Physiologic Responses of Mammalian Oocytes and Embryos under Thermal
Stress: An Overview
Jung-Kai Tseng
1,2,3,Nen-Wen Lo
4,
Perng-Chih Shen
5,
Hsin-I Chiang
3,
Yang-Kwang Fan
3, Chien-Hong Chen
6, and Jyh-Cherng Ju
3,7,8,9,10*1 School of Optometry, Chung Shan Medical University, 110 Sec. 1, Jianguo N. Rd., Taichung 402, Taiwan, ROC
2 Department of Ophthalmology, Chung Shan Medical University Hospital, 110 Sec. 1, Jianguo N. Rd., Taichung 402, Taiwan, ROC
3Department of Animal Science, National Chung Hsing University, 250 Kuokuang Rd., Taichung 402, Taiwan, ROC
4Department of Animal Science and Biotechnology, Tunghai University. 181, Sec. 3, Taichung Harbor Road, Taichung 407, Taiwan, ROC
5 Department of Animal Science, National Pingtung University of Science and Technology, Neipu, Pingtung 912,Taiwan
6Animal Technology Laboratories, Agriculture Technology Research Institute,
Hsinchu City 300, Taiwan, ROC
7Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan ROC.
8Core Laboratory for Stem Cells, Medical Research Department, China Medical University Hospital, 2 Yude Rd., Taichung 40447, Taiwan ROC.
9Agriculture Biotechnology Center, National Chung Hsing University, 250, Kuokuang Rd., Taichung 402, Taiwan, ROC
10Department of Biomedical Informatics, College of Computer Science, Asia University, Taichung, Taiwan, ROC
* Corresponding author: Jyh-Cherng Ju, PhD, Professor,
Phone:+886-4-2286-2799 Fax: +886-4-2284-0265 or 886-4-2286-2199 E-mail: [email protected]
Running title: heat shock responses in oocytes and embryos
Abstract
Animals respond to environmental stresses by a global adaptation and adjustment to their physiologic homeostasis in order to eliminate most harmful changes and survive from the insults. This adaptation is the summation of all 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
responses from their building blocks, cells with their associated cellular machineries,
in response to the undesired environmental clues. It has been known that heat shock proteins (hsps), some of which are molecular chaperones presenting in all organisms, are a group of universal cellular proteins to oppose environmentally induced denaturation of many other proteins. These proteins function to assist in folding of newly synthesized proteins and maintain pre-existing proteins in a stable conformation without aggregation under stress conditions, which are essential for thermal adaptation in prokaryotes and eukaryotes, including mammalian cells. Although thermobiology has been one of the active fields of study in cell biology or cancer therapy, there is less information available for the adaptation or responses to thermal stresses in embryonic cells such as oocytes and embryos. Previous studies have shown that a short-term heat shock (HS) impacts the developmental competence of embryos during the early phase of apoptosis and the alteration of intracellular calcium concentrations of matured porcine oocytes. Cleavage and blastocyst rates declined while the Ca2+-releasing ability of matured oocytes was enhanced by a short duration (2 h) of HS, but declined after prolonged heat exposure. Taken together, the mechanisms of physiologic adaptation in response to thermal stress in oocytes and embryos are complex processes. HS can cause multiple changes to the oocyte and developing embryos such as enzymatic reactions, ionic influxes, DNA structure and cytoskeleton reorganization, as well as changes in the ooplasmic [Ca2+]i after various intensities of HS. These phenomena may be critical parameters to evaluate their developmental competence. Delicate equilibrium between the deleterious effects and thermotolerance of oocytes, embryos and even the whole animal adapting to HS is one of the decisive factors in determining their fate during the course of development.
Keywords: adaptation; embryo; oocyte; apoptosis; heat shock; hsp 70
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1. Introduction
Reduced fertility and increased embryo mortality of domestic animals by heat stress in hot seasons are prevailing in the tropical and subtropical regions, where high ambient temperature associated with high humidity reduces feed intake, growth rate and reproductive performance of animals. The deleterious effects on animals are caused by multiple factors such as suboptimal environment and management, as well as age and animal species (5). One of the major factors responsible for the reduced reproductive performance in domestic animals is heat stress caused by elevated ambient temperatures and environmental humidity. Therefore, improving reproductive efficiency under thermal insults is a major challenge for livestock breeding in tropical and subtropical countries, where the animals have actually developed a certain degree of physiological adaptation to the temperature.
However, the mechanism of heat-induced deleterious effects on oocyte or embryo viability is still unclear. Many in vitro and in vivo studies have examined the effects of prolonged heat stress, mimicking the extreme hyperthermic conditions during hot seasons, on the heat shock protein 70 (hsp 70), apoptosis, developmental 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
competence and calcium-releasing capacity of matured oocytes. For instance, a further investigation to analyze the sensitivity of nuclei or cytoplasm to heat shock will be discussed later in this review.
2. The Effect of Heat Shock on Reproductive Performance of Animals
In most mammalian species, heat stress has deleterious effects on physiological functions (13). Exposure of animal to a hot environment causes body temperature increase (11). An elevated ambient temperature also causes a decrease in the length and intensity of estrus by disturbing ovarian function, in addition to decreasing embryonic subsequent development and pregnancy rates (41, 42). Due to the insulating properties of the animals’ body, heat stress to an animal is different from the direct impact of elevated temperature on an oocyte (102). However, homeostatic thermoneutrality under a sub-optimal ambient temperature can be maintained temporarily. Continuous hyperthermia can increase core body temperature when animals lose their thermal homeostasis (1, 25, 27, 35, 40, 85, 86). It is common that the cattle are exposed to 3-5 h of hyperthermic stress under field conditions during summer in tropical or subtropical regions. The duration of extreme hyperthermia may range from 1 to 4 h at 41-42 °C, which might exceed the capacity of the animals to regulate their body temperature (85). It has been shown that heat stress changed the dynamics of follicular development in cattle. The size of follicles was enlarged and the follicular waves were hastened by 2 days (99). The endocrine status (38) and the uterine environment (86) in cows could also contribute to the thermosensitivity of oocytes and embryos.
Elevated ambient temperatures may directly impair maturing and preovulatory oocytes. Mammalian oocytes and pre-implantation embryos are known to be sensitive to heat stress. The pre-ovulatory oocyte was found to be one of the stages of cattle oocytes most sensitive to an elevated ambient temperature (within 12 days before oestrus) (53). Similar observations on heat sensitivity of oocytes and embryos were reported in pigs (80) and sheep (28). The most vulnerable stages are during ovulation, fertilization, within 2 days after fertilization, and at the first cleavage division (31, 102). Heat shocking to bovine oocytes for 1 h at 40 to 42 °C has no deleterious effects on subsequent blastocyst formation after in vitro fertilization (IVF) (56). However, blastocyst rates decrease when heat shock sustained for 12 h at 41°C (33). The developmental competence of the treated oocytes is severely reduced following a 45-min exposure to 43 °C (57). To sum up, the effect of hyperthermic conditions on the viability and developmental competence of oocytes and embryos depends upon both the temperature and the duration of exposure.
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3. Cellular Changes and Molecular Implication of Oocytes/Embryos after Heat Shock
3.1. Alterations of the Chromatin and Cytoskeleton
It has been reported that 50–90% of mouse oocytes are parthenogenetically activated and 6% of them developed into blastocysts when the oocytes are exposed to an elevated temperature in the dissected oviduct (6, 15, 16, 64). However, the activation-like chromatin alterations are not as typical as those shown during the normal progression of activated oocytes. In a normally activated oocyte, the chromosomes are decondensed into prepronuclear structures and then swelled into full-sized pronuclei in later development. In contrast, the heat-shocked MII oocytes only exhibited a similar decondensation of the chromosomes into the chromatin-like structures, but the structures further segregated into several subgroups representing one of the adverse effects on the oocytes under hyperthermia (58).
In addition, the microtubules structure is sensitive to both low and high environmental temperatures. The exposure of oocytes to room temperature or a lower temperature, for as short as 1 or 5 min causes depolymerization of the meiotic spindle (2, 97). With elevated temperatures, Baumgartner and Chrisman (7, 8) found that maternal heat stress causes disruption of the meiosis I spindle in maturing mouse oocytes, which results in disruption of meiosis II and polar body extrusion. The heat shock depolymerizes spindle microtubules of pig and bovine oocytes and the spindle size is reduced significantly in the HS-groups compared to the non-HS control groups. On the contrary, the pericytoplasmic microtubules are polymerized gradually after HS treatment, which usually formed microtubules arrays irradiating out from where the chromosomes are located (58, 59). Hyperthermia has also been found to affect the microtubule organizing center (MTOC) of several types of cultured cells, such as in Drosophila. Similarly, when murine cytotoxic T lymphocytes are heated to 41-42.5 ℃ for 30 min, disorganization or disruption of microtubule T arrays occurred (63, 101). Suzuki et al. (92, 93) demonstrated that with only a short time HS (42 °C for 30 min) to mature bovine oocytes, the ultrastructure of the vitelline membrane was changed from a normal microvillus-dominated pattern to a mixed pattern of normal microvilli with lamellipodia-like enlargement of cytoplasmic protrusions. When cattle and porcine oocytes were exposed to elevated temperatures, depolymerization of the spindle was prominent and the microfilament integrity was changed (59, 94). Presumably, these phenomena may be might have been due to a rapid depolymerization or disruption of the cytoskeleton, which may result in abnormal chromosome separation after fertilization or activation. (Fig. 1)
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3.2. Heat Shock Proteins and Heat Shock Responses
Heat shock proteins (hsps) are a set of evolutionary conserved proteins, some of which are constitutively expressed while others whose expression is induced in response to environmental and physiological stresses such as thermal or oxidative stress. A large group of hsps is molecular chaperones whose biological role is to maintain the unfolded, newly-synthesized proteins and allow them to traverse biological membranes or different cellular compartments such as the endoplasmic reticulum (ER) and mitochondria. Chaperones also prevent proteins from denaturing and help with renaturing during and after stresses (4).
Generally, the hsp family can be grouped into several subfamilies based on molecular weights, which include: hsp100, hsp90, hsp70, hsp60, small heat shock proteins (shsps; including hsp27 and αB-crystalline), and immunophilins (14, 24, 56). In fact, the family of heat shock proteins or molecular chaperones are expanding. Hsps can be classified into two major groups; the constitutive and the inducible forms. The hsps, synthesized at a constant rate without stimulation by stresses, are termed ‘constitutive’ hsps or heat shock cognates (hsc). The other group of the hsp family is designated as “inducible” hsps. Inducible hsps are expressed in different cell types by various stresses, such as temperatures (65) or toxic stimuli (3, 51). Constitutive hsps play important biological roles on directing folding of newly synthesized proteins (22) and transport of proteins across cell compartments (62); whereas the inducible forms, such as the hsp70 family, are expressed by stressed cells, and are believed to prevent cellular proteins from denaturation (4), aggregation, and to facilitate functional restoration of denatured proteins (81) caused by adverse environmental or thermal insults (49, 54, 79, 84). Hsps mediate the refolding or degradation of stress-damaged proteins, thus protecting cells from potential deleterious effects and promoting cell recovery. The accumulation of hsps in cells is associated with an increased resistance
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to cytotoxic injury by subsequent insults which would otherwise be lethal, a phenomenon known as thermotolerance (55, 76). Overexpression of hsp70 has been found to be sufficient for survival of cultured cells after thermal challenge (81). Two-cell bovine embryos could respond to an elevated temperature by increasing synthesis of a heat-inducible form of hsp70 (32, 34).
3.3. Activation of Mitogen-Activated Protein Kinase (MAPK) by Heat Shock
HS activates the major signaling transduction pathways involving MAPKs, extracellular signal-regulated kinase, stress-activated protein kinase 1 (SAPK1)/c-Jun N-terminal kinase (JNK), and SAPK2/p38. MAPK is activated in a cascade of phosphorylation reactions in which MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is itself phosphorylated and activated by a MAPKK kinase (MAPKKK). Heat-regulated activation of the MAPK has 3 distinct mechanisms which are involved ERK, JNK and p38 pathways. HS activation of JNK and p38 requires the participation of specific upstream kinases, the MAPKK (MKK4/7 and MKK3/6) and the MAPKKK (apoptosis signal-regulating kinase 1, ASK1). The role of ASK1 in the activation of p38 is not specific to HS (23). In the case of oxidative stress, it has been shown that ASK1 is the MAPKKK responsible for the activation of MKK3/6 and MKK4/7 (52). However, ASK1 has also been attributed a role in determining the cell fate, such as survival, proliferation and differentiation (69). Overexpression of wild-type or the constitutively active mutant can either induce either apoptosis or differentiation (20, 21, 48, 52, 61). JNK-mediated phosphorylation is implicated in the modification of the activity of several molecules in the apoptotic pathway. JNK-mediated phosphorylation of the antiapoptotic Bcl-2 proteins Bcl-2 and Bcl-XL, a modification suggested by some to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
neutralize their protective function, may also be expected to promote the mitochondrial release of cytochrome c (9). Among the downstream kinases of p38, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) and several transcription factors ATF2 have been identified (36, 50, 89; Fig. 1). Immunofluoresence microscopic analysis demonstrates that p38 is present in the nucleus and cytoplasm (87, 96). Activation of p38 phosphorylates ATF2 to cause increased reporter gene expression. The p38 MAPK pathway may increase gene expression by a general effect on transcription, by the activation of specific transcription factors, or by posttranscriptional regulation including mRNA processing, nuclear export, mRNA stability, translation, and protein stability (87).
4. Apoptosis of the Oocyte/Embryo after Heat Shock
Apoptosis, or programmed cell death, is one of the important processes for normal development and may play a critical role in eliminating cells that are abnormal, damaged, or misplaced during mammalian embryonic development (44, 47, 73). Apoptosis is a highly-regulated process involving condensation of nuclear chromatin, cytoplasmic shrinkage, membrane blebbing, nuclear fragmentation and finally formation of apoptotic bodies. One of the early intracellular events during apoptosis is the activation of a family of proteases called caspases (Fig. 1). These are cysteine proteases that cleave aspartic acid residues.
Several molecular markers including caspase-3, cytochrome c, and Bcl-2 associated Bax are available for measuring apoptosis of cells and embryos. Annexin V-labeling has been used as an early sign of apoptosis on the cell membrane, in which phosphatidylserine (PS) flips over the outer surface of the membrane (68). Nuclear chromatin integrity can be labeled by terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), which represents an 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
irreversible or later consequence of chromatin degradation by endogenous DNase (12). The occurrence of apoptosis in bovine embryos as determined by TUNEL staining has been shown to be developmentally regulated. Spontaneous apoptosis was first observed in bovine embryos at the 8- to 16-cell stage (18, 70). More studies on spontaneous apoptosis have been done in bovine embryos, and some studies reported various degrees of apoptosis were observed around 8–16-cell (< 50%) to morula (60-80%) stages of IVP cow embryos, as well as in vivo-derived morulae (approximately 50% of embryos) [18, 39, 46]. However, no spontaneous apoptotic processes were observed in in vivo-developing embryos before embryonic genome activation (EGA) (26), not even before the blastocyst stage [46, 60, 67]. Apparently, differential exhibition of spontaneous apoptosis for in vivo-derived and in vitro-derived embryos has been observed. For most mammalian species such as mice, pigs or cattle,
apoptosis can be artificially triggered by culture systems or specific environmental clues. For instance, chemical staurosporine was reported to induce apoptosis in mouse (98) and bovine (71) embryos around and after embryonic genome activation (EGA). Interestingly, no apoptosis signal was observed in in vitro-derived human embryos before compaction (43, 100) which was likely due to the improved culture system or quality of the harvested oocytes and embryos for observation.
Although apoptosis is known to occur in preimplantation embryos, there are few studies on extrinsic or intrinsic control systems for activation of apoptosis in preimplantation embryos or the ontogeny of such systems. Moreover, it is not unclear whether heat shock, which can induce apoptosis in many cell lines through activation of acid sphingomyelinase (45, 83, 95), also induces apoptosis in preimplantation embryos.
Apoptosis, identified by TUNEL and caspase activity (82, 88), was observed in preimplantation bovine embryos subjected to 41 to 42 °C heat shock in a time-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
dependent manner. Heat shock is a cellular stress associated with embryonic loss in
vivo (29, 85, 86) and in vitro (31, 33, 57). It can induce apoptosis as determined by
TUNEL reaction. In addition, further evidence showed an increase in activity of group II caspases in TUNEL-positive blastomeres in bovine embryos (82). Heat-induced apoptosis was first found at the developmental stage that was coincident with embryonic genome activation in the cow (74), suggesting it might be possible that heat-induced apoptosis is dependent on transcriptionally-controlled events in the embryo. Taken together, since the heat stress-inducing temperatures caused characteristic signs of apoptosis, it is suggested that apoptosis could be induced in preimplantation embryos exposed to maternal hyperthermia at 40 and 41 °C.
5. Heat-Induced Adaptation in Thermotolerance
Previous exposure to mildly elevated temperatures induce the expression and accumulation of heat shock proteins. As a result, the resistance to a second severe elevation in temperature is enhanced in virtually all organisms. Similarly, in mouse and bovine in vivo-produced embryos, a mild temperature increase (40 °C, 1 h) induced thermotolerance in the embryos and increased their ability to survive after a subsequent severe challenge (42 to 43 °C for 1 to 2 h) (30, 82).
The biochemical mechanisms by which mild heat shock prevents apoptosis induced by a more severe heat shock presumably involves hsp70 (10, 66, 76, 77, 78, 90). Induction of thermotolerance with a parallel increase of hsps prevents heat-induced apoptosis in several cell lines (17, 76, 91). Overexpression of hsp70 can inhibit JNK activation by various stimuli, including HS, sorbitol, tumor necrosis factor (TNF), UV light, and H2O2 (37, 77). The mechanism by which hsp70 exerts its antiapoptotic action is not completely understood, but it can block multiple points along the apoptotic pathway. Hsp70 and hsp72 inhibit protease caspase 3 or poly-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
(ADP-ribose) polymerase cleavage indicating that hsps could block the apoptotic processes (17, 77). Hsp70 also blocks cytochrome c release from mitochondria (78), processing of inactive procaspase-3, and activation of JNK (Fig. 1) (77). In addition, disruption of murine heat shock factor 1 gene increased heat-induced apoptosis (72). The bovine embryo can produce higher amounts of hsp70 in response to heat shock as early as the 2-cell stage (19, 32, 34).
A direct heat treatment in vitro can eliminate the possible confounding factors from endometrium and conceptuses, such as endocrine involvement (38) or uterine secretions (85, 86), which may indirectly affect oocyte viability following heat stress. Additionally, the endocrine status (38) and the uterine environment (85, 86) in cows could also contribute to the thermosensitivity of oocytes and embryos.
Conclusion
In general, alteration of intracellular calcium concentration, apoptosis, hsp70 expression, and in vitro development of oocytes and embryos have been evaluated intensively. The sensitivity of the nucleus and the cytoplasm of matured pig oocytes to heat shock are different from those of other cell types. Heat shock-induced apoptosis may be amplified during early embryonic development, by which in vitro blastocyst rates are significantly compromised. The changes in developmental competence of in vitro-matured oocytes after heat shock are not directly correlated to the expression of hsp70 in the oocytes. Apoptotic signals detected by TUNEL can only be observed in the cleavage stage embryos but not in MII oocytes, which require further investigation. Interestingly, Ca2+-releasing capacity of matured pig oocytes could be enhanced by a short duration of heat shock, but it declined after prolonged exposure to heat shock and/or in vitro culture. High intracellular Ca2+ release may 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
have disturbed intracellular Ca2+ homeostasis which results in the decline of subsequent development. The differential Ca2+ -releasing capacity of heat-shocked and non-heat-shocked matured oocytes revealed evident physiological changes. The development of matured oocytes or embryos could be enhanced by a short duration of heat shock. Further investigation is required to clarify the effect of heat shock on the nucleus and the ooplasm by extending period of heat shock.
Acknowledgments
This work is partially supported by grants from the National Science Council (95-2313-B-005-034-MY3; 101-2313-B-039-010-MY3) and China Medical University and Hospital (DMR-103-104).
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