Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study.
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In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.
Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.
Animals↗
The culture of 12- and 13-day rat embryos using continuous and noncontinuous gassing of rotating bottles.
A technique for the culture of 12- and 13-day rat embryos is presented. The culture method described utilizes the opening of the extraembryonic membranes together with a simple bottle rotator during incubation to facilitate tissue oxygenation. This method was compared with a more elaborate device that enabled constant gassing during incubation. Best results were obtained with 12-day embryos cultured for 24 hr in closed bottles. Thereafter, there was a marked falloff in embryonic development in culture. Optimal medium conditions were 25% rat serum in tissue culture medium with a gas phase of 60% O2, 5% CO2, 35% N2. The culture method described allows for larger numbers of embryos to be cultured more simply than previous methods and should be valuable to workers wishing to study embryos in the more advanced stages of organogenesis.
Animals↗
Placental lactogen secretion in the mouse: in vitro responses and ovarian and hormonal influences.
In vivo and in vitro methodologies were utilized to study the regulation of placental lactogen (PL) secretion in the mouse. Chorioallantoic placental explants obtained from midpregnant (day 10 of gestation) C3H/HeN or Swiss Webster mouse conceptuses behaved differently in culture than explants of chorioallantoic placentas obtained later in pregnancy. Midpregnant placental explants maintained or showed an increasing amount of PL output per day for 5-6 days of culture, while gestationally older placental explants demonstrated a progressive decline in PL output per day over the 5-6-day culture period. On selected days of gestation, bilaterally ovariectomized C3H/HeN or Swiss Webster mice were shown to have significantly higher serum PL concentrations than sham operated mice. Reduction of serum progesterone concentrations by unilateral ovariectomy and/or partial lutectomy had no significant effect on serum PL concentrations. Progesterone significantly reduced PL production by midpregnant placental explants from both C3H/HeN and Swiss Webster mice, whereas 17 beta-estradiol did not significantly affect PL release in vitro. PL production by mouse chorioallantoic placentas has gestational specific characteristics in vitro and is responsive to regulatory influences of the maternal ovaries and progesterone.
Animals↗
Amino acids can induce nondisjunction during maturation of mammalian oocytes in vitro.
Certain chromosomal disorders in mammalian embryos are traceable to meiotic errors during oocyte maturation. This report evaluates the influence of amino acids on meiotic maturation in vitro of oocytes from pigs, hamsters, and rats. The results indicate that maturing porcine oocytes respond not only to 1-glutamine (1-gln) but also to 1-isoleucine (1-ileu) in complex or chemically defined media by exhibiting significantly (P less than .05) increased incidences of nondisjunction when compared with oocytes in control medium. Nondisjunction was highly correlated (r = 0.981) with dose of 1-gln in porcine oocytes (incidences of maturing oocytes exhibiting nondisjunction were 19.3%, 39.7%, 41.5%, 66.2%, and 88.5% at 1-gln concentrations of 0 mM, 0.5 mM, 1.0 mM, 3.0 mM and 10.0 mM, respectively). Hamster oocytes also exhibited significantly (P less than .05) increased nondisjunction when cultured in medium containing 1-gln (52% of maturing cumulus-enclosed oocytes exhibited nondisjunction in medium with 3.0 mM 1-gln vs. 6.3% in control medium). Denuded hamster oocytes also responded to 1-gln (35% exhibited nondisjunction in 4.0 mM 1-gln, 8% in control medium). In contrast the incidence of nondisjunction in rat oocytes was not increased significantly by 1-gln over a concentration range of 0-12.0 mM. This study demonstrates that maturing oocytes respond to certain environmental conditions by undergoing chromosomally abnormal maturation. Specifically, amino acids can induce nondisjunction when present in elevated concentrations during oocyte maturation. The amino acid influence not only was dose dependent for porcine oocytes but also was species specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
The response of transplants of cultured fetal mouse ovaries in kidneys of ovariectomized adult mice.
Whole ovaries from 16-day fetal mice were cultured for 6 to 20 days and then transplanted to the kidneys of ovariectomized adult mice where they remained for one to four weeks. After three weeks in the host's kidneys, many follicles developed within the transplants and became vesiculated. Many of the oocytes within these follicles had formed the first metaphase spindle of meiosis and several had completed the first polar body. Host mice bearing transplants that contained vesiculated follicles showed uterine stimulation and keratinization of their vaginae. However, ovaries that had been in culture for more than ten days before transplantation showed a limited response to the gonadotropins and never matured sufficiently to stimulate the host's reproductive tract. No ovulations occurred in any of the transplants.
Animals↗
Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding.
Preimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD-1 and B6CBA. After removal of the zona pellucida by treatment with pronase, FITC-lectins were bound to the embryonic cell surfaces at either 4 degrees C or 37 degrees C. Both morula and blastocyst stage embryos bound the following lectins, FITC-ConA, FITC-WGA, FITC-RCAII and FITC-RCAI. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B6CBA embryos bound less FITC-ConA and FITC-WGA than the corresponding Swiss CD-1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4 degrees C and 37 degrees C for FITC-ConA, FITC-RCAII, and FITC-RCAI. While lectins bound at 4 degrees C showed a pattern of continuous labeling, the same lectin at 37 degrees C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC-WGA bound at 4 degrees C and 37 degrees C demonstrated continuous labeling of embryos at both temperatures. FITC-fucose binding protein did not bind to Swiss CD-1 embryos. The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.
Animals↗
The effects of phenylalanine on cultured rat embryos.
The offspring of mothers with untreated classic phenylketonuria (PKU) have shown a high frequency of microcephaly, mental retardation, pre- and postnatal growth retardation, and birth defects. The aim of this study was to determine whether phenylalanine (phe) is the teratogenic agent in maternal PKU. To observe the direct effects of phe on organogenesis, embryos of 9.5-day pregnant rats were cultured for 48 h in the presence of phe at concentrations of 0.1 to 6.0 mM. Within this range no morphological abnormalities occurred in exposed embryos, when compared to control embryos. However there was a reduction (P less than or equal to 0.05) in embryonic protein content and somite number at the highest concentration of phe (6.0 mM). This does not preclude the longer-term effects of phe at later stages of gestation. To examine phe transport into the embryo in response to elevated serum phe levels, 3H-phe uptake studies were undertaken. These showed that 3H-phe from the culture serum is incorporated rapidly into the free amino acid pools and embryonic protein. At serum concentrations of 1.4 mM or higher, phe saturation occurs in the cellular pools of the embryo. Amino acid analysis of the exocoelomic fluid showed that when embryos were cultured for 48 h in serum containing 3.45 mM phe, the total amino acid concentration was maintained near the control levels (16 mM). Of this, 27% (4.26 mM) was contributed by phe, and all other amino acids, except methionine, were decreased with respect to control levels.
Animals↗
An assessment of the effects of cyclophosphamide and sodium valproate on the viability of preimplantation mouse embryos using the fluorescein diacetate test.
The fluorescein diacetate test is a measure of both esterase enzyme activity and membrane integrity and it has been shown to be useful in assessing the viability of a variety of cells cultured in vitro, mouse embryos after freezing and thawing, and mouse embryos grown under inadequate culture conditions. In this study, the effects were examined of cyclophosphamide and sodium valproate administered respectively to pregnant inbred CBA mice 60 h after fertilization, on the viability of 84-h blastocysts. Cyclophosphamide 20 and 40 mg/kg bodyweight and sodium valproate 90 mg/kg significantly increased the number of nonviable blastocysts. Cyclophosphamide 4 mg/kg and sodium valproate 23 and 45 mg/kg did not adversely affect blastocyst viability. The intensity of embryo fluorescence correlated well with the subsequent development of the embryos in culture (r = 0.863; p less than 0.001). The test had a sensitivity of 83% and a specificity of 100%. Exposure of embryos to fluorescein diacetate under the conditions of this experiment did not adversely influence the subsequent postimplantation development of 84-h blastocysts cultured in vitro for a further 120 h. These findings suggest that the fluorescein diacetate test is a simple, rapid, and nontoxic procedure that may be useful in assessing the viability of preimplantation embryos after exposure to embryotoxic drugs and chemicals.
Animals↗
Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos.
The effects of cyclophosphamide (CPA), administered to pregnant inbred CBA/Ca mice 60 h after copulation, on cell number, mitotic index, chromosome structure, histone synthesis, and DNA synthesis of 84-h blastocysts, and the subsequent development of these blastocysts cultured for a further 120 h in vitro are described. Cyclophosphamide 4, 20, and 40 mg/kg significantly increased the number of chromosomally aberrant cells, chromosomal aberrations, and chromosome breaks in the blastocysts. Chromosomal rearrangements were significantly increased in the CPA 20 and 40-mg/kg treated groups, and in the 40-mg/kg group the number of cells with ring chromosomes was significantly increased. Histone synthesis and DNA synthesis were significantly inhibited in the CPA 20 and 40-mg/kg treated groups. Blastocyst cell number in each of the treated groups was less than the controls. On subsequent culture in vitro, significantly fewer embryos in the CPA 20 and 40-mg/kg groups hatched, attached, developed trophoblast outgrowths, and expanded their inner cell masses. However, the differentiation of inner cell mass into ectoderm and endoderm was impaired by all three doses of the drug. These results demonstrate that CPA administered to pregnant mice 60 h after copulation has a clastogenic effect and interferes with synthesis of DNA and histones in the preimplantation embryo, and that the drug inhibits the subsequent development and differentiation of these embryos. Cytogenetic analysis of preimplantation embryos might be a useful adjunct to the existing methods in the evaluation of the embryotoxicity of drugs and chemicals.
Animals↗
Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen.
The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.
Animals↗
Cardiovascular defects in rat embryos cultured on serum from rats chronically exposed to phenytoin.
Headfold-stage rat embryos were cultured for 48 hours on serum from rats chronically exposed to phenytoin for periods as long as from conception until 11 months of age. Serum from phenytoin-exposed rats caused approximately 50% of the cultured rat embryos to develop cardiovascular defects as compared to 12% for controls. These morphological abnormalities included hemorrhaging of blood vessels within the embryo, pericardial edema, and absence of yolk sac circulation. Neither serum glucose nor phosphate levels nor serum osmolality were appreciably affected by phenytoin treatment. However, serum protein concentration was reduced in rats exposed to phenytoin as compared to controls. An absence of the serum protein hemopexin was associated with the reduction in serum protein levels but did not appear to be responsible for the observed cardiovascular defects.
Animals↗
Asbestos and embryonic development.
Chrysotile asbestos was administered to pregnant CD-1 mice in their drinking water and to Day 4 blastocysts during culture. Administration to pregnant mice did not affect embryo survival. In vitro administration of asbestos did not interfere with embryo implantation upon transfer of blastocysts to recipient females but did result in a decrease in postimplantation survival. These studies did not demonstrate asbestos to be teratogenic whether administered to the dam or to the blastocyst in vitro.
Animals↗
Abnormalities induced in cultured rat embryos by hyperthermia.
Rat embryos were explanted at nine and one-half days of gestation and cultured for 48 hours in rotating bottles containing rat serum and a gas phase, at temperatures of 38, 40, 40.5 and 41 degrees C. The embryo cultured at 40.5 degrees C were retarded and many of them were abnormal, and at 41 degrees C, all the embryos were malformed and retarded. The most frequent abnormalities occurring at both these temperatures were microcephaly and oedema of the pericardium. Development of the embryos cultured at 40 degrees C was similar to that of the controls at 38 degrees C, and superficially they appeared to be normal. However, measurement of the head dimensions, and separate determinations of head and body protein contents showed that the 40 degrees C embryos were microcephalic.
Animals↗
The induction of axial blisters in the chick embryo by trypan blue.
The hemorrhage, blister formation, and rumplessness observed in the chick embryo following treatment with trypan blue may be due to (1) increased ventricular blood pressure or (2) to necrosis and edema in the caudal region of the embryo by inhibiting nutrient utilization. To test the role of increased ventricular blood pressure in the induction of caudal blisters, primitive streak to 7-somite chick embryos were cut in half, separating the upper presumptive heart region from the lower presumptive trunk and tail regions. Each half was then explanted on media containing trypan blue for 24 hours. In intact embryos treated with 0.04 mM trypan blue the frequency of blisters in the posterior region was 80.8%. The blisters usually appeared on both sides of the neural tube, below or in the region of the last few somites. In transected embryos treated with trypan blue, the frequency of blisters in the posterior halves which had beating hearts was 2.6%. However, the frequency of blisters in the posterior halves which were not connected to beating hearts was 47.8%. In some cases the blisters were found in posterior halves in which the rump was not well developed or present at all. Thus, we may conclude that: (1) direct connection between the heart and the rump is not necessary for the induction of caudal blisters. (2) The presence of a well-differentiated rump is also not necessary for blister formation. We suggest that trypan blue acts directly on organs or structures found in the caudal region of the chick embryos.
Abnormalities, Drug-Induced↗
Ultrastructural analysis of malformations of the embryonic neural axis induced by in vitro hyperglycemic conditions.
Neural tube defects are the most common malformations associated with diabetic pregnancies. Although the teratogenic effects of excess glucose have been investigated in in vivo and in vivo studies, a cellular basis for neural tube defects has not been elucidated. We used rat embryo culture to study the organogenesis period of development, with excess d-glucose added to the serum medium to induce neural tube anomalies. Light and electron microscopic examination of control 12-day-old embryos grown 48 hours in culture revealed blastlike cells with few organelles or cellular processes. Twelve-day-old embryos cultured in excess d-glucose had advanced cellular maturation with differentiation, including the presence of free polysomes and copious cell processes, regardless of whether they had an open neural tube. Cytoarchitectural changes such as decreased numbers of mitotic figures with mitotic cells in the mantle layer were focally distributed throughout the neural epithelium but with predominance at the site of failed closure. In vivo studies failed to demonstrate neural processes in day 12 normal embryos. Fourteen-day-old embryos grown in utero also had foci of cell processes in the neural tube but to a much lesser degree than that observed in the in vitro day 12 glucose-exposed embryos. The cellular aberrations in the excess d-glucose-treated embryos are characteristic of a premature maturational change. Since they are present in excess d-glucose-exposed embryos with or without failure of neural tube closure, these maturational and cytoarchitectural changes may contribute to the cellular basis for neural tube defects.
Animals↗
In vitro teratogenicity of ethylenethiourea in the rat.
We have demonstrated that ethylenethiourea (ETU) is a potent teratogen to the rat embryo developing in vitro. Sprague Dawley rat embryos were explanted on gestation day 10 and cultured for 48 hours in the presence of 40-200 micrograms/ml ETU. This resulted in a dose-related inhibition of growth and differentiation as assessed by crown-rump length, protein and DNA content, and somite number and in an increase in the frequency of abnormalities. A variety of anomalies was produced, including fluid accumulation in the brain (hydrocephalus), decreased mandibular size, decreased telencephalon size, abnormal dorsiflexion, as well as subectodermal blisters on the tail and limb buds and maxilla. Frank malformations have been observed at these same sites--hydrocephalus, brachygnathia, kyphosis, limb and tail defects, cleft palate--in the term fetus in vivo. The presence of abnormal fluid accumulation in the embryos--distended neural tube and subectodermal blisters--suggesting that the osmotic environment of the embryo had been altered by ETU exposure. Osmolality of the exocoelomic fluid (ECF) surrounding the embryo was measured after 48 hours of exposure to a concentration of ETU that caused nearly a 100% incidence of subectodermal blisters. ECF osmolality was found to be significantly lower than that of control embryos. Lowered osmolality would cause water to move out of the ECF, presumably causing the observed fluid accumulation in the embryo. It is speculated that altered osmotic balance and localized edema in the embryo are contributory steps in the formation of defects after ETU exposure.
Abnormalities, Drug-Induced↗
Developmental toxicity of aflatoxin B1 in the rodent embryo in vitro: contribution of exogenous biotransformation systems to toxicity.
Aflatoxin B1 (AFB1) is a known carcinogen and developmental toxin in various species of mammals, birds, amphibians, and fish. AFB1 requires metabolic activation (biotransformation) to the 2,3-epoxide metabolite for carcinogenicity; however, it is unknown if biotransformation is a prerequisite for AFB1 embryotoxicity. Cultured day 10 rat embryos were exposed to AFB1 alone and AFB1 in the presence of cofactors and hepatic S9 fractions from adult male rats induced with either phenobarbital or 3-methylcholanthrene. Under these different culture conditions qualitatively similar patterns of malformation were seen in all embryos exposed to AFB1. At culture concentrations of 15 microM or greater, AFB1 produced abnormalities in neural tube development in a concentration-dependent manner. The presence of hepatic S9 fractions had no effect on the ability of AFB1 to produce dysmorphogenesis in vitro or on the spectrum of malformations elicited. However, the addition of hepatic S9 fractions did greatly enhance the embryolethality of AFB1. This enhancement was greater with phenobarbital- than 3-methylcholanthrene-preinduced hepatic S9 fractions. Our results suggest that separate chemical mediators may be responsible for the embryolethal and dysmorphogenic effects of AFB1 observed in day 10 rat embryos in vitro. We found that the embryolethality of AFB1, but not the dysmorphogenicity, could be greatly modulated by exogenous biotransformation.
Abnormalities, Drug-Induced↗