Published December 2, 2014
PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature1,2 F. F. Paula-Lopes,*† R. S. Lima,† R. A. Satrapa,† and C. M. Barros†3 *Institute of Environmental Sciences, Chemistry and Pharmacology, Federal University of Sao Paulo, Diadema, SP, Brazil; and †Department of Pharmacology, Institute of Bioscience, University of Sao Paulo State, 18618-970, Botucatu-SP, Brazil
ABSTRACT: High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater
for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.
Key words: bovine, embryo, heat stress, oocyte, thermotolerance © 2013 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2013.91:1143–1153 doi:10.2527/jas2012-5802 INTRODUCTION
1Based on a presentation at the Physiology and Endocrinology Symposium titled “The Current Status of Heat Shock in Early Embryonic Survival and Reproductive Efficiency” at the Joint Annual Meeting, July 15–19, 2012, Phoenix, Arizona, with publication sponsored by the Journal of Animal Science and the American Society of Animal Science. 2The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil, for funding and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasilia, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasilia, Brazel), and FAPESP (Sao Paulo, Brazil) for fellowships. 3Corresponding author:
[email protected] Received August 30, 2012. Accepted November 21, 2012.
Heat stress can be defined as the sum of forces external to a homeothermic animal that shifts core body temperature from the resting state (Yousef, 1984), causing physiologic, metabolic, cellular, and molecular changes. Mammals are homeothermic animals that maintain a constant internal body temperature by the balance between the amount of metabolic heat produced and heat dissipation to the environment (Hansen, 2004). Breeds of cattle have evolved to be adapted for different environments. Bos indicus cattle are more resistant to increased temperature and humidity than the majority of Bos taurus breeds (Adeyemo et al., 1979; Bennett et al., 1985; Hammond et al., 1996,1998). Such genetic
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variation in resistance to heat stress results from the ability of thermotolerant breeds to regulate body temperature (Adeyemo et al., 1979; Bennett et al., 1985; Hammond et al., 1996, 1998; Gaughan et al., 1999). In addition, however, there are indications that breed differences in thermal resistance extend to the cellular level (Malayer and Hansen, 1990; Kamwanja et al., 1994; Paula-Lopes et al., 2003). Genetic selection for high milk yield decreases the thermoregulatory ability in lactating animals exposed to heat stress (Berman et al., 1985). Lactating dairy cows are more susceptible to heat stress because increased metabolic heat production associated with lactation can lead to hyperthermia. Therefore, the magnitude of the deleterious effects of heat stress on fertility is more pronounced in high-producing dairy cows (Al-Katanani et al., 1999), whereas fertility of heifers is usually not affected by heat stress (Badinga et al., 1985). High environmental temperature observed during the hot months of the year reduces fertility in lactating dairy cows (Dunlap and Vincent, 1971; Badinga et al., 1985; Al-Katanani et al., 1999; Pires et al., 2002). In studies conducted in Florida, conception rates of lactating Brown Swiss, Jersey, and Holstein cows decreased from 52 to 32% as maximum air temperature increased from 23.9 to 32.2°C during the summer (Badinga et al., 1985). Similarly, in Brazil, pregnancy rates of Holstein cows housed in free stalls were reduced from 71.2% in the winter to 45.7% in the summer (Pires et al., 2002). Summer heat stress depression in fertility is a multifactorial problem that affects physiological and cellular functions in several tissues. Heat stress compromises follicular growth (Badinga et al., 1993; Wolfenson et al., 1995), hormonal secretion (Wolfenson et al., 1995; Roth et al., 2000), uterine blood flow (Roman-Ponce et al., 1978), and endometrial function (Malayer et al., 1988). Additionally, it causes a reduction in oocyte developmental potential (Al-Katanani et al., 2002; Paula-Lopes et al., 2008) and preimplantation embryonic development (Ealy et al., 1993; Paula-Lopes and Hansen, 2002b). The cellular mechanisms triggered by increased temperatures in bovine oocytes and embryos are not well known, nor are the factors involved in thermal resistance to stress. This review discusses the role of cattle genotype and thermoprotective molecules on oocyte and preimplantation embryo resistance to increased temperature. An improved knowledge of these mechanisms and the identification of new genes responsible for cellular thermotolerance may allow the development of strategies to increase cell survival in the face of various types of stress. The terms heat stress and heat shock will be used to describe in vivo and in vitro exposure to increased temperatures, respectively. In vivo heat stress studies reflect the degree of hyperthermia imposed on each animal consid-
ering environmental and animal factors, such as temperature, humidity, genotype, and thermoregulatory ability. In contrast, heat shock experiments indicate the direct effect of physiological (i.e., 40 to 41.5°C) and nonphysiological (i.e., > 41.5°C) temperatures on oocyte and embryo function that can also be modulated by time of exposure and genotype. A majority of the heat shock studies described herein were conducted with slaughterhouse-derived oocytes. Although these oocytes were of variable genotype, most of the cows were crossbred Bos indicus (Rivera et al., 1999; Paula-Lopes et al., 2008, 2010; Risolia et al., 2011). Breed Differences in Thermal Resistance Bos indicus and certain Bos taurus breeds, such as Senepol and Romosinuano (tropically-adapted Bos taurus), are more resistant to increased temperature and humidity than breeds that evolved in Europe, such as Angus and Holstein (Block et al., 2002; Barros et al., 2006). In general, Bos indicus cows (i.e., Zebu breeds) have greater thermoregulatory ability than Bos taurus (i.e., European breeds). The deleterious effect of heat stress on fertility is affected by cattle genotype. The average depression of fertility associated with heat stress is greater for Bos taurus than Bos indicus breeds (Turner, 1982). The genetic variation in resistance to heat stress results from the ability of thermotolerant breeds to regulate body temperature (Adeyemo et al., 1979; Bennett et al., 1985; Hammond et al., 1996, 1998; Gaughan et al., 1999). In addition, there are indications that breed differences in thermal resistance extend to the cellular level. There is evidence that Bos indicus oocytes, embryos, lymphocytes, endometrium, and oviductal cells carry greater heat-tolerance than Bos taurus cells. Embryos produced by insemination of Brahman oocytes with Angus sperm are more thermotolerant than embryos produced by insemination of Holstein oocytes with Angus semen (Block et al., 2002). In contrast, there are no differences in thermotolerance between Holstein × Brahman embryos and Holstein × Angus embryos (Block et al., 2002). Similarly, Saito et al. (2008; Table 1) and Satrapa et al. (2011a) found that heat shock (41.0°C for 12 h, 96 h post insemination) significantly decreased the development of bovine embryos produced from Holstein oocytes fertilized with either Gir (Bos indicus) or Holstein sires (Bos taurus) to the blastocyst stage. These results were interpreted to indicate that contribution of the oocyte has a more crucial role determining the genetic ability of an embryo to resist the effects of heat shock than the contribution of the spermatozoa. In contrast, Eberhardt et al. (2009) observed that embryos produced by insemination of Holstein oocytes with Nelore semen were more resistant to increased temperature in culture compared with embryos produced by
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Table 1. Number of oocytes, rates of cleavage, and rates of development to morula, blastocyst, and hatched blastocyst for Holstein oocytes fertilized with Gir or Holstein sires and cultured at 39°C or heat stressed for 12 h at 41°C Item
39°C
41°C
39°C
41°C
Cow Sire
Holstein Gir 302 78.0 54.3 40.1 22.8
Holstein Gir 299 77.2 55.5 34.7* 15.7*
Holstein Holstein 300 75.0 54.0 37.6 19.9
Holstein Holstein 307 76.0 55.7 30.6* 13.9*
Oocyte, n Cleavage, % Morula, % Blastocyst, % Hatched blastocyst, %
*The heat stress affected negatively the blastocyst formation and hatching rates when compared with the control group (39°C; P < 0.05).
insemination of Holstein embryos with Angus semen. This result indicated that the breed of the sire could influence the genetic thermotolerance capacity of the embryo. However, in that study, the limited number of bulls used per breed (n = 2 bulls/breed) may have influenced the results obtained by Eberhardt et al. (2009), so that the effect of individual bull may have prevailed over the effect of breed (Bos indicus or Bos taurus) on the development of thermotolerance. In fact, in vitro blastocyst production can vary from 0 to 36%, depending on the bull used (Parrish et al., 1986; Lacandra et al., 1993). In addition, Palma and Sinowatz (2004), when testing the capacity of 63 bulls to produce in vitro embryos, found great variation among the bulls in cleavage (36.3 to 93.4%) and blastocyst rates (6.9 to 51.2%). The lethal effects of a nonphysiological heat shock of 45°C for 3 h on lymphocyte viability were greater for lymphocytes from Angus heifers than for lymphocytes from Brahman or Senepol heifers. Exposure of phytohemagglutinin-treated lymphocytes to a less severe heat shock (i.e., nonphysiological heat shock of 42°C) for 12 h caused a greater reduction in viability of Angus lymphocytes than Brahman or Senepol lymphocytes. In contrast, 42°C for 12 h reduced proliferation of phytohemagglutinin-treated lymphocytes to the same degree regardless of breed (Kamwanja et al., 1994). Exposure of bovine lymphocytes to a physiological heat shock (41.0°C for 9h) increased the proportion of apoptotic cells. Heat-induced apoptosis was less for Brahman and Senepol lymphocytes than for Angus and Holstein lymphocytes (Paula-Lopes et al., 2003). Breed differences in resistance to increased temperature are also observed in reproductive tissues. Exposure to a nonphysiological heat shock of 43°C increased secretion of nondialysable [3H]-labeled macromolecules by cultured oviductal tissues from both oviducts of Brahman cows and from the oviduct contralateral to the side of ovulation of Holstein cows but decreased secretion by the oviduct ipsilateral to ovulation in Holstein cows. Nonphysiological heat shock of 43°C also caused
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an increase in [3H]-thymidine incorporation by endometrial explants from Brahman cows and suppressed incorporation in explants from Holstein cows (Malayer and Hansen, 1990). However, there was no effect of moderate heat shock (41°C) or breed on [3H]-leucine incorporation into proteins secreted by oviductal or endometrial explants from Holstein, Brahman, Angus, and Senepol cows (Paula-Lopes et al., 2003). The molecular basis for genetic differences in cellular resistance to increased temperatures in Bos indicus and Senepol cattle is not known. It is possible that cells from Bos indicus cows have more heat shock protein (HSP)70 and, therefore, are better able to stabilize and refold damaged proteins (Hartl and Martin, 1992). However, although heat shock increased intracellular HSP70 in lymphocytes (Kamwanja et al., 1994) and endometrial tissue (Malayer and Hansen, 1990), these responses were not altered by breed. Heat-Induced Damage of Bovine Germinal Vesicle-Stage Oocytes: Effects on Oocyte Developmental Competence Germinal vesicle (GV)-stage oocytes remain in the antral follicle environment for 42 d (Lussier et al., 1974). During this period, animals exposed to adverse environmental conditions may reach body temperatures above 40 to 41°C (Putney et al., 1989; Ealy et al., 1993;Wolfenson et al., 1993; Rivera and Hansen, 2001), compromising oocyte function before maturation. Indeed, developmental competence of bovine GV-stage Holstein oocytes was affected by increased temperature (Rocha et al., 1998; Al-Katanani et al., 2002; Gendelman et al., 2010). The proportion of Holstein GV-stage oocytes that developed to the blastocyst stage after fertilization was less in the warm season as compared with the cool season (Al-Katanani et al., 2002). In a study that used ultrasound-guided follicular aspiration (i.e., oocyte pickup; OPU) to collect Holstein and Brahman oocytes during the cool and hot seasons, the percentage of Holstein oocytes with normal morphology decreased from 80% in the cool season to 25% in the hot season. The percentage of in vitro-fertilized oocytes that developed to the 8-cell, morula, and blastocyst stages was greater during the cool season (44.4% 8-cell, 34.2% morula, and 29% blastocysts) than the hot season (1.1% 8-cell, 0% morula, and 0% blastocyst; Rocha et al., 1998). In contrast to the susceptibility of Holstein oocytes to heat stress, there was no effect of season on the proportion of Brahman oocytes that displayed normal morphology or that developed to the 2-cell, 8-cell, morula, or blastocyst stage (Rocha et al., 1998). This result indicated that either i) the thermoregulatory ability of Brahman cows reduces maternal hyperthermia compared with Holstein cows; ii)
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oocytes from Brahman cows contain intrinsic mechanisms that allow survival after exposure to increased temperature; or iii) a combination of both mechanisms occurs. There is evidence that heat stress exerts both immediate and delayed effects on GV-stage oocyte developmental competence. A pioneering study demonstrated that exposure of Bos taurus cows to summer heat stress reduced conception rates from summer to early autumn (Badinga et al., 1985), indicating a delayed effect of heat stress on reproductive function. Another study indicated that seasonal reductions in Holstein oocyte competence were recovered 2 to 3 estrous cycles after the end of summer (Roth et al., 2001). These findings narrowed down the heat stress window, indicating that increased temperature can damage the follicle and oocyte reserve that began growing during the warm period. Immediate and delayed effects of severe heat stress were evaluated in Bos indicus animals (i.e., Gir) exposed to environmental chambers at 38°C and 80% relative humidity (RH) during the day and 30°C and 80% RH during the night for a 28-d period (Torres-Júnior et al., 2008). Although there was no immediate effect of heat stress on reproductive function, exposure of Gir cows to severe heat stress exerted a delayed deleterious effect on follicular growth and GV-stage oocyte developmental competence to the blastocyst stage (Torres-Júnior et al., 2006). This study indicated that follicles and oocytes from thermotolerant Bos indicus breeds could be compromised by severe environmental conditions. Moreover, oocyte competence was not recovered after an approximate 4-mo period after stress. Therefore, it is possible that preantral follicleenclosed oocytes are more susceptible to heat stress than antral follicle oocytes. In vitro experiments demonstrated the direct effect of increased temperature on crossbred Bos indicus GV-stage oocytes (Payton et al., 2004; Lima, 2012). The development of in vitro models to study direct temperature effects on GV-stage oocytes is challenging because removal of the oocyte from the antral follicle induces spontaneous meiotic progression to the Metaphase II (MII) stage (Pincus and Enzmann, 1935). One approach to overcome this problem would be to use pharmacological tools, such as meiotic inhibitors, to maintain oocytes at the GV stage. Payton et al. (2004) evaluated the direct effects of increased temperature on slaughterhouse-derived GV-stage oocytes cultured in the presence of the p34cdc2/cyclin-dependent kinase inhibitor, roscovitine (50 μM). Heat shock at 41°C for 6 and 12 h reduced embryonic development to the 8- to 16-cell stage and to the blastocyst stage, respectively (Payton et al., 2004). Similarly, culture of crossbred Bos indicus oocytes in butyrolactone-containing meiotic arrest medium at 41°C for 14 h did not affect cleavage rate but reduced embryonic development to the blastocyst stage on d 8 and 9 after fertilization (Lima, 2012).
Heat-Induced Damage during Oocyte Maturation: Effects on Oocyte Developmental Competence Exposure of bovine oocytes to increased temperature during oocyte maturation compromises oocyte function. The fully-grown oocyte that reached approximately 110 μm is transcriptionally inactive (Hyttel et al., 1997) and does not increase synthesis of thermoprotective molecules, such as HSP70, after exposure to heat shock (Edwards and Hansen, 1997). This inability of oocytes to mount a complete heat shock response may explain oocyte sensitivity to increased temperature. Some of the first evidence that heat stress compromised oocyte maturation came from in vivo experiments. Exposure of Holstein heifers to heat stress at 42°C for 10 h between estrus and AI decreased the number of normal embryos compared with control cows maintained at 24°C (Putney et al., 1989). Heat-induced damage of bovine oocytes during maturation has been well characterized in oocytes exposed to heat shock. Edwards and Hansen (1996) demonstrated that heat-induced reduction in developmental competence of crossbred Bos indicus oocytes was greater when heat shock was applied during the first 12h in vitro maturation (IVM, i.e., 0 to 12 h IVM) than the last 12 h IVM (i.e., 12 to 24 h IVM; Edwards and Hansen, 1996). This 0- to 12-h IVM heat shock model has been widely used to evaluate the effects of heat shock during oocyte maturation. Recently, a study mimicked the circadian rhythm of vaginal temperature in lactating Holstein-Friesian cows during the hot season (Nabenishi et al., 2011). Exposure of bovine oocytes to this heat shock scheme (39.5°C for 5 h, 40°C for 5 h, 40.5°C for 6 h, and 40°C for 4 h) reduced cleavage and blastocyst rates (Nabenishi et al., 2011). Exposure of crossbred Bos indicus oocytes to severe and nonphysiological heat shock (44°C during 0- to 12-h IVM) blocked subsequent cleavage and preimplantation embryonic development (Paula-Lopes et al., 2008). Similar studies indicated that exposure of oocytes to a nonphysiological severe temperature of 43°C for short periods of 45 and 60 min during IVM did not block but reduced blastocyst and expanded blastocyst rates (Ju et al., 1999). A lesssevere heat shock of 42°C for 0- to 12-h IVM also reduced development to the blastocyst stage (Edwards and Hansen, 1996; Roth and Hansen, 2004). A physiological or moderate heat shock of 40°C (Roth and Hansen, 2004) or 41°C (Paula-Lopes et al., 2008) for 0- to 12-h IVM reduced the proportion of oocytes that cleaved at d 3 and developed to the blastocyst stage at d 8 after insemination. However, oocyte exposure to a moderate heat shock (40.5°C and 41.5°C) for short periods of 30 and 60 min during IVM did not affect development to the blastocyst stage (Ju et al., 1999). Therefore, in vitro studies indicated that heat shock reduces developmental competence of crossbred Bos indicus oocytes. Moreover, the oocyte response to increased
Heat stress in bovine oocytes and emb
temperature is a function of stress intensity measured by temperature and exposure time. The mechanisms by which increased temperature affects oocyte physiology are not completely understood. However, it has been shown that, depending on oocyte thermotolerance as well as the severity of stress, increased temperature can cause reversible or irreversible cellular damage in different cell structures and organelles (Ju et al., 2005; Roth and Hansen, 2005). Such effects can trigger adaptive and (or) cellular death responses (Paula-Lopes and Hansen, 2002a). Considering that apoptosis is the major process responsible for the reduction in oocyte number throughout mammalian female reproductive lifetime (Morita and Tilly, 1999; Tilly, 2001), it is possible that this form of cell death has an essential role in oocytes exposed to stressful conditions. Cellular Alterations Induced by Increased Temperature in Bovine Oocytes Heat-induced cellular damage in bovine oocytes can be observed in the cytoplasmic and nuclear compartments. However, there is evidence that oocyte cytoplasm is more susceptible to the adverse effects of increased temperature than the nucleus (Shen et al., 2010). Nuclear transfer studies in Bos indicus and Bos taurus oocytes indicated that exposure of donor cell nuclei to heat shock did not affect embryonic development in either Bos indicus or Bos taurus oocytes. However, exposure of receptor ooplasm to heat shock decreased developmental competence of Bos taurus oocytes (Shen et al., 2010). Cytoplasmic Alterations. Cytoskeletal disruption is among the heat-induced cytoplasmic changes that occur in bovine oocytes. Exposure of bovine oocytes to increased temperature affected microtubule and microfilament organization (Ju and Tseng, 2004; Tseng et al., 2004; Ju et al., 2005; Roth and Hansen, 2005). The lack of cytoskeleton organization has been shown to impair transport and distribution of cytoplasmic organelles and chromosome segregation, thereby compromising oocyte fertilization and cleavage (Tseng et al., 2004; Ju et al., 2005). The pericellular actin ring and trans-zonal actin processes were decreased in crossbred Bos indicus oocytes matured at 41°C (Roth and Hansen, 2005) and 41.5°C (Tseng et al., 2004). Actin microfilaments are responsible for cortical granule translocation to the cortical region of the oocyte during maturation (Wessel et al., 2002). Although GV-stage oocytes show cortical granule aggregates (Type I), GV breakdown promotes cortical granule dispersion and translocation to the oolemma (Type III). Exposure of GV-stage (Payton et al., 2004) and maturing oocytes (Edwards et al., 2005) to 41°C heat shock increased the proportion of oocytes that had a Type III cortical granule distribution, indicating that heat shock hastened cytoplas-
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mic maturation kinetics and induced oocyte aging. The percentage of mouse oocytes carrying incomplete cortical granules migration during IVM was greater in oocytes matured at 40°C than 37°C (Wang et al., 2009). High temperature affects the organized αβ-tubulin heterodimer structure. Exposure of slaughterhouse-derived oocytes to increased temperature caused meiotic spindle disorganization so that Metaphase I and II microtubules become deformed and chromosomes misaligned (Tseng et al., 2004; Roth and Hansen, 2005). Meiotic spindle size also decreased as the duration of heat shock (42°C) increased from 1 to 4 h (Ju et al., 2005). Changes in the size and morphology of the meiotic spindle indicated that heat shock affected microtubule polymerization and depolymerization. Such changes most likely compromise chromosome separation during fertilization and subsequent embryonic division. Microtubule disruption also affects cytoplasmic organelle transport such as oocyte mitochondrial distribution (Sun et al., 2001). Mitochondria are targets of the deleterious effects induced by high temperature. Exposure of crossbred Bos indicus GV-stage (Lima, 2012) and maturing oocytes (Ispada et al., 2011) to heat shock reduced oocyte mitochondrial activity. Similarly, exposure of high- and low-quality cumulus-oocyte complexes (COC) to heat shock reduced cumulus cell mitochondrial membrane potential regardless of COC quality (Paula-Lopes et al., 2010). Changes in oocyte mitochondrial activity may be associated with activation of the apoptotic cascade. Exposure of crossbred Bos indicus oocytes to heat shock during IVM increased the proportion of oocytes with high Group II caspase (i.e., Caspases 2, 3, and 7) enzymatic activity (Roth and Hansen, 2004), but this increase was not observed in GV-stage oocytes (Lima, 2012). Heat shock also decreased oocyte total protein synthesis (Curci et al., 1987; Edwards and Hansen, 1996, 1997). Nuclear Alterations. Reduced nuclear maturation is one of the changes caused by heat shock in bovine oocytes. Exposure of crossbred Bos indicus GV-stage (Payton et al., 2004) or maturing oocytes (Roth and Hansen, 2005; Paula-Lopes et al., 2008) to 41°C heat shock decreased the proportion of oocytes that reached MII stage after IVM. In these experiments, heat shock blocked meiotic progression by increasing the proportion of Metaphase I oocytes. However, heat shock of 41°C was previously shown to accelerate nuclear maturation kinetics, increasing the proportion of MII oocytes after 16 to 18 h of maturation (Edwards et al., 2005). Another nuclear change observed in oocytes subjected to heat shock is the DNA fragmentation characteristic of apoptosis. Heat shock during the first 12 to 14 h IVM increased the proportion of oocytes positive for TUNEL (terminal deoxynucleotidyl transferase dUTP nick end la-
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beling) assay indicative of DNA fragmentation (Roth and Hansen, 2004; Ispada et al., 2010). Experiments conducted with crossbred Bos indicus oocytes also indicated that moderate heat shock (41°C) during the first 12 h IVM reduced oocyte developmental competence. However, heat shock had no effect on the proportion of oocytes in late apoptosis stages, as determined by plasma membrane permeability alterations (Paula-Lopes et al., 2008). Moderate heat shock reduced nuclear maturation, cleavage, and blastocyst rates (Paula-Lopes et al., 2008). Exposure of bovine oocytes to a severe nonphysiological heat shock (44°C) for 12 h also reduced nuclear maturation, as observed after moderate heat shock. However, this stress increased the proportion of oocytes positive for apoptosis and necrosis and completely blocked cleavage and embryonic development from reaching the blastocyst stage (Paula-Lopes et al., 2008). Effects of Increased Temperature on Early Embryonic Development In Vitro Heat stress is capable of impairing sperm production in males, reducing conception rates in females, and causing embryonic loss (Thatcher and Hansen, 1993; Hansen et al., 2001), which is likely associated with major adverse effects of heat stress during early embryo development, especially in the preimplantation stage (Ealy et al., 1993). During the first cleavage divisions, the embryo genome is still inactive, and throughout this period of low transcriptional activity, the embryo is vulnerable to many forms of stress, including excessive heat (Paula-Lopes and Hansen, 2002a,b). Al-Katanani and Hansen (2002) determined in vitro that increased temperature reduces embryo survival during early stages of development. The effects of heat stress on embryonic mortality decreases as pregnancy progresses (Biggers et al., 1987; Ealy and Hansen, 1994; Edwards and Hansen, 1997; Hansen, 1999; Edwards et al., 2001) and are minimized by 3 d after estrus in cows (Ealy et al., 1993). Several studies indicated that Bos indicus embryos are more thermotolerant than Bos taurus embryos in vitro. Paula-Lopes et al. (2003) reported that cultured embryos (>8-cell stage) from a heat-tolerant breed (Brahman) were more likely to develop to the blastocyst stage after exposure to heat shock (41°C for 6 h) than embryos from heat-sensitive breeds (i.e., Holstein and Angus). Similar results have been found by others comparing Zebu (i.e., Nelore) with European (i.e., Holstein; Barros et al., 2006) or crossbred breeds (Bos indicus vs. Bos taurus; Barros et al., 2002; Eberhardt et al., 2009) and by comparing Brahman and Romosinuano with Angus cattle (Hernández-Céron et al., 2004).
In vitro experiments in which bovine embryos were exposed to a temperature similar to that experienced by lactating dairy cows in a heat stress condition in Florida (38.5 to 40.5°C for 8 d of in vitro culture; Rivera and Hansen, 2001) or in southeast Queensland, Australia (39.5 to 41.0°C during the first 48 h of in vitro culture; Sugiyama et al., 2007), produced low blastocyst rates and confirmed the negative effects of high temperatures at certain times of the year on embryo yield. Silva et al. (2013) cultured embryos from Angus and Nelore cows at 40.0°C for 12 h, beginning 96 h after fertilization, and transferred blastocysts to crossbred recipients. The pregnancy rates after transfer were 29.4% (15/51) for nonstressed Nelore embryos, 29.0% (11/38) for Nelore embryos after heat shock, 21.4% (6/28) for nonstressed Angus embryos and 7.1% (1/14) for Angus embryos after heat shock. These results indicated that Bos indicus embryos were better able to survive increased temperature at early stages of development and were more capable of establishing pregnancy after heat shock than Bos taurus embryos. Adaptive changes that provide thermotolerance are likely derived from changes in gene expression or activity in biochemical systems that control cellular functions against aggression, such as high temperatures (Edwards et al., 2001). Silva et al. (2013) investigated the expression of some genes related to early embryonic development and implantation (PLAC8, CDX2, HSF1, and COX2) in Nelore and Jersey embryos collected 96 h postinsemination and subjected to heat shock at 41°C for 6 h. In both Nelore and Jersey breeds, PLAC8 and CDX2 expression was less in pools of blastocysts submitted to heat stress than control groups. In addition, the expression of these genes was greater in blastocysts of Nelore control group than in those of Jersey control group. Heat stress decreased the expression of HSF1 in the Nelore heat stress group relative to all other groups. The expression of COX2 differed significantly between Jersey heat stress group and Nelore groups (control and heat stress) but did not differ within Jersey groups. The authors concluded that heat stress affects the bovine embryo by decreasing the developmental rate and decreasing the expression of important developmental genes. Furthermore, these effects of heat stress were more evident in Bos taurus embryos than in Bos indicus embryos. Thermoprotective Factors on Bovine Oocytes and Embryos Several approaches to mitigate the deleterious effects of heat stress in animal breeding have been described (Hansen et al., 1992; Hansen and Aréchiga, 1999; Hansen et al., 2001). Thus, the focus of this section is to present molecules whose thermoprotective actions minimize
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heat-induced cellular and functional damage in bovine oocytes and embryos. The deleterious effects of heat shock on bovine oocytes may be regulated by growth factors, such as IGF-I. It has been shown that IGF-I has autocrine, paracrine, and endocrine actions in cellular metabolism, proliferation, growth, and differentiation (Voss and Rosenfeld, 1992; Baker et al., 1993; Yakar et al., 1999). Exposure of dairy cows to heat stress decreased serum IGF-I, leading to impairment of oocyte quality (De Rensis and Scaramuzzi, 2003). Recent studies indicated that IGF-I plays a thermoprotective role in GV-stage and IVM oocytes. Exposure of GV-stage oocytes to 41°C reduced oocyte mitochondrial activity, increased percentage of apoptotic oocytes and reduced cleavage and blastocyst rates (Lima, 2012). However, addition of 12.5 ng/mL IGF-I reduced these deleterious effects rescuing GV-oocyte cellular functions and developmental capacity. Similarly, 100 ng/mL IGF-I reduced the detrimental effects of heat shock during the first 14 h of IVM. Although heat shock during IVM reduced mitochondrial activity and increased the percentage of apoptotic oocytes, IGF-I was able to reverse these effects (Ispada et al., 2010, 2011). However, in those studies, the concentration of 100 ng/mL IGF-I during IVM was unable to rescue oocytes from the negative effects of heat shock on blastocyst development (Risolia et al., 2011). It is possible that in this study, IGF-I exerted only a partial beneficial effect on heat-shocked oocytes because the IGF-I dose used (i.e., 100 ng/mL) was much greater than the physiologic IGF-I concentration present in the follicular fluid. It has been demonstrated that the concentration of free IGF-I in the follicular fluid of dominant and subordinate follicles is approximately 12 ng/mL (Ginther et al., 2002). This may explain the better oocyte response to this physiologic dose as compared with 100 ng/mL IGF-I. The beneficial effects of IGF-I on embryonic development during heat stress have been demonstrated in several species. Addition of IGF-I to the culture medium improved morula and blastocyst rates (Herrler et al., 1998; Moreira et al., 2002; Block et al., 2003; Satrapa et al., 2011a), increased blastocyst cell number (Moreira et al., 2002) and reduced the incidence of apoptosis in bovine embryos (Moreira,et al., 2002; Jousan and Hansen, 2004; Bonilla et al., 2011). Moreover, supplementing the culture medium with IGF-I improved blastocyst resistance to high temperature, as indicated by the increase in pregnancy rate after embryo transfer to Holstein cows exposed to summer heat stress (Block et al., 2003). When evaluating the expression of the IGF system in oocytes and blastocysts from Nelore and Holstein cows, Satrapa et al. (2011b) observed that expression of IGF-I and II, their receptors (i.e., IGFR-I and IGFR-II, respectively), and IGFBP-2 and IGFBP-4 were greater in oocytes from Holstein cows than Nelore cows. However,
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the expression of pregnancy-associated plasma protein A, which metabolizes IGFBP, was significantly greater in Nelore than Holstein oocytes. In addition, IGF-I was more highly expressed in heat-shocked compared with nonheatshocked blastocysts in Nelore and Holstein breeds, and this effect was more evident in Nelore cattle. Although protein expression was not assessed, these results based on gene expression indicated that the greater bioavailability of IGF in Nelore oocytes and blastocysts may contribute to the superior tolerance of Nelore to heat stress compared with Holstein cattle. Heat-induced oocyte DNA fragmentation (TUNELpositive) can also be reversed by supplementation of IVM medium with molecules that inhibit the apoptotic cascade, such as the Group II caspase inhibitor, z-DEVD-fmk (Roth and Hansen, 2004). This molecule inhibited the negative effects of heat shock on cleavage and blastocyst rates (Roth and Hansen, 2004). Another apoptosis inhibitor, sphingosine-1-phosphate (S1P), is a sphingolipid metabolite that blocks the proapoptotic effects of ceramide (Roth and Hansen, 2004). Addition of S1P to the maturation medium blocked the deleterious effects of heat shock on meiotic progression and apoptosis and increased the proportion of heat-shocked oocytes that cleaved and reached blastocyst stage (Roth and Hansen, 2004). SUMMARY AND CONCLUSIONS Summer heat stress depression in fertility is a multifactorial problem that affects physiological and cellular functions in several tissues. Heat stress compromises follicular dynamics, hormonal secretion, and oocyte and embryonic function. Oocytes at the GV-stage and maturation period, as well as the early preimplantation embryo undergo cellular and molecular changes induced by increased temperature. Such alterations compromise the events required for successful fertilization and preimplantation embryonic development. The deleterious effects of heat stress on reproductive function may be modulated by several factors such as cattle genotype, molecules of thermoprotection, and the intensity of stress. Indeed, several experiments demonstrated that cattle genotype plays a major role in tolerance to stress. Whereas the greater thermoregulatory ability of Bos indicus breeds has been long established, the superior cellular resistance of Bos indicus oocytes and embryos to heat shock has been characterized more recently. Bovine oocyte cellular damage caused by increased temperature can be detected in different cellular compartments, such as the oocyte cytoplasmic and nuclear regions. There is evidence that heat shock reduced oocyte progression to the MII stage, impaired microtubule and microfilament organization, decreased oocyte mitochondrial function, induced cell death, and compro-
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mised oocyte developmental competence. Heat-induced disruption of microtubules and microfilaments alters the distribution of cellular structures, such as cortical granules and mitochondria, besides affecting chromosome segregation during fertilization and cell division. Heat shock also increases oocyte DNA fragmentation and decreases mitochondrial activity, indicating activation of the heat-induced mitochondrial apoptotic pathway in bovine oocytes. There are also indications that the oocyte cytoplasm is more susceptible to heat shock than the nucleus. Although heat shock reduced developmental competence of Bos taurus oocyte cytoplasm, it had no effect on Bos taurus and Bos indicus oocyte nuclei. Increased temperature also damages the early bovine embryo, reducing blastocyst development and altering expression of developmental important genes such as PLAC8, CDX2, and IGF-I. These effects of heat stress were more evident in Bos taurus than in Bos indicus embryos. As we improve our understanding of the cellular mechanisms that contribute to heat stress resistance of Zebu cattle, we should be able to develop strategies to minimize the effects of increased temperatures on less thermotolerant breeds. Several approaches have been employed to alleviate the low fertility associated with heat stress. Molecules such as growth factors and apoptosis inhibitors have been shown to exert thermoprotective actions in bovine oocytes and embryos. For example, IGF-I rescued several cellular functions and improved developmental competence of bovine oocytes and embryos subjected to heat shock. Moreover, IGF-I bioavailability seems to be greater in Bos indicus oocytes and embryos than Bos taurus. Even though these molecules can be considered as potential candidates to modulate the effects of increased temperature, a better understanding of the mechanisms involved in thermoprotection are necessary for development of new strategies to alleviate the effects of heat stress in reproductive function. LITERATURE CITED Adeyemo, O., E . Heath, B. K. Adadevoh, J. Steinbach, and E. A. Olaloku. 1979. Some physiological and behavioral responses in Bos indicus and Bos taurus heifers acclimatized to the hot humid seasonal equatorial climate. Int. J. Biometeorol. 23:231–241. Al-Katanani, Y. M., and P. J. Hansen. 2002. Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development. Mol. Reprod. Dev. 62:174–180. Al-Katanani, Y. M., F. F. Paula-Lopes, and P. J. Hansen. 2002. Effect of season and exposure to heat stress on oocyte competence in Holstein cows. J. Dairy Sci. 85:390–396. Al-Katanani, Y. M., D. W. Webb, and P. J. Hansen. 1999. Factors affecting seasonal variation in 90 day non-return rate to first service in lactating Holstein cows in a hotclimate. J. Dairy Sci. 82:2611–2615. Badinga, L., R. J. Collier, W. W. Thatcher, and C. J. Wilcox. 1985. Effects of climatic and management factors on conception rate of dairy cattle in subtropical environment. J. Dairy Sci. 68:78–85.
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