Differentiation in vitro of mouse embryos to the stage of early somite

Mouse blastocysts continuously differentiate in vitro to the early somite stage with reconstituted rat tail collagen as the substrate for the attachment. In order for this to occur, it appears that two differentiation barriers must be overcome. The first, the formation of egg cylinders from the inner cell mass, can be overcome by incubating embryos in heat-inactivated fetal calf serum. The second, the formation of the early somite from the presomite stage, can be overcome by replacing fetal calf serum with human cord serum.Mouse blastocysts were initially incubated with calf serum in Eagle's minimum essential medium. After shedding the zona pellucida, the denuded blastocysts lay flat on the surface of the collagen. Soon thereafter, trophoblastic cells invaded the underlying collagen leaving the rounded inner cell mass protruding from the surface of the collagen. By replacing calf serum in the medium with fetal calf serum the inner cell mass differentiated into endoderm and ectoderm to form an egg cylinder.The egg cylinder rapidly became elongated and formed extraembryonic and embryonic regions. However, the embryonic region shrank from this point on in the fetal calf serum, and the resulting yolk sac formation did not contain the embryo proper. When fetal calf serum was replaced with human cord serum at the end of the egg cylinder stage (equivalent to embryos of about 7.5 days gestation) neural tissue, cardiac chambers, and somites were formed.     

Effects of hyperthermia and X-irradiation on mouse stromal tissue

The sensitivity of normal stroma to heat, irradiation and heat combined with irradiation has been studied using the tumour bed effect (TBE) assay. Irradiation before implantation led to a TBE. This TBE was dose dependent below 15 Gy, the TBE remaining relatively constant above 15 Gy. The interval (0-90 days) between irradiation and tumour implantation did not influence the magnitude of the TBE. Hyperthermia with large heat doses (45-60 min at 44 degrees C) before implantation may lead to a TBE. The interval between hyperthermia and tumour implantation proved to be very important. Our results show that the recovery from heat-induced stromal damage is very rapid. When the interval between hyperthermia and tumour implantation is 10 days or longer, no TBE could be observed. Irradiation combined with large heat doses (30-60 min at 44 degrees C) decreased the radiation-induced TBE. However, the combination of irradiation with mild heat treatments (15 min at 44 degrees C) could lead to a larger TBE than after irradiation alone. When hyperthermia was given prior to irradiation, the interval between heat and irradiation proved to be very important. With large intervals (21 days or longer) the TBE values were about the same as with irradiation alone. When heat was given after irradiation it always reduced the irradiation-induced TBE.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.     

In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage.

The sequential processes of in vitro development of whole mouse embryos were classified by stages according to the in vivo criteria of E. Witschi (1972, “Biology Data Book,” Part II: “Rat,” L. Altman and D. S. Dittmer, eds., 2nd ed., Vol. 1, pp. 178–180, Federation of American Societies for Experimental Biology, Bethesda, Md.) and K. Theiler (1972, “The House Mouse,” Springer-Verlag, Berlin/New York). The mouse embryos which developed in vitro in each day of culture were then classified into stages according to the characteristics of mouse embryos developed in vivo. A series of 10 blastocysts were inoculated into 35-mm plastic culture dishes (30–50 blastocysts per experiment). Developing embryos were scored on the fourth, sixth, and eighth days and classified into stages. Among the total of 118 blastocysts cultured in three repeated experiments, 100 mouse embryos had attached and developed in culture dishes. Ninety-four percent of the attached mouse embryos developed to the early egg cylinder stage after 4 days of incubation, and 87% grew to the stage of late egg cylinder after 6 days of culture. An average of 62% of the embryos reached the early somite stage with heart beating after 8 days in culture with frequent medium change. In two separate experiments single mouse blastocysts were placed individually in culture dishes in 2 ml of culture medium. The development of each embryo was followed every day. Each of 10 blastocysts had attached in its respective culture dish and had developed to the early egg cylinder stage after 4 days of culture. About 50 to 70% of each of these 20 individually isolated mouse embryos developed in vitro to the early somite stage after 8 days of culture.     

Long-term and transgenerational effects of in vitro culture on mouse embryos

The mouse is a convenient model to analyze the impact of in vitro culture (IVC) on the long-term health and physiology of the offspring, and the possible inheritance of these altered phenotypes. The preimplantation period of mammalian development has been identified as an early ‘developmental window’ during which environmental conditions may influence the pattern of future growth and physiology. Suboptimal culture media can cause severe alterations in mRNA expression in the embryo, which are associated with embryo quality reduction. In addition, the embryonic epigenetic reprogramming may also be severely affected by IVC, modifying epigenetic marks particularly in imprinted genes and epigenetically sensitive alleles. These altered epigenetic marks can persist after birth, resulting in adult health problems such as obesity, increased anxiety and memory deficits. Furthermore, some epigenetic modifications have been found to be transmitted to the offspring (epigenetic transgenerational inheritance), thereby providing a suitable model to asses risks of cross-generational effects of perturbing early embryo development. This review will highlight how preimplantation environment changes can not only affect developmental processes taking place at that time, but can also have an impact further, affecting offspring health and physiology; and how they may be transmitted to the next generation. We will also analyze the emerging role of epigenetics as a mechanistic link between the early environment and the later phenotype of the developing organism.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

The response of mouse skin to combined hyperthermia and X-rays.

The effects of combined hyperthermia and X-irradiation were studied in the skin of the mouse ear. Ears were heated for 1 hour by immersion in a waterbath at temperatures ranging from 37 degrees C--43 degrees C. These heat treatments had little visible effect alone, but when combined with X-rays, enhanced the radiation response. Enhancement depended on the degree of heating. When heat was given immediately after X-rays, the radiation dose to cause a given skin reaction had to be reduced by about 10 per cent for 37 degrees C and about 40 per cent of 43 degrees C. The timing and sequence of the two treatments were important. Heat after X-rays was less effective than heat before X-rays. When heat followed X-rays, the enhancing effect was lost completely if the interval exceeded 4 hours. When heat preceded X-rays, the effect was lost more slowly, depending on temperature. The implications of this for the treatment of cancer by combined therapy are discussed.     

Embryotoxic effects of sodium arsenite and sodium arsenate on mouse embryos in culture

Embryotoxic effects of two inorganic arsenic compounds, sodium arsenite (Asi) and sodium arsenate (Asa), on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with three to five somites exposed to 1-40 microM Asi or to 10-400 microM Asa were cultured for 48 hours and their development was compared with that of control embryos. Asi proved to be teratogenic between 3 and 4 microM and embryolethal at higher concentrations; Asa had similar activity but at concentrations ten times higher than for Asi. Both compounds produced a growth retardation and a similar pattern of defects. Growth retardation was indicated by a statistically significant reduction in crown-rump length, head length, and yolk sac diameter. Abnormal embryos were characterized by hypoplasia of the prosencephalon with open neural tube, hydropericardium, somite abnormalities, and failure of development of limb buds and sensory placodes. These results confirm that both Asa and Asi are embryotoxic compounds and that the Asi activity occurs at concentrations ten times lower than for Asa. Our results suggest that in humans both of these compounds may be involved in part of "unaccountable" early abortions and malformations claimed to be due to the toxicity of heavy metals.     

The early development of the lymphatic system in mouse embryos.

The early development of the lymphatic system was studied in embryos of an inbred strain of the laboratory mouse. During the first stage of its development the system is represented by a more or less regular series of small and blind-ending outgrowths of the major embryonic veins which develop in a cranio-caudalward direction from the jugular to the pelvic region. As a result of differences in growth rates of adjacent anatomical structures this series of early lymphatic primordia becomes subdivided into 4 singular primordia and 12 groups of primordia. After the constituents of each group of early primordia have fused, 16 isolated lymphatic plexuses (sacs) are formed of which 14 are in bilaterally symmetric and 2 are in a median line position: i.e. bilaterally: (1) the jugulo-axillary lymph sac situated lateral to the anterior cardinal vein and dorsal to the primitive ulnar vein and its major branch, the external mammary vein, (2) the paratracheal lymph plexus situated medial to the anterior cardinal vein, (3) the internal thoracic lymph plexus situated lateral to the thoracic part of the posterior cardinal vein, (4) the thoracic ducts situated medial to the thoracic part of the posterior cardinal vein, (5) the lumbar lymph plexus situated dorso-lateral to the abdominal part of the posterior cardinal vein, (6) the subcardinal lymph plexus and (7) the iliac lymph plexus situated ventro-lateral to the abdominal part of the posterior cardinal vein; and in the median line: (8) the subtracheal lymph plexus situated at the confluence of the pulmonary veins and (9) the mesenteric lymph plexus situated near the confluence of the splenic and the superior mesenteric veins. Except for some openings at the jugulo-subclavian confluence all connections with the veins disappear. From the primordia extensions grow out centrifugally. They invade the surrounding tissues and, in part, fuse with similar sprouts of adjacent primordia. In this way a continuous system of lymph truncs is formed that opens into the venous system at the jugulo-subclavian confluence.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Effects of acute X-irradiation on pre-implantation embryos and on the implantation reaction in the mouse

Summary Pregnant mice were treated on the 1st, 2nd, and 3rd day of pregnancy by a single dose of 300 R X-rays. Uterine dissections at day 6 p.c. topographically revealed decrease of the implantation sites from 9.67 per female in the controls to 8.00 in females irradiated on day 1, to 6.63 in females irradiated on day 2, and to 7.00 in females irradiated on day 3 p.c. Among a number of 22 implantations after irradiation on day 1, 19 after irradiation on day 2 and 11 after irradiation on day 3, however no living embryo could be detected on histological examination. The degree of damage as indicated by the total resorptions was highest (94,7%) after irradiation on day 2 p.c., and lowest (31,8%) after irradiation on day 1 p.c. Since the decidual cell reaction was either unaffected or only slightly reduced after irradiation on day 2 p.c. as indicated by cytomorphological criteria and the alkaline phosphatase reaction, not maternal effects but direct effects only of the irradiation on the embryo must account for embryonic deaths.     

Effects of human diabetic serum on the in vitro development of early somite rat embryos

High levels of glucose, beta-hydroxybutyrate (B-HOB), and acetoacetate are known to have embryotoxic and teratogenic effects on rat embryos in culture, especially when added concomitantly to the culture medium. We studied the effects of human serum from different types of diabetes mellitus on the in vitro development of 10 1/2-day-old rat embryos cultured for 48 hours. We used serum from type I diabetes with and without ketoacidosis and type II diabetes either untreated or treated with insulin or with daonil. Type I diabetes without ketoacidosis increased the rate of malformations to 27% vs. 11% in controls. Serum from type I diabetes with ketoacidosis further increased the malformation rate to 44%. The rate of malformations induced by serum of type II diabetes was dependent on the treatment. It was relatively low among embryos cultured on serum from untreated (16%) or treated with daonil (19%) and rose to 27% among embryos cultured on serum from type II diabetes treated with insulin. No significant correlation was found between the rate of malformations and the concentrations of glucose, B-HOB, acetoacetate, and HbA1c in all diabetic sera except serum from type I diabetes with ketoacidosis. We may therefore conclude that for most types of diabetes in humans, neither the high blood glucose concentrations nor the high levels of ketone bodies seem to be the main reason for the high rate of malformations. However, we used cultured rat embryos, and the effects on the human embryo may be different. The results of studies on various experimental animal models in diabetes teratogenicity seem to have only partial relevance to the human situation.     

Heat-shock induced tolerance to the embryotoxic effects of hyperthermia and cadmium in mouse embryos in vitro

Mammalian embryos growing in vitro are harmed by short elevations in the culture temperature. However, a relatively mild hyperthermic exposure can induce thermotolerance, a transient state of resistance to the effects of a subsequent heat exposure. The present study examines the induction of tolerance to heat and cross-tolerance to another teratogen, cadmium, in day 8 CD-1 mouse embryos in vitro. The ability of a mild heat pretreatment (5 min at 43 degrees C) to partially protect embryos against an embryotoxic heat exposure (20 min at 43 degrees C) was demonstrated. The frequency of death was reduced from 43% to 20%, abnormal branchial arches from 44% to 13.2%, and retarded turning from 22% to 5% in pretreated embryos. Other malformations, such as small forebrains and microphthalmia, were not affected, and the rate of exencephaly was significantly increased. The same heat pretreatment (5 min at 43 degrees C) was also found to reduce the damaging effects of a subsequent exposure to 1.75 microM cadmium. In the absence of pretreatment, cadmium caused 55% embryo deaths and 87% malformations, but prior heat exposure caused significant reductions in these frequencies to 29% and 55%. The total morphological score was higher in the pretreated group, as were the measurements of the yolk sac diameter, crown-rump length, and head length. Thus, embryos that have developed resistance to hyperthermia are also partially protected against the harmful effects of a second teratogen, cadmium. The response of the embryo to elevated temperatures may be involved in the development of tolerance to a variety of stresses.     

Effects of secondary bile acids on the in vitro development of early somite rat embryos

Effects of secondary bile acids--lithocholic (LCA) and deoxycholic (DCA)--on the in vitro development of early somite (10.5 days old) rat embryos were studied. It was shown that an addition to the culture medium of 0.1 mM LCA (final concentration) resulted in 9% growth-retarded and 12% malformed embryos when the duration of exposure was 24 h. When treatment with LCA was prolonged to 48 h, the rate of growth retardation increased to 18% and that of malformations to 40% versus 0.5% for both parameters observed in controls. This could be interpreted as a reversible or time-dependent effect of LCA on the in vitro development of the mammalian embryo. Culture of embryos in medium with 0.5 mM DCA resulted in 22% of growth retardation and 50% of malformations. DCA in 0.1 mM final concentration had only slight and statistically nonsignificant effects. Retardation of growth development could be demonstrated by a decrease in crown-rump length and the number of somites. Among malformed embryos, abnormalities in the development of the neural tube and exencephaly were the most common types of malformations. Abnormalities as well as growth retardation were accompanied by significant pathological changes in structure and perhaps in function of the endodermal visceral yolk sac cells. It could be suggested that secondary bile acids when present in pathophysiological concentrations can affect the embryonic development by direct inhibitory effects and that these effects may be time and dose dependent.     

Detection of sperm antigens on mouse ova and early embryos.

Serum and colostrum antibodies against mouse sperm were developed in two rabbits after systemic and mammary gland immunizations. Indirect immunofluorescence utilizing fluorescein-labeled goat antisera against rabbit IgG and IgA, respectively, indicated that both immune serum (IS) and colostrum (IC) compared with control samples caused intensive staining of the acrosome and tail of sperm. Absorption of IS and IC with mouse serum and the spleen, kidney, liver, and brain of male mice did not reduce the strength or the pattern of staining reaction on sperm. The absorbed IS reacted with cell surfaces of oocytes, unfertilized ova, zygotes, two-cell and four- to eight-cell fertilized ova, and blastocysts. The absorbed IC, however, reacted only with the four- to eight-cell embryo and blastocyst. Further absorption of the IS with mouse ovary removed the reaction with unfertilized ova and the one- to two-cell fertilized ova, but the staining of later embryo stages was unaffected. Therefore, it appears that specific rabbit anti-sperm antibodies are detecting two cell-membrane antigens on mouse embryos: one originating from the ovary and the other arising after fertilization.     

Effect of maternal parity on aneuploidy in early mouse embryos.

An attempt to evaluate the incidence of chromosomal aneuploidy in mouse blastocysts recovered from females of various ages and parity levels revealed an insignificant regression coefficient for aneuploidy on age of the female and its square, and an insignificant correlation coefficient for aneuploidy with the number of previous offspring born to the dam. However, significant regression coefficients were obtained for aneuploidy on parity of the dam and its square. These results indicate that not only does aneuploidy increase with parity level, but the rate of increase accelerates as parity level increases. Possible explanations for the increase in aneuploidy and its detrimental effect on reproductive efficiency were discussed.     

The antiteratogenic effects of hypo- and hyperthermia in trypan blue-treated chick embryos

Fertile White Leghorn chicken eggs (N = 174) were incubated under optimum conditions until the embryos had reached Hamburger-Hamilton stage 12 (about 48 hr). At that time, 20 μl of 1% trypan blue solution, dissolved in 0.85% NaCl (wt/vol) was injected through the yolk sac into the liquid yolk found just under the embryo. After injection, the eggs were separated into groups and returned to the incubator under control conditions (38°C), or at temperatures lower (35°C) or higher (41°C) than optimum.After an additional 24 hr of incubation, the embryos incubated at 35°C (N = 53) exhibited significantly fewer caudal hematomas (P < 0.02) than did embryos incubated at 38°C (N = 51). Similarly, embryos incubated at 41°C (N = 40) also exhibited significantly fewer caudal hematomas (P < 0.05) than did their corresponding (38°C) controls (N = 30). There was no significant difference between the 35°C group and their controls, or the 38°C group and their controls, in embryonic dry weight, dry weight of the area vasculosa, or crown-rump length. The only other significant difference detected between groups was a very slight but significant (P < 0.0005) decrease in Hamburger-Hamilton stage (0.4 stage unit) between embryos incubated at 35°C and the corresponding controls.Since incubation temperatures either above or below optimum result in a marked reduction in the teratogenic response to trypan blue treatment, we conclude that there exists a temperature optimum for the development of caudal hematomas.