Association of heat shock protein 70 with semen quality in boars

Animal Reproduction Science 63 (2000) 231–240

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,∗ a

Department of Comparative Medicine, Pig Research Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC b Department of Applied Biology, Pig Research Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC c Department of Animal Science, National Chung-Hsing University, 250 Kuo-Kwang Rd., Taichung 402, Taiwan, ROC d Department of Animal Resources, Pig Research Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC e Council 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 protein (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 proximal 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).

0378-4320/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 0 ) 0 0 1 7 5 - 5

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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 sensitive 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 inseminated 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 decreasing 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. Raab et al. (1995) observed the presence of HSP70 in the ejaculated sperm of mammals including boars. Sarge (1995) indicated that the expression of HSP70 in male germ cells could be induced at a lower temperature than that in somatic cells under in vitro culture conditions, implying that male germ cells are more heat sensitive than somatic cells. Owing to the ability of HSP70 to protect cells against heat stress, this study attempts to determine whether or not the level of HSP70 increases during heat stress; if not, whether or not semen quality decreases is also examined. Therefore, in addition to determining seasonal and breed effects on the levels of HSP70 in ejaculated boar spermatozoa, this work also investigates the relationship between HSP70 level and semen quality.

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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×g for 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, 500 ␮l of the supernatant were mixed with an equal volume of Laemmli’s buffer and saved for later analysis. Proteins from 3×106 spermatozoa or 10 ␮l 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 containing 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) and ␤-tubulin (Amersham; diluted 1:500) followed 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 containing 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. For quantitative analysis of the constitutive (HSC70) and the inducible (HSI70) forms of HSP70, total proteins from 3×106 spermatozoa were Western blotted and the optical densities of HSP70 and ␤-tubulin bands on nitrocellulose membranes were determined using a densitometer (Molecular Dynamics, Sunnyvale, CA, USA; software was ImageQuant). The levels of HSC70, and HSI70 were normalized using ␤-tubulin as the covariate, and their sum was given as the level of HSP70.

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Table 1 Grouping of samples by level of heat shock protein 70 Group 1 Level of total HSP70

2

>M+1 S.D. M+0.5 S.D.

3

4

M >M−0.5 S.D.

5

6

M−1 S.D.

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 The concentration, motility and morphology of the sperm in the semen samples were determined. Table 2 lists these semen quality traits together with the level of HSP70. A significant seasonal difference was observed in all the traits evaluated (P<0.05). Semen

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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 and ␤-tubulin (b). Each lane contains total proteins from 3×106 spermatozoa except lane 1 which contains molecular weight standards. HSC70 and HSI70 are the constitutive and inducible form of 70 kDa heat shock protein, respectively.

quality in cool season was significantly better than that during the hot season in all three breeds of boars. The semen quality of Landrace boars was better than Duroc and Yorkshire boars in both seasons (P<0.05). The level of HSP70 during the hot season was significantly lower than that during the cool season (P<0.05). Notably, the interactions between season and breed in all the traits were insignificant (data not shown; P>0.05).

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Table 2 Least squares means of semen quality traits and level of HSP70 in boar spermatozoaa Traits

Cool season

Hot season

Duroc

Landrace

Yorkshire

Duroc

Landrace

Yorkshire

Motility (%) Normal sperm (%) PPDb (%) DPDc (%) Abnormal sperm (%) Sperm concentration (×108 /ml)

80.5±1.2 a 55.1±1.9 c 4.6±0.6 bc 13.0±1.3 c 27.3±1.8 c 2.38±0.19 b

82.6±1.5 a 70.7±2.4 a 3.2±0.7 c 6.5±1.6 de 19.6±2.3 d 1.75±0.24 c

80.5±1.7 a 63.3±2.6 b 5.7±0.8 b 2.5±1.8 e 28.5±2.6 bc 2.03±0.27 bc

58.3±1.2 c 37.0±1.8 e 7.7±0.6 a 21.6±1.2 a 33.8±1.8 ab 3.14±0.19 a

71.1±1.5 b 53.3±2.3 c 4.6±0.7 b 17.0±1.5 b 25.1±2.2 cd 2.45±0.23 b

58.9±1.7 c 45.9±2.5 d 6.6±0.8 ab 8.1±1.7 d 39.0±2.5 a 2.28±0.26 c

HSP70d

1.53±0.05 a 1.40±0.06 a 1.42±0.08 a

1.18±0.05 b 1.00±0.07 b 1.00±0.08 b

a

Means within the same row with different letters differ significantly (P<0.05). PPD: sperm with proximal plasma droplets. c DPD: sperm with distal plasma droplets. d HSP70: level of 70 kDa heat shock protein, which was normalized optical density by using ␤-tubulin as covariate with values of arbitrary unit. b

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 calculated (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 Sample size Motility Normal sperm Sperm with proximal plasma droplets Sperm with distal plasma droplets Abnormal sperm Sperm concentration ∗

P<0.05. P<0.01.

∗∗

Cool season 113 0.00 0.10 −0.05 −0.11 −0.03 −0.12

Hot season 112 0.14 0.15 −0.20∗ −0.08 −0.04 0.48∗∗

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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 Motility (%) Normal sperm (%) PPDc (%) DPDd (%) Abnormal sperm (%) Sperm concentration (×108 /ml)

33 75.9±2.5 ab 61.6±3.9 a 3.4±1.1 b 8.3±2.4 b 27.0±2.9 b 2.60±0.30 a

29 77.9±2.6 a 59.1±4.2 a 4.7±1.2 ab 8.4±2.6 b 27.9±3.1 ab 2.78±0.32 a

41 75.4±2.2 ab 56.5±3.5 ab 4.6±1.0 ab 11.7±2.2 ab 27.1±2.6 b 2.52±0.27 a

51 72.5±2.0 ab 54.7±3.2ab 5.8±0.9 ab 14.0±2.0 ab 25.4±2.4 b 2.60±0.24 a

38 70.5±2.3 b 48.0±3.7 bc 5.9±1.0 ab 14.1±2.3 ab 31.7±2.7 ab 2.44±0.28 a

33 58.5±2.5 c 39.5±3.9 c 7.2±1.1 a 17.9±2.4 a 35.4±2.9 a 1.58±0.30 b

HSP70e

2.07±0.03 a 1.62±0.03 b 1.39±0.02 c 1.15±0.02 d 0.93±0.02 e 0.63±0.03 f

a

Mean 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. b Means within the same row with different letters differ significantly (P<0.05). c PPD: sperm with proximal plasma droplets. d DPD: sperm with distal plasma droplets. e HSP70: level of 70 kDa heat shock protein, which was normalized optical density by using ␤-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. Percentages of normal sperm, motility, and sperm concentration are common criteria for evaluating semen quality (Den Daas, 1992; Colenbrander et al., 1993). Many studies have demonstrated that heat stress during the summer decreases semen quality in boars (Cheng and Wung, 1974; Koh et al., 1976; Kennedy and Wilkins, 1984; Liu et al., 1994; Kuo et al., 1997). Our detection of significant seasonal differences in all the semen quality traits implies that semen quality can be used as an indicator for thermotolerance in pigs (Table 2). Although its reason is unknown, a significantly lower level of HSP70 in the spermatozoa

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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-AID23(1) and 87AST-1.5-AID-14(12)) and the National Science Council (Grant No. NSC862123-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. Hammersdtedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl. 11, 73–88.

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