on Mount Sannine, Lebanon. Females have larger body sizes than males and males have relatively larger heads than females. Females reach maturity at 56 mm snout-vent length. They spend at least six months in hibernation, from October to March. Adult females emerge from hibernation in April. Fertilization occurs mid-May and gestation lasts for 8-10 weeks. All females collected on the same date had embryos at the same embryonic developmental stage. Females produced 1-4 embryos. There is a significant positive relationship between female body size and number of embryos. Parturition lasts for two weeks and new-borns achieve adult size within about ten months.     

Fecundity of Litomosoides carinii (Nematoda, Filarioidea) in vivo and in vitro

Several parameters concerning the reproduction of Litomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18 +/- 2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7-8 weeks p.i. the females were 71 +/- 6 mm long and on average contained 308 X 10(3) embryos/female, of which 19% were pathologically altered. In the middle of patency 16-20 weeks p.i. the females had grown up to 100 +/- 11 mm in length and now contained 509 X 10(3) embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a female L. carinii in vivo of around 20 X 10(3) microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.     

Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos

This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P<0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P> or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.     

Embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in chickens: effects of dose and embryonic stage on hatchability and growth

Chicken embryos (Gallus domesticus) were injected with 0, 8, 20 or 50 ng tetrachlorodibenzo-p-dioxin (TCDD) per egg at embryonic day (ED) 4, 8 or 12 to investigate the effects of differential periods of sensitivity to TCDD exposure. At hatch, all chicks were weighed, sexed and examined macroscopically to identify possible malformations. Liver, bursa, heart and spleen masses were recorded from a number of chicks. The remaining chicks were raised until 6 weeks of age and body and organ masses, plasma concentrations of thyroid hormones, triglycerides and glucose were measured. Dose and stage during embryonic development at which injection was performed affected hatchability. Fifty nanogram of TCDD was highly toxic for 4-day-old chicken embryos. TCDD was less toxic for chicken embryos of 8- and especially 12-days old. One-day-old chick and organ weights were not different between TCDD doses at all injection days. However, injection performed at ED4 or ED8 with 20 and 50 ng, respectively, significantly depressed post-hatch body mass gain. Moreover, body mass gain in males was more depressed than in females. The delayed growth in TCDD treated chickens was accompanied by changes in T(3)/T(4) ratio that at some ages were significantly higher compared to control animals. No pronounced changes in plasma triglycerides or glucose concentrations during postnatal life were observed. Absolute and relative organ masses of 6-week-old chickens showed no remarkable changes.     

胚胎冷冻技术应用于抗菌肽转基因小鼠的保种传代

目的研究胚胎冷冻在抗菌肽转基因FVB小鼠保种传代中的应用。方法对6~8周正常雌性FVB小鼠进行超排分别与雄性杂合子抗菌肽转基因FVB小鼠交配,收集2-cell胚胎,进行胚胎冷冻。1周后进行胚胎复苏移植,通过PCR方法对仔代鉴定。结果冻存胚胎140枚,复苏获得存活胚胎98枚,移植85枚,产仔38只,获得阳性后代12只。结论通过胚胎冷冻技术保种及复苏移植技术可对抗菌肽转基因小鼠进行传代。     

Development of enucleated mouse oocytes reconstituted with embryonic nuclei

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.     

Delayed development in Fischer's pygmy fruit bat, Haplonycteris fischeri, in the Philippines

A long delay in post-implantation embryonic development was detected in Fischer's pygmy fruit bats (palaeotropical fruit bats of the suborder Megachiroptera), the first time such a delay has been demonstrated outside the bat suborder Microchiroptera. Samples of bats were obtained from the Philippines over 5 years, and reproductive tracts were preserved and examined using standard histological techniques. Most parous female pygmy bats were impregnated in June, within a few weeks of parturition, and the embryos underwent superficial implantation at the anterior end of the uterus contralateral to the previously gravid uterus. Shortly thereafter, the rate of embryonic growth slowed tremendously for up to 8 months. During the period of delay, the mean length of the embryoblast increased only from 280 microns to 520 microns. In March of the following year, the developmental rate increased, and the embryos completed development in the next 3 months. The 8-month delay gives these bats a gestation period of 11.5 months, the longest known in bats. Most nulliparous females become pregnant at an age of 3-5 months, and their embryos entered a similar delay that terminated in March or April, after 2-6 months of delay. Males showed signs of fertility throughout the entire year, but testis volume was highest during May, June and July, at about the time when most females become receptive.     

Early estrogen exposure induces abnormal development of Fundulus heteroclitus

Many chemicals released into the environment exhibit estrogenic activity, having the potential to disrupt development and the functioning of the endocrine system. In order to establish a model system to study the effects of such environmental chemicals on aquatic animals, we examined the effects of a natural estrogen, 17 beta-estradiol (E(2)), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8), and 10(-6) M E(2) throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner, and fry treated with 10(-6) M E(2) and 10(-8) M E(2) were dead by two weeks and 12 weeks after hatching, respectively. More than 85% of fry treated with 10(-8) M E(2) showed malformations: i.e., eye extrusion, crooked vertebral column, faded lateral-stripe pattern eight weeks after hatching. Body weight and head and body lengths were significantly reduced in E(2)-treated fry when compared to controls. Ossification was not completed in vertebrae, cranial bones, and other bones in fry treated with 10(-8) M E(2) even 12 weeks after hatching. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E(2) were 100% female eight weeks after hatching. The present results demonstrate that exogenous estrogen induced death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones, which would result in shorter body and head lengths and in malformed vertebrae leading to a hunchback condition.     

Identification of variables contributing to superovulation efficiency for production of transgenic prairie voles (Microtus ochrogaster)

ABSTRACT: BACKGROUND: The prairie vole (Microtus ochrogaster) is an emerging animal model for biomedical research because of its rich sociobehavioral repertoire. Recently, lentiviral transgenic technology has been used to introduce the gene encoding the green fluorescent protein (GFP) into the prairie vole germline. However, the efficiency of transgenesis in this species is limited by the inability to reliably produce large numbers of fertilized embryos. Here we examined several factors that may contribute to variability in superovulation success including, age and parentage of the female, and latency to mating after being placed with the male. METHODS: Females produced from 5 genetically distinct breeder lines were treated with 100 IU of pregnant mare serum gonadotrophin (PMSG) and immediately housed with a male separated by a perforated Plexiglas divider. Ovulation was induced 72 hr later with 30IU of human chorionic gonadotropin (hCG) and 2 hrs later mating was allowed. RESULTS: Superovulation was most efficient in young females. For example, females aged 6-11 weeks produced more embryos (14 +/- 1.4 embryos) as compared to females aged 12-20 weeks (4 +/- 1.6 embryos). Females aged 4-5 weeks did not produce embryos. Further, females that mated within 15 min of male exposure produced significantly more embryos than those that did not. Interestingly, there was a significant effect of parentage. For example, 12 out of 12 females from one breeder pair superovulated (defined as producing 5 or more embryos), while only 2 out of 10 females for other lines superovulated. CONCLUSIONS: The results of this work suggest that age and genetic background of the female are the most important factors contributing to superovulation success and that latency to mating is a good predictor of the number of embryos to be recovered. Surprisingly we found that cohabitation with the male prior to mating is not necessary for the recovery of embryos but is necessary to recover oocytes. This information will dramatically reduce the number of females required to generate embryos for transgenesis in this species.     

Increased gelling agent concentration promotes somatic embryo maturation in hybrid larch (Larix × eurolepsis): a 2-DE proteomic analysis

An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by comparing embryos incubated in media with a high gellan gum concentration (8 g l(-1) ) and the standard concentration (4 g l(-1) ) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l(-1) medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l(-1) of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in 'carbohydrate metabolism', 'genetic information processing' or 'environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l(-1) of gellan gum.     

In vitro culture of silver fox embryos

This experiment was designed to establish in vitro culture methods for silver fox embryos in order to develop the methods for evaluation of the post-thaw viability of frozen embryos in future studies. Artificially inseminated silver fox females were killed humanely on predetermined days after insemination and oviducts and uteri were flushed for embryos. The embryos were cultured in modified TCM 199 or in the same medium supplemented with silver fox oviductal tissue suspension for varying periods, from 6 days to 3 weeks. A total of 60 embryos was recovered. Only embryos beyond the 8-cell stage up to expanded blastocysts developed in vitro (28 % of all embryos). Early stage blastocysts developed most reliably and were of the best quality.     

Transient expression of transglutaminase C during prenatal development of human muscles.

Tissue transglutaminase (TGase C, TGase II) is known to participate in cellular processes during morphogenesis, differentiation, and development of various prenatal tissues and organs. The expression of TGase C during myoblast proliferation and attachment to external laminae was examined by immunohistochemical (IH) localization at 5-12 weeks of developmental stages of prenatal human muscle in 23 embryos. IH detection using a monospecific antibody to TGase C showed a prominent expression of TGase C in muscle cells as stage- and spatial-specific patterns during an early embryonal period. The myoblasts of intervertebral, tongue, and limb muscles, attached to adjacent cartilaginous skeletons or fibrous fascia, showed a pronounced expression of TGase C at 5-6, 6-7, and 7-8 weeks after fertilization, respectively. The most intense activity of TGase C was observed in some cardiac myoblasts infiltrating into endocardial mesenchyme at 6-7 weeks after fertilization. Although weak staining was detected until 14 weeks after fertilization, the level of TGase C expression in all muscles was significantly decreased after 6-7 weeks, with the exception that the smooth muscle cells of blood vessels and gastrointestinal tract showed diffusely intense staining of TGase C between 5 and 12 weeks after fertilization. Western blotting analysis of the cellular extracts of pooled samples showed a single strong band at 80 kD at 6 weeks after fertilization. This band became weaker after 8-10 weeks of prenatal development. These findings of transient expression of TGase C, which coincides with the development of myoblast anchoring and differentiation, suggest that TGase C plays a role in myoblast attachment to the extracellular laminae during the early embryonal period.     

Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 degrees C

The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI-) in fresh and stored embryo for 6 h or 24 h did not differ significantly (P > 0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI (R = 0.87) (P < 0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.     

Dominant lethal assay of some hair-dye components in random-bred male rats.

Male rats were exposed to maximally tolerated doses of 5 hair-dye components in a dominant lethal test. Each component was tested at 3 dosage levels with 15 random-bred male rats per level. The highest dose, selected on the basis of subacute toxicity testing, generally reduced weight gains without being lethal. Freshly prepared solutions were injected i.p. at 1 ml/kg 3 times a week for 10 weeks. Rats injected with dimethylsulfoxide and triethylenemelamine served as solvent and positive controls, respectively. A majority of rats survived the treatment at the levels tested and were mated to two virgin females each per week for 2 weeks. The females were sacrificed at midterm of pregnancy and examined for live and dead implants. Dominant lethality was evaluated on the basis of 4 criteria: dead implants per pregnant female, dead implants per total implants, proportion of females with one or more dead implants, and proportion of females with two or more dead implants. 2-Nitro-p-phenylenediamine, 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate produced negative responses, whereas m-phenylenediamine and 4-nitro-o-phenylenediamine induced weak dominant lethality in the first trial. On retesting these weakly positive components, both m-phenylenediamine and 4-nitro-o-phenylenediamine produced negative responses.     

Artificial insemination in the mouse with special reference to the effect of sperm numbers on the conception rate and litter size (author's transl)

Mature female mice (ICR-JCL), 8 to 12 weeks of age, were artificially inseminated at 8:30-9:30 a. m. on the day of estrus vaginal smear (about 3-7 hr after ovulation) with 3.18 X 10(6), 1.83 X 10(6) and 1.15 X 10(6) sperms from four, two and one cauda epididymidis, respectively, of adult males which were suspended in 50 microliter of a modified Krebs-Ringer-bicarbonate solution and incubated at 37 degrees C was under 5% CO2 in air for an hour. Immediately after insemination, pseudopregnancy induced by an artificial penis and a vaginal tampon. Out of 13 females inseminated with 3.18 X 10(6) sperms, 9 females showed placental signs and 8 of them gave birth to the mean 10.5+/-2.20 (M+/-S.D.) young at term. Four of 13 females having received 1.83 X 10(6) sperms became pregnant giving birth to the mean 4.3+/-2.1 (M+/-S.D.) young at term. On the other hand, 5 out of 6 females failed to become pregnant following insemination with 1.15 X 10(6) sperms, and only one showed a placental sign and gave birth to twelve young at term. It is concluded that the conception rate and litter size are both dependent on the number of sperms inseminated and that more than 3 X 10(6) sperms are necessary to get the conception rate and the litter size comparable to those in natural mating.     

MNNG-induced mutations in the adult gill and hepatopancreas and in embryos of rpsL transgenic zebrafish

To evaluate the feasibility of a mutagenicity assay using adult rpsL transgenic zebrafish, 4- to 8-month-old females were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0, 15 or 30 mg/L in a water bath for 2 h). At 2 weeks after exposure, MNNG showed a concentration-dependent significant increase in mutant frequency (MF) of 8 x 10(-5), 18 x 10(-5), and 51 x 10(-5), respectively, in the gill. DNA sequencing revealed that 60-74% of the induced mutations were G:C to A:T transitions, consistent with the known mutagenic effects of MNNG. A marginal but significant increase in MF was observed in the hepatopancreas only in the group exposed to 30 mg/L, with the induction of some G:C to A:T transitions. A time-course of the appearance of mutations was determined in fish treated with 15 mg/L MNNG. In both, the gill and hepatopancreas, a higher MF was observed at 3 weeks than at 2 weeks, suggesting that an expression time of at least 3 weeks is preferable for the assay. When embryos (29 h post-fertilization) were exposed to MNNG (0, 50, and 150 mg/L) for 1 h, MFs increased significantly with an increase in the concentration of MNNG (5 x 10(-5), 40 x 10(-5), and 144 x 10(-5), respectively) at 3 days after exposure. G:C to A:T transitions were the predominant mutations, and these occurred at the same sites in the rpsL gene as in adult tissues. Thus, MNNG induces typical mutations in the gill and hepatopancreas of adult fish, and in embryos, suggesting that the rpsL zebrafish is a useful tool for monitoring genotoxicity caused by water-borne mutagens.     

舟山及其邻近岛屿獐种群的初步研究

舟山海岛上的獐(Hydropotes inermis),定海县志(1923)已有简要记录。作者在1978年调查时发现,舟山岛及其邻近较大的岛屿上普遍有獐,1976年海岛獐单位面积产量,5倍于浙江大陆邻近10个县的产量(盛和林,1981)。为进一步合理利用和发展獐资源,继而对舟山岛獐的种群结构繁殖和种群增长等问题进行研究,同时又对邻近10多个岛屿进行了访问调查,兹将初步结果报道如下。     

Toxic and teratologic effects of verapamil on early chick embryos: evidence for the involvement of calcium in neural tube closure

Toxic and teratologic effects of verapamil, a calcium antagonist, on chick embryos explanted at stage 8 (four-somite stage) and cultured for 6-8 hours were investigated. In general, embryos responded to verapamil in a dose-related manner. Concentrations lower than 2 micrograms/ml had no apparent effect on the development of embryos. A concentration of 15 micrograms/ml significantly increased the incidence of embryos (approximately 80% of viable embryos) with neural tube closure defects and less numerous somites. Higher concentrations (e.g., 30 micrograms/ml) were embryotoxic and over 90% of the embryos were either severely malformed or dead after 8 hours of incubation. Compared to controls, verapamil-treated neuroepithelial cells had smoother apical surfaces and less conspicuous microfilament bundles. The deleterious effects of verapamil (15 micrograms/ml) could be reversed by subculturing the affected embryos, within 3 hours of treatment, on nutrient medium alone or on nutrient medium containing 25 micrograms/ml chlorotetracycline (CTC), a calcium agonist, the latter being more effective provided that treatment did not exceed 4 hours. Exposure of the developing neuroepithelium to 15 micrograms/ml verapamil for 3-4 hours resulted in a significant reduction in free Ca2+ levels, as revealed by the pyroantimonate precipitation method, throughout neuroepithelial cells. Overall results suggest that verapamil causes neural tube closure defects by reducing intracellular free Ca2+ levels, thereby relaxing apical microfilament bundles of developing neuroepithelial cells.