In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.     

Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Transfer of cultured bovine embryos

A total of 126 bovine embryos were surgically collected from 16 superovulated donor heifers 5 days after estrus and randomly selected for either immediate transfer to synchronized recipients or $\text{in}$$\text{vitro}$ culture at 37°C for 24 hours and subsequent transfer. Twenty-four of 56 (42.8%) embryos maintained for 24 hours in Ham's F10 medium supplemented with 10% heat treated fetal calf serum (HTFCS) and transferred to 32 recipients produced live calves. Survival of 70 noncultured embryos transferred to 35 recipients was 55.7% (39 calves). The percentages of recipients that were diagnosed pregnant at 42 days with cultured and control embryos were 59.4% ($\text{19}\text{32}$) and 74.3% ($\text{26}\text{35}$), respectively. No statistical difference was observed between the $\text{in}$$\text{vitro}$ cultured and control embryos for viability following transfer to recipient females.In a second study, Day 7 embryos maintained in Ham's F10 medium supplemented with 10% HTFC serum for various culture periods were tested for viability following nonsurgical transfer to recipient females. A total of $\text{1}\text{5}$, $\text{1}\text{3}$ and $\text{0}\text{4}$ embryos cultured for 24, 48 and 72 hours, respectively, resulted in pregnant recipients following transfer.     

Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis.

The intracellular distribution of ¹¹⁵cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling $\text{in}\text{vivo}$ - $\text{in}\text{vitro}$ experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.     

Commercial splitting of bovine embryos

Independent studies were undertaken at Alberta Livestock Transplants (ALT) and at Select Embryos, Inc. (SEI) to develop procedures for splitting bovine embryos. At both locations embryos were recovered seven days after the onset of estrus and superovulation. Initially, survival after splitting was evaluated by culture $\text{in}$$\text{vitro}$ for 18 to 24 hours. Culturing half or demi-embryos without a zona pellucida at ALT resulted in 15% survival compared to 35% survival when both halves were in separate zonae. Culturing demi-embryos on a monolayer of luteal cells at SEI did not improve survival $\text{in}$$\text{vitro}$. In fertility trials, best results were obtained at ALT when both demi-embryos within separate zonae were nonsurgically transferred into separate uterine horns of the same recipient (55% pregnancy rate) and at SEI when one demi-embryo was surgically transferred into the uterine horn ipsilateral to the corpus luteum (65% pregnancy rate). Culturing demi-embryos more than 4 hours reduced fertility at both locations. Splitting embryos was a worthwhile addition to the commercial ET programs and further trials are in progress to improve survival $\text{in}$$\text{vitro}$ and pregnancy rates.     

Cell surface antigens of totipotent mouse teratocarcinoma cells grown in vivo: their relation to embryo, adult, and tumor antigens.

Cell surface antigens on mouse embryonal carcinoma (or teratocarcinoma) cells were investigated by means of a syngeneic antiserum prepared against small-size embryoid bodies from the ascites form of the OTT 6050 transplantable teratoma. These embryoid bodies consist of embryonal carcinoma cells which are usually covered by a yolk-sac-like epithelium. The choice of immunogen was based on the previous demonstration [Mintz, B., and Illmensee, K. (1975) Proc. Nat. Acad. Sci. USA72, 3585–3589] that embryonal carcinoma cells from this specific source are euploid, developmentally totipotent, and completely reversible to normalcy. In indirect immunofluorescence tests, anti-embryoid-body serum reacted with both cell types of the immunogen and with two in vitro lines of embryonal carcinoma cells. Absorption of antiserum with a pure yolk sac carcinoma derived from the epithelial component of the embryoid bodies enabled assessment of reactivity with the embryonal carcinoma component of the immunogen: The absorption revealed that some antigens recognized on the embryonal carcinoma cells were shared by the yolk sac epithelial cells but that some antigens were present only on the embryonal carcinoma cells. The antigens were not shared by sperm, which failed to fluoresce with unabsorbed antiserum and were ineffective when tested as absorbents of antiserum reactivity against embryoid body target cells. Unfertilized eggs also failed to fluoresce. Preimplantation embryos gave immunofluorescence evidence of some antigens shared with embryonal carcinoma cells (and some with yolk sac cells) during cleavage, and in the blastocyst on both inner cell mass and trophoblast. Postimplantation embryos were also antigen-positive (at least through Day 6) in immunofluorescence tests on endoderm as well as ectoderm cells. Absorption of the antiserum with various normal adult tissues showed substantial cross-reactivity, especially with ovary and testis. Other tumors were tested, but only hepatoma cells grown in vitro were reactive, thereby indicating lack of any general tumor recognition in the antiserum. The above results with syngeneic immunizations demonstrate that known totipotent teratocarcinoma cells possess surface molecules which, while not universal on normal cells or tumors, are shared with many other tissues, including developmentally plastic cells of early embryos, developmentally restricted cells of later embryos, and various adult tissues. Immunofluorescence tests of cleavage-stage (Day 2) embryos from matings of $\text{+}\text{t}{}^{12}\text{×}\text{+}\text{t}{}^{12}$ heterozygotes, yielding 40% mutant $\text{t}{}^{12}\text{t}{}^{12}$ homozygotes lethal on Day 3, were uniformly positive on all the embryos, including mutants and normals. Therefore, under these conditions, no evidence was adduced to support the hypothesis that surface components required for normal early development might be coded by the wild-type allele of t¹².     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Poly(adenosine diphosphate ribose) synthesis by isolated nuclei of Xenopus,laevis embryos: Invitro elongation of invivo synthesized chains

We have studied the synthesis of poly(ADP-ribose) by nuclei isolated from $\text{Xenopus}\text{laevis}$ embryos at different stages of development. Determination of the total chain length of poly(ADP-ribose) molecules by hydroxylapatite column chromatography generally gave higher values than when the radioactive portions of these molecules, synthesized $\text{in}\text{vitro}$, were measured by poly(ethyleneimine)-cellulose thin layer chromatography, after snake venom phosphodiesterase digestion. The results show that most of the poly(ADP-ribose) synthesized $\text{in}\text{vitro}$ is a covalent elongation of molecules previously initiated $\text{in}\text{vivo}$.     

Teratogenicity of acetaldehyde invitro: Relevance to the fatal alcohol syndrome

Day 10 rat embryos grown $\text{in}$$\text{vitro}$ showed significant retardation in growth and development when culture media contained acetaldehyde. A concentration-response range for acetaldehyde-induced embryotoxicity was defined, from no effect at 5μM to complete lethality at 100μM. The relative teratogenicity of ethanol and acetaldehyde, and the potential roles of these compounds in producing the Fetal Alcohol Syndrome are discussed.Despite intensive investigation into alcohol teratogenicity, the mechanism that produces the Fetal Alcohol Syndrome (FAS) remains unknown. Observed anomalies may result from direct embryonic exposure to ethanol or one of its metabolites, or from some indirect effect such as altered placental function or maternal nutritional status. Use of $\text{in}$$\text{vitro}$ techniques allows study of direct embryonic exposures in the absence of indirect influences. Under such conditions, ethanol has been found to exert direct embryotoxicity (1). Rat embryos, grown as cultured explants and subjected to ethanol concentrations of 32.5 or 65mM, were retarded in growth and development when compared to untreated controls. In this paper, we report direct embryotoxic effects of acetaldehyde, the primary metabolite of ethanol, at concentrations as low as 25μM.Acetaldehyde teratogenicity has not been extensively studied. Veghelyi et al. (2) and Lambert, Papp and Nishiura (3) employed a combination of ethanol and disulfiram (an inhibitor of acetaldehyde-oxidizing enzymes). Teratogenic effects exceeded expectations based upon assumption of an additive interaction between these two compounds, and were attributed to elevated maternal blood acetaldehyde. O'Shea and Kauffman (4,5) and Dreosti et al. (6) administered acetaldehyde to pregnant animals by injection. Treatment resulted in retarded growth and development, decreased DNA synthesis, and increased frequencies of malformation and resorption. While these studies imply a role for acetaldehyde in alcohol-induced teratogenesis, indirect effects through altered maternal or placental factors cannot be eliminated. We present here the first concentration-response data for direct ebryonic exposure to acetaldehyde.     

An organ culture system for study of fetal lung development

Fetal rat lungs placed in $\text{in}$$\text{vitro}$ organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development $\text{in}$$\text{vivo}$. Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system $\text{in}$$\text{vitro}$ were similar to those produced $\text{in}$$\text{vivo}$ in fetal lung at 21 days gestation.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

Comparison of invivo and invitro uterine contractility in the ewe

Uterine contractions were observed $\text{in}$$\text{vivo}$ by laparotomy and exposure of the uterus. Ten hours after the beginning of estrus, an average of 39 contractions per 10 min originated in the posterior ends of the uterine horns and moved toward the oviducts, while an average of, 13 contractions originated in the anterior ends of the horns and moved toward the cervix. Two days later (58 hr after the beginning of estrus), the contractions had changed in origin and direction; only 6 contractions originated in the posterior ends of the horns and moved anteriorly, while 39 originated in the anterior ends and moved posteriorly.Experiments were done to determine whether the change in origin of contractions $\text{in}$$\text{vivo}$ was reflected in the contractility of strips of myometrium in a tissue bath. The number and amplitude of contractions were recorded from strips of circular and of longitudinal myometrium taken from the posterior and anterior ends of the uterine horns at 10 and 58 hr after the beginning of estrus. The myometrial strips contracted approximately 3 to 4 times per minute regardless of the time after the beginning of estrus, the end of the uterine horn from which the tissue was taken, or whether the contracting muscle was circular or longitudinal. Thus, the physiological mechanisms that controlled the number and origin of uterine contractions $\text{in}$$\text{vivo}$ did not maintain that control over myometrial tissue $\text{in}$$\text{vitro}$.     

The effect of visible light on the regeneration of rhodopsin

Both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$, increased exposure to visible light decreases the regenerability of the visual pigment. Isolated opsin irradiated with increasing periods of white light decreased in pigment formation yields on combination with 9- or 11-$\text{cis}$ retinal. The yield of regeneration of the visual pigment extracted from albino rats depended on the amount of light to which the animal had been exposed. Animals exposed to normal room light demonstrated lower regeneration yields than dark-reared animals, but these yields increased on dark adaption. Opsin from animals exposed to sunlamps did not regenerate any pigment. On dark adaption, the pigment yields increased but the opsin level remained below that for the control group.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

Improved Development of “Rat Egg-Cylinders” <Emphasis Type="Italic">in vitro</Emphasis> as a Result of Fusion of the Heart Primordia

EMBRYOS at the stage of formation of the primitive streak (“egg-cylinders”) are highly susceptible to teratogenic agents in vivo because of the numerous complex and closely integrated events occurring at this time¹. If an in vitro system is used for the study of teratogenesis or other aspects of embryogenesis, then development should be as near normal as possible. In a study by New and Daniel of explanted rat egg-cylinders (7.5 and 8.5 days old) cultured in homologous serum, development was usually retarded by the formation of a double heart resulting from a failure of the paired heart primordia to fuse². These embryos rarely developed a yolk sac circulation and none turned (the process of axial rotation by which the embryo adopts the characteristic foetal position). Embryos with double hearts had also been obtained previously by Goss who applied pressure between the heart primordia to prevent their fusion³. New and Daniel suggested that in their embryos in vitro the failure of the heart primordia to fuse might be an effect of mechanical stresses resulting from the abnormal shape of the yolk sac. Attempts which I have made to remove these stresses by altering the shape of the yolk sac have resulted in even more irregular development or early death of the embryo. By using “true” serum⁴ or plasma instead of serum as the nutrient medium, however, more normal development in vitro has now been achieved.     

Regulation of the in vitro synthesis of E. coli ribosomal protein L12.

The $\text{in}\text{vitro}$ DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized $\text{in}\text{vitro}$ from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene.