[Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro]. / Deistvie rostovykh factorov FGF2 i IGF2 na razvitie partenogeneticheskikh c'mbrionov myshei in utero i in vitro.

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro.

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.

Prolonged development of normal and parthenogenetic postimplantation mouse embryos in vitro.

Parthenogenetic mammalian embryos show reduced placental development and do not develop beyond the 25-somite stage. But non-parthenogenetic embryos in culture, without a functional placenta, can develop to 40 somites or more. We have therefore examined the possibility that parthenogenetic embryos might also show prolonged development in culture. After parthenogenetic activation and diploidization, 23% of CBA and 56-58% of hybrid (CBAxC57BL/6) F1 mouse eggs developed in culture to blastocysts. When transferred to pseudopregnant recipients: 60% of the CBA blastocysts implanted and 26% of these developed to somite stage embryos; 71-72% of the hybrid blastocysts implanted and 11-17% of these developed to somite stage embryos. Improved development of postimplantation embryos explanted into culture at about the 15-20 somite stage was obtained by opening the visceral yolk sac (without exteriorizing the embryo). All the normal (non-parthenogenetic) embryos cultured in this way developed to more than 35 somites and many reached 45-55 somites. Under the same conditions, 11/17 diploid parthenogenetic CBA embryos developed in culture to more than 35 somites and 5 of these reached 45 somites; and 9/28 diploid parthenogenetic (CBAxC57BL/6) F1 embryos developed to 35 somites or more and 5 of these reached 45 somites. The size and protein content of the parthenogenetic embryos after culture was less than that of the normal embryos of equivalent stages. These results raise new possibilities for the analysis of parthenogenesis and genomic imprinting, including studies of the effects of adding to the culture medium specific growth factors and demethylating agents.

Effects of the anti-progestin RU 38486 on rat embryos growing in culture.

RU 38486 is a steroid currently manufactured as an abortifacient. In view of the lack of conclusive information about teratogenic risk, it is current practice for most human embryos surviving RU 38486 to be surgically aborted. In this study the effects were examined of different concentrations of RU 38486 on postimplantation rat embryos in culture. No statistically significant effects were observed until the doses reached x 7 the 'standard' dose (x 1). The experiments provide no evidence of a teratogenic effect of RU 38486 at the levels currently used in clinical practice.

The culture of postimplantation embryos.

Culture methods for postimplantation embryos are now widely used in studies of embryo physiology, growth and development. Available methods support growth and development of rat and mouse embryos at all stages of organogenesis. The best results are obtained from embryos between head fold and early limb bud stages; overall growth and differentiation of these embryos in vitro is almost identical to that in vivo. Some examples of the application of postimplantation embryo culture to specific lines of study are given and some possible future developments of the technique are discussed.

Whole embryo culture, teratogenesis, and the estimation of teratologic risk.

Mammalian whole-embryo culture systems are now widely used and have proved useful in many studies of normal and abnormal development. The main advantages of these systems are that they allow precise control of experimental conditions and can often provide information unobtainable from in vivo studies; the main disadvantages are the rather short period of embryonic development that can be supported in culture and the present restriction of the techniques to very few species. The possibility of using whole-embryo culture systems for screening for new teratogenic agents remains controversial, but there are indications that the systems may have more potential in this area than has sometimes been supposed.

Effects of temporary cooling, and of different explantation and storage conditions, on the subsequent development of post-implantation rat embryos in vitro.

Rat embryos explanted at head fold stage were stored under various levels of hypothermia prior to culture. The storage media were Hanks' Balanced Salt Solution (BSS), 50% rat serum with 50% Dulbecco's Modification of Eagle's Medium (standard medium), or 100% rat serum. The media were gassed with 5% O2/5% CO2/90% N2 or 20% O2/5% CO2/75% N2. Subsequent development of embryos after storage at temperatures between 10 degrees C and 30 degrees C for 5 hr in Hanks' BSS, or for 5-10 hr in standard medium or serum, was similar to that of controls. Some embryos developed well even after storage for 48 hr in standard medium. Development was poorer after storage at 0 degrees C or 5 degrees C, and after storage at all temperatures in ungassed Hanks' or standard medium (pH greater than 8.0). Differences in oxygen level had little effect. For routine explantation at room temperature in (ungassed) phosphate-buffered saline solutions such as Hanks', it is recommended that the delay before transferring the embryos to the culture incubator not exceed 2-3 hr.

An in vivo/in vitro evaluation of the teratogenic action of excess vitamin A.

Pregnant rats of CFHB strain were injected 81/2 days postcoitum with a 1% suspension of retinoic acid (RA) in arachis oil to give 20 mg RA per kg body weight. Control rats were injected with arachis oil only. After 26 hours, one uterine horn was removed from each rat and the embryos cultured in serum from untreated rats. The embryos in the other horn were allowed to continue development in vivo. After a further 48 hours the cultures were terminated and the second uterine horn removed from each rat. This provided four groups of embryos for comparison: (1) embryos from RA-treated rats, (2) cultured embryos from RA-treated rats, (3) embryos from control rats, and (4) cultured embryos from control rats. The results showed that the effects of the teratogen on the cultured embryos were similar to those on the embryos allowed to continue development for the same period in the mother. In both groups RA reduced protein synthesis, inhibited somite and limb bud formation, and caused various neural tube defects, particularly microcephaly and abnormalities in the closure of the anterior and posterior neuropores.

Teratogenic action of hypolipidemic agents: an in vitro study with postimplantation rat embryos.

Two hypolipidemic agents known to be embryotoxic and teratogenic in vivo were found also to cause developmental abnormalities when added to the culture serum of rat embryos growing in vitro. However, development was normal when the embryos were grown in hypolipidemic serum (low in cholesterol and triglycerides) prepared from rats dosed with the hypolipidemic agents. The results indicated that it is a direct action of the agents on the embryos, and not the hypolipidemia, which is harmful to embryonic development.

Culture of mouse embryos during neurulation.

A comparison between static versus rotator culture systems and a variety of media (rat serum, new born calf serum, DMEM and Waymouth's) was made in an attempt to promote in vitro growth of mouse embryos from the beginning of neurulation (headfold stage) to the closure of the neural tube and formation of the limb buds (48 h). The results demonstrate that good development can be achieved for 48 h using a rotator system and that 80% of embryos cultured on rotators show growth and differentiation similar to that obtained for the same time period in vivo. Static cultures are less successful and embryos grown in this system show lower protein content and somite numbers than those maintained on rotators. Undiluted rat serum is superior to all other media tested and supports better growth and development as monitored by total protein and developmental abnormalities.

In-vitro development of the rat parietal yolk sac.

Rat embryos were cultured with the parietal yolk sac and other membranes intact. The development of the parietal yolk sac after 24 h in vitro was assessed by (1) the increase in size, (2) the morphology of the cellular components, and (3) the development of the embryo within the yolk sac. Development in vitro most resembled that in vivo when the embryos and membranes were explanted and cultured in rat serum diluted 1 : 1 with Hanks' or Waymouth's medium. High levels of oxygen were also beneficial. Culture in undiluted serum or under low oxygen concentrations resulted in very poor expansion of the yolk sac, abnormal trophoblastic giant cell morphology and stunted embryonic development.

Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos.

Rat embryos, 9 1/2 days old, cultured with a 5% or 10% O2 gas phase underwent normal or near-normal cranial neurulation; however, culture at 20% or 40% O2 resulted in abnormal morphogenesis of the cranial neural folds from the 9-somite stage onwards, and the brain tube frequently failed to close. Normal morphogenesis was characterized by a narrowing V-shaped profile, development of a slightly concave neuroepithelial surface, and formation of a sharp mediad curvature of the most lateral region prior to midline apposition and fusion. These morphogenetic events were related to cellular changes within the neuroepithelium, namely cell death, onset of neural crest cell migration, and loss of apical microfilament bundles from the most lateral cells. In 20% and 40% O2-cultured embryos, failure of curvature of the neuroepithelium was associated with failure or retardation of the related cellular changes; it may therefore have been due to the maintenance of an excessive rigidity which opposed the forces involved in bringing about the final stage of brain-tube formation. Mitochondria in normal (low O2 and in vivo) embryos were of the anaerobic type, having few cristae; in high O2-cultured embryos they were of the characteristic aerobic type, indicating an adaptation to the abnormal environment.

A rotating bottle culture method with continuous replacement of the gas phase.

A 'rotator' culture method is described which provides a continuous flow of oxygenating gas to cultures in rotating bottles. The system maintains constant O2 and CO2 levels in the culture medium throughout the incubation period. It also provides a more stable pH than systems with sealed culture bottles.

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.

Growth of opossum embryos in vitro during organogenesis.

Opossum embryos, explanted between primitive streak and late fetal stages, were grown in culture for periods of 20-30 H. Many of the explants had a good heartbeat and blood circulation in embryo and yolk sac after 12 h, and a few after 24 h. Growth of the embryos included formation of the neural tube and body flexures, increase in the number of somites, differentiation of the limbs and digits, and development of the amnion and allantois. Embryos explanted during the last day of gestation showed persistent and vigorous body movements in culture, particularly of the forelimbs, head and tongue.

Development of a placental blood circulation in rat embryos in vitro.

Rat embryos explanted with their membranes at head-fold stage (9 1/2 days gestation) formed an allantoic placenta which enlarged in culture and developed a foetal blood circulation. Embryos explanted at early somite stages (10 1/2 days) also formed a growing allantoic placenta but only after removal of most of the ectoplacental trophoblast. Assays of total protein in the embryo and placenta suggested that, in the absence of a maternal blood circulation to the placenta, embryo and placenta compete for the respiratory and nutritional resources obtained through the yolk-sac.