Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 _________________________________________ 4Corresponding author: paula.lopes29@unifesp.br Phone:+55(11) 3319-3300; Fax: +55(11) 4043-6428 Received: July 3, 2012 Accepted: August 13, 2012 Heat stress induced alteration in bovine oocytes: functional and cellular aspects F.F. Paula-Lopes1,2,4, R.S. Lima2,3, P.H.B. Risolia1,2, J. Ispada1, M.E.O.A. Assumpção3, J.A. Visintin3 1Institute of Environmental Sciences, Chemistry and Pharmacy, Federal University of Sao Paulo, Diadema, SP, Brazil. 2Institute of Biosciences, Sao Paulo State University, Botucatu, SP, Brazil. 3Department of Animal Reproduction, University of São Paulo, São Paulo, SP, Brazil. Abstract High environmental temperatures observed during the hot months of the year reduce fertility in lactating dairy cows. Summer heat stress depression in fertility is a multifactorial problem that affects physiological and cellular functions in several tissues. It has been shown that in addition to compromise follicular development, hormonal secretion, endometrial and embryonic function, heat stress also markedly reduces oocyte developmental ability. Oocyte susceptibility to elevated temperature can be detected during the germinal vesicle (GV) and oocyte maturation periods. In vivo (heat stress) and in vitro (heat shock) experiments indicated that exposure of bovine oocytes to elevated temperature affects the events required for successful oocyte maturation, fertilization and preimplantation embryonic development. This heat- induced decrease in oocyte function occurs due to a series of cellular alterations that affects nuclear and cytoplasmic compartments of the bovine oocyte. However, thermoprotective molecules such as growth factors and apoptosis inhibitors, which rescue heat- induced oocyte damage and developmental competence, can reverse these cellular changes. Therefore, identification of thermoprotective molecules can be considered as an alternative to modulate the effects of elevated temperature in reproductive function. Keywords: bovine, fertility, heat stress/shock, oocyte. Introduction Effects of heat stress on animal fertility Heat stress can be defined as the sum of external forces acting on a homeothermic animal to shift body temperature from the resting state (Yousef, 1984), causing physiologic, metabolic, cellular and molecular changes. Bovine are homeothermic animals which regulate internal body temperature by the balance between the amount of metabolic heat produced and heat dissipation to the environment in order to maintain constant body temperature (Hansen, 2004). Bos indicus animals (Zebu) have greater thermoregulatory ability than Bos taurus (European). Such thermoregulatory efficiency of Bos indicus cattle is due to lower internal heat production and/or higher heat dissipation to the environment. Thus, these breeds are more resistant to hyperthermia (Adeyemo et al., 1979; Bennett et al., 1985; Hammond et al., 1996, 1998). Genetic selection for high milk yield decreases the thermoregulatory ability in animals exposed to heat stress (Berman et al., 1985). Lactating cows are more susceptible to heat stress since high metabolic heat production associated with lactation predispose 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 temperatures observed during the hot months of the year reduce fertility in lactating dairy cows (Dunlap and Vincent, 1971; Badinga et al., 1985; Al-Katanani et al., 1999; Pires et al., 2002). Exposure of lactating cows to high temperature and humidity increases internal body temperature, resulting in heat stress and reduced pregnancy rates. 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 summer (Badinga et al., 1985). Al-Katanani et al. (1999) reported that 90-day non-return rate to the first service was lower when air temperature was higher than 20°C on day -10, day of insemination (day 0) and day +10 (Al-Katanani et al., 1999). Similarly, in Brazil, pregnancy rates of Holstein cows housed in free stall 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. It has been shown that in addition to compromise 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), endometrial (Malayer et al., 1988) and embryonic (Ealy et al., 1993; Paula-Lopes and Hansen, 2002b) function, heat stress also causes a great reduction in oocyte developmental potential (Al-Katanani et al., 2002; Paula-Lopes et al., 2008). The cellular mechanisms triggered by elevated temperature in bovine oocytes are not well known, as well as the molecular events associated with oocyte Paula-Lopes et al. Heat-induced damage in oocytes. 396 Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 death or survival in response to stress. This review aims to characterize the effects of elevated temperature in bovine oocytes, especially the cellular changes triggered in response to stress. The terminology heat stress and heat shock will be used to describe increased in vivo and in vitro temperatures, respectively. Effects of elevated temperature on oocyte function and developmental competence In vivo studies Bovine oocyte function is highly compromised in heat stressed animals (Rocha et al., 1998; Al-Katanani et al., 2002; Gendelman et al., 2010). Susceptibility of bovine oocytes to elevated temperature can be detected at the germinal vesicle (GV)-stage and during oocyte maturation (Fig. 1). Exposure of Holstein cows to heat stress reduced GV oocyte developmental competence as indicated by a reduction in subsequent embryonic development (Rocha et al., 1998; Al-Katanani et al., 2002). A pioneering study conducted by Putney et al. (1989) demonstrated that exposure of Holstein heifers to environmental chamber heat stress of 42°C for 10 h during oocyte maturation decreased the number of normal embryos as compared to control (24ºC; Putney et al., 1989). Figure 1. Representative images illustrating bovine GV (A) and MII (B) oocytes stained with Hoechst 33342. Arrows indicate the DNA and asterisk the polar body. The deleterious effect of heat stress on oocyte function has been well characterized in seasonal studies. Rocha et al. (1998) conducted experiments in which Bos taurus (Holstein) and Bos indicus (Brahman) oocytes collected during hot and cold season were subjected to in vitro maturation and fertilization. The percentage of oocytes classified as normal, fertilized and that reached the 8-cell stage was reduced for Bos taurus oocytes collected in the hot season as compared to the cold season. Moreover, no embryos reached morula or blastocyst stages. In contrast, developmental potential of Bos indicus oocytes was not affected by season (Rocha et al., 1998). Similarly, summer seasonal conditions reduced oocyte developmental competence in heifers, repeat-breeder and peak lactation Holstein cows. Such negative effect was more pronounced in repeat-breeder cows (Ferreira et al., 2011). Exposure of dairy cows to summer heat stress reduced conception rates from the summer to early autumn (Badinga et al., 1985), suggesting a delayed effect of heat stress on reproductive function. Heat stress caused a seasonal reduction in oocyte competence, which was recovered 2-3 estrous cycles after the end of summer (Roth et al., 2001). These findings indicated that heat stress damaged the pool of follicles and oocytes that began their growth during the warm period. Roth et al. (2001) conducted a study to characterize the delayed effect of heat stress and the rescue of oocyte quality by removal of summer damaged follicles. Holstein cows exposed to summer heat stress were subjected to follicular aspiration (OPU) on day 4 of the cycle (control group) or days 4, 7, 11 and 15 of the cycle (treated group) during four estrous cycles in the autumn cooler months. Cleavage and blastocyst rates did not differ between groups for Paula-Lopes et al. Heat-induced damage in oocytes. Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 397 oocytes collected during the first two estrous cycles. However, cleavage rate and preimplantation embryonic development increased in cycles 3 and 4 of the treated group as compared to control (Roth et al., 2001). Immediate and delayed effects of severe heat stress (environmental chamber: 38°C and 80% relative humidity (RH) during the day and 30ºC and 80% RH during the night) were also evaluated in Bos indicus animals (Gir; Torres-Júnior et al., 2008). Heat stress caused no immediate effects on reproductive function. However, there was a decrease in the proportion of embryos that reached blastocyst and expanded blastocyst stages for oocytes collected after the stress period (Torres-Júnior et al., 2008). Although Bos indicus breeds are known to be heat tolerant this study indicated that severe heat stress could also damage oocytes. Moreover, it is possible that preantral follicle- enclosed oocytes are more susceptible to heat stress than antral follicle oocytes. In vitro studies The direct effect of elevated temperature on GV and matured oocyte has been demonstrated in experiments conducted in vitro. GV-stage oocytes reside in the antral follicle environment for 42 days (Lussier et al., 1974) and during this period may be exposed to daily body temperature fluctuations above 40-41°C (Putney et al., 1989; Ealy et al., 1993; Wolfenson et al., 1993; Rivera and Hansen, 2001) compromising oocyte function prior to maturation. Development of in vitro models to study direct temperature effects on GV oocytes is challenging since removal of the oocyte from antral follicle induces spontaneous meiotic progression to metaphase II (MII) stage (Pincus and Enzmann, 1935). One approach in order to by pass this problem would be to use pharmacological tools such as meiotic inhibitors. Payton et al. (2004) evaluated the direct effects of elevated temperature on GV oocytes cultured in the presence of the meiotic inhibitor p34cdc2/cyclin dependent kinase (roscovitine-50 µm). Heat shock at 41°C for 6 and 12 h reduced embryonic development to the 8-16 cell stage and blastocyst stage, respectively (Payton et al., 2004). Similarly, culture of bovine oocytes in meiotic inhibition medium at 41°C for 14 h did not affect cleavage rate but reduced embryonic development to the blastocyst stage on days 8 and 9 after fertilization (Lima, 2012). The intensity of stress can be considered as a function of temperature and exposure time. Exposure of bovine oocytes to severe (42-44°C; not physiologically relevant temperature; Edwards and Hansen, 1996; Roth and Hansen, 2004b; Paula-Lopes et al., 2008) or moderate (40-41°C) temperatures (Edwards and Hansen, 1996; Roth and Hansen, 2004a; Paula-Lopes et al., 2008) during the first 12 h of IVM (0-12 h IVM) blocked or reduced preimplantation embryonic development, respectively. Moderate heat shock (40- 41°C) for 0-12 h during IVM reduced the proportion of day 3 cleaved embryos and day 8 blastocyst (Roth and Hansen, 2004a; Paula-Lopes et al., 2008). Moderate heat shock (40.5°C and 41.5°C) for a short period of time (30 and 60 min) during oocyte maturation did not affect development to the blastocyst stage (Ju et al., 1999). However, exposure of oocytes to severe heat shock of 43°C for short periods of 45 and 60 min during IVM reduced blastocyst and expanded blastocyst rates (Ju et al., 1999). Cellular alterations induced by elevated temperature in bovine oocytes The mechanisms by which elevated temperature affects oocyte physiology are not completely understood. However, it has been shown that depending upon stress intensity, elevated 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 response (Paula- Lopes and Hansen, 2002a). Considering that apoptosis is the major process responsible for the reduction in oocyte number on mammalian reproductive female lifetime (Morita and Tilly, 1999; Tilly, 2001) is possible that this form of cell death has an essential role in oocytes exposed to stress conditions. Heat-induced cellular damage in bovine oocytes can be observed in biological membranes as well as cytoplasmic and nuclear compartments (Table 1). However, there is evidence that oocyte cytoplasm is more susceptible to the adverse effects of elevated temperature than the nucleus (Shen et al., 2010). Nuclear transfer studies in Bos indicus and Bos taurus oocytes indicated that exposure of donor cell nucleus to heat shock did not affect embryonic development in both Bos indicus and Bos taurus oocytes. However, exposure of receptor ooplasm to heat shock decreased developmental competence of Bos taurus oocytes (Shen et al., 2010). Paula-Lopes et al. Heat-induced damage in oocytes. 398 Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 Table 1. Cellular alterations induced by elevated temperature in GV-stage and maturing oocytes. Cellular compartment Structure Alterations References GV-stage oocytes Maturing oocytes Cytoplasm Actin filaments ------------------- Reduction in peri-cellular and trans-zonal actin polymerization. (Ju and Tseng, 2004; Tseng et al., 2004; Roth and Hansen, 2005). Microtubules ------------------- Microtubule disorganization. (Ju and Tseng, 2004; Tseng et al., 2004; Ju et al., 2005; Roth and Hansen, 2005). Cortical granules (CG) Increased CG translocation to oolemma. Increased CG translocation to oolemma. (Payton et al., 2004; Edwards et al., 2005). Mitochondria Reduction in mitochondrial activity. Reduction in mitochondrial activity. (Ispada et al., 2011; Lima, 2012). Proteins Reduction in total protein synthesis. Group II caspase activity was not affected. Reduction in total protein synthesis. Increased group II caspase activity. (Curci et al., 1987; Edwards and Hansen, 1996; Edwards and Hansen, 1997). (Roth and Hansen, 2004a; Lima, 2012). Nucleus Meiotic spindle Reduction in meiotic maturation (MII stage). ------------------- Reduction in meiotic maturation (MII stage). Reduction in meiotic spindle size. (Payton et al., 2004; Roth and Hansen, 2005; Paula-Lopes et al., 2008). (Ju et al., 2005). DNA Increased DNA- fragmentation (TUNEL- positive). Increased DNA- fragmentation (TUNEL- positive). (Roth and Hansen, 2004a; Ispada et al., 2010; Lima, 2012). RNA Amount of total RNA was not affected. Amount of poly (A) RNA was not affected. (Payton et al., 2011; Gendelman and Roth, 2012). Membrane Lipids Increased saturated fatty acid. ------------------- ------------------- Phosphatidylserine translocation to outer leaflet of the plasma membrane (Annexin-V). (Zeron et al., 2001). (Kalo and Roth, 2011). Paula-Lopes et al. Heat-induced damage in oocytes. Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 399 Cytoplasmic alterations Cytoskeletal alterations are among the heat- induced cytoplasmic changes that occur in bovine oocytes. Elevated temperature affects microtubules and microfilaments organization (Ju and Tseng, 2004; Tseng et al., 2004; Ju et al., 2005; Roth and Hansen, 2005). These alterations compromise the transport and distribution of cytoplasmic organelles as well as chromosomes separation during fertilization and cleavage (Tseng et al., 2004; Ju et al., 2005). The pericellular actin ring and trans-zonal actin processes were decreased in oocytes matured at 41 (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). While GV oocytes show cortical granules aggregates (type I), GV breakdown promotes cortical granules dispersion and translocation to the oolemma (type III). Exposure of GV (Payton et al., 2004) and maturing (Edwards et al., 2005) oocytes to 41°C increased the proportion of oocytes that had type III cortical granules distribution, suggesting that heat shock hastened cytoplasmic maturation kinetics and induced oocyte aging. In contrast, the percentage of mice oocytes carrying incomplete cortical granules migration during IVM was higher in oocytes matured at 40°C as compared to 37°C (Wang et al., 2009). High temperature affects the organized αβ- tubulin heterodimer structure. Exposure of oocytes to elevated temperature caused meiotic spindle disorganization so that metaphase I and II microtubules become deformed and chromosomes are unaligned (Tseng et al., 2004; Roth and Hansen, 2005). There was also a reduction in meiotic spindle size as the duration of the heat shock at 42°C increased from 1 to 4 h (Ju et al., 2005). Changes in size and morphology of the meiotic spindle indicated that heat shock affected microtubule polymerization and depolymerization. Such changes probably compromise chromosome separation during fertilization and subsequent embryonic division. Microtubules disruption also affects cytoplasmic organelles transport such as oocyte mitochondrial distribution (Sun et al., 2001). Mitochondria are also targets to the deleterious effects induced by high temperature. Exposure of GV-stage (Lima, 2012) and maturing (Ispada et al., 2011) bovine oocytes to heat shock reduced oocyte mitochondrial activity. Similarly, exposure of high and low quality cumulus-oocyte complexes (COCs) to heat shock reduced cumulus cells mitochondrial membrane potential regardless of COCs quality (Paula-Lopes et al., 2010). Changes on oocyte mitochondrial activity may be associated with activation of apoptotic cascade. It has been shown that exposure of oocytes to heat shock during IVM increased the proportion of oocytes with high group II caspase (caspases 2, 3 and 7) enzyme activity (Roth and Hansen, 2004a), however such increase was not observed in GV-stage oocytes. Nuclear alterations Depression of nuclear maturation is one of the changes caused by heat shock in bovine oocytes. Exposure of 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 following IVM. In these experiments, heat shock blocked meiotic progression by increasing the proportion of metaphase I (MI) oocytes. However, in one study, heat shock at 41°C accelerated nuclear maturation kinetics increasing the proportion of MII oocytes after 16-18 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 12-14 h IVM increased the proportion of TUNEL- positive oocytes (Roth and Hansen, 2004a; Ispada et al., 2010). Experiments conducted with crossbred Bos indicus oocytes indicated that moderate heat shock (41°C) during the first 12 h of IVM reduced oocyte developmental competence. Such heat shock reduced nuclear maturation, cleavage and blastocyst rates (Paula-Lopes et al., 2008). However, there was no heat shock effect on the proportion of oocytes in late apoptosis stages as determined by plasma membrane permeability alterations (carbocyanine nucleic acid staining; Paula-Lopes et al., 2008). Exposure of bovine oocytes to severe heat shock (44°C) for 12 h also reduced nuclear maturation similarly to the observed response to moderate heat shock. However, there was an increase in the proportion of oocytes positive for apoptosis and necrosis as well as blockage of cleavage and embryonic development to the blastocyst stage (Paula-Lopes et al., 2008) suggesting that severe heat shock fully impaired Bos indicus oocyte developmental competence. Thermoprotective factors on bovine oocytes Heat stress in lactating dairy cows has great economic impact in warm climate dairy industry. However there are few strategies available to prevent this problem. Environmental modifications such as fans and sprinklers allow only a partial improvement in herd reproductive efficiency resulting in high costs to the dairy farm (Hansen et al., 1992; Hansen, 2004). Although such environmental changes reduce cow body temperature and increase milk yield, the effects on summer-related infertility are limited. Thus, development of alternatives to prevent the negative effects of heat stress on reproduction of dairy cows is of Paula-Lopes et al. Heat-induced damage in oocytes. 400 Anim Reprod, v.9, n.3, p.395-403, Jul./Sept. 2012 major importance in hot climates. Several approaches to mitigate the deleterious effects of heat stress in animal breeding have been previously described (Hansen et al., 1992, 2001; Hansen and Aréchiga, 1999). Thus, the focus of this topic is to present thermoprotective molecules that act by minimizing heat-induced cellular and functional damage in bovine oocytes. The deleterious effects of heat shock in bovine oocytes may be regulated by growth factors such as insulin-like growth factor I (IGF-I). IGF-I exerts autocrine, paracrine and endocrine actions in cellular metabolism, proliferation, growth and differentiation (Voss and Rosenfeld, 1992; Baker et al., 1993; Yakar et al., 1999). It has been shown that exposure of dairy cows to heat stress decreased serum IGF-I levels leading to impairment of oocyte quality (De Rensis and Scaramuzzi, 2003). Recent studies indicated that IGF-I plays a themoprotective role in GV-stage and maturing oocytes. Exposure of GV oocytes to heat shock at 41°C reduced mitochondrial activity, increased the 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 of heat shock rescuing oocyte functions. Similarly, 100 ng/ml IGF-I during IVM reversed the detrimental effects of heat shock on oocyte mitochondrial activity and heat- induced apoptosis (Ispada et al., 2010, 2011). However, in these studies the concentration of 100 ng/ml IGF-I during IVM was unable to rescue the negative effects of heat shock on blastocyst development (Risolia et al., 2011). Heat-induced oocyte DNA fragmentation (TUNEL positive) can also be reversed by supplementation of IVM medium with molecules that inhibit the apoptotic cascade, such as group II caspase inhibitor z-DEVD-fmk (Roth and Hansen, 2004a). This molecule inhibits the negative effects of heat shock on cleavage and blastocyst rates (Roth and Hansen, 2004a). Another apoptosis inhibitor molecule, sphingosine-1-phosphate (S1P), is an sphingolipid metabolite that blocks the pro-apoptotic effects of ceramide (Roth and Hansen, 2004a). Addition of S1P to the maturation medium blocked the deleterious effects of heat shock on meiotic progression, apoptosis and increased the proportion of heat-shocked oocytes that cleaved and reached blastocyst stage (Roth and Hansen, 2004a). Summary High microenvironmental temperature of the reproductive tract compromises follicular dynamics, hormonal secretion, oocyte and embryonic function. Oocytes at the GV-stage and maturation period undergo cellular changes induced by elevated temperature. These changes reduce the efficiency of physiological processes involved in oocyte growth and maturation, fertilization and preimplantation embryonic development. Bovine oocyte cellular damage caused by elevated temperature can be detected in different cellular compartments such as oocyte cytoplasmic and nuclear regions. The oocyte cytoskeleton is highly affected by high temperature. 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 suggesting activation of the heat-induced mitochondrial apoptotic pathway in bovine oocytes. Several approaches have been employed to alleviate the low fertility associated with heat stress. Recently, molecules such as IGF-I, caspase inhibitors and sphyngolipid S1P were identified as thermoprotective factors in bovine oocytes. These factors rescued several cellular functions damaged by high temperatures and increased oocyte developmental competence. Therefore, identification and characterization of cellular thermoprotective molecules can be considered as an alternative to modulate the effects of elevated temperature in reproductive function. 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