Association of heat shock protein 70 with
semen quality in boars
S.Y. Huang
a,c, Y.H. Kuo
b, Y.P. Lee
c, H.L. Tsou
d, E.C. Lin
d,
C.C. Ju
e, W.C. Lee
a,∗aDepartment of Comparative Medicine, Pig Research Institute Taiwan,
P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC
bDepartment of Applied Biology, Pig Research Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC cDepartment of Animal Science, National Chung-Hsing University,
250 Kuo-Kwang Rd., Taichung 402, Taiwan, ROC
dDepartment of Animal Resources, Pig Research Institute Taiwan,
P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC
eCouncil of Agriculture, Executive Yuan, Taipei, Taiwan, ROC
Received 10 August 1999; received in revised form 22 May 2000; accepted 16 June 2000
Abstract
This study attempted to clarify the relationship between the levels of 70 kDa heat shock pro-tein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7
Yorkshire) boars (mean age=25.2±2.2 months) were examined. Three to four ejaculates per boar,
collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proxi-mal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than
those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the
hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that
protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly
lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low
correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest
∗Corresponding author. Tel.:+886-37-672352/ext. 587; fax:+886-37-692806.
E-mail address:[email protected] (W.C. Lee).
that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Heat shock protein 70; Semen quality; Season; Boar
1. Introduction
In most mammals, spermatogenesis deteriorates at an elevated testis temperature even only reaching as high as body temperature (Chowdbury and Steinberger, 1964; Vandemark and Free, 1970). This phenomenon implies that spermatogenetic cells are extremely sensi-tive to heat stress. Although not affecting semen volume or gel weight, heat stress decreases sperm motility and percentage of normal sperm and increases the percentage of abnormal sperm and sperm with aged acrosome (McNitt and First, 1970; Christenson et al., 1972; Wettemann et al., 1976). Conception rates declined in sows that were either artificially in-seminated or were naturally mated with heat stressed boar (Wettemann et al., 1976, 1979). Wettemann and Desjardins (1979) indicated that heat stress suppressed maturation of sperm cell, subsequently decreasing the sperm output and semen quality. In the subtropical climate of Taiwan, the semen quality of boars generally decrease during the summer, ultimately de-creasing reproductive performance (Cheng and Wung, 1974; Liu et al., 1994; Kuo et al., 1997).
Cells or multi-cell organisms respond to heat or other stresses by inducing or increasing the synthesis of a group of unique proteins commonly referred to as heat shock proteins or HSPs (Lindquist, 1986; Lindquist and Craig, 1988; Welch, 1992). Although the exact functions of HSPs are still unclear, one of their roles is to prevent the organisms from adverse environmental impacts (Li and Laszlo, 1985; Pelham, 1986; Welch, 1992). Many studies have demonstrated that HSPs, particularly the abundantly expressed 70 kDa HSP (HSP70), play important roles in acquired thermotolerance (Li and Laszlo, 1985; Lindquist, 1986; Subjeck and Shyy, 1986; Sanchez and Lindquist, 1990; Nover, 1991; Sanchez et al., 1992), and HSP70 has been suggested to function as an indicator of thermotolerance in cells (Craig and Gross, 1991; Leung et al., 1996). According to recent vertebrate-related studies, HSP70 plays important roles in many stresses in multiple-cell organisms and protects lizards from heat stress (Ulmasov et al., 1992; Lyashko et al., 1994; Malysheva et al., 1994; Marber et al., 1995; Plumier et al., 1995). Whether or not HSP70 could function as an indicator of thermotolerance in livestock requires further study.
2. Materials and methods
2.1. Experimental animals, semen collection and quality evaluation
Twenty-nine normal, mature boars (13 Duroc, 9 Landrace, and 7 Yorkshire) from an AI center located in northern Taiwan were used. The average age was 25.2±2.2 months.
Three to four samples per boar were collected during cool (average temperature range was 15.0±0.3 to 22.2±0.4◦C) and hot (average temperature range was 23.7±0.3 to 32.0±0.4◦C) seasons. A phase contrast microscope was used to evaluate semen quality traits including sperm motility, sperm concentration, percentages of normal sperm, sperm with persistent proximal plasma droplets, sperm with persistent distal plasma droplets, and abnormal sperm (Kuo et al., 1997).
2.2. Gel electrophoresis
For protein analysis, the spermatozoa were washed with an extender and centrifuged at 800×gfor 1 min. The pellets were then lysed in Laemmli’s sample buffer (Laemmli, 1970), boiled for 5 min and then stored at−20◦C until further analysis. In addition, 500ml of the supernatant were mixed with an equal volume of Laemmli’s buffer and saved for later analysis.
Proteins from 3×106 spermatozoa or 10ml of seminal fluid were separated by 9%
SDS-PAGE according to Laemmli (1970). The molecular standards were purchased from BioRad (Hercules, CA, USA). After electrophoresis, the gels were stained with 0.1% Coomassie brilliant blue R-250 for 60 min, followed by destaining with a solution con-taining methanol and acetic acid until the background was clear.
2.3. Western blot analysis and quantitation of heat shock protein 70
Western blot analysis was performed according to a procedure described elsewhere (Lee et al., 1996; Huang et al., 1999). After electrophoresis, the gels were blotted with monoclonal antibodies against mouse HSP70/72 (clone N27F3-4, Stressgen, Victoria, Canada; diluted 1:1000 with TTBS containing 1% gelatin) andb-tubulin (Amersham; diluted 1:500) fol-lowed by biotinylated goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St. Louis, MA, USA; diluted 1:5000). The membranes were then developed with a buffer con-taining nitro blue tetrazolium and 5-bromo-4-chloro-3-indoly phosphate (BioRad). The color of immunocomplex was displayed at a proper intensity and within a linear range.
Table 1
Grouping of samples by level of heat shock protein 70
Group
2.4. Grouping of samples by level of heat shock protein 70
All the samples were grouped according to the mean (M) and one-half standard deviation (S.D.) of HSP70 level in order to evaluate how HSP70 affects semen quality traits. Six groups were defined as shown in Table 1.
2.5. Statistical analysis
The extent to which season and breed affect semen quality traits, level of HSP70 (sum of constitutive and inducible form of HSP70) was analyzed by adopting the GLM procedure of SAS (SAS Institute, 1989). The statistical model included season (S), breed (B), S×B, and boar (S×B). The effect of HSP70 grouping was also analyzed by the GLM procedure. The extent to which the effects differed was determined by the least squares means method. Correlation coefficients between levels of HSP70 and semen quality traits were Pearson correlation calculated by the CORR procedure of SAS (SAS Institute, 1989).
3. Results
3.1. Protein profile and qualitative characterization of heat shock protein 70 in boar spermatozoa
This study analyzed the sperm protein contents of 29 boars using SDS-PAGE. Fig. 1 displays typical protein profiles of boar spermatozoa. Coomassie blue staining of the gels revealed more than 50 proteins with molecular weights ranging from 30 to 100 kDa (Fig. 1a). In addition, different breeds or different seasons did not apparently differ in the overall protein profile of spermatozoa. Furthermore, an anti-mouse HSP70/72 monoclonal antibody was used to detect the level of constitutive and inducible form of HSP70 in boar spermatozoa (Fig. 1b). Both forms of HSP70 could be easily detected with a commercial antibody. However, no HSP70 was detected in the seminal fluid (data not shown).
3.2. Seasonal and breed effects on semen quality and level of heat shock protein 70 in boar spermatozoa
Fig. 1. Protein profiles and immunoblotting analysis of boar spermatozoa. Samples from cool (Lanes 2, 4 and 6) and hot season (Lanes 3, 5 and 7), and different breeds of boar (Duroc: lanes 2 and 3; Landrace: lanes 4 and 5; Yorkshire: lanes 6 and 7) were subjected to SDS-PAGE, followed by staining with Coomassie blue (a). In a separate experiment, proteins in polyacrylamide gel were transferred to a nitrocellulose membrane and blotted with monoclonal antibody against HSP70 andb-tubulin (b). Each lane contains total proteins from 3×106spermatozoa except lane 1 which contains molecular weight standards. HSC70 and HSI70 are the constitutive and inducible form of 70 kDa heat shock protein, respectively.
Table 2
Least squares means of semen quality traits and level of HSP70 in boar spermatozoaa
Traits Cool season Hot season
aMeans within the same row with different letters differ significantly (P<0.05). bPPD: sperm with proximal plasma droplets.
cDPD: sperm with distal plasma droplets.
dHSP70: level of 70 kDa heat shock protein, which was normalized optical density by usingb-tubulin as covariate with values of arbitrary unit.
3.3. Correlation between level of heat shock protein 70 and porcine semen quality
The correlation coefficients between level of HSP70 and semen quality traits were cal-culated (Table 3), and no significant correlation was found between levels of HSP70 and semen quality traits in either cool season or hot season. Therefore, a different approach was employed to evaluate the relationship between HSP70 and semen quality traits. The samples were grouped according to their HSP70 level and the semen quality traits were compared at different HSP70 levels.
The levels of HSP70 for all the samples collected ranged from 0.34 to 3.06 with a mean value of 1.27 and a standard deviation of 0.46. Thus, HSP70 levels of 1.73, 1.5, 1.27, 1.04, 0.81 were used as cutting points for grouping. The HSP70 levels in the six groups were Group 1>1.73, 1.73>Group 2>1.5, 1.5>Group 3>1.27, 1.27>Group 4>1.04, 1.04>Group 5>0.81, and 0.81>Group 6. Table 4 lists the semen quality traits for the six groups, indicating that the level of HSP70 and semen quality appeared to have a good correlation. The correlation for boars with a spermatozoa HSP70 level higher than 1.04 (Groups 1–4) was not obvious.
Table 3
Correlation coefficients of level of HSP70 and semen quality traits in boars
Traits Cool season Hot season
Sample size 113 112
Motility 0.00 0.14
Normal sperm 0.10 0.15
Sperm with proximal plasma droplets −0.05 −0.20∗
Sperm with distal plasma droplets −0.11 −0.08
Table 4
Least squares means of semen quality traits among groups with different levels of HSP70 in boar spermatozoaa,b
Traits Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Number of samples 33 29 41 51 38 33
Motility (%) 75.9±2.5 ab 77.9±2.6 a 75.4±2.2 ab 72.5±2.0 ab 70.5±2.3 b 58.5±2.5 c
aMean and standard deviation of total HSP70 was 1.27 and 0.46. The cut points of HSP70 level for grouping were 1.73, 1.5, 1.27, 1.04, 0.81 as defined in Section 2.
bMeans within the same row with different letters differ significantly (P<0.05). cPPD: sperm with proximal plasma droplets.
dDPD: sperm with distal plasma droplets.
eHSP70: level of 70 kDa heat shock protein, which was normalized optical density by usingb-tubulin as covariate with values of arbitrary unit.
However, for Groups 5 and 6 with a HSP70 level lower than 1.04, sperm motility, percentage of normal sperm, and sperm concentration significantly decreased and abnormal sperm and sperm with plasma droplets increased.
4. Discussion
The 70 kDa heat shock protein is the most abundant and highly conserved HSPs in all organisms studied so far (Lindquist and Craig, 1988). In eukaryotic cells, the expression of HSP70 is encoded by a multigene family and can be divided into constitutively expressed and stress-inducible form (Lindquist and Craig, 1988; Welch, 1992). In pigs, at least four genes regulate the expression of HSP70 (Nunes et al., 1993). van Laack et al. (1993) has demonstrated that HSP70 is present in cardiac and longissium muscle, liver, spleen, kidney, and adrenal glands of pig. Our Western blot analysis using a commercial HSP70/72 antibody revealed that both constitutive and inducible forms of HSP70 were appeared in ejaculated spermatozoa from boars of different breeds (Fig. 1b). Ejaculated spermatozoa are highly differentiated cells and lack the biosynthetic machinery to cope with adverse environmental impacts (Stewart et al., 1984; Hammersdtedt et al., 1990). The HSP70 proteins found in spermatozoa must therefore be synthesized during spermatogenesis before the formation of mature spermatozoa.
during hot season (Table 2) implies that boars cannot effectively respond to a high ambient temperature during the hot season by increasing the expression of HSP70. Grouping the samples according to the level of HSP70 allowed us to observe a correlation between the level of HSP70 in spermatozoa and semen quality, implying that HSP70 might significantly affect the heat stress response in boars.
The relationship between the level of HSP70 and economic traits in pigs has seldom been mentioned. Although van Laack et al. (1993) failed to find a relationship between meat quality and the expression of either form of HSP70, the promoter region of HSP70.2 gene seemed to influence meat quality (Schwerin et al., 1996) and birthweight (Maak et al., 1998). Kamaruddin et al. (1996) observed the presence of HSP70 in the spermatozoa of normal mature bulls and its important role in sperm–oocyte interaction. Under this situation, HSP70 possibly acts as a receptor to ligands on the zona pellucida (Kamaruddin et al., 1998). Whether or not a similar phenomenon occurs in boar spermatozoa requires further investigation.
In conclusion, our study has demonstrated that both constitutive and inducible forms of HSP70 are present in ejaculated spermatozoa of boars. In addition, both the level of HSP70 in spermatozoa and semen quality traits of boars significantly decline during the hot season. In addition, the level of HSP70 in spermatozoa appears to be correlated with semen quality in pigs. Nevertheless, the physiological role of HSP70 in thermotolerance of pigs requires further study.
Acknowledgements
The authors would like to thank the Council of Agriculture (Grant Nos. 85AST-1.14-AID-23(1) and 87AST-1.5-AID-14(12)) and the National Science Council (Grant No. NSC86-2123-B-059-016-A20) of the Republic of China for financially supporting this research. Ms. Hui-Chin Chen, Ms. Jin-Lin Wang, Ms. Hui-Lan Chang and Mr. Jung-Chang Chiang are also appreciated for their technical support. Drs. Pauline H. Yen and Shyh-Hwa Liu are commended for carefully reviewing this manuscript.
References
Cheng, S.P., Wung, S.C., 1974. A study of seasonal changes in the boar semen characteristics. Research Report Animal Industry Research Institute, Taiwan Sugar Corporation 1973–1974, pp. 89–103 (In Chinese, with English abstract).
Chowdbury, A.K., Steinberger, E., 1964. A quantitative study of the effect of heat on germinal epithelium of rat testes. Am. J. Anat. 115, 509–524.
Christenson, R.K., Teaque, H.S., Grifo Jr., A.P., Roller, W.L., 1972. The effect of high environmental temperature on the boar. Ohio Swine Research and Information Report, Ohio Agricultural Research and Development Center, Wooster, OH, USA, pp. 19–23.
Colenbrander, B., Feitsma, H., Grooten, H.J., 1993. Optimizing semen production for artificial insemination in swine. J. Reprod. Fertil. Suppl. 48, 207–215.
Craig, E.A., Gross, C.A., 1991. Is HSP70 the cellular thermometer? TIBS 16, 135–140.
Den Daas, N., 1992. Laboratory assessment of semen characteristics. Anim. Reprod. Sci. 28, 87–94.
Huang, S.Y., Kuo, Y.H., Lee, W.C., Tsou, H.L., Lee, Y.P., Chang, H.L., Wu, J.J., Yang, P.C., 1999. Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa. Theriogenology 51, 1007–1016.
Kamaruddin, M., Basrur, P.K., King, W.A., 1998. Inhibition of bovine in vitro fertilization by HSP70 antibody. Theriogenology 49, 287.
Kamaruddin, M., Kroetsch, T., Basrur, P.K., King, W.A., 1996. Heat shock protein 70 in bovine semen. Biol. Reprod. 54 (Suppl.), 112.
Kennedy, B.W., Wilkins, J.N., 1984. Boar, breed and environmental factors influencing semen characteristics of boars used in artificial insemination. Can. J. Anim. Sci. 64, 833–843.
Koh, T.J., Crabo, B.G., Tsou, H.L., Graham, E.F., 1976. Fertility of liquid boar semen as influenced by breed and season. J. Anim. Sci. 42, 138–144.
Kuo, Y.H., Huang, S.Y., Lee, Y.P., 1997. Effects of breed and season on semen characteristics of boars in subtropical area. J. Chin. Soc. Vet. Sci. 23, 114–122 (In Chinese, with English abstract).
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee, W.C., Lin, K.Y., Chiu, Y.T., Lin, J.H., Cheng, H.C., Huang, H.C., Yang, P.C., Liu, S.K., Mao, S.J.T., 1996. Substantial decrease of heat shock protein 90 in ventricular tissues of two sudden-death pigs with hypertrophic cardiomyopathy. FASEB J. 10, 1198–1204.
Leung, S.M., Senisterra, G., Ritchie, K.P., Sadis, S.E., Lepock, J.R., Hightower, L.E., 1996. Thermal activation of the bovine HSC70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer. Cell Stress Chaperones 1, 78–89.
Li, G.C., Laszlo, A., 1985. Thermotolerance in Mammalian Cells: A possible role for heat shock protein. In: Atkinson, B.G., Walden, D.B. (Eds.), Changes in Eukaryotic Gene Expression in Response to Environmental Stress. Academic Press, Orlando, FL, USA, pp. 227–254.
Lindquist, S., 1986. The heat shock response. Annu. Rev. Biochem. 55, 1151–1191. Lindquist, S., Craig, E.A., 1988. The heat shock proteins. Annu. Rev. Genet. 22, 63–77.
Liu, S.H., Kuo, Y.H., Yang, T.S., Lee, K.H., 1994. Relationship between characteristics of serum reproductive hormones and reproductive performance in Yorkshire boars during hot summer months. J. Chin. Soc. Anim. Sci. 23, 33–42 (In Chinese, with English abstract).
Lyashko, V.N., Vikulova, V.K., Chernicov, V.G., Ivanov, V.I., Ulmasov, K.A., Zatsepina, O.G., Evgen’ev, M.B., 1994. Comparison of the heat shock response in ethnically and ecologically different human populations. Proc. Natl. Acad. Sci. U. S. A. 91, 12492–12495.
Maak, S., Petersen, K., Von Lengerken, G., 1998. Association of polymorphisms in the porcine HSP 70.2 gene promoter with performance traits. Anim. Genet. 29 (Suppl.), 71.
Malysheva, E.V., Zamotrinskii, A.V., Malyshev, I.Y., 1994. Role of heat shock proteins in the formation of stress resistance in different animal strains. Bull. Exp. Biol. Med. 118, 689–691.
Marber, M.S., Mestril, R., Chi, S.H., Sayen, M.R., Yellon, D.M., Dillmann, W.H., 1995. Overexpression of the rat inducible 70-kDa heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 95, 1446–1456.
McNitt, J.I., First, N.L., 1970. Effects of 72-h heat stress on semen quality in boars. Int. J. Biometer. 14, 373–380. Nover, L., 1991. Heat shock response. CRC Press, Boca Raton, FL, USA.
Nunes, M., Yerle, M., Dezeure, F., Gellin, J., Chardon, P., Vaiman, M., 1993. Isolation of four HSP70 genes in the pig and localization on chromosomes 7 and 14. Mammalian Genome 4, 247–251.
Pelham, H.R.B., 1986. Speculations on the functions of the heat shock and glucose-regulated proteins. Cell 46, 959–961.
Plumier, J.C.L., Ross, B.M., Currie, R.M., Angelidis, C.E., Kazlaris, H., Kollias, G., Paagoulatos, G.N., 1995. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J. Clin. Invest. 95, 1854–1860.
Raab, L.S., Polakoski, K.L., Hancock, L.W., Hamilton, D.W., 1995. Characterization of the heat shock protein P70 in rat spermatogenetic cells. Mol. Reprod. Develop. 40, 186–195.
Sanchez, Y., Lindquist, S.L., 1990. HSP104 is required for induced thermotolerance. Science 248, 1112–1115. Sanchez, Y., Taulien, J., Borkovich, K.A., Lindquist, S.L., 1992. HSP104 is required for tolerance to many forms
Sarge, K.D., 1995. Male germ cell-specific alteration in temperature set point of the cellular stress response. J. Biol. Chem. 270, 18745–18748.
SAS Institute, 1989. SAS User’s Guide: Statistics, Release 6.3 Edition. SAS Institute Inc., Cary, NC, USA. Schwerin, M., Langhammer, M., Matthes, W., Dietl, G., 1996. Additive genetic effects of the RYR1 and HSP70.2
loci upon stress susceptibility in swine. Anim. Genet. 27 (Suppl. 2), 106.
Stewart, T.A., Bellve, A.R., Leder, P., 1984. Transcription and promoter usage of the myc gene in normal somatic and spermatogenic cells. Science 226, 707–710.
Subjeck, J.R., Shyy, T.T., 1986. Stress protein systems of mammalian cells. Am. J. Physiol. 250 (Cell Physiol. 19), C1–C17.
Ulmasov, K.A., Shammakov, S., Karaev, K., Evgenve, M.B., 1992. Heat shock proteins and thermoresistance in lizards. Proc. Natl. Acad. Sci. U. S. A. 89, 1666–1670.
Vandemark, N.L., Free, M.J., 1970. Temperature effects. In: Johnson, A.D., Gomes, W.R., Vandemark, N.L. (Eds.), The Testes, Vol. 3. Academic Press, New York, USA, pp. 233–312.
van Laack, R.L.J.M., Faustman, C., Sebranek, J.G., 1993. Pork quality and the expression of stress protein HSP70 in swine. J. Anim. Sci. 71, 2958–2964.
Welch, W.J., 1992. Mammalian stress response: cell physiology, structure/function of stress proteins and implications for medicine and disease. Physiol. Rev. 72, 1063–1081.
Wettemann, R.P., Desjardins, C., 1979. Testicular function in boars exposed to elevated ambient temperature. Biol. Reprod. 20, 235–241.
Wettemann, R.P., Wells, M.E., Johnson, R.K., 1979. Reproductive characteristics of boars during and after exposure to increased ambient temperature. J. Anim. Sci. 49, 1501–1505.
