Patent Application: US-91336101-A

Abstract:
the invention relates to a method to detect male antifertility problems based on the determination of latent phospholipid hydroperoxide glutathione peroxidase .

Description:
phgpx as the major component of the sperm mitochondrial capsule had so far escaped attention , since as such it is enzymatically inactive , as it generally is in mature spermatozoa prepared from the tail of the epididymis ( tab . 1 ). it is neither reactivated by glutathione in the low millimolar range as used under conventional test conditions . high concentrations of thiols ( 0 . 1 m 2 - mercaptoethanol or dithiothreitol ), which in the presence of guanidine fully dissolve the capsule , regenerate a significant phgpx activity , as measured after elimination of denaturating and reducing agents ( tab . 1 ). in fact , the specific activities thus obtained from mitochondrial capsules exceed , by a factor of 20 , the highest values ever measured , i . e . in spermatogenic cells . nevertheless , this extreme phgpx activity is still low compared to its content in phgpx protein . based on the specific activity of pure phgpx , the reactivated enzyme would be equivalent to less than 3 % of the capsule protein , whereas the 2d - electrophoresis suggests a phgpx protein content of at least 50 %. it is worth noting that the same reductive procedure does not increase the specific activity of phgpx in spermatogenic cells from testicular tubules ( tab . 1 ). the switch of phgpx from a soluble active enzyme to an enzymatically inactive structural protein thus occurs during final differentiation of spermatozoa . the alternate roles of phgpx , being either a glutathione - dependent hydroperoxide reductase or a structural protein , are not necessarily unrelated . one of the features common to all glutathione peroxidases is a selenocysteine residue which together with a tryptophan and a glutamine residue forms a catalytic triad ( 15 , 16 ). therein the selenol group of the selenocysteine residue is dissociated and highly activated by hydrogen bonding to reduce hydroperoxides with high rate constants . the reaction product , a selenenic acid derivative , r - seoh , will readily react with thiols , e . g . gsh , to form an intermediate with a selenadisulfide bridge between enzyme and substrate , r - se - s - g , from which the ground state enzyme can be regenerated by a second gsh . phgpx is unique among the glutathione peroxidases in several respects : i ) it usually is monomeric having its active site freely accessible at the surface ; this facilitates interaction with bulky substrates . ii ) arginine residues surrounding the active site and specifically binding glutathione in most types of glutathione peroxidases are lacking in phgpx ( 16 ); correspondingly , its specificity for the reducing substrate is less pronounced ( 16 ). it therefore can be envisaged that oxidized phgpx may form diselenide or selenadisulfide bridges with exposed seh or sh groups of proteins ( 16 ) including phgpx itself , and this process , possibly followed by se / ss , sh / ses , or sh / sese exchange reactions , will create cross - linked protein aggregates . this ability of phgpx might become particularly important if cells are exposed to hydroperoxides at extremely low concentration of glutathione , as is documented for late states of spermatogenesis ( 17 - 20 ). fig3 is to mimick the oxidative events occurring during sperm maturation . short term exposure of soluble proteins derived from spermatogenic cells to moderate h 2 o 2 concentrations in the absence of gsh yields a variety of phgpx - containing high molecular weight aggregates . undoubtedly , therefore , phgpx , by means of its intrinsic enzymatic potential , can catalyse oxidative protein aggregation using protein thiols as alternate substrates . during sperm maturation , phgpx thereby transforms itself into an enzymatically inactivated structural protein . this view , however , is not to imply that phgpx could not depend on additional proteins when building up the highly organized architecture of the spermatozoal midpiece . our findings require a fundamental reconsideration of the role of selenium in male fertilty . the intriguing predominance of the selenoprotein phgpx in the male reproductive system has so far been believed to reflect the necessity to shield germ line cells from oxidative damage by hydroperoxides and reactive oxygen species derived therefrom ( 11 , 17 , 21 , 22 ). this concept still merits attention with regard to the mutagenic potential of hydroperoxides and probably holds true for the early phases of spermatogenesis where phgpx is still present as an active peroxidase ( 6 , 21 ). at this stage related activities reported for phgpx or other glutathione peroxidases , e . g . silencing lipoxygenases ( 23 ), dampening the activation of nf b ( 24 ) or inhibiting apoptosis ( 25 ), may also be relevant . in later stages of spermatogenesis characterized by a shift of the redox status resulting in loss of gsh ( 18 - 20 , 26 ), the ability of phgpx to use protein thiols as alternate substrates opens up new perspectives of redox regulation which remain to be explored . in the mature spermatozoon phgpx has experienced a pronounced metamorphosis now being a major constituent of the keratinuous material embedding the mitochondrial helix . it appears revealing that precisely this architectural pecularity in the midpiece of spermatozoa shows gross structural alterations in selenium deficiency . we therefore assume that the mechanical instability of the midpiece observed in selenium deficiency is a consequence of an impaired phgpx biosynthesis . this view implies that it is not the antioxidant capacity of phgpx which is crucial for male fertility but its ability to utilize hydroperoxides to build an indispensable structural element of the spermatozoon . any shortage of phgpx during sperm maturation , be it due to selenium deficiency , other reasons of inhibited biosynthesis or inhibition of activity should therefore result in disturbed sperm midpiece architecture and , in consequence , loss of fertilization potential of sperm . this conclusion was further corroborated by determination of reactivated phgpx in sperm of individuals with documented fertility problems . the latter were divided into three groups : depending on whether i ) intrauterine sperm injection ( iui ) or ii ) conventional in - vitro - fertilisation ( ivt - et ) was still successful or iii ) intracytoplasmatic sperm injection was required ( icsi ). as shown in fig4 , the phgpx values differed markedly between these groups . while the iui group displayed values close to normal , phgpx in the icsi group was almost absent , the ivf - et group ranking in between . the reasons of the diverse phgpx content being unknown , the data reveal that markedly reduced phgpx content in sperm is incompatible with normal male fertility . similarly , there is a strong correlation between “ typical ” sperm appearance ( fig5 ) and “ fast ” moving sperm with phgpx content ( fig6 ). this correlation , however , shows marked scattering of data indicating that phgpx content of sperm is not the only reason of abnormal shape and motillity of sperm . it should also be pointed out that the sperm samples were taken from individuals without any obvious disease suggesting that extremely reduced phgpx levels are well tolerated . taken together , the ovservation that phgpx builds up an esssential structure of sperm and that its content in sperm correlates with the fertilization potential leads to the inventive concept to use the phgpx content of sperm as a predictive parameter for the necessary measures to overcome male fertility problems . to this end , it appears necessary to reactivate the phgpx contained in sperm in order to estimate its content by either immunological methods or by any of the established determinations of its specific activity . ( 28 , 29 ). spermatozoa of four month old wistar rats ( about 300 grams of body weight ) were collected by squeezing cauda epididymis and vas deferens in phosphate buffer saline ( pbs ) and by centrifugating at 600 × g for 10 minutes . cell and sperm pellets were layered on a discontinous 45 %, 70 % and 95 % percoll gradient and centrifugated at 300 × g for 20 min . spermatogenic cells stacked on top of the gradient , while spermatozoa separated into the 70 % percoll layer . cells from seminiferous epithelium were prepared as follows ( 26 ): testes were deprived of albuginea , seminiferous tubules were cut into small pieces in pbs containing 0 . 250 mg / ml collagenase , and incubated twice 25 ° c . for 15 min . cells then were filtered through a stainless steel screen ( 140 μm pore ), washed in pbs and centrifugated at 300 × g for 10 min . sperm mitochondrial capsule was prepared according to calvin et al . ( 1 ): sperms were resuspended in 0 . 05 m tris - hcl ph 8 . 0 at the concentration of 10 6 cells / ml and treated with trypsin ( 0 . 2 mg / ml ) for 10 minutes . after stopping the protease action with trypsin inhibitor ( 0 . 5 mg / ml ) and sds ( 10 mg / ml ) sperms were centrifugated at 1 , 500 × g for 10 minutes . pellets were resuspended in 0 . 05 m tris - hcl , ph 8 . 5 containing 1 % sodium dodecyl sulphate ( sds ), and 0 . 2 mm dtt and kept under continuous stirring for 30 minutes . following centrifugation at 4 , 500 × g for 15 min , the resulting supernatant was layered on a 1 . 6 m sucrose cushion . after centrifugation for 20 min at 18 , 000 × g in a swinging rotor , sperm capsules were collected as a band at the top of the sucrose cushion , washed in tris - hcl , ph 8 . 0 and spun at 140 , 000 × g . electrophoresis was performed according to laemmli under either reducing (+ 2 − mercaptoethanol ) or non - reducing conditions . proteins were blotted onto nitrocellulose , probed with an antigen - purified rabbit antibody raised against pig heart phgpx and detected by biotinylated anti rabbit igg and streptoavidin alkaline phosphatase complex . 100 μg of the mitochondrial capsule material was dissolved in 400 μl of a solution containing of 7 m urea , 2 m thiourea , 4 % chaps , 40 mm dtt , 20 mm tris base and 0 . 5 % ipg buffer ( pharmacia ) and focused in an ipg - phor ( pharmacia ) 2 - d gel electrophoresis system at 20 ° c . by stepwise increasing voltage up to 5000 v but not exceeding a current of 30 μa per ipg strip . the ph gradient was non - linear from 3 - 10 or linear from 3 - 10 or 6 - 11 . the focused ipg strips were then equilibrated for sds electrophoresis ( 10 min each ) with a solution containing 60 mm dtt in 6 m urea , 30 % glycerol , 0 . 05 m tris - hcl buffer ph 8 . 8 and in the same buffer where dtt was substituted by 250 mm iodoacetamide . after sds - electrophoresis ( 12 % polyacrylamide ) the gels were stained with coomassie . coomassie - stained spots were cut out from the gels , neutralized with ( nh 4 ) hco 3 , destained with 400 μl 50 % acetonitrile / 10 mm ( nh 4 ) hco 3 and dried in a speed vac concentrator . protein digestion was done overnight using 2 ng / μl sequencing grade trypsin ( promega ) in 50 mm ( nh 4 ) hco 3 ( boehringer , mannheim ). the resulting peptides were extracted twice with 60 % acetonitrile / 40 % h 2 o / 0 . 1 % tfa . extracts were combined and lyophilized in the speed vac concentrator . peptide digests were desalted on small rp18 - columns , eluted with saturated α - hydroxy - 4 - cyano - cinnamic acid and loaded directly onto the maldi target ( 27 ). reflectron maldi mass spectra were recorded on a reflex ™ maldi / tof - mass spectrometer ( bruker - franzen - analytik , bremen ). the ions were excellerated at 20 kv and reflected at 21 . 3 kv . spectra were externally calibrated using the monoisotopic mh + ion from two peptide standards . 100 - 200 laser shots were summed up for a single mass spectrum . mass identification was performed with ms - fit ( http :// falcon . ludwig . ucl . ac . uk / ucsfhtml / msfit . htm ). alternatively , protein spots from 1 . 5 mm 2d - gels were digested with modified trypsin ( promega , sequencing grade ) in 25 mm ( nh 4 ) hco 3 overnight at 37 ° c . the digests were extracted twice and dried as before and reconstituted in 10 μl water . peptides were separated on a reversed - phase capillary column ( 0 . 5 mm × 150 mm ) with a gradient of acetonitrile in 0 . 1 % formic acid / 4 mm ammonium acetate at a flow rate of 5 μl / min and collected manually . aliquots of 5 μl were spotted onto biobrene - treated glass fiber filters and sequenced on an applied biosystems 494a sequencer with standard pulsed - liquid cycles . before n - terminal sequencing , proteins were blotted from polyacrylamide gels onto pvdf membranes for 16 h at ph 8 . 3 ( 25 mm tris - hcl , 192 mm glycine ) and 100 ma ( 30 v ). when applicable , phgpx was also identified by activity measurement according to ( 28 ) using the specific substrate phosphatidylcholine hydroperoxide . fig1 presence of phgpx in the mitochondrial capsule of spermatozoa . a , mitochondrial capsule prepared by trypsination and centrifugation according to ( 1 ) at 80 , 000 fold magnification . b , the same preparation as shown in a , but after exposure to 0 . 1 m 2 - mercaptoethanol for 15 min at 4 ° c . contamination of the capsule material by mitochondria is evident from the presence of mitochondrial ghosts . c , sds gel electrophoresis of proteins extracted from capsule material ( see methods ) by treatment with 0 . 1 m 2 - mercaptoethanol , 0 . 1 m tris - hcl , ph 7 . 5 , and 8 m guanidine hcl . left lane is stained with coomassie , right lane demonstrates presence of phgpx by western blotting . fig2 showws the analysis of the composition of the mitochondrial capsule of spermatozoa a , 2d - electrophoresis of purified dissolved capsule material . proteins were focused in a linear ph - gradient from 3 to 10 ( horizontal direction ), then reduced , amidocarboxymethylated , subjected to sds - electrophoresis , and stained with coomassie . maldi - tof analysis of the visible spots identified the following proteins ( swissprot data base ): spot 1 - 7 phgpx ( mw 19 443 ; pi 8 . 27 ; acc . no . 544434 ); spots 8 and 9 , outer dense fiber protein ( mw 27351 ; pi 8 . 36 ; acc . no . p21769 ); spots 10 and 11 , voltage - dependent anion channel - like protein ( mw 31720 ; pi 7 . 44 ; acc . no . 540011 ); spot 12 , “ stress - activated protein kinase ” ( mw 48107 ; pi 5 . 65 ; acc . no . 493207 ); spot 13 , glycerol - 3 - phosphate dehydrogenase ( mw 76479 ; pi 5 . 86 ; acc . no . p35571 ). b , maldi - tof spectrum ( overview ) of tryptic peptides obtained from phgpx as found in spot 3 . abscissa , mass / charge ratio of the peptide fragments ; ordinate , arbitrary units of intensity ; numbers at mass signals , identified peptides in the phgpx sequence ( see insert for position numbers ); t , trypsin - derived fragments . c , compilation of tryptic phgpx fragments identified in spots 1 - 7 by maldi - tof . vertical lines designate potential tryptic lag cleavage sites . dark blocks , identified typical cleavage products ; shadowed blocks , masses resulting from incomplete cleavage or equivocally assignable to different fragments ( e . g . 3 - 9 and 63 - 69 ). fig3 shows the formation of phgpx - containing aggregates from spermatogenetic cells by h 2 o 2 in the absence of gsh . spermatogenic cells were homogenised in 0 . 1 m tris - hcl , 6 m guanidine - hcl , 0 . 5 μg / ml pepstatin a , 0 . 7 μg / ml leupeptin and 5 mm 2 - mercaptoethanol at ph 7 . 5 and 4 ° c . after centrifugation at 105 , 000 × g for 30 min , excess reagents were removed by gel permeation using nap 5 columns equilibrated with 10 mm tris - hcl , 0 . 15 m nacl , 1 mm edta and 0 . 1 % triton x - 100 , ph 7 . 5 . immediately ( t 0 ) and 15 min after ( t 15 ) the addition of 75 μm h 2 o 2 aliquots of the mixture ( 0 . 05 mg of protein ) were withdrawn and subjected to electrophoresis under ( a ) reducing and ( b ) non reducing conditions . after blotting on nitrocellulose , phgpx was detected by specific antibodies . fig4 shows the phgpx specific activity in extracts ( 0 . 1 % triton x - 100 and 0 . 1 m 2 - mercaptoethanol of human sperm . correlation between this parameter and therapeutic appproach in cases of couple infertility . fig5 shows the relationship between phgpx specific activity and number of “ typical ” sperms per milliliter of semen . “ typical ” is a morphological parameter of sperm evaluation . fig6 shows the relationship between phgpx specific activity and number of “ fast ” sperms per milliliter of semen . “ fast ” is a parameter of sperm mobility . c solubilisation / reduction was carried out in 0 . 1m tris - hcl , 6m guanidine - hcl , 0 . 5 μg / ml pepstatin a , 0 . 7 μg / ml leupeptin and 2 - mercaptoethanol as indicated at ph 7 . 5 and 4 ° c . for 10 min low molecular weight compounds were removed before activity determination by a nap 5 cartridge . 1 . calvin , h . i ., cooper , g . w . & amp ; wallace , e . evidence that selenium in rat sperm is associated with a cysteine - rich structural protein of the mitochondrial capsules . gamete res . 4 , 139 - 149 ( 1981 ). 2 . brown , d . g . & amp ; burk , r . f . selenium retention in tissues and sperm of rats fed a torula yeast diet . j . nutr . 103 , 102 - 108 ( 1973 ). 3 . wu , a . s . h ., oldfield , j . e ., shull , l . r . & amp ; cheeke , p . r . specific effect of selenium deficiency on rat sperm . biol . reprod . 20 , 793 - 798 ( 1979 ). 4 . wallace , e ., calvin , h . i ., ploetz , k . & amp ; cooper , g .- w . functional and developmental studies on the role of selenium in spermatogenesis . in : combs , g . f ., levander , o . a ., spallholz , j . e ., and oldfield , j . e . 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