Summary
1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation.

2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta.

3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice.

4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac.

5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate.

6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac.

7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds.

8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.

Whole-embryo culture and the study of mammalian embryos during organogenesis.

The embryonic development of mammalian neural tube defects.

Objective. —To determine if exposure to hot tub, sauna, fever, or electric blanket during early pregnancy was associated with an increased risk for neural tube defects (NTDs). Design. —Prospective follow-up study. Setting. —Mostly private obstetric practices, primarily in New England. Participants.—A cohort of 23491 women having serum alpha-fetoprotein screening or an amniocentesis were identified. Complete exposure and outcome information was available for 97% of these women. Outcome Measures. —Relative risks (RRs) were used to compare incidence of NTD in those exposed to heat with those who were not exposed to any heat. Crude RRs were calculated directly from the data. Unconfounded RRs were calculated using logistic regression. Results. —Women reporting any heat exposure (sauna, hot tub, fever, or electric blanket) in early pregnancy had a crude risk of their fetuses developing NTD of 1.6 (95% CI [confidence interval], 0.9 to 2.9). Women reporting exposure to sauna, hot tub, or fever in early pregnancy had a crude risk of their fetuses developing NTD 2.2 times that of women without heat exposure (95% CI, 1.2 to 4.1). For hot tub use, the crude RR was 2.9 (95% CI, 1.4 to 6.3); for sauna, 2.6 (95% CI, 0.7 to 10.1); for fever, 1.9 (95% CI, 0.8 to 4.1); and for electric blanket, 1.2 (95% CI, 0.5 to 2.6). Multivariate adjusted RRs for individual heat sources, after controlling for maternal age, folic acid supplements, family history of NTD, and exposure to other heat sources, were for hot tub use, 2.8 (95% CI, 1.2 to 6.5); sauna, 1.8 (95% CI, 0.4 to 7.9); fever, 1.8 (95% CI, 0.8 to 4.1); and electric blanket, 1.2 (95% CI, 0.5 to 2.6). When only hot tub, sauna, and fever were considered and the women's exposure to each tallied, compared with no heat exposure, the RR for NTDs increased from 1.9 (95% CI, 0.9 to 3.7) after one type of heat exposure to 6.2 (95% CI, 2.2 to 17.2) after two types of heat exposure. Conclusions. —Exposure to heat in the form of hot tub, sauna, or fever in the first trimester of pregnancy was associated with an increased risk for NTDs. Hot tub exposure appeared to have the strongest effect of any single heat exposure. Exposure to electric blanket was not materially associated with increased risk for NTDs. ( JAMA . 1992;268:882-885)

Maternal Heat Exposure and Neural Tube Defects

Interest in pattern regulation arose with the first experiments of embryology almost a century ago. The hypotheses of His and Weismann (reviewed by Wilson, 1896) predicted that the fertilized egg behaves as a mosaic of autonomously differentiating parts, due to either the invisible promorphology of its cytoplasm (His) or the preexisting architecture of its chromosomes (Weismann). Roux’s first experiments in 1888 seemed to support these hypotheses: he pricked a frog egg (Rana esculenta) at the two-cell stage with a hot needle so as to inactivate one cell, hence half the egg, and found the other cell continuing to divide and in many cases developing into a half-embryo, usually a right or left half. Thus, the active cell failed to adjust its development to compensate for its defective neighbor, at least under these experimental conditions. The frog egg appeared to meet the definition of a mosaic egg, that is, one showing no pattern regulation.

Mechanisms Regulating Pattern Formation in the Amphibian Egg and Early Embryo

Although hyperthermia is teratogenic in birds, all the common laboratory animals, farm animals, and primates and satisfies defined criteria as a teratogen, its study as a human teratogen has been neglected. Homeothermic animals, including humans, can experience body temperature elevations induced by febrile infections, heavy exercise and hot environments which exceed the thresholds (1.5-2.5 degrees C elevation) which are known to cause a syndrome of embryonic resorptions, abortions, and malformations in experimental animals. Hyperthermia is particularly damaging to the central nervous system, and if a threshold exposure occurs at the appropriate stages of embryonic development, exencephaly, anencephaly, encephalocoele, micrencephaly, microphthalmia, neurogenic talipes, and arthrogryposis can be produced in a high proportion of exposed embryos, the incidence and type of defect depending on the species and strain within species, the stage of development, and the severity of hyperthermic exposure. Other defects which can be induced experimentally include exomphalos, hypoplasia of toes and teeth, renal agenesis, vertebral anomalies, maxillary hypoplasia, facial clefting, cataract, coloboma, and heart and vascular defects. Proliferating cells are particularly sensitive to temperature elevations, resulting in arrest of mitotic activity and immediate death of cells in mitosis with threshold elevations (1.5-2.5 degrees C) and delayed death of cells probably in S phase with higher elevations (3.5 degrees C). In general, lower temperature elevations (2.5 degrees C) require longer durations of elevation to cause defects than a simple spike at a higher elevation (4.5 degrees C). The death of cells is largely confined to the brain and in the day 21 guinea pig embryo to the alar regions of the brain. Cell death probably accounts for most of the defects in the central nervous system, but microvascular disturbances leading to leakage, oedema and haemorrhage, placental necrosis, and infarction are other known effects of hyperthermia; and these are probably involved in the pathogenesis of many defects of the heart, limbs, kidneys, and body wall. Recent experiments have demonstrated protection of rat embryos in culture against a known teratogenic exposure by a brief nonteratogenic exposure given at least 15 min earlier. This protection is associated with the synthesis of heat-shock proteins, and temporary arrest of the cell proliferative cycle. Hyperthermia appears to be capable of causing congenital defects in all species and may act alone or synergistically with other agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperthermia as a teratogen: A review of experimental studies and their clinical significance

Pregnant guinea-pigs were exposed to an environmental temperature of 42·0–42·5 °C for 1 h on day 21 of gestation. Their embryos were removed at periods from 45 min of heating to 48 h following exposure. Histological preparations of embryos showed clumping of nuclear chromatin and subsequent death of cells which were at about the stage of mitosis. Affected cells were particularly numerous in the central nervous system. Further mitotic activity was inhibited for 6–8 h. Squash preparations of the telencephalon at 1 h after heating showed an increase from 3 to 86 % in the number of mitotic cells showing damage in the form of nuclear clumping; this number fell progressively to 30% by 24 h after heating. The proportion of cells in various stages of mitosis changed considerably at 1–8 h after heating, but had returned to pre-heating values by 24 h. The proportion of cells in prophase fell markedly, while the proportion of metaphase cells was doubled at 4 h after heating, indicating blocks to the cell generation cycle before prophase and in metaphase.

Mitotic cell death and delay of mitotic activity in guinea-pig embryos following brief maternal hyperthermia

Guinea-pigs were exposed to a temperature of 41.5–42.5°C for 1h daily on days 20–23 of gestation. Fetuses from control and from heated mothers were studied at 30, 35, 41, 48, 55 and 62 days gestation and post-natal offspring at 69, 76, 83, 90, 104, 118, 132, 146 and 160 days after conception. Body weights, whole and regional brain weights, and the dry matter, DNA (an index of cellularity) and cholesterol (an index of myelination) contents of these brain regions were measured. There was frequently a small but variable deficit in the body weight of heated offspring. A significant reduction of whole brain weight was found in the heated groups. The deficit in brain weight was detected 7 days after the heat-stress and remained at about the same relative level until maturity. The DNA and cholesterol concentrations were the same in control and heated brains, but the absolute amounts were less in the latter which resembled proportional, normally shaped miniatures of the control brains. Hyperthermia is known to disrupt cellular proliferations, and since it was given during neurogenesis it is concluded that the size which might be attained by the brain is largely determined by the extent of early neuronal proliferation.

R. C. Mulley

Papers

Mitotic cell death and delay of mitotic activity in guinea-pig embryos following brief maternal hyperthermia

Growth and development of the brain in normal and heat‐retarded guinea‐pigs