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Phospholipid–Hydroperoxide Glutathione Peroxidase in Sperm
208
[18]
SELENOPROTEINS
[18] Phospholipid–Hydroperoxide Glutathione Peroxidase in Sperm By ANTONELLA ROVERI, LEOPOLD FLOHE´ , MATILDE MAIORINO, and FULVIO URSINI Introduction The phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene (gpx-4) was found to be highly expressed in postpubertal mammalian testis.1 Mature testis was reported to have the highest PHGPx activity of all mammalian tissues investigated.2 There it is found associated, in part at least, with mitochondria,3 which complies with a tissue-specific transcription of the PHGPx gene into an mRNA encoding a protein with a mitochondrial leader peptide.4,5 Biosynthesis of PHGPx in testis occurs predominantly in round spermatids.6,7 Correspondingly, specific PHGPx activity in testis appears to parallel the thickness of the spermatid layer.6 The hormonal regulation of testicular PHGPx activity1 was shown to be indirect; Leydig cell-derived testosterone stimulates the seminiferous epithelium and thereby augments the number of spermatids that produce PHGPx.6 Despite the abundance of PHGPx in spermatids, PHGPx activity is practically undetectable in mature spermatozoa by conventional activity assays.8 Surprisingly, however, PHGPx protein could easily be detected by Western blotting in reduced and sodium dodecyl sulfate (SDS)-solubilized sperm proteins.9 This enzymatically inactive PHGPx protein was found to make up at least 50% of the keratinous material embedding the mitochondrial helix in the midpiece of sperm mitochondria.9 It accounts for most, if not all, of the selenium content in the midpiece of spermatozoa and thus is the real “mitochondrial capsule selenoprotein,” a term that had 1
A. Roveri, A. Casasco, M. Maiorino, P. Dalan, A. Calligaro, and F. Ursini, J. Biol. Chem. 267, 6142 (1992). 2 A. Roveri, M. Maiorino, and F. Ursini, Methods Enzymol. 233, 202 (1994). 3 M. Maiorino, A. Roveri, and F. Ursini, in “Free Radicals: From Basic Science to Medicine” (G. Poli, E. Albano, and M. U. Dianzani, eds.), p. 412. Birkh¨auser Verlag, Basel, 1993. 4 T. R. Pushpa-Rekha, A. L. Burdsall, L. M. Oleksa, G. M. Chisolm, and D. M. Driscoll, J. Biol. Chem. 270, 26993 (1995). 5 M. Arai, H. Imai, D. Sumi, T. Imanaka, T. Takano, N. Chiba, and Y. Nakagawa, Biochem. Biophys. Res. Commun. 227, 433 (1996). 6 M. Maiorino, J. B. Wissing, R. Brigelius-Floh´ e, F. Calabrese, A. Roveri, P. Steinert, F. Ursini, and L. Floh´e, FASEB J. 12, 1359 (1998). 7 K. Mizuno, S. Hirata, K. Hoshi, A. Shinohara, and M. Shiba, Biol. Trace Elem. Res. 74, 1112 (2000). 8 R. Brigelius-Floh´ e, K. Wingler, and C. M¨uller, Methods Enzymol. 347, [9], 2002 (this volume). 9 F. Ursini, S. Heim, M. Kiess, M. Maiorino, A. Roveri, J. Wissing, and L. Floh´ e, Science 285, 1393 (1999).
METHODS IN ENZYMOLOGY, VOL. 347
C 2002 by Academic Press. Copyright All rights of reproduction in any form reserved. 0076-6879/02 $35.00
[18]
PHGPx IN SPERM
209
misleadingly been introduced for the “sperm mitochondria-associated cysteinerich protein” (SMCP).10 The process transforming the enzymatically active selenoperoxidase into a structural protein during late spermatogenesis remains largely elusive. Obviously, the transformation results from oxidative cross-linking of PHGPx with itself and/or other proteins via Se–S and/or S–S bonds, because PHGPx protein can be solubilized and PHGPx activity can be recovered from the capsule material by drastic reductive procedures.9 The change in enzymatic activity and physical properties of PHGPx during spermatogenesis is a typical example of “moonlighting,” as has also been described, for example, for proteins making up the eye lens.11,12 This moonlighting certainly indicates a dual role of PHGPx in male reproduction; the enzyme may protect the rapidly dividing spermatogenic cells from potentially harmful oxidants, whereas it later becomes part of a structural element essential for the function of mature spermatozoa. The latter biological role of PHGPx appears at least as important for male fertility as its presumed antioxidant activity; interestingly, it is precisely the morphological alteration and mechanical instability of the midpiece material that lead to the impaired fertilization potential of selenium-deficient spermatozoa.13,14 Measurement of Phospholipid-Hydroperoxide Glutathione Peroxidase in Human Spermatozoa As outlined in the introduction, PHGPx is found in spermatozoa mainly in the midpiece, where it is part of the “mitochondrial capsule.” The enzyme is catalytically inactive in this form. The measurement of specific activity therefore requires a “rescue” procedure. The solubilization–reactivation is efficiently carried out in the presence of high concentrations of thiols and guanidine. The accuracy and reproducibility of specific activity measurements require (1) the complete and reproducible solubilization of all proteins of the sperm and (2) the careful removal of guanidine and of the thiols used for solubilization–reactivation, because they compete with glutathione in the peroxidase reaction, thus leading to an underestimation of the activity. Specimen Collection Human sperm are collected by masturbation between days 3 and 7 of abstinence. On each sample sperm count, morphological and functional parameters are immediately evaluated. Sperm are centrifuged at 300g for 10 min at 4◦ . Pellets are 10
L. Cataldo, K. Baig, R. Oko, M. A. Mastrangelo, and K. C. Kleene, Mol. Reprod. Dev. 45, 320 (1996). 11 J. Piatigorski, Prog. Ret. Eye Res. 17, 145 (1998). 12 C. J. Jefferey, Trends Biol. Sci. 24, 8 (1999). 13 M. Maiorino, L. Floh´ e, A. Roveri, P. Steinert, J. Wissing, and F. Ursini, BioFactors 10, 251 (1999).
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SELENOPROTEINS
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washed twice in phosphate-buffered saline (PBS). Washed pellets can be stored at −20◦ for a few weeks without any apparent loss of activity. Sample Preparation The pellet is solubilized in 0.1 M Tris-HCl, 6 M guanidine hydrochloride, 0.1% (v/v) Triton X-100, 0.1 M 2-mercaptoethanol, pH 7.4 (solubilization buffer; SB). Sperm count in SB is 15–25×106cell/ml. Solubilization is carried out by vortex mixing. In the standardized procedure three periods of 30 sec of vortexing are used with a pause in ice of 1 min between each of them. The absence of any visible pellet after centrifugation ensures a thorough solubilization. The protein concentration thus obtained in the SB ranges between 0.5 and 1.0 mg/ml. Solubilized samples can be stored in this buffer at −20◦ and, under these conditions, PHGPx activity is stable for several days without any obvious loss of activity. Sample Preparation for Activity Measurement NAP 10 desalting columns (Amersham Pharmacia Biotech, Uppsala, Sweden) are equilibrated just before use in 0.1 M Tris-HCl, 5 mM glutathione (GSH), 0.1% (v/v) Triton X-100, 5 mM EDTA, pH 7.4 (elution buffer; EB). A sample containing 0.30–0.40 mg of solubilized sperm proteins is diluted to exactly 1 ml with SB and loaded on the NAP 10 column. After the sample has entered the gel bed, elution is carried out with 1.5 ml of EB according to the manufacturer instructions. The buffer exchange procedure usually needs to be repeated twice in order to assure a complete removal of 2-mercaptoethanol. Activity Measurement Activity is measured in 2.2 ml of EB to which glutathione reductase (GSSG reductase, 0.6 IU/ml) and 0.030 mM NADPH are added. The mixture is incubated for 5 min at 25◦ and, after recording the basal rate of NADPH oxidation, the reaction is started by adding 30 μM phosphatidylcholine hydroperoxide (PC-OOH) prepared as previously described.15 PHGPx activity is measured from the time course of absorbance decrease at 340 nm (ε = 6.22 mM−1 cm−1 for NADPH). The basal rate, although negligible, is subtracted from the enzyme activity rate. PHGPx activity is calculated from the regression curve obtained from three different amounts of sample.8 A good sample volume-to-activity correlation (R, 0.995 ± 0.004) is reproducibly found. 14
L. Floh´e, R. Brigelius-Floh´e, M. Maiorino, A. Roveri, J. Wissing, and F. Ursini, in “Selenium: Its Molecular Biology and Role in Human Health” (D. Hatfield, ed.), p. 273. Kluwer Academic Publisher, 2001. 15 M. Maiorino, C. Gregolin, and F. Ursini, Methods Enzymol. 186, 448 (1990).
[18]
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Specific Activity Protein measurement is carried out by the Lowry procedure on the sample after elution from the NAP 10 column. Protein concentration is calculated by linear regression of activity data obtained with increasing amounts of sample (from 0.02 to 0.12 ml). Care is always taken that the amount of protein recovered corresponds to that loaded onto the column. The PHGPx measurements by this procedure of 30 normal subjets resulted in 200.5 ± 50.2 nmol/min×mg protein. Comments on Procedure Homogeneous PHGPx from pig heart was used to test the effect of the procedure on the activity of the enzyme. A constant loss of 25–30% of activity was observed to take place during the procedure. This is apparently due to the presence of guanidine. Because no further loss of activity was observed both over a 30-min incubation in the presence of guanidine at room temperature and after storage for weeks at −20◦ , it is apparent that guanidine does not progressively denature the protein but simply slows down, for an unknown reason, the catalytic activity. Because guanidine is useful to ensure a complete and reproducible solubilization of sperm, which is crucial for precision of the measurement, the decrease in activity was considered acceptable and not affecting the precision of the analysis when different samples are compared. 1. To optimize solubilization of sperm a relatively large dilution of the sperm samples must be used (<25×106 sperm/ml). 2. The amount and concentration of solubilized proteins to be loaded on buffer exchange column must be kept relatively constant (±20%) in order to optimize the recovery from the column and the reproducibility of the results. 3. The removal of 2-mercaptoethanol should be performed as carefully as possible. It is advisable to use the 5,5 -dithio-bis(2-nitrobenzoic acid) (DTNB) reaction for calibrating the column elution volumes. Futhermore, a linear correlation between different volumes of sample and measured activities gives a valuable index of the absence of thiols interfering with the measurement of activity. Discussion Determination of PHGPx in sperm may be considered a possible way to characterize hitherto poorly understood disturbances of male fertility. The idea that PHGPx is of pivotal importance for sperm architecture and function9 has been supported by partially successful gene disruption in mice.16 Nine chimeric male 16
M. Conrad, U. Heinzelmann, W. Wurst, G. W. Bornkamm, and M. Brielmeier, in “7th International Symposium on Selenium in Biology and Medicine,” October 1–5, 2000, Venice, Italy. [Abstract]
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SELENOPROTEINS
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mice having at least 50% PHGPx+/– cells produced 190 offspring homozygous for PHGPx. It is clear that the PHGPx+/– cells did not contribute to the germ line. Interestingly, the testes of the chimeric mice displayed mosaic-like disturbances, with a few spermatozoa displaying morphological alterations reminiscent of those observed in selenium-deficient rats, for example, fuzzy or broken midpieces, disoriented tails, or even isolated heads and tails.17,18 Reduced PHGPx content in sperm can therefore be expected to result in disturbed sperm morphology and function, as does selenium deficiency. Whether selenium shortage may affect PHGPx synthesis and sperm function in humans remains to be demonstrated. In rodents, male fertility was observed to be affected only on long-lasting selenium deprivation for several generations.17–20 These findings are not surprising if PHGPx is indeed the selenoprotein responsible for sperm integrity, because (1) testis tends to retain selenium even under severe selenium restriction21 and (2) PHGPx responds poorly to selenium deprivation.22–24 It can therefore hardly be anticipated that minor variations of selenium intake affect testicular PHGPx synthesis and fertility in men. It can nevertheless be envisaged that a reduced content of functional PHGPx in human testes due to genetic, hormonal, or severe nutrional deficiencies is etiologically linked to certain forms of male fertility problems. Acknowledgments The preparation of this article was supported by the Deutsche Forschungsgemeinschaft (Grant F161/12-1) and the Volkswagenstiftung (ZN548).
17
A. S. H. Wu, J. E. Oldfield, P. D. Whanger, and P. H. Weswig, Biol. Reprod. 8, 625 (1973). A. S. H. Wu, J. E. Oldfield, L. R. Shull, and P. R. Cheeke, Biol. Reprod. 20, 625 (1979). 19 E. Wallace, G. W. Cooper, and H. I. Calvin, Gamete Res. 4, 389 (1983). 20 E. Wallace, H. I. Calvin, and G. W. Cooper, Gamete Res. 4, 377 (1983). 21 D. Behne, T. Hofer, R. von Bersworat-Wallrabe, and W. Egler, J. Nutr. 112, 1682 (1982). 22 F. Weitzel, F. Ursini, and A. Wendel, Biochim. Biophys. Acta 1036, 88 (1990). 23 R. Brigelius-Floh´ e, Free Radic. Biol. Med. 27, 951 (1999). 24 L. Floh´ e, E. Wingender, and R. Brigelius-Floh´e, “Oxidative Stress and Signal Transduction” (H. J. Forman and E. Cadenas, eds.), p. 415. Chapman & Hall, New York, 1997. 18
[18]
SELENOPROTEINS
[18] Phospholipid–Hydroperoxide Glutathione Peroxidase in Sperm By ANTONELLA ROVERI, LEOPOLD FLOHE´ , MATILDE MAIORINO, and FULVIO URSINI Introduction The phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene (gpx-4) was found to be highly expressed in postpubertal mammalian testis.1 Mature testis was reported to have the highest PHGPx activity of all mammalian tissues investigated.2 There it is found associated, in part at least, with mitochondria,3 which complies with a tissue-specific transcription of the PHGPx gene into an mRNA encoding a protein with a mitochondrial leader peptide.4,5 Biosynthesis of PHGPx in testis occurs predominantly in round spermatids.6,7 Correspondingly, specific PHGPx activity in testis appears to parallel the thickness of the spermatid layer.6 The hormonal regulation of testicular PHGPx activity1 was shown to be indirect; Leydig cell-derived testosterone stimulates the seminiferous epithelium and thereby augments the number of spermatids that produce PHGPx.6 Despite the abundance of PHGPx in spermatids, PHGPx activity is practically undetectable in mature spermatozoa by conventional activity assays.8 Surprisingly, however, PHGPx protein could easily be detected by Western blotting in reduced and sodium dodecyl sulfate (SDS)-solubilized sperm proteins.9 This enzymatically inactive PHGPx protein was found to make up at least 50% of the keratinous material embedding the mitochondrial helix in the midpiece of sperm mitochondria.9 It accounts for most, if not all, of the selenium content in the midpiece of spermatozoa and thus is the real “mitochondrial capsule selenoprotein,” a term that had 1
A. Roveri, A. Casasco, M. Maiorino, P. Dalan, A. Calligaro, and F. Ursini, J. Biol. Chem. 267, 6142 (1992). 2 A. Roveri, M. Maiorino, and F. Ursini, Methods Enzymol. 233, 202 (1994). 3 M. Maiorino, A. Roveri, and F. Ursini, in “Free Radicals: From Basic Science to Medicine” (G. Poli, E. Albano, and M. U. Dianzani, eds.), p. 412. Birkh¨auser Verlag, Basel, 1993. 4 T. R. Pushpa-Rekha, A. L. Burdsall, L. M. Oleksa, G. M. Chisolm, and D. M. Driscoll, J. Biol. Chem. 270, 26993 (1995). 5 M. Arai, H. Imai, D. Sumi, T. Imanaka, T. Takano, N. Chiba, and Y. Nakagawa, Biochem. Biophys. Res. Commun. 227, 433 (1996). 6 M. Maiorino, J. B. Wissing, R. Brigelius-Floh´ e, F. Calabrese, A. Roveri, P. Steinert, F. Ursini, and L. Floh´e, FASEB J. 12, 1359 (1998). 7 K. Mizuno, S. Hirata, K. Hoshi, A. Shinohara, and M. Shiba, Biol. Trace Elem. Res. 74, 1112 (2000). 8 R. Brigelius-Floh´ e, K. Wingler, and C. M¨uller, Methods Enzymol. 347, [9], 2002 (this volume). 9 F. Ursini, S. Heim, M. Kiess, M. Maiorino, A. Roveri, J. Wissing, and L. Floh´ e, Science 285, 1393 (1999).
METHODS IN ENZYMOLOGY, VOL. 347
C 2002 by Academic Press. Copyright All rights of reproduction in any form reserved. 0076-6879/02 $35.00
[18]
PHGPx IN SPERM
209
misleadingly been introduced for the “sperm mitochondria-associated cysteinerich protein” (SMCP).10 The process transforming the enzymatically active selenoperoxidase into a structural protein during late spermatogenesis remains largely elusive. Obviously, the transformation results from oxidative cross-linking of PHGPx with itself and/or other proteins via Se–S and/or S–S bonds, because PHGPx protein can be solubilized and PHGPx activity can be recovered from the capsule material by drastic reductive procedures.9 The change in enzymatic activity and physical properties of PHGPx during spermatogenesis is a typical example of “moonlighting,” as has also been described, for example, for proteins making up the eye lens.11,12 This moonlighting certainly indicates a dual role of PHGPx in male reproduction; the enzyme may protect the rapidly dividing spermatogenic cells from potentially harmful oxidants, whereas it later becomes part of a structural element essential for the function of mature spermatozoa. The latter biological role of PHGPx appears at least as important for male fertility as its presumed antioxidant activity; interestingly, it is precisely the morphological alteration and mechanical instability of the midpiece material that lead to the impaired fertilization potential of selenium-deficient spermatozoa.13,14 Measurement of Phospholipid-Hydroperoxide Glutathione Peroxidase in Human Spermatozoa As outlined in the introduction, PHGPx is found in spermatozoa mainly in the midpiece, where it is part of the “mitochondrial capsule.” The enzyme is catalytically inactive in this form. The measurement of specific activity therefore requires a “rescue” procedure. The solubilization–reactivation is efficiently carried out in the presence of high concentrations of thiols and guanidine. The accuracy and reproducibility of specific activity measurements require (1) the complete and reproducible solubilization of all proteins of the sperm and (2) the careful removal of guanidine and of the thiols used for solubilization–reactivation, because they compete with glutathione in the peroxidase reaction, thus leading to an underestimation of the activity. Specimen Collection Human sperm are collected by masturbation between days 3 and 7 of abstinence. On each sample sperm count, morphological and functional parameters are immediately evaluated. Sperm are centrifuged at 300g for 10 min at 4◦ . Pellets are 10
L. Cataldo, K. Baig, R. Oko, M. A. Mastrangelo, and K. C. Kleene, Mol. Reprod. Dev. 45, 320 (1996). 11 J. Piatigorski, Prog. Ret. Eye Res. 17, 145 (1998). 12 C. J. Jefferey, Trends Biol. Sci. 24, 8 (1999). 13 M. Maiorino, L. Floh´ e, A. Roveri, P. Steinert, J. Wissing, and F. Ursini, BioFactors 10, 251 (1999).
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SELENOPROTEINS
[18]
washed twice in phosphate-buffered saline (PBS). Washed pellets can be stored at −20◦ for a few weeks without any apparent loss of activity. Sample Preparation The pellet is solubilized in 0.1 M Tris-HCl, 6 M guanidine hydrochloride, 0.1% (v/v) Triton X-100, 0.1 M 2-mercaptoethanol, pH 7.4 (solubilization buffer; SB). Sperm count in SB is 15–25×106cell/ml. Solubilization is carried out by vortex mixing. In the standardized procedure three periods of 30 sec of vortexing are used with a pause in ice of 1 min between each of them. The absence of any visible pellet after centrifugation ensures a thorough solubilization. The protein concentration thus obtained in the SB ranges between 0.5 and 1.0 mg/ml. Solubilized samples can be stored in this buffer at −20◦ and, under these conditions, PHGPx activity is stable for several days without any obvious loss of activity. Sample Preparation for Activity Measurement NAP 10 desalting columns (Amersham Pharmacia Biotech, Uppsala, Sweden) are equilibrated just before use in 0.1 M Tris-HCl, 5 mM glutathione (GSH), 0.1% (v/v) Triton X-100, 5 mM EDTA, pH 7.4 (elution buffer; EB). A sample containing 0.30–0.40 mg of solubilized sperm proteins is diluted to exactly 1 ml with SB and loaded on the NAP 10 column. After the sample has entered the gel bed, elution is carried out with 1.5 ml of EB according to the manufacturer instructions. The buffer exchange procedure usually needs to be repeated twice in order to assure a complete removal of 2-mercaptoethanol. Activity Measurement Activity is measured in 2.2 ml of EB to which glutathione reductase (GSSG reductase, 0.6 IU/ml) and 0.030 mM NADPH are added. The mixture is incubated for 5 min at 25◦ and, after recording the basal rate of NADPH oxidation, the reaction is started by adding 30 μM phosphatidylcholine hydroperoxide (PC-OOH) prepared as previously described.15 PHGPx activity is measured from the time course of absorbance decrease at 340 nm (ε = 6.22 mM−1 cm−1 for NADPH). The basal rate, although negligible, is subtracted from the enzyme activity rate. PHGPx activity is calculated from the regression curve obtained from three different amounts of sample.8 A good sample volume-to-activity correlation (R, 0.995 ± 0.004) is reproducibly found. 14
L. Floh´e, R. Brigelius-Floh´e, M. Maiorino, A. Roveri, J. Wissing, and F. Ursini, in “Selenium: Its Molecular Biology and Role in Human Health” (D. Hatfield, ed.), p. 273. Kluwer Academic Publisher, 2001. 15 M. Maiorino, C. Gregolin, and F. Ursini, Methods Enzymol. 186, 448 (1990).
[18]
PHGPx IN SPERM
211
Specific Activity Protein measurement is carried out by the Lowry procedure on the sample after elution from the NAP 10 column. Protein concentration is calculated by linear regression of activity data obtained with increasing amounts of sample (from 0.02 to 0.12 ml). Care is always taken that the amount of protein recovered corresponds to that loaded onto the column. The PHGPx measurements by this procedure of 30 normal subjets resulted in 200.5 ± 50.2 nmol/min×mg protein. Comments on Procedure Homogeneous PHGPx from pig heart was used to test the effect of the procedure on the activity of the enzyme. A constant loss of 25–30% of activity was observed to take place during the procedure. This is apparently due to the presence of guanidine. Because no further loss of activity was observed both over a 30-min incubation in the presence of guanidine at room temperature and after storage for weeks at −20◦ , it is apparent that guanidine does not progressively denature the protein but simply slows down, for an unknown reason, the catalytic activity. Because guanidine is useful to ensure a complete and reproducible solubilization of sperm, which is crucial for precision of the measurement, the decrease in activity was considered acceptable and not affecting the precision of the analysis when different samples are compared. 1. To optimize solubilization of sperm a relatively large dilution of the sperm samples must be used (<25×106 sperm/ml). 2. The amount and concentration of solubilized proteins to be loaded on buffer exchange column must be kept relatively constant (±20%) in order to optimize the recovery from the column and the reproducibility of the results. 3. The removal of 2-mercaptoethanol should be performed as carefully as possible. It is advisable to use the 5,5 -dithio-bis(2-nitrobenzoic acid) (DTNB) reaction for calibrating the column elution volumes. Futhermore, a linear correlation between different volumes of sample and measured activities gives a valuable index of the absence of thiols interfering with the measurement of activity. Discussion Determination of PHGPx in sperm may be considered a possible way to characterize hitherto poorly understood disturbances of male fertility. The idea that PHGPx is of pivotal importance for sperm architecture and function9 has been supported by partially successful gene disruption in mice.16 Nine chimeric male 16
M. Conrad, U. Heinzelmann, W. Wurst, G. W. Bornkamm, and M. Brielmeier, in “7th International Symposium on Selenium in Biology and Medicine,” October 1–5, 2000, Venice, Italy. [Abstract]
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SELENOPROTEINS
[18]
mice having at least 50% PHGPx+/– cells produced 190 offspring homozygous for PHGPx. It is clear that the PHGPx+/– cells did not contribute to the germ line. Interestingly, the testes of the chimeric mice displayed mosaic-like disturbances, with a few spermatozoa displaying morphological alterations reminiscent of those observed in selenium-deficient rats, for example, fuzzy or broken midpieces, disoriented tails, or even isolated heads and tails.17,18 Reduced PHGPx content in sperm can therefore be expected to result in disturbed sperm morphology and function, as does selenium deficiency. Whether selenium shortage may affect PHGPx synthesis and sperm function in humans remains to be demonstrated. In rodents, male fertility was observed to be affected only on long-lasting selenium deprivation for several generations.17–20 These findings are not surprising if PHGPx is indeed the selenoprotein responsible for sperm integrity, because (1) testis tends to retain selenium even under severe selenium restriction21 and (2) PHGPx responds poorly to selenium deprivation.22–24 It can therefore hardly be anticipated that minor variations of selenium intake affect testicular PHGPx synthesis and fertility in men. It can nevertheless be envisaged that a reduced content of functional PHGPx in human testes due to genetic, hormonal, or severe nutrional deficiencies is etiologically linked to certain forms of male fertility problems. Acknowledgments The preparation of this article was supported by the Deutsche Forschungsgemeinschaft (Grant F161/12-1) and the Volkswagenstiftung (ZN548).
17
A. S. H. Wu, J. E. Oldfield, P. D. Whanger, and P. H. Weswig, Biol. Reprod. 8, 625 (1973). A. S. H. Wu, J. E. Oldfield, L. R. Shull, and P. R. Cheeke, Biol. Reprod. 20, 625 (1979). 19 E. Wallace, G. W. Cooper, and H. I. Calvin, Gamete Res. 4, 389 (1983). 20 E. Wallace, H. I. Calvin, and G. W. Cooper, Gamete Res. 4, 377 (1983). 21 D. Behne, T. Hofer, R. von Bersworat-Wallrabe, and W. Egler, J. Nutr. 112, 1682 (1982). 22 F. Weitzel, F. Ursini, and A. Wendel, Biochim. Biophys. Acta 1036, 88 (1990). 23 R. Brigelius-Floh´ e, Free Radic. Biol. Med. 27, 951 (1999). 24 L. Floh´ e, E. Wingender, and R. Brigelius-Floh´e, “Oxidative Stress and Signal Transduction” (H. J. Forman and E. Cadenas, eds.), p. 415. Chapman & Hall, New York, 1997. 18