Heat-induced alterations in embryonic cytoskeletal and stress proteins precede somite malformations in rat embryos.

Previous work from this laboratory has demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development 24 hr after exposure and subsequent thoracic skeletal malformations in neonates. The purpose of the present study was to examine the effects of in vitro heat shock on de novo protein synthesis and on cytoskeletal protein levels in developing rat embryos. Explanted GD10 embryos were exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure (0-27 hr). embryos were labeled with 35S-methionine and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. The 70-kD protein was identified as a eukaryotic stress protein and the presence of this protein was detected between 2 and 27 hr posttreatment. Western blot analysis was used to detect quantitative changes in total actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments). Immediately following exposure, a reduction of total vimentin to minimal detectable levels was observed in heat-treated embryos. Levels of total vimentin remained depressed for more than 2 hr and gradually returned to control levels 4-8 hr postexposure. No change in total actin or tubulin was detected in treated embryos. The data demonstrate that heat-induced alterations in proteins comprising intermediate filaments occur concomitantly with the induction of stress proteins and precede aberrant somite morphology. These alterations in embryonic proteins may help elucidate the mechanism(s) by which skeletal malformations are produced.

Induction of hsp72 in heat-treated rat embryos: a tissue-specific response.

Previous studies have demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development in rat embryos 24 hr after exposure and in thoracic skeletal malformations in neonatal rats examined 3 days postpartum. The production of abnormal somites was correlated with the location of skeletal elements that developed from the affected somites. Heat has also been shown to induce changes in genetic expression whereby new proteins are synthesized and the expression of constituent proteins may be repressed. In the present study, heat-induced alterations in protein synthesis during rat organogenesis that may be associated with previously observed malformation was investigated. GD10 rat embryos were exposed in utero to a heat treatment previously demonstrated to produce skeletal malformations; maternal core temperature was raised and maintained at 42-42.4 degrees C for 5 min. In addition, explanted GD10 embryos were cultured in vitro and exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure, embryos were labeled with 35S-methionine and processed for SDS-PAGE. In both in vivo and in vitro heat-treated embryos, a transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. Actinomycin D studies were conducted to determine whether transcription of new mRNA was required for the enhanced synthesis of the 70- and 90-kD proteins in heat-treated embryos. Results from these studies demonstrated that the expression of these proteins was transcriptionally regulated. The 70-kD protein was identified, using Western blot analysis, as a eukaryotic inducible stress protein (hsp72), and the presence of this protein was detected between 2 and 27 hr post-treatment. Immunohistochemical results indicated that following heat shock, hsp72 accumulates in the neuroectodermal tissues of the embryos. The data demonstrate that although heat-induced expression and accumulation of the hsp72 precedes aberrant somite morphology, the lack of hsp72 accumulation in the somite mesoderm may explain the sensitivity of this tissue to heat.

Relationship between abnormal somite development and axial skeletal defects in rats following heat exposure.

The effects of in vivo heat exposure on gestation day (GD) 10 rat embryos were evaluated on GD 11 to determine the relationships between morphological sequelae following in vivo and in vitro exposures and between effects detected on GD 11 and those observed in postnatal day (PND) 3 pups. Anesthetized rats were exposed to 42 degrees C in a warm air incubator until their rectal temperatures reached 41 degrees C or until a rectal temperature of 42-42.5 degrees C had been maintained for 5 minutes. Heat-exposed embryos exhibited a significant decrease in growth parameters including head length, somite number, and protein content/embryo versus controls. These changes correlated well with in vitro effects from an earlier study (G.L. Kimmel et al., '93). Among the morphological endpoints which were slightly delayed in development were the caudal neural tube, branchial bars, forelimb and hindlimb. The only effect on the embryos that could not be explained as a transient delay in development induced by heat was the induction of unsegmented somites. Additional embryos were exposed to 42 degrees C for 15-20 min in vitro and examined specifically for unsegmented somites, which were observed in 47% of embryos exposed to 42 degrees C in vivo or in vitro. This phenomenon was observed in somites 9-20, i.e., those that give rise to cervical and thoracic vertebrae and ribs. These results correlated well with the axial skeletal malformations observed in PND 3 pups exposed to the same heat treatment (C.A. Kimmel et al., '93).

Skeletal development following heat exposure in the rat.

The effects of gestation day (GD) 10 heat exposure in the rat were studied to determine the temperature-response relationship for the induction of skeletal and other defects. Conscious pregnant rats (Experiment 1) were exposed to various temperatures in a warm air chamber. Body temperature was measured using a rectal probe, and these measurements were confirmed as representing core body temperature in separate animals using telemetric procedures. Those animals whose core body temperature was raised to 41-41.9 degrees C had over 90% malformed pups (examined at postnatal day (PND) 3), and a 25% reduction in the percent of live pups per litter. Animals whose temperature was raised to 39.2-40.9 degrees C had a low incidence of pups with similar types of malformations. The primary types of malformations were of the axial skeleton, consisting of fusions and other abnormalities of the ribs and vertebral elements, and a decrease in the total number of ribs and centra. The acute maternal effects of these temperature increases were signs of heat exhaustion during and 1-2 hr after exposure, but there were no permanent changes in weight gain or other signs. When temperatures were raised to > or = 42 degrees C, all maternal animals died. In a second study (Experiment 2), pregnant rats (from a different supplier) were anesthetized to determine the effect of reducing maternal stress and were exposed to heat as in Experiment 1. Those animals whose core body temperature was raised to 42-42.5 degrees C for 5 min had pups with similar responses to those in Experiment 1 at 41-41.9 degrees C, although the reduction in litter size was not as great. Animals whose temperature was raised to 41 degrees C had a much lower incidence of pups with similar defects, and animals whose temperature was raised to 43 degrees C did not survive. A more detailed analysis of the skeletal defects in Experiment 2 showed rib and vertebral malformations that appear to be related to the stage of somite development at the time of exposure.

Embryonic development in vitro following short-duration exposure to heat.

Gestation day (GD) 10 rat embryos (10-12 somites) were exposed in vitro for 10 to 25 minutes at 42 or 43 degrees C and evaluated 24 hrs later for alterations in growth and specific morphological parameters, using a modified Brown-Fabro (Brown and Fabro: Teratology, 24:65-78, '81) scoring system that allowed evaluation of development relative to gestational age. At 42 degrees C, crown-rump length appeared to be particularly sensitive, responding to only 10 mins exposure. A 15-min exposure resulted in decreased total protein, somite number and morphological score. No system was uniquely sensitive, since all parameters demonstrated some degree of response. Rather, systems affected were those that would be developing most rapidly at this time in gestation. At 43 degrees C, all of the parameters measured were affected by a 10-min exposure. These results demonstrate alterations in vitro after much shorter exposure periods than previously reported on GD10, which may be due, in part, to the use of a modified scoring system that permitted the evaluation of graded individual end point changes relative to gestational age. The response patterns demonstrated a clear temperature- and exposure duration-dependency, with a shift from a more shallow duration-response curve to a more dramatic inhibition of development as temperature increased from 42 degrees C to 43 degrees C.