Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Heat shock affects cell cycling in the neural plate of cultured rat embryos: a flow cytometric study

The effects of heat shock on cell cycling in the mammalian neuroectoderm were determined by applying heat shocks to cultured rat embryos at the neural plate stage, as part of a study on the teratogenic effects of heat shock on neural development. The heat shocks had been characterized previously (Walsh et al.: Teratology 36:181-191, 1987) with respect to their effects on the gross morphological development of the rat embryos. The effects on cell cycling were observed in DNA histograms of neural plate cells recorded in a flow cytometer after staining with DAPI. The mild heat shock (42 degrees C for 10 min) arrested cells at entry to S phase. The teratogenic heat shock (43 degrees C for 7.5 min) arrested cells at entry to S phase also but for a longer time and inhibited cycling through S phase. After each arrest, a synchronized peak of cells later entered S phase and progressed through the cycle. The induced-thermotolerance heat shock, which was the mild heat shock followed after an interval by the teratogenic heat shock, showed that pre-treatment with the mild heat shock reduced the magnitude of the response to the teratogenic heat shock. The cell-cycle inhibitor ICRF 159 showed the effects on cycling rates of the heat-shock treatments. The arrest of cells at entry to S phase by heat shock may function to prevent cells entering DNA synthesis under non-optimal conditions. We report estimates of proportions of non-proliferative cells in the neural plate of the rat embryos.     

Morphogenesis after heat shock during the cell cycle ofLymnaea: A new interpretation

Summary The effect of a heat shock (37.0–38.0°C, 10 min) during the third and fourth cleavage cycles ofLymnaea was investigated. The sensitivity with respect to the duration of the cell cycle and morphogenesis appeared to be periodic. The cycle extension curve has three maxima: at the beginning of the cycle, at the G₂-phase, and at prometaphase. With regard to morphogenesis, the eggs become sensitive shortly before cleavage, when cleavage cannot be delayed any more.In eggs treated at the morphogenetically sensitive stages, mitotic abnormalities caused by an incomplete separation of the chromosomes during treatment were observed. Some cells were lethally affected, and the division chronology was abnormal in some embryos.It is concluded that heat shock disturbs a process relevant to the cell cycle. If applied before metaphase, an extension of the cell cycle permits a complete recovery and morphogenesis remains unaffected. If applied at metaphase or later, cell division is not delayed, but mitosis is seriously disturbed. This irreversible damage is the cause of abnormal morphogenesis. The type of malformation depends on the prospective significance of the affected blastomeres.     

Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos

Although sensitive to various disrupters, pre-implantation embryos possess some cellular cytoprotective mechanisms that allow continued survival in the face of a deleterious environment. For stresses such as heat shock, embryonic resistance increases as development proceeds. Present objectives were to determine whether (1) arsenic compromises development of pre-implantation bovine embryos, (2) developmental changes in embryonic resistance to arsenic mimic those seen for resistance to heat shock, and (3) developmental patterns in induction of apoptosis by arsenic are correlated with similar changes in resistance of embryos to inhibitory effects of arsenic on development. Bovine embryos produced by in vitro fertilization were exposed at the two-cell stage or at day 5 after insemination (embryos > or = 16-cells in number) to either sodium arsenite (0, 1, 5, or 10 microM) or heat shock (exposure to 41 degrees C for 0, 3, 4.5, 6, or 9 hr). Arsenic induced apoptosis and increased group 2 caspase activity for embryos at the > or = 16-cell stage, but not for embryos at the two-cell stage. In contrast to these developmental changes in apoptosis responses, exposure to arsenic reduced cell number 24 hr after exposure for both two-cell embryos and embryos > or = 16-cells. Similarly, the percentage of embryos that developed to the blastocyst stage at day 8 after fertilization was reduced by arsenic exposure at both stages of development. Heat shock, conversely, reduced development to the blastocyst stage when applied at the two-cell stage, but not when applied to embryos > or = 16-cells at day 5 after insemination. In conclusion, arsenic can compromise development of bovine pre-implantation embryos, the temporal window of sensitivity of embryos to arsenic is wider than for heat shock, and cellular cytoprotective responses that embryos acquire for thermal resistance are not sufficient to cause increased embryonic resistance to arsenic exposure. It is likely that despite common cellular pathologies caused by arsenic and heat shock, arsenic acts to reduce development in part through biochemical pathways not activated by heat shock. Moreover, the embryo does not acquire significant resistance to these perturbations within the time frame in development examined.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Modification of actions of heat shock on development and apoptosis of cultured preimplantation bovine embryos by oxygen concentration and dithiothreitol

Preimplantation embryos exposed to elevated temperatures have reduced developmental competence. The involvement of reactive oxygen species in these effects has been controversial. Here we tested hypotheses that (1) heat shock effects on development and apoptosis would be greater when embryos were cultured in a high oxygen environment (air; oxygen concentration = approximately 20.95%, v/v) than in a low oxygen environment (5% oxygen) and (2) that these effects would be reversed by addition of the antioxidant dithiothreitol (DTT). Heat shock of 41 degrees C for 9 hr reduced development of two-cell embryos and Day 5 embryos to the blastocyst stage embryos when in high oxygen. There was no effect of heat shock on development when embryos were in low oxygen. Furthermore, induction of TUNEL-positive cells in Day 5 embryos by heat shock only occurred when embryos were in high oxygen. Addition of DTT to two-cell embryos either did not reduce effects of a heat shock of 41 degrees C for 15 hr on development or caused slight protection only. In contrast, treatment of Day 5 embryos with DTT reduced effects of heat shock on development and apoptosis. In summary, oxygen tension was shown to be a major determinant of the effects of heat shock on development and apoptosis in preimplantation bovine embryos. Protective effects of the antioxidant DTT were stage specific and more pronounced at later stages of development.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock

During normal development in D. melanogaster, messenger RNAs for three of the seven heat shock proteins (hsp83, hsp28 and hsp26) accumulate in adult ovaries and are abundant in embryos until blastoderm. The three mRNAs appear to originate in nurse cells and subsequently pass, during stages 10-11, into the oocyte. Little if any of the four other heat shock mRNAs is present in unshocked ovaries or embryos at any time examined. Pre-blastoderm embryos fail to accumulate these heat shock mRNAs even if subjected to heat shock. The accumulation in normal oogenesis of mRNAs for only three of the seven heat shock proteins indicates the existence of differential, possibly multiple controls of heat shock gene expression, and suggests that heat shock proteins hsp83, hsp28 and hsp26 function in the oocyte or early embryo.     

Factors influencing the heat shock response of Xenopus laevis embryos

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.     

Insulin-like growth factor-I as a survival factor for the bovine preimplantation embryo exposed to heat shock

Insulin-like growth factor-I (IGF-I) is a survival factor for preimplantation mammalian embryos exposed to stress. One stress that compromises preimplantation embryonic development is elevated temperature (i.e., heat shock). Using bovine embryos produced in vitro as a model, it was hypothesized that IGF-I would protect preimplantation embryos by reducing the effects of heat shock on total cell number, the proportion of blastomeres that undergo apoptosis, and the percentage of embryos developing to the blastocyst stage. In experiment 1, embryos were cultured with or without IGF-I; on Day 5 after insemination, embryos >or=16 cells were cultured at 38.5 degrees C for 24 h or were subjected to 41 degrees C for 9 h followed by 38.5 degrees C for 15 h. Heat shock reduced the total cell number at 24 h after initiation of heat shock and increased the percentage of blastomeres that were apoptotic. Effects of heat shock were less for IGF-I-treated embryos. Experiment 2 was conducted similarly except that embryos were allowed to develop to Day 8 after insemination. The percentage reduction in blastocyst development for heat-shocked embryos compared with those maintained at 38.5 degrees C was less for embryos cultured with IGF-I than for control embryos. Heat shock reduced the total cell number in blastocysts and increased the percentage of blastomeres that were apoptotic, whereas IGF-I-treated embryos had increased total cell number and a reduced percentage of apoptosis. Taken together, these results demonstrate that IGF-I can serve as a survival factor for preimplantation bovine embryos exposed to heat shock by reducing the effects of heat shock on development and apoptosis.     

Development profile of the heat shock response in early embryos of Drosophila

Drosophila melanogaster embryos reared at 22 degrees C were subjected to a mild heat shock (40 min at 37 degrees C) at various ages in order to determine whether there are changes in the heat shock response during embryogenesis. The effects of the heat shock were measured by assaying (1), subsequent developmental abnormalities (2), developmental time (3), hatchability, and (4), the ability to synthesize the heat shock proteins as assayed by 35S-methionine pulse labeling followed by protein separations using both one-and two-dimensional polyacrylamide gel electrophoresis. Our data show that, first, proteins with molecular weights similar to those of six of the seven major heat shock proteins are normally found in the embryo at control temperatures (22 degrees C); second, that the pregastrula embryo (stages 2-6) is not capable of displaying any aspect of the heat shock response upon treatment, although it may possess all of the so-called heat shock proteins; third, that the complete heat shock response is acquired very rapidly by early gastrula embryos; and fourth, that the heat shock treatment brings about developmental delays and/or abnormalities, depending on the developmental stage of the embryo at the time of the treatment. These developmental abnormalities appear to stem from the failure of early embryos to completely inhibit their synthesis of non-heat-shock proteins. In the light of these findings, it becomes important not to base conclusions about the putative presence of a heat shock response in a particular tissue or developmental stage solely on the presence or absence of the heat shock proteins.     

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

Effects of heat shock on in vitro development and intracellular oxidative state of bovine preimplantation embryos

We investigated the effects of heat shock on developmental competence of bovine embryos and intracellular oxidative state. After in vitro fertilization, embryos were exposed to heat shock at 41 degrees C for 6 hr on days 0, 2, 4, and 6, respectively. On day 2, cleavage rate was not significantly different in all groups. However, the percentage of embryos developing to blastocyst stage after exposure to heat shock on day 0 (18.8 +/- 4.3%) and day 2 (23.6 +/- 3.7%) were significantly decreased compared with control (37.5 +/- 4.0%), day 4 (40.0 +/- 7.4%), and day 6 (38.1 +/- 2.0%). In addition, the total cell number of blastocysts was significantly decreased by heat shock on day 0 (107.5 +/- 6.6) and day 2 (112.8 +/- 5.7) compared with the control (143.2 +/- 9.4). To evaluate intracellular oxidative state by heat shock, embryos exposed to heat shock on days 0, 2, 4, and 6 were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescence of oxidized DCHFDA by reactive oxygen species (ROS) was detected under fluorescent microscope. The intensity of fluorescence was significantly increased when embryos were exposed to heat shock on days 0 and 2. However, heat shock on day 4 and day 6 did not increase the fluorescence intensity. These results indicate that (1) heat shock to earlier stage embryos causes a decrease in development to blastocysts and cell proliferation and (2) the decrease in development by heat shock could be involved in an increase of intracellular oxidative stress. Mol. Reprod. Dev. 67: 77-82, 2004.     

Impact of ecologically relevant heat shocks on Hsp developmental function in the vetigastropod Haliotis asinina

Heat shock proteins (Hsps) are essential for cellular maintenance, normal differentiation and morphogenesis, and protection against a range of environmental stresses. It is unknown which of these roles takes precedence when they are required simultaneously. Here we examined the impact of thermal stress on the complex developmental expression patterns of HasHsp70 and HasHsp90A in the vetigastropod Haliotis asinina. We find that near-lethal heat shocks do not alter the spatial demarcation of Hsp expression despite such treatments impacting on the external character of the embryos. Using a suite of molecular markers that are both coexpressed with the Hsps (i.e. in ventrolateral ectoderm and prototroch) and expressed in tissues that have lower (basal) Hsp expression (e.g. serotonergic nervous system and shell gland), we determined that Hsp-expressing tissues do not incur markedly less thermal damage than adjacent tissues. To explore the relationship of Hsp expression with sensitivity of specific cell territories to heat shock, we focused on the formation of the prototroch, a tissue where HasHsp70 and HasHsp90A are coexpressed. By heat shocking at specific developmental stages, we determined that the most sensitive period of prototroch development is during its early specification and differentiation, which overlaps with the time the Hsps are expressed at their highest levels in these cells. This correlation is consistent with heat shock impairing the function of Hsps in regions of the H. asinina embryo undergoing morphogenesis.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Summary Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41° C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37° and 41° C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Response to heat shock of different sea urchin species

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.