TY - JOUR
AU - Khatib, H.
AB - Abstract For the mammalian embryo to successfully complete development, it must not only incur proper timing of internal machinery, but also protect itself from potentially harmful external stimuli. These stimuli, ranging from chemical to temperature flux, can result in defects in processes regulating gamete production and quality, as well as early embryonic development. To counterbalance these potential detriments, the mammalian cell has complex machinery consisting of heat shock factors and proteins that prevent protein misfolding and malfunction. Heat shock protein (HSP) genes have become a growing topic to understand the mechanisms of successful gamete formation and embryonic development, critical factors for livestock fertility. In addition, HSP have become a focus in understanding how external stimuli during the in vitro embryo production process may have a developmental impact. To further elucidate these mechanisms, it has become a necessity for more in-depth functional studies on HSP using technologies such as RNA interference and antibody use during embryo culture. Through these studies we can gain a more comprehensive perspective of HSP function and importance during early development. In addition, information from these studies may provide critical markers for improved fertility and development. INTRODUCTION Factors ranging from temperature flux to chemical exposure could detrimentally affect embryonic development, and as such, there must be internal systems to protect and maintain cell homeostasis (Mathew and Morimoto, 1998; Schlesinger, 1990). For early mammalian development, sources of stress, such as excessive heat, can affect many aspects including gametogenesis, early embryonic development, fetal and placental growth, and lactation (Hansen, 2009). To combat stress, the cell has developed a complex system for protection. This system consists of specific proteins, known as heat shock proteins (HSP), which appear to be produced to counteract the exposure of the cell to external stimuli (Schlesinger, 1990). Functionally, HSP serve as molecular chaperones in the mammalian cell for the protection, preservation, and restoration of protein function (Schlesinger, 1990). External stimuli shown to induce the expression of HSP genes in embryos include, but are not limited to, viruses, heavy metal exposure, chemicals, light, and temperature flux (Collins and Hightower, 1982; Hales et al., 2005; Korhonen et al., 2009; Mortensen et al., 2010; Pinsino et al., 2011). In terms of cell survival, this is critical because external stimuli can cause misfolding or aggregation of proteins within the cell to potentially toxic levels (Outeiro et al., 2006; Akerfelt et al., 2010). Protein misfolding leads to a cascade of effects on other functions such as oxidative balances, energy production, and mitochondrial integrity (Gregersen and Bross, 2010). These consequences could ultimately result in cell death (Davenport et al., 2008). Therefore, HSP could have a relatively large impact on the success of the early developing embryo and a key part to normal development. Organization and Regulation of HSP There are numerous HSP within the mammalian cell system and, as such, are organized into groups according to molecular weight. Overall, the 6 groups of HSP are divided into the following families: HSP40, HSP60, HSP70, HSP90, HSP100, and small HSP (HSPB) families, which can work independently or jointly to address protein aggregation and misfolding (Mathew and Morimoto, 1998). In addition, specific roles may be assigned to individual families as both members of HSP70 and HSP90 have been implicated in protein degradation pathways (Mathew and Morimoto, 1998). Heat shock proteins are regulated through heat shock factors (HSF; Akerfelt et al., 2007, 2010). Within the mammalian system, HSF work to both maintain normal development and address stress through functions within 4 groups (HSF1, –2, –3, and –4; Akerfelt et al., 2010; Bjork and Sistonen, 2010). Functionally, HSF bind to heat shock elements that are present upstream of HSP transcriptional start sites (Wu, 1995). Interestingly, evidence indicates that these HSF function in unique ways to regulate HSP during development. Heat shock factor 1 binds to an upstream regulatory region in the promoter of HSP genes to cause activation of transcription (Anckar and Sistonen, 2007). Ostling et al. (2007) demonstrated that both HSF1 and HSF2 bind to the upstream promoter region of the HSP gene HSP70. Interestingly, the binding affinity of HSF2 was dependent on the presence of HSF1, indicating a complex interplay between HSF (Ostling et al., 2007). This interplay was also noted with HSF4, which can influence the binding of HSF1 at specific sites (Fujimoto et al., 2008). Functionally, HSF4 binds to intronic and distal portions of protein-coding genes to alter the chromatin structure of specific target genes (Fujimoto et al., 2008). Therefore, not only can HSF alter expression of HSP, but they also alter conformation and subsequent function of methylation sites. Thus, HSF can influence both directly or indirectly response to external stimuli by either targeting HSP or altering binding affinity for other factors. Developmentally, HSF have been implicated in early development roles. Christians et al. (2000) reported that Hsf1–/– female mice produced oocytes that were able to complete initial development (through fertilization) but then failed to develop once transferred to the uterus. Similarly, Hsf2–/– female mice incurred increased embryonic lethality during late gestation (Wang et al., 2003). In terms of fertility, this study also found that female Hsf2–/– mice had reduced litter sizes and male Hsf2–/– mice had reduced testicular size and sperm counts (Wang et al., 2003). Generation of mutant knockouts have allowed for further evidence of not only the specific effects of certain HSP genes, but also an understanding of their imperative role during development. Timing of HSP Expression Although HSF play important roles in regulation of HSP, there are other factors that determine HSP function in an organism. Namely, HSP can be broken into 2 further categories: constitutive and inducible. Although associated with a stress response, HSP can be present in unstressed binding to nascent polypeptides and functioning as a molecular chaperone (Hendrick and Hartl, 1995). Developmentally, altering constitutively expressed HSP could have dramatic effects on an organism. Turturici et al. (2009) reported constitutive expression of HSP70 in unstressed mouse A6 mesangioblast cells. In addition, they reported that Ku factor binds to the proximal promoter of HSP70 to enhance the expression of this gene under unstressed conditions, whereas HSF1 binds under stressed conditions (Turturici et al., 2009). Functionally, stable knockdown of HSP70 in these cells resulted in decreased cell proliferation (Turturici et al., 2009). In addition to proliferation, roles for HSP in mitosis during spermatogenesis have been discussed (Eddy, 1999), thus providing evidence for roles outside of the stress response. Although some HSP genes are expressed throughout development, others are activated by external stimuli classifying them as inducible. One example of an inducible HSP gene was shown in developing rat brain, where it was found that when exposed to 10 to 50 mg/kg of morphine once per day for 10 d, the relative expression of HSP70 increased (Loones et al., 2000; Ammon-Treiber et al., 2004). The complex nature of the 2 expression-based categories can be observed during early embryonic development. Loones et al. (2000) detected constitutively expressed heat shock cognate 70 and HSP90β in embryonic d 9.5 mouse brains, but inducible HSP70 and HSP90α were not detected. However, expression of both HSP90α and HSP70 were detected by embryonic d 15.5 indicating temporal expression during development (Loones et al., 2000). Thus, HSP can display both time- and stimuli-dependent expression to ensure survival of an organism. Roles of HSP during Gametogenesis For early mammalian development, sources of stress can start as early as gametogenesis, with potential downstream effects. Across several mammalian species, evidence of heat stress response mediated by HSP has been observed within the female. Temperature has been long established as a critical influence on bovine in-vitro embryo production. Lenz et al. (1983) reported the optimal culture temperature to be approximately 39°C. When exposed to temperatures higher than this (i.e., 40° and 41°C) for the first 12 h during maturation, developmental competence of the bovine oocyte decreased (Roth and Hansen, 2004). Namely, increased apoptosis via terminal deoxynucleotidyltransferase (dUTP) nick end labeling was detected in oocytes from the 41°C group in comparison with the control group (Roth and Hansen, 2004). The percentage of cleaved oocytes developing to blastocyst was also noted as being slightly reduced (Roth and Hansen, 2004). In addition to the internal characteristics of the oocyte, cumulus cells surrounding the bovine oocyte have also been reported to increase expression of HSP70 and HSP71 when heat shock occurs during maturation (Edwards and Hansen, 1997). Thus, the authors suggested another example of a mechanism in which the supporting nature of cumulus cells involves HSP in a protective manner. Similarly, a study by Mortensen et al. (2010) found sensitivity of the equine oocyte to heat during late oocyte maturation. Interestingly, heat-shocked oocytes showed greater expression of HSPA1 indicating an induced HSP response due to the stress (Mortensen et al., 2010). In another model species, the mouse, chronic heat shock has been shown to cause a disruption in oocyte maturation as well (Kim et al., 2002). In the male, thermoregulation is critical for proper spermatogenesis, because even minor changes in temperature can cause drastic and damaging effects. An early study by Chowdhury and Steinberger (1970) demonstrated that rat testes exposed to increased temperatures had abnormal pachytene dividing spermatocytes and young spermatids. Evidence has mounted for the individual importance of HSP during sperm formation because loss of the small HSP ODF1/HSBP10 caused infertility in mice (Yang et al., 2012). In addition, Kaneko et al. (1997) reported that expression of the HSPa41 in numerous mouse tissues, the greatest expression was found in testes. Later research established that although HSPa41–/– female mice appeared normal, males had increased incidence of infertility (Held et al., 2011). In pigs, expression of HSP was found to occur during development from the immature testis to the mature testis, indicating developmental stage-specific expression during spermatogenesis (Huang et al., 2005). For example, HSP70 has been implicated to have roles involved in the acrosome reaction and capacitation which occurs during fertilization and is necessary for formation of the preimplantation embryo (Kamaruddin et al., 2004). Preimplantation Embryonic Development and HSP Although the precise functions of HSP in the embryo still remain unclear, studies have begun to recognize their importance in successful development of the embryo. In a study with mice by Christensen et al. (2010), HSP60 ablation resulted in early embryonic lethality. In another study, reduction of HSP70 with either antibody or small interfering RNA (siRNA) was associated with increased oocyte development and maturation in mice (Liu et al., 2010). This study found that, by knocking down HSP70, there was an increase in oocytes transitioning from germinal vesicle to Metaphase II stage (Liu et al., 2010). In addition, HSP70 knockdown resulted in increased Bmp5 and Gdf9 expression, 2 genes with known roles in oocyte development (Liu et al., 2010). Besides HSP, alterations of genes in the heat shock pathway show developmental importance. Mice lacking Faf1, involved in apoptosis and heath shock pathways, incurred embryonic lethality (Adham et al., 2008). Although mice are a traditional laboratory model, studies on HSP have expanded to livestock species. In a study of bovine embryos, increased abundance of HSP showed association with apoptosis (Fear and Hansen, 2011). Recently, we used a bovine in vitro embryo production (IVP) system to determine association between gene expression and developmental status. That is, bovine embryos were developed in vitro and graded on morphological characteristics. Those showing expected blastocyst morphological characteristics after 8 d of culture were compared with those that arrested in development between d 5 and 8 of culture and deemed to be degenerating. This system has been used in prior studies uncovering candidate genes for development, as presented in Huang and Khatib (2010) and Huang et al. (2010). Zhang et al. (2011) found significant associations between expression of specific HSP and degeneration in the preimplantation embryo. Of the 17 HSP genes assayed, gene expression fold-differences ranged from 1.5 to 7.6 between the blastocyst and degenerate embryos. Of particular interest was the observation of specific patterns within certain HSP families. For example, all members of the HSP40 family were found to be more highly expressed in the degenerate embryos. In contrast, a majority of the HSP70 family members showed greater expression in blastocysts, indicating specific roles of the HSP during early embryonic development (Zhang et al., 2011). An association between HSP abundance and embryonic development has also been shown in buffalo embryos. In a study by Sharma et al. (2012), amounts of HSP70 were assessed between fast-growing and slow-growing IVP buffalo embryos. Expression analysis showed that, although HSP70 expression was present in oocytes and all preimplantation embryo stages in the fast growing embryo group, it was not present past the oocyte stage in slow-growing embryos (Sharma et al., 2012). Thus, those embryos that are retarded in growth may not have active HSP70 to help protect from cellular stress and apoptosis. These results provide an example that these proteins are critical for the in vitro embryo as there are numerous potential sources of external stress during growth. Potential Stressors of IVP For a livestock embryo, one of the biggest sources of stress may come from the manipulations occurring during IVP (Pribenszky et al., 2010). During this process, gametes are co-incubated for the subsequent development into embryos within a laboratory environment to eventually be transferred back into a recipient animal. It is during this period that the embryo may endure stimuli including, but not limited to, temperature flux, differing CO2 and O2 concentrations, and lighting changes (Pribenszky et al., 2010). All of these factors serve as potential stressors and, as such, studies have been completed to assess HSP abundance between in vivo and in vitro embryo production. Indeed, differences between HSP have been found between in vivo and in vitro embryos for multiple species. For example, levels of HSP70 members were increased in porcine (McElroy et al., 2008) and equine (Mortensen et al., 2010) embryos produced by IVP. In a recent study, our laboratory performed a comparative genomic analysis of in vivo and in vitro produced embryos using RNA-Sequencing technology (Driver et al., 2012). Numerous genomic differences were found, including a variety of alternative splicing events, differential gene expression, and differential expression of biological pathways. This is of particular interest because the embryos were morphologically of similar stage and quality at the time of grading, and yet showed transcriptomic differences emphasizing the importance of assessing embryos beyond morphological characteristics. In the study of Zhang et al. (2011), our lab found several HSP genes to be differentially expressed between blastocysts and degenerate embryos. To test whether or not the expression of these genes was biased because of IVP conditions, expression of 5 genes that had shown greater than 2-fold difference between blastocyst and degenerates were assessed between in vivo and in vitro embryos (Fig. 1). The level of increase between in vivo and in vitro embryos varied for these genes as DNAJC19, DNAJC24, and HSPB1 showed 1.3-, 1.6-, and 1.64-fold increases, respectively. The genes DNAJC15 and DNAJC27 showed greater differences, having 2.6- and 5.6-fold increases, respectively, between in vivo and in vitro embryos (Fig. 1). Both of these genes showed the greatest fold change between blastocyst and degenerate embryos with a 7.6- and 4.3- fold increase, respectively (Fig. 1). Thus, there may be a potential pattern between stress of the in vitro process and detriment to development of the embryo for specific HSP genes. In addition, developmentally important genes may have sensitivity to external influences such as IVP. However, the question remains whether or not alterations in HSP levels would have long-term effects on development. Figure 1. View largeDownload slide Fold increase in expression (±SE) of heat shock protein genes in blastocyst vs. degenerate embryos and in vivo versus in vitro bovine embryos. Expression of DNAJC15, DNAJC19, DNAJC27, and HSPB1 was greater in the in vitro embryos whereas expression of DNAJC24 was greater in the in vivo embryos (light gray bars). All examined genes were highly expressed in degenerate embryos compared with blastocysts (dark gray bars). Genes assayed were DnaJ homolog, subfamily c, member 15 (DNAJC15); DnaJ homolog, subfamily c, member 19 (DNAJC19); DnaJ homolog, subfamily c, member 24 (DNAJC24); DnaJ homolog, subfamily c, member 27 (DNAJC27); and heat shock protein β-1 (HSPB1). Figure 1. View largeDownload slide Fold increase in expression (±SE) of heat shock protein genes in blastocyst vs. degenerate embryos and in vivo versus in vitro bovine embryos. Expression of DNAJC15, DNAJC19, DNAJC27, and HSPB1 was greater in the in vitro embryos whereas expression of DNAJC24 was greater in the in vivo embryos (light gray bars). All examined genes were highly expressed in degenerate embryos compared with blastocysts (dark gray bars). Genes assayed were DnaJ homolog, subfamily c, member 15 (DNAJC15); DnaJ homolog, subfamily c, member 19 (DNAJC19); DnaJ homolog, subfamily c, member 24 (DNAJC24); DnaJ homolog, subfamily c, member 27 (DNAJC27); and heat shock protein β-1 (HSPB1). Alternative Splicing of HSP: Critical during Stress? In addition to expression levels, patterns of alternative splicing for HSP were also associated with early embryonic degeneration. Alternative splicing research has become an area of interest because it provides insight on the RNA processing and protein functions produced from a single gene. Studies have shown that 1 gene can produce multiple proteins with altered properties ranging from binding affinity to intracellular localization (Stamm et al., 2005). Protein function can differ between isoforms and cause subsequent detriment to development. For example, in the mouse, 2 isoforms of RET receptor kinase have been implicated in specific functions because mice lacking the RET51 isoform have kidney malformation and lack enteric ganglia in the colon (de Graaff et al., 2001). Studies have indicated that stressors, such as heat shock on a cell, may halt activities such as pre-mRNA splicing (Biamonti and Caceres, 2009). However, appropriate splicing for HSP90a, HSP90b, and HSP27 has been shown to occur in human cells under heat shock, indicating that HSP may escape this shutdown (Jolly et al., 1999; Biamonti and Caceres, 2009). Thus, the multiple isoforms of HSP may be critical in aiding the cell during times of stress and should be characterized further to understand their presence and role during early development. Although our laboratory established differential expression among HSP genes, only a select number of genes showed evidence of alternative splicing (Zhang et al., 2011). Four genes (DNAJC5, DNAJC15, DNAJC24, and DNAJB12) had multiple isoforms and as such were assayed further for differential isoform abundance between embryo populations (Table 1). Using quantitative real-time PCR, the abundance of the individual variants was measured, thereby providing insight on the processing of HSP between developmentally different embryos (Table 1). Of these variants, all were increased in the degenerate embryos, thereby providing evidence that specific RNA isoforms may be associated with developmental differences during early embryonic development. However, further analysis should be pursued to determine potential biological roles that distinguish the variants functionally. Table 1. Differential expression of the splice variants of heat shock protein genes in blastocyst vs. degenerate embryos and in vivo vs. in vitro bovine embryos1 Gene  Splice variant, bp  Blastocyst vs. degenerate  In vivo vs. in vitro  DNAJC5  667  1.672  1.04    725  Not present  1.123    1049  2.532  1.48  DNAJC19  475  Not expressed  Not expressed (in vivo), Lowly expressed (in vitro)    536  Lowly expressed  2.04  DNAJC24  628  Lowly expressed  Lowly expressed (in vivo), Not expressed (in vitro)    2172  3.222  2.433    1893  Not expressed  Not expressed  DNAJB12  1220  1.952  1.12  Gene  Splice variant, bp  Blastocyst vs. degenerate  In vivo vs. in vitro  DNAJC5  667  1.672  1.04    725  Not present  1.123    1049  2.532  1.48  DNAJC19  475  Not expressed  Not expressed (in vivo), Lowly expressed (in vitro)    536  Lowly expressed  2.04  DNAJC24  628  Lowly expressed  Lowly expressed (in vivo), Not expressed (in vitro)    2172  3.222  2.433    1893  Not expressed  Not expressed  DNAJB12  1220  1.952  1.12  1The 4 genes tested were DnaJ homolog subfamily C member 5 (DNAJC5), DNAJ homolog subfamily C member 19 (DNAJC19), DnaJ homolog subfamily C member 24 (DNAJC24), and DnaJ subfamily b member 12 (DNAJB12). 2Expression is greater in degenerate embryos that arrested in development between the morula to blastocyst transtition compared with blastocyst embryos. 3Expression is greater in the in vivo embryos. Remaining values are greater in the in vitro embryos. View Large Table 1. Differential expression of the splice variants of heat shock protein genes in blastocyst vs. degenerate embryos and in vivo vs. in vitro bovine embryos1 Gene  Splice variant, bp  Blastocyst vs. degenerate  In vivo vs. in vitro  DNAJC5  667  1.672  1.04    725  Not present  1.123    1049  2.532  1.48  DNAJC19  475  Not expressed  Not expressed (in vivo), Lowly expressed (in vitro)    536  Lowly expressed  2.04  DNAJC24  628  Lowly expressed  Lowly expressed (in vivo), Not expressed (in vitro)    2172  3.222  2.433    1893  Not expressed  Not expressed  DNAJB12  1220  1.952  1.12  Gene  Splice variant, bp  Blastocyst vs. degenerate  In vivo vs. in vitro  DNAJC5  667  1.672  1.04    725  Not present  1.123    1049  2.532  1.48  DNAJC19  475  Not expressed  Not expressed (in vivo), Lowly expressed (in vitro)    536  Lowly expressed  2.04  DNAJC24  628  Lowly expressed  Lowly expressed (in vivo), Not expressed (in vitro)    2172  3.222  2.433    1893  Not expressed  Not expressed  DNAJB12  1220  1.952  1.12  1The 4 genes tested were DnaJ homolog subfamily C member 5 (DNAJC5), DNAJ homolog subfamily C member 19 (DNAJC19), DnaJ homolog subfamily C member 24 (DNAJC24), and DnaJ subfamily b member 12 (DNAJB12). 2Expression is greater in degenerate embryos that arrested in development between the morula to blastocyst transtition compared with blastocyst embryos. 3Expression is greater in the in vivo embryos. Remaining values are greater in the in vitro embryos. View Large HSP and Fertility: Potential Markers for Development It is clear that HSP have been implicated as important factors in early embryonic development and, as such, may serve as strong markers for fertility traits. This has already been demonstrated in part, by a number of studies in cattle. For livestock, fertility is critical for production because infertility can cause imminent financial strain due to costs of rebreeding and lost production. Therefore, determining potential markers for improved fertility has been a continual process. It is of interest to note that genetic differences have been determined between breeds of cattle that show increased tolerance to high temperatures (i.e., thermotolerance; Paula-Lopes et al., 2003). Currently, there is growing evidence for the association between specific HSP and the ability for cattle to counteract temperature challenges. Basirico et al. (2011) reported that cows heterozygous for a cytosine deletion in the 3ʹ-untranslated region of the inducible HSP70.1 resulted in increased viability of peripheral blood mononuclear cells after heat shock at 43°C for 1 h. Increased abundance of HSP70.1 mRNA and protein were also detected, indicating increased tolerance to heat stress (Basirico et al., 2011). Specific polymorphisms within HSP70 were also associated with improved heat stress tolerance in Chinese Holstein cattle (Li et al., 2011). Therefore, marker selection using this gene may help improve the ability of a cow to endure increased temperatures. By accomplishing this, the effects of heat stress on fertility may be minimized and breeding programs could be more successful. However, further work is necessary to determine the most influential markers for improvement of fertility traits. In addition to associations with thermotolerance, HSP70 has been implicated in specific fertility traits. Rosenkrans et al. (2010) reported an association between HSP70 polymorphisms and calving traits in Brahman cattle. Interestingly, all of these polymorphisms were within the promoter region indicating that alterations in HSP70 function could reduce fertility. As such, these mutations may serve as markers for improved calving rate. In addition to genes within the HSP70 family, those within the HSP40 family have also been implicated in fertility. Recently, we reported significant associations between SNP within 2 bovine HSP40 genes (DNAJC15 and DNAJC27) with fertilization rate and blastocyst development in vitro (Zhang et al., 2011). Given that both of these genes also showed differential expression between developed and arrested in vitro embryos, these findings indicate that HSP have a strong relationship with development and should be characterized further for marker assisted selection in cattle breeding schemes. Future Studies to Characterize the Importance of HSP within the Embryo Although the evidence for the importance of HSP during early development has been surmounting, there is a need to pursue further studies to characterize their direct function. One method that has gained increasing popularity for assessing single-gene function on early embryonic development is known as RNA interference (RNAi). Using preexisting machinery within the mammalian cell, siRNA fragments can be introduced into a 1-cell embryo. The siRNA will target specific gene transcripts and degrade them before translation, thus knocking down function of the gene product. Developmental observations can be conducted during in vitro maturation to assess whether knockdown of that specific gene product has significant effects on the preimplantation embryo. Multiple studies have reported using this system with success (Lee et al., 2009; Tesfaye et al., 2010; O'Meara et al., 2011; Wang et al., 2011). We have used this system to assess functional effects of 2 imprinted genes, CDKN1C and PHLDA2, on early embryonic development. Silencing of CDKN1C caused decreased blastocyst development when knocked down with siRNA (A. Driver, H. Khatib, unpublished data), whereas silencing of PHLDA2 produced dosage-sensitive effects that improved embryonic development (A. Driver, H. Khatib, unpublished data). Thus, RNAi technology provides the opportunity to functionally investigate the roles of candidate genes in the developing embryo. At present, no RNAi experiments in livestock embryos have been reported for HSP, leaving a large opportunity for growth and expansion on our knowledge of HSP within the embryo. In addition to RNAi, use of antibodies during embryo culture can provide insight on HSP function. A recent study by Olexikova et al. (2010) added an antibody against HSP70 to determine functional effects in the developing rabbit embryo. Embryos were then exposed to either control or hyperthermic conditions to determine roles of HSP70 during the heat shock response. Interestingly, their analysis used more structural characteristics (i.e., lipid composition, nucleoar area) allowing for a more direct phenotypic analysis of depleted heat shock function. Studies like this could provide more direct insight on the physiological impacts of external stimuli on embryo development and provide a noninvasive method of depleting a HSP to determine subsequent functional effects on development. SUMMARY AND CONCLUSIONS Overall, HSP have been shown to have functional effects during both gametogenesis and early embryonic development. Because these factors are key components to livestock fertility, it would be of interest to further pursue HSP function and potential opportunity as a marker for improved or hindered development. 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Google Scholar CrossRef Search ADS PubMed  Footnotes 1 Based 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. 2 This study was supported by Hatch grant No. 142-PRJ16JH from the USDA National Institute of Food and Agriculture to the University of Wisconsin-Madison. American Society of Animal Science
TI - PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Heat shock proteins: Potentially powerful markers for preimplantation embryonic development and fertility in livestock species,
JF - Journal of Animal Science
DO - 10.2527/jas.2012-5928
DA - 2013-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/physiology-and-endocrinology-symposium-heat-shock-proteins-potentially-HiOYYuS8kf
SP - 1154
EP - 1161
VL - 91
IS - 3
DP - DeepDyve
ER -