Abstract
Hormone sensitive lipase (HSL) is a triacylglycerol hydrolase and cholesterol esterase that is essential for male fertility. The aim of the study was to investigate the distribution of C-60G polymorphism of HSL gene and alleles in fertile and infertile males living in Hamadan, Iran. In addition, lipase activity was determined in these two groups. The HSL genotype was determined by PCR-RFLP and the lipase activity was detected by turbidometery in 164 fertile and 169 infertile males. A significant difference in HSL genotype distribution was observed between groups (χ2 = 8.1, df = 2, p = 0.017). Infertile males showed a higher percentage of CC as well as a lower percentage of CG and GG genotype compared with fertile individuals. The presence of CC to CG + GG genotype conferred a 2.4-fold risk for male infertility (odds ratio = 2.4 (1.3 - 4.5), p = 0.006). In addition, lipase activity was remarkably higher (p < 0.001) in fertile males (94 ± 66 U/L) compared to the infertile subjects (50.6 ± 49 U/L). This suggests that genetic variation of HSL may be a risk factor for male infertility.
Introduction
Lipid components have an important role in the sperm capacitation and function [Sebastian et al. Citation1987]. Lipids are essential for sperm maturation, viability, and functioning. Alterations in lipid composition have been reported in infertile males [Lenzi et al. Citation1996; Tavilani et al. Citation2008]. Approximately 50 percent of cases of infertility among couples are related to male factors. Infertility is a multifactorial disease in which many genetic and environmental factors are involved [Poongothai et al. Citation2009]. The contribution of some genetic polymorphisms to male infertility have been documented [Stouffs et al. Citation2011] and studies on polymorphism of genes involved in metabolism of lipids in the male reproductive tract have been reported [Peterlin et al. Citation2006].
The human hormone sensitive lipase (HSL) has been mapped to chromosome 19q13.1-13.2 [Talmud et al. Citation1998]. HSL plays a crucial role in lipid metabolism and hydrolyzes di- and triacylglycerol, cholesterol ester, and retinyl ester. This 84 kDa enzyme is highly expressed in adipose tissue as well as in heart and skeletal muscle, pancreatic β-cells, placenta, adrenal glands, ovary, and testis [Haemmerle et al. Citation2003]. Two somatic and testicular isoforms of HSL have been identified [Yeaman Citation2004; Holst et al. Citation1996]. Hsl-deficient male knockout mice [Chung et al. Citation2001; Mairal et al. Citation2002] are infertile and show decreased testis weights, reduced sperm motility, and count [Osuga et al. Citation2000; Hermo et al. Citation2008]. Fertility can be rescued by expression of Hsl [Wang et al. Citation2004; Vallet-Erdtmann et al. Citation2004].
To date, several polymorphisms within the human HSL gene have been detected [Garenc et al. Citation2002]. The Arg 309 Cyc polymorphism is associated with an increase of serum cholesterol and type 2 diabetes [Shimada et al. Citation1996]. HSL i6(CA)n repeat polymorphism is associated with type 2 diabetes and obesity [Magré et al. Citation1998]. The C-60 G polymorphism is the most extensively studied single nucleotide polymorphism of the HSL gene. This polymorphism is located in the promoter region of the HSL gene and it is possible that the substitution of C with G nucleotide result in a decrease in HSL gene expression [Talmud et al. Citation2001]. The C-60 G polymorphism in the promoter of HSL is associated with body composition and waist circumference [Garenc et al. Citation2002; Carlsson et al. Citation2006]. In vitro, the promoter variant C-60 G exhibits 40% decreased promoter activity [Talmud et al. Citation1998]. In addition, the C-60 G allele male carriers present lower levels of fasting non-esterified fatty acid and higher levels of low density lipoprotein cholesterol [Talmud et al. Citation2001]. Since the alteration of HSL activity has previously been reported in C-60G polymorphism and the different sperm lipid composition has been shown in fertile and infertile males [Lenzi et al. Citation1996; Tavilani et al. Citation2008], the differential distribution of C-60 G polymorphism of HSL likely contributes to male fertility. The issue of whether C-60 G polymorphism of HSL is a risk factor for infertility has not been systemically addressed and little information is available about distribution of C-60 G polymorphism in fertile and infertile males. The aim of this study was to investigate the distribution of C-60 G polymorphism of the HSL gene as well as lipase activity in fertile and infertile males living in Hamadan, Iran.
Results
The semen profiles from infertile samples categorized as asthenozoospermia (n = 66) and teratoasthenozoospermia (n = 103), are summarized in . The distribution of CC, CG, and GG genotypes (and alleles) was determined by PCR as shown in in 164 fertile and 169 infertile individuals. The results are summarized in . The homozygous CC genotype was observed in 129 fertile males (78.7%) whereas heterozygous CG and homozygous GG genotypes were detected in 32 (19.5%) and 3 (1.8%) individuals, respectively. In contrast, the wild type CC genotype was observed in 152 (89.9%) infertile men, while 16 subjects (9.5%) were genotyped as CG and only 1 infertile participant was genotyped with GG (0.6%). Semen parameters showed no notable difference for different HSL genotypes or alleles in infertile individuals. Moreover, the mean lipase activity was markedly higher (p < 0.001) in fertile subjects (94 ± 66 U/L) in comparison to the infertile individuals (50.6 ± 49 U/L).
Figure 1. The PCR-RFLP products for HSL C-60G polymorphism on agarose 2%. The size of products were determined in comparison with Roche DNA molecular marker V. Homozygote CC 271 bp, homozygote GG 182 and 89 bp, and heterozygote CG 271, 182, and 89 bp.
Table 1. Basic parameters of semen sample in total infertile, asthenozoospermia, and teratoasthenozoospermia males.
Table 2. The distribution of HSL genotypes (C-60G) and alleles and odds ratio of fertile (n = 164) and infertile (n = 169) males using χ2 test and regression logistics analysis.
Data analysis showed a statistical difference in the overall distribution of the HSL genotypes and alleles in fertile and infertile subjects (χ2 = 8.1, df = 2, p = 0.017 and χ2 = 8.34, df = 1, p = 0.004, respectively). The presence of the CC genotype compared to the CG + GG genotype conferred a 2.4-fold risk for male infertility (odds ratio = 2.4 (1.3 – 4.5), p = 0.006). However, the distribution of HSL genotypes and alleles did not significantly differ in teratoasthenozoospermic and asthenozoospermic males ().
Table 3. Distribution and odds ratio of HSL genotypes and alleles among fertile, asthenozoospermia, and teratoasthenozoospermia males.
Discussion
The present study aimed to investigate the distribution of C-60G HSL genotypes in fertile and infertile males. Our results showed that the distribution of C-60G HSL genotypes and alleles in fertile subjects significantly differed from that of infertile individuals. A higher percentage of CG and GG genotype carriers were observed in fertile individuals compared to the infertile group.
The C-60G polymorphism is located in the promoter region of the HSL gene and previous studies have shown that the sequence between −86 and −57 is required for the HSL expression [Grober et al. Citation1997]. However, the importance of the C-60G polymorphism in male infertility is not well understood. Our results showed that the presence of the CC polymorphism increased the risk of infertility 2.4-fold compared to those individuals with the CG and GG genotype. It appears that the CG and GG genotypes provided protection against male infertility. The significance of various genotypes of HSL in the male reproductive tract has not been fully determined [Kabbaj et al. Citation2001], although, the role of testicular isoform HSL in male fertility has been reported. A review of the literature on the importance of HSL in male reproduction showed that HSL deficiency resulted in testicular defect and infertility in mice [Osuga et al. Citation2000; Hermo et al. Citation2008]. In addition, expression of Hsl in postmeiotic germ cells of Hsl deficient mice, induced normal fertility in these animals [Wang et al. Citation2004; Vallet-Erdtmann et al. Citation2004]. Therefore, it can be concluded that HSL is necessary for spermatogenesis. Furthermore, lipid accumulation was detected in testicular cells in the absence of HSL activity highlighting the important role of this enzyme in lipid metabolism in the male reproductive tract [Haemmerle et al. Citation2002].
The results of the study presented above showed that the lipase activity was higher in the fertile group as compared to infertile individuals (p < 0.001). We have also previously shown a difference in the lipid profile of spermatozoa between fertile and infertile males [Tavilani et al. Citation2006; Tavilani et al. Citation2007]. Collectively, it can be suggested that the different lipid composition of spermatozoa of fertile and infertile men might be partially due to the different lipase activity in these two groups, as observed here. Undoubtedly, the measurement of HSL activity instead of determining total lipase activity would be a useful tool to associate various genotypes of C-60G polymorphism in the HSL gene with HSL activity. In conclusion, our findings showed that distribution of HSL genotypes and alleles was different in fertile and infertile individuals. We suggest that genetic variation of HSL may be a risk factor for male infertility.
Materials and Methods
Blood specimens were collected from 164 fertile and 169 infertile men. All fertile individuals had children and were age-matched (29 – 40 y) with infertile males. Infertile individuals were selected from patients who were admitted to the Fatemieh Fertility Clinic (Hamadan University of Medical Sciences). All infertile subjects had abnormal spermogram and those with known infertility causes such as varicocele, abnormal karyotype, or Y-chromosome microdeletions were excluded from the study. Individuals with diabetes, thyroid disease, and those taking lipid lowering drugs also were excluded. Written informed consent for participation was obtained and the project was approved by the Research Ethics Committee of Hamadan University of Medical Sciences. Semen samples of infertile males were collected by masturbation following 3 days of abstinence. After liquefaction, semen volume, sperm concentration (haemocytometer), total sperm count, morphology (Papanicolaou staining method), and motility grades (a: rapid progressive, b: slow progressive, c: non-progressive, and d: immotile) were determined using World Health Organization [WHO Citation1999] standard procedures.
DNA analysis
Genomic DNA was extracted from peripheral blood leukocytes using standard method [Bartlett and Stirling 2003]. C-60G polymorphism of the HSL gene were detected by PCR using the forward, 5'-gagggaggaggggctatgggt-3' and the reverse, 5'-tccctgggctgggactactgg-3' primers, as previously described [Garenc et al. Citation2002]. The PCR product was digested with Rsal restriction endonuclease and subjected to electrophoresis on a 2% agarose gel. The presence of the homozygote C-60 allele (without Rsa1 restriction site) produced a 271 bp product whereas the homozygote G-60 allele (with the Rsa1 restriction site) yielded two bands of 89 bp and 182 bp. The heterozygotes for C-60G allele resulted in three (89 bp, 182 bp, and 271 bp) products.
Lipase activity
Plasma total lipase activity was determined by turbidometeric method using a commercially available kit (Li 188, Randox Laboratories Ltd., Antrim, UK).
Statistical Analysis
The allelic frequencies were calculated by the gene counting method. The χ2 test was used to verify the agreement of the observed genotype frequencies with those expected according to the Hardy–Weinberg equilibrium. The genotypes and HSL allele frequencies were compared between fertile and infertile subjects using the χ2 test. Student's t-test was performed to determine difference in mean lipase activity between two groups. The results were presented as Mean ± SD and statistical significant difference was assumed at the p < 0.05 level.
Acknowledgments
This research was carried out as part of Akram Vatannejad's dissertation research and was supported by Hamadan University of Medical Sciences.
Declaration of interest: The authors claim no declarations of interest.
References
- Bartlett, J.M.S., and Stirling, D. (2003) Methods in Molecular Biology, Vol. 226: PCR Protocols, Second edition, Humana Press, NJ. pp. 29–30.
- Carlsson, E., Johansson, L.E., Ström, K., Hoffstedt, J., Groop, L., Holm, C., and Ridderstråle, M. (2006) The hormone-sensitive lipase C-60G promoter polymorphism is associated with increased waist circumference in normal-weight subjects. Int J Obes (Lond) 30:1442–1448.
- Chung, S., Wang, S.P., Pan, L., Mitchell, G., Trasler, J., and Hermo, L. (2001) Infertility and testicular defects in hormone-sensitive lipase-deficient mice. Endocrinology 142: 4272–4281.
- Garenc, C., Pérusse, L., Chagnon, Y.C., Rankinen, T., Gagnon, J., Borecki, I.B., (2002) The hormone-sensitive lipase gene and body composition: the HERITAGE Family Study. Int J Obes Relat Metab Disord 26:220–227.
- Grober, J., Laurell, H., Blaise, R., Fabry, B., Schaak, S., Holm, C., and Langin, D.. (1997) Characterization of the promoter of human adipocyte hormone-sensitive lipase. Biochem J 328:453–461.
- Haemmerle, G., Zimmermann, R., Hayn, M., Theussl, C., Waeg, G., Wagner, E., (2002) Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem 15: 4806–4815.
- Haemmerle, G., Zimmermann, R., and Zechner, R. (2003) Letting lipids go: hormone-sensitive lipase. Curr Opin Lipidol 14: 289–297.
- Hermo, L., Chung, S., Gregory, M., Smith, C.E., Wang, S.P., El-Alfy, M., (2008) Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility. Mol Reprod Dev 75:565–577.
- Holst, L.S., Langin, D., Mulder, H., Laurell, H., Grober, J., Bergh, A., (1996) Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase. Genomics 35:441–447.
- Kabbaj, O., Holm, C., Vitale, M.L., and Pelletier, R.M. (2001) Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig. Biol Reprod 65:601–612.
- Lenzi, A., Picardo, M., Gandini, L., and Dondero, F. (1996) Lipids of the sperm plasma membrane: from polyunsaturated fatty acids considered as markers of sperm function to possible scavenger therapy. Hum Reprod Update 2:246–256.
- Magré, J., Laurell, H., Fizames, C., Antoine, P.J., Dib, C., Vigouroux, C., (1998) Human hormone-sensitive lipase: genetic mapping, identification of a new dinucleotide repeat, and association with obesity and NIDDM. Diabetes 47:284–286.
- Mairal, A., Melaine, N., Laurell, H., Grober, J., Holst, L.S., Guillaudeux, T., (2002) Characterization of a novel testicular form of human hormone-sensitive lipase. Biochem Biophys Res Commun 291:286–290.
- Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A., (2000) Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad. Sci USA 18:787–792.
- Peterlin, B., Zorn, B., Volk, M., and Kunej, T. (2006) Association between the apolipoprotein B signal peptide gene insertion/deletion polymorphism and male infertility. Mol Hum Reprod 12:777–779.
- Poongothai, J., Gopenath, T.S., and Manonayaki, S. (2009) Genetics of human male infertility. Singapore Med J 50:336–347.
- Sebastian, S.M., Selvaraj, S., Aruldhas, M.M., and Govindarajulu, P. (1987) Pattern of neutral and phospholipids in the semen of normospermic, oligospermic and azoospermic men. J Reprod Fertil 79:373–378.
- Shimada, F., Makino, H., Hashimoto, N., Iwaoka, H., Taira, M., Nozaki, O., (1996) Detection of an amino acid polymorphism in hormone-sensitive lipase in Japanese subjects. Metabolism 45:862–864.
- Stouffs, K., Vandermaelen, D., Tournaye, H., Liebaers, I., and Lissens, W. (2011) Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages. Reprod Biomed Online 22:65–71.
- Talmud, P.J., Palmen, J., Luan, J., Flavell, D., Byrne, C.D., Waterworth, D.M., (2001) Variation in the promoter of the human hormone sensitive lipase gene shows gender specific effects on insulin and lipid levels: results from the Ely study. Biochim Biophys Acta 29:239–244.
- Talmud, P.J., Palmen, J., and Walker, M. (1998) Identification of genetic variation in the human hormone-sensitive lipase gene and 5' sequences: homology of 5' sequences with mouse promoter and identification of potential regulatory elements. Biochem Biophys Res Commun 27:661–668.
- Tavilani, H., Doosti, M., Abdi, K., Vaisiraygani, A., and Joshaghani, H.R. (2006) Decreased polyunsaturated and increased saturated fatty acid concentration in spermatozoa from asthenozoospermic males as compared with normozoospermic males. Andrologia 38:173–178.
- Tavilani, H., Doosti, M., Nourmohammadi, I., Mahjub, H., Vaisiraygani, A., Salimi, S., and Hosseinipanah, S.M. (2007) Lipid composition of spermatozoa in normozoospermic and asthenozoospermic males. Prostaglandins Leukot Essent Fatty Acids 77:45–50.
- Tavilani, H., Goodarzi, M.T., Doosti, M., Vaisi-Raygani, A., Hassanzadeh, T., Salimi, S., and Joshaghani, H.R. (2008) Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa. Reprod Biomed Online 16:649–656.
- Vallet-Erdtmann, V., Tavernier, G., Contreras, J.A., Mairal, A., Rieu, C., Touzalin, A.M., (2004) The testicular form of hormone-sensitive lipase HSLtes confers rescue of male infertility in HSL-deficient mice. J Biol Chem 279:42875–42880.
- Wang, S.P., Chung, S., Soni, K., Bourdages, H., Hermo, L., Trasler, J., and Mitchell, G.A. (2004) Expression of human hormone-sensitive lipase (HSL) in postmeiotic germ cells confers normal fertility to HSL-deficient mice. Endocrinology 145:5688–5693.
- WHO (1999) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 4th edn, Cambridge University Press, Cambridge, UK.
- Yeaman, S.J. (2004) Hormone-sensitive lipase–new roles for an old enzyme. Biochem J 379 (Pt 1):11–22.