Patent Application: US-63679603-A

Abstract:
this invention relates to a novel form of the phgpx protein , the sperm nuclei glutathione peroxidase as well as portions thereof playing a role in mammalian spermatogenesis , and to the nucleic acids encoding the same . the invention further relates to vectors containing said nucleic acid and to host cells transformed by these vectors . furthermore the invention comprises antibodies specific for the above proteins / peptides as well as the use of the proteins / peptides in the diagnosis or therapy of male infertility .

Description:
a 34 kd selenoprotein isolated from rat testes was identified as a specific sperm nuclei glutathione peroxidase ( sngpx ) having properties similar to those of phgpx . the determination of its primary structure by analysis of the first n - terminal amino acids , a data base search , polymerase chain reaction , and sequencing of the cdna showed that its n - terminal sequence is different from that of phgpx . this sequence encoded by an alternative exon in the first intron of the phgpx gene shows more than 50 % homology to the protamine sequences and contains a nuclear localization signal . in rats , sngpx is highly expressed in the nuclei of late spermatids where it is the only selenoprotein present . its appearance coincides with dna rearrangements resulting in highly condensed chromatin stabilized by crosslinked protamine thiols . in selenium - depleted rats in which the concentration of sngpx has been reduced to one third of the normal level the chromatin condensation is severely defective . we have shown that sngpx acts as a protamine thiol peroxidase responsible for disulfide crosslinking by reduction of reactive oxygen species . its dual function in chromatin condensation and in the protection of sperm dna from oxidation is necessary to ensure male fertility and sperm quality . in a first step the 34 kd selenoprotein was purified from the srs nuclei of late spermatids of rats which had been fed a selenium - adequate diet . following treatment of the surfaces of these nuclei with the detergent ctab it was found that the 34 kd protein was the only selenoprotein within these nuclei as can be seen from the autoradiograph in fig1 lane 3 . for the selenium concentration and thus for the 34 kd selenoprotein in ctab treated srs nuclei a relatively high value of about 60 μmol / kg of dry weight was determined which far exceeded the selenium concentration in the nuclear fractions of other rat tissues such as for example the liver ( 14 μmol se / kg of dry weight ) and of brain ( 8 μmol se / kg of dry weight ). a protein fragment analysis by means of maldi - ms after tryptic digestion showed a considerable similarity between the 34 kd protein and another selenoprotein , namely phgpx , one of the four glutathione peroxidases identified up to now which catalyze the reduction of peroxide by oxidation of glutathione ( 12 , 15 - 17 ). the similarity became more striking in an experiment by finding out that the 34 kd protein reacts with an anti - phgpx antibody as represented in the first lane of fig1 . it also inhibited the glutathione peroxidase activity and , similar to phgpx , catalyzed the reduction of various peroxides such as for example hydrogen peroxide , t - butylhydroperoxide and phosphatidylcholine hydroperoxide ( 12 ). its specific activity was about 3000 u / mg protein when the latter was used as the substrate . due to its localization this was called sperm nuclei glutathione peroxidase ( sngpx ). during the transfer of the sperms from the testicle to the epididymis two thirds of the 34 kd selenoprotein were converted to smaller proteins having molecular weights of 24 , 22 and 20 kd , as may be seen from lanes 2 and 4 in fig1 . this truncation is associated with a shift in the ph value from an extremely basic value of more than 10 for the 34 kd protein to a value of about 7 . 5 for the 20 kd product ( fig2 ). reduction of the mass , however , had no effect on the enzymatic properties of the processed proteins . the analysis of the n - terminal sequence of the 34 kd compound showed that it started with the amino acids sraaargrkr . data base searches revealed that the protein was unknown up to now . however , if the search was extended to all available genomic sequences and these were transformed into all reading frames we ended up with a similar sequence in the first intron of the murine phgpx gene . an alternative exon responsible for the formation of the novel selenoprotein was identified in murine and rat testis rna using a cdna amplification with primers derived from the available dna and protein sequence information ( fig3 ). the sequence of this exon in mouse and rat and the presumable corresponding exon in human and pig is represented in fig3 b . it encodes an arginine - rich sequence immediately following the first methionine . information as to the function of said sequence was obtained from an experiment wherein two gfp fusion genes were constructed . for the first , the whole alternative exon of the murine sequence , and for the second the sequence subsequent to the second methionine were fused to gfp . after transfection of both constructs into two mammalian cell lines the subcellular localizations of the two fusion proteins were determined by means of gfp fluorescence . only the largest gfp fusion protein had entered the nucleus indicating that the n - terminal arginine - rich sequence contains a nuclear localization signal ( data not shown ). a northern blot analysis of different tissues of the mouse probed with the ea sequence of sngpx or with the total sequence of phgpx ( fig4 ) showed that sngpx is expressed only in the testis . this was confirmed by measuring the distribution of 75 se labeled proteins in the murine organism . in this case sngpx was detected only in testis and sperms as has been found earlier for rats ( 5 ), wherein this fact indicates a specific function for this selenoenzyme . the occurrence of sngpx in late stages of spermatogenesis coincides with the packaging of the dna with protamines and the stabilization of the resulting highly condensed chromatin by crosslinking of protamine disulfides . ( 8 ). this process is induced by reactive oxygen species ( ros ) ( 18 ) and thus is analogous to glutathione oxidation and peroxide reduction catalyzed by glutathione peroxidases . together with the finding that the glutathione concentration in spermatids during the late stages of spermatid development markedly decreases ( 19 ) this suggests that the selenoenzyme could be capable of using protamine cysteine residues as reducing agents thus acting as a protamine thiol reductase . therefore the sperm nuclear condensation was examined in selenium - deficient rats in which the concentration of selenium and thus of sngpx in ctab - treated srs nuclei was decreased to 20 μmol se / kg of dry weight as compared to a value of 60 μmol se / kg of dry weight in seleniumadequate animals . the nuclei of both groups were stained with acridine orange making it possible to distinguish between double and single stranded nucleic acids . since sperm dna can be denatured by heat only prior to but not after protamine sulfide crosslinking , by means of an acridine orange stain the thiol disuflide state during chromatine condensation of mammalian sperm nuclei can be monitored after the treatment ( 14 ). staining revealed that almost all sperm cells obtained form the vas deferens of the selenium - deficient rats were incompletely condensed ( fig5 ). in vitro experiments using sperm nuclei of seleniumadequate rats showed that the condensed state was lost during the reduction of protamine disulfides with dithiothreitol and could be reestablished by addition of hydrogen peroxide . the recondensation was blocked by addition of bromosulfophthaleine , an inhibitor of phgpx ( 20 ), or by an excess of another thiol in the form of gsh . it may be concluded from these findings that sngpx is involved in protamine sulfide crosslinking . the 34 kd selenoprotein found in testis and sperm ( 5 ) has now been identified as a specific sperm nuclei glutathione peroxidase having properties similar to those of phgpx . it is encoded by the phgpx gene , however , in contrast thereto it is expressed exclusively in testis and starts with an arginine - rich n - terminal sequence encoded by an alternative exon . this sequence contains a nuclear localization signal causing sngpx to be the only selenoprotein able to enter the sperm nucleus . another indication as to the significance of this sequence is the fact that is shows more than 50 % homology to the sequences of the protamines . protamines are small basic proteins which are arginine - and cysteine - rich and replacing the histones during sperm maturation . it has been known that they bind to dna via their polyarginine region and it is very likely that sngpx is associated with dna in a similar manner . by our findings that sngpx acts as a protamine thiol peroxidase responsible for the formation of crosslinked protamine disulfides and thus for the stabilization of condensed sperm nuclei , and that a decrease in sngpx levels results in severe defects in chromatin condensation , another important role of selenium has been established . the process of chromatin condensation seems to be important not only for the maturation of the sperm cells but also for the fertility and generation of progeny . in humans , a high correlation between regular sperm condensation and the in vitro fertilization rate has been observed ( 22 ). in vitro experiments in mice show that a condensed sperm nucleus having a stable matrix is required to ensure a normal fertilization and embryonic development ( 23 ). thus , the sngpx dependent protamine thiol oxidation appears to play a key role in male fertility and reproduction . as soon as the sperm reaches the caput epididymis and the condensation process is completed sngpx is partially processed to smaller proteins having the same enzymatic activity but neutral ph values indicating that the basic arginine - rich n - terminal sequence has been lost . accordingly , sngpx and the processed proteins fulfill two functions : first , the oxidation of protamines by sngpx bound to full length dna and , second , protection of sperm dna against oxidative damage by the enzyme and the forms resulting therefrom which can be more efficient in ros degradation since they are not bound to dna . it has been demonstrated in several studies that the quality of human sperms decreases with increasing oxidative damages to the dna . since , however , a limited generation of ros is required for protamine thiol crosslinking dna damages due to excessive amounts of ros adversely affect the health of the progeny ( 24 ). consequently , a determination of the sngpx state of the sperm nucleus can be of importance for establishing the sperm quality . it has been suggested that phgpx plays a role in chromatin condensation ( 25 ) and in the protection of the sperm dna against oxidative damage ( 26 ). however , phgpx is expressed only in a cytosolic and a mitochondrial form and is mainly membrane - bound in sperms ( 25 ). this discrepancy could be solved by the identification of sngpx as the only selenoprotein present in spermatid nuclei and by its characterization as a protamine thiol peroxidase . it has been known that selenium plays a role in various processes in the male reproductive system . phgpx acts as a structural component in the mitochondrial capsule ( 27 ) and thus is required for flagellum formation whereas in the nuclei sngpx and its processed products are involved in chromatin condensation and in the protection of the germ line against oxidative damage . in addition , selenium has also been reported to be required for testosterone biosynthesis in a manner not identified to date ( 4 ). it will be of much interest to find to what level defects in the formation and function of selenoproteins contribute to male fertility disorders . the following examples merely serve as an illustration and should not be construed as limiting the scope of the present invention . rats were subjected for several generations either a selenium deficient diet containing 2 - 5 μg se / kg or a selenium - adequate diet consisting of the basal diet with 300 μg se / kg , added in the form of sodium selenite . in the tracer experiments the animals were labeled in vivo by injection of 35 mbq / μm se ) in the form of sodium selenite . the composition of the diet and the treatment of the animals have been described elsewhere ( 6 ). in the tracer experiments with mice , adult mice were subjected to the basal diet for two weeks and then labeled by injecting a dose of 3 mbq 75 se selenite . the selenium concentrations of tissues and testicular fractions were determined by instrumental neutron activation analysis as described earlier elsewhere ( 9 ). in the tracer experiments , the 75 se activity in the tissues and tissue fractions was measured by means of a nai ( ti ) drill hole detector connected to a four channel analyzer ( canberra , frankfurt , germany ). the 75 se labeled selenoproteins separated by sds page were examined by means of autoradiography using a light sensitive imaging sheet ( fuji , bas 1000 , raytest , straubenhardt , germany ). following decapsulation the testes were homogenized in a fourfold volume of a buffer a ( 20 mm mes , 0 . 31 m sucrose , 3 mm mgcl 2 , 0 . 1 % triton x - 100 , 50 mm benzamide hcl , 0 . 1 mm pmsf , ph 6 . 0 - 6 . 1 ). after centrifugation at 1000 × g the sonication - resistant nuclei ( srs nuclei ) of late spermatids were isolated as already described earlier ( 10 ) by using a b15 branson cell disruptor ( heinemann , schwäbisch - gmünd , germany ). after incubation with buffers b ( 1 % ctab , 50 mm tris , 20 mm dtt , ph 8 ) and c ( 1 % chaps , 50 mm tris , 20 mm dtt , ph 8 ), several times washing with buffer d ( 50 mm tris , 20 mm dtt , ph 8 ), extraction with nacl solution ( 1 m nacl in 50 mm tris , 2 % β - mercapto ethanol , ph 8 ) and centrifugation at 1000 × g the supernatant was distilled against aqua dest . and the 34 kd protein was separated from other proteins by means of sds page . after sds page separation the 34 kd band was cut from the gel and a tryptic digestion was carried out as described ( 11 ). the digest was extracted with 1 % trifluoroacetic acid in acetonitrile . the fragment masses were determined by means of a modified bruker reflex iii equipped with delayed extraction . peptide search (“ protein identification by peptide mass data ”, embl , heidelberg ) was used for mass identification . the glutathione peroxidase activity was determined spectrometrically at 340 nm as described ( 12 ) by using hydrogen peroxide or t - butylperoxide as substrates . the phgpx activity was measured by phosphatidylcholine hydroperoxidase which was used as a specific substrate . the 34 kd protein isolated by sds page was transferred to a pvdf membrane ( biometra , göttingen , germany ) and probed with a purified anti - rat phgpx antibody . the 34 kd protein was isolated by means of sds page , transferred to a pvdf membrane ( biometra , göttingen , germany ) and stained with amido black . an n - terminal sequencing was carried out with respect to the bands of interest using an abi 494 procise edman sequencer ( pe biosystems , foster city , calif , usa ) after the bands had been excised . the amino acids were analyzed in the form of pth derivatives . the sequencing data obtained in this manner were compared to the sequences in data bases using the “ blast search ” at ncbi . the 3 ′ race experiments were performed in the geneamp system 2400 thermal cycler ( perkin elmer , norwalk , conn , usa ). for subsequent sequencing the respective pcr product was cloned into vector pcr2 . 1 - topo ( invitrogen , groningen , netherlands ) mouse sngpx : polya + rna was isolated from murine testes using the polyatract mrna isolation system iv ( promega , madison , wis , usa ). adapter - bound testis cdna was synthesized according to the instructions of the manufacturer ( clontech , palo alto , calif . 2 + -, usa ). the alternative exon was amplified by means of 3 ′ race using primer phgpx ea - fl ( gggacgctgcagacagcgcggcggatcc ) and advantage 2 polymerase ( clontech ). rat sngpx : the data base searches using the sequence of the alternative exon ea of mouse resulted in an est clone of rat ( gi : 3399319 ) which was identical to exon i of phgpx from rat and to ea of mouse . the total rna was isolated from homogenized testes by the acidic guanidinium thiocyanate - phenol - chloroform process ( 13 ). an rt - pcr was carried out by means of the super script one step rt - pcr system ( life technologies , gibco brl , karlsruhe , germany ) using the sngpx specific primer ( atgggccgcgcggccg ) and the primer derived from the rat phgpx exon 7 ( cggcaggtccttctctatcacctg ). human sngpx : a presumable ea for the human 34 kd was found following a comparison of the derived amino acid sequence with the human phgpx gene ( nucleotides 770 - 964 of gene af 060973 ). a 3 ′ race was carried out with adapter - ligated testis cdna ( clontech ) using the sngpx specific primer 1 ( atgggccgcgcgggcgcaggctccc ) and primer 2 ( cttgcgaccggagatccacgaatgtccc ) and advantage 2 polymerase ( clontech ). only the last 14 nucleotides from exon ea and the transition point to phgpx were obtained . a search in the est database resulted in an est clone ( gi : 2754408 ) covering the ea sequence , and in another est clone ( gi : 6703705 ) containing the presumable starting methionine . pig sngpx : as for the human sngpx a presumable alternative exon was obtained from the pig phgpx gene ( sus scrofa accession number : x76088 ) by comparison of the derived amino acids . it encodes an arginine - rich sequence following the presumable translational start . the transition to phgpx is ambiguous , however , the nucleotide sequence is identical to that of humans . the total rna was isolated from homogenized tissue using the peqgoldtrifast ( peqlab , erlangen , germany ). 20 μg of total rna were applied per lane , blotted onto hybondn + ( amersham pharmacia , freiburg , germany ) and with the coding region of the alternative exon was probed with phgpx total cdna . two n - terminal gfp fusion proteins were generated by means of overlap pcr . one contained the complete coding region of the alternative exon , the other started at the second presumable translational start . the primer pairs :( gatctctagaccggcgggcatgggccgcgcg ) ( gfp - ea - f1 ) and ( gctcctcgcccttgctcaccaagcccaggaactcggagc ) ( gfp - ea - r2 ) as well as ( gatctcagactcgccggatggagcccattcc ) ( gfp - ea - f2 ) and gfp - ea - r2 were used for the amplification of the n - terminal portions for the longer or the shorter version , respectively , by using pmc42 as a template . ( gctccgagttcctgggcttggtgagcaagggcgaggagc ) ( gfp - f3 ) and ( cctctacaaatgtggtatgg ) ( gfp - r1 ) were used to generate the gfp portion using pegfp - n1 as a template . an overlap pcr was performed by combining the products with the outermost primers . all pcrs were carried out in the perkin elmer geneamp system 2400 thermal cycler . the two products were cloned into pcdna3 via xhii / xbai and transiently transfected into nih3t3 and hela cells . an abnormal chromatin structure which is evaluated hereby is quantitatively determined by means of flowcytometric measurement of the metachromatic shift of green ( dsdna ) to red ( denatured , ssdna ) of the acridine orange ( a / o ) fluorescence . the shift is expressed by the function alpha t ( greek αt ) representing the ratio of the red to the total fluorescence intensity ( red + green ) ( darzynkiewicz et al ., 1975 ) thereby representing the amount of denatured ssdna with respect to the total cellular dna . in the scsa the αt for each spermatozoon in a sample was calculated , and the results were expressed as the mean value ( x αt ), the standard deviation ( sd αt ) of the αt distribution and as the percentage of cells having a high αt value referred to as cells outside the main population ( comp ) (% comp αt ) representing the cells having an excess of ssdna . the range of the at values obtained is expressed as a range from 0 to 1024 fluorescence channels . samples of thawed sperm were diluted with tne buffer ( 0 . 15 m nacl , 0 . 01 m tris - hcl , 1 mm edta , ph 7 . 4 ) and diluted in polypropylene tubes to a final sperm concentration of approx . 2 × 10 6 / ml . aliquots of the diluted sperm ( 0 . 2 ml ) were deep - frozen immediately in liquid nitrogen steam ( ln 2 ) and then transferred to an ultracold freezer (− 80 ° c .) and stored therein until the fcm examination ( spano et al ., 1999 ). the samples were codified and a blind fcm analysis was carried out . after thawing on crushed ice the sperm cells were subjected to partial denaturation in situ and stained with ao ( merck ). for this purpose , the sperm samples were mixed with 0 . 4 ml of detergent solution having a low ph ( 0 . 17 % triton x - 100 , 0 . 15 m nacl , and 0 . 08 n hcl , ph 1 . 4 ). after 30 seconds the cells were stained by 1 . 2 ml of a solution ( 0 . 1 m citric acid , 0 . 2 m na 2 hpo 4 , 1 mm edta , 0 . 15 m nacl , ph 6 . 0 ) containing 6 μm / ml of ao . the stained samples were divided in two halves and examined within 3 - 5 minutes following ao staining . the samples were examined by means of a facstar plus flowcytometer ( becton dickinson immunochemistry systems , san josé , calif ., usa ) equipped with standard optics . after excitation with an ar ionic laser ( innova 90 , coherent , santa clara , calif ., usa ) adjusted to 488 nm and 200 mw , a / o intercalated into double stranded dna shows a green fluorescence ( 530 ± 30 nm ) while ao bound to single stranded dna fluoresces red (& gt ; 630 nm ). a total of 10 , 000 results were measured with each sample with a flow rate of about 200 cells / sequences . the fluorescence stability of the flow cytometer was monitored daily using standard beads ( fluoresbrite plain yg 1 . 0 μm ; polysciences inc ., warrington , pa , usa ). equivalent instrumental equipment was used for all samples . a distribution diagram analysis of the raw data was performed using cellquest version 3 . 1 ( becton dickinson ) wherein each point of the coordinate of the red - green fluorescence intensities evaluates each spermatozoon individually . the results accumulating in the left lower corner correspond to cellular fragments from the sample and were excluded form the examination ( see fig6 and 7 for the evaluation ). 1 . behne d ., duk . m ., and w . elger w . 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