Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.     

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.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Thermotolerance of IVM‐derived bovine oocytes and embryos after short‐term heat shock

A series of experiments were designed to study the effect of elevated temperatures on developmental competence of bovine oocytes and embryos produced in vitro. In experiment 1, the effect of heat shock (HS) by a mild elevated temperature (40.5°C) for 0, 30, or 60 min on the viability of in vitro matured (IVM) oocytes was tested following in vitro fertilization (IVF) and culture. No significant difference was observed between the control (39°C) and the heat‐treated groups in cleavage, blastocyst formation, or hatching (P > 0.05). In experiment 2, when the HS temperature was increased to 41.5°C, neither the cleavage rate nor blastocyst development was affected by treatment. However, the rate of blastocyst hatching appeared lower in the HS groups (13% in control group vs. 3.9% and 5.6% in 30 min and 60 min, respectively; P < 0.05). When IVM oocytes were treated at 43°C prior to IVF (experiment 3), no difference was detected in blastocyst and expanded blastocyst development following heat treatment for 0, 15, or 30 min, but heat treatment of oocytes for 45 or 60 min significantly reduced blastocyst and expanded blastocyst formation (P < 0.05). In experiment 4, the thermotolerance of day 3 and day 4 bovine IVF embryos were compared. When embryos were pre‐treated with a mild elevated temperature (40.5°C) for 1 hr, and then with a higher temperature (43°C) for 1 hr, no improvement in thermotolerance of the embryos was observed as compared to those treated at 43°C alone. However, a higher thermotolerance was observed in day 4 than day 3 embryos. In conclusion, treatment at 43°C, but not 40.5°C or 41.5°C significantly reduced oocyte developmental competence. An increase in thermotolerance was observed from day 3 to day 4 of in vitro embryonic development, which corresponds to the maternal to zygotic transition of gene expression in bovine embryos. Mol. Reprod. Dev. 53:336–340, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Development of normal mice after microinjection of round spermatids into oocytes stored at room temperature for one day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 hr still retained full developmental potential. In this study, we stored mouse COCs and denuded oocytes (DOs) at room temperature for 24 hr and activated these oocytes with 10 mM SrCl(2) or injected the oocytes with round spermatids. We found that DOs were better than COCs when stored at room temperature for 1 day and more normal oocytes were obtained when COCs were stored in more H-CZB medium at room temperature for 1 day. The rates of normal oocytes were significantly different after preservation with three schemes (90.01%, 55.81%, and 86.70%, P < 0.05). Our results also indicated that oocytes stored at room temperature for 1 day were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei [PN]) after microinjection of round spermatid nuclei, and that the existence of cumulus cells (CCs) during oocyte storage did not significantly influence the early cleavage but had a detrimental effect on later embryo development and full-term development. After fertilization, most embryos developed to two-cell stage after being cultured for 24 hr, and the development rates of four- to eight-cell embryos between two experiments were similar. However, the rates of morula/blastocyst formation were significantly different (47.44% and 26.27%, respectively, P < 0.05). The birth of four healthy pups from stored DOs indicated that the storage of DOs at room temperature for 1 day might become a practical procedure in mammalian reproduction.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of ¹²⁵I-insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of ¹²⁵I-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between ¹²⁵I-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress. © 1996 Wiley-Liss, Inc.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley-Liss, Inc.     

Relationships between the completion of first cleavage and the chromosomal complement,sex, and developmental rates of bovine embryos generated in vitro

One thousand eighty-four two-cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24,30,40,48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight-cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the “early” cleaving (24 and 30 hpi) and “late” cleaving (40–62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight-cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X- and Y-bearing sperm within the zygote or to very early differential expression of genes derived from X- and Y-bearing sperm. © 1993 Wiley-Liss, Inc.     

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU⁺ and Ki67⁺ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.