WO2000010594A1 - The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives - Google Patents
The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/16—Masculine contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention is related to the use of glial cell line-derived neurotrophic factor (GDNF) family-related compounds, such as glial cell line-derived neurotrophic factor (GDNF) , GDNF-like factors and/or other compounds which act like GDNF on its receptor and/or co-receptors, for regulating and studying spermatogenesis, for inhibiting the differentiation of sperm cells, for developing male contraceptives or as a male contraceptive as well as their use for manufacturing male contraceptive compositions.
- GDNF glial cell line-derived neurotrophic factor family-related compounds, such as glial cell line-derived neurotrophic factor (GDNF) , GDNF-like factors and/or other compounds which act like GDNF on its receptor and/or co-receptors, for regulating and studying spermatogenesis, for inhibiting the differentiation of sperm cells, for developing male contraceptives or as a male contraceptive as well as their use for manufacturing male contraceptive compositions.
- GDNF
- Spermatogenesis is a complex sequence of events involving both hormonal regulators and local interactions between Sertoli and germ cells (Bellve, A. R. & Zhang, W. J., Reprod. Fertil. 85, 771-793 (1989); Skinner, M. K. , Endocr. Rev. 12, 45-72 (1991); Pescovitz, 0. H., et al . , Trends Endocrinol . Metab. 5, 126-131 (1994). Parvinen, M., et al . , J. Cell Biol . 117, 629-41 (1992), Djakiew, D., et al . , Biol. Reprod. 51, 214-21 (1994), Cancilla, B.
- the differentiation of sperm cells or spermatogenesis can be divided in three phases. In the first phase of the spermatogenesis undifferentiated stem cells from the germ line, the spermato- gonia, are divided mitotically and differentiate into sper- matocytes .
- Spermatogonia are very resistant against different types of damages and after damage spermatogenesis starts from said cells. At present, it is not known by which mechanism the spermatogonia are maintained and secondly by which signals they are differentiated into spermatocytes .
- Spermatogonia are located in the peripheral parts of the seminiferous tubules and are in contact with the Sertoli cells, which represent the regulating cells of the seminiferous tubules and are classified as somatic cells of the organism.
- the spermatogonia are at first differentiated into primary and thereafter into secondary spermatocytes, whereby their chromosomes become haploid by two meiotic cell divisions after which they are called spermatides.
- the spermatides mature into spermazoa or mature sperm cells.
- the luteinizing hormone, the follicle stimulating hormone and testosterone are the most important hormonal regulators of spermatogenesis.
- the cells of the germ line in the seminiferous tubules of testis are not direct targets of the hormonal regulation but the hormonal regulation is transmitted by the Sertoli cells. They modulate the hormonal signals into paracrine signals which are believed to be signal molecules and growth factors, such as stem cell factor (c-kit ligand) , insulin like growth factor- 1 and three members of the transforming growth factor- ⁇ family (transforming growth factor- ⁇ , activin and inhibin) (Bellve A. R. & Zhang . J., Reprod. Fertil. 85, 771-793 (1989); Skinner, . K. , Endocr. Rev.
- the spermatogenetic cells can also send the signals back to the Sertoli cells (Bellve, A. R. & Zhang W. J., Reprod. Fertil. 85, 771-793 (1989); Skinner, M. K. , Endocr. Rev. 12, 45-72 (1991); Pescovitz, 0. H., et al . , Trends Endocrinol . Metab. 5, 126-131 (1994)).
- This is indicated for example by the fact that nerve growth factor and fibroblast growth factor- 1 are expressed in the cells of the germ line and the p75 nerve growth factor receptor and fibroblast growth factor receptor are located in the Sertoli cells. Hints of auto- crine regulation of spermatogenesis also exist.
- Bone morphoge- netic protein 8a and 8b and their receptors are expressed in the same spermatogenetic cells (Zhao, G. Q., et al . , Gene Dev . 10, 1657-1669 (1996); Zhao, G. Q., et al . , Dev. 125, 1103-1112 (1998)) .
- the functional evidence for the autocrine regulation is still lacking.
- GDNF glial cell line-derived neurotrophic factor
- the glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor- ⁇ superfamily (Lin, L. F., et al., Science 260, 1130-1132 (1993)).
- GDNF maintains the dopaminergic, noradrenergic, cholinergic and motor neurons in the nervous system and also protects peripheral parasympa- thetic ciliary and sensory nerve cells (Lin, L.F., Neural Notes. 2, 3-7, (1996).
- GDNF is expressed also in other tissues, such as embryonic kidney and testis (Suvanto, P., et al .
- mice European Journal of Neuro- science 8, 816-822, (1996); Trupp M, et al . , Journal of Cell Biology 130, 137-148, (1996)).
- the role of GDNF as a regulator of the differentiation of the kidney has been shown with gene knockout or gene deletion techniques in mice (Pichel, J.G., et al., Nature 382, 73-75 (1996). Because said mice die as newborn, it has not been possible to study with said deletion gene model - or other experimental models - the function of GDNF in spermatogenesis, which is initiated much later, only after birth. Studies with different model systems indicate that there is a multitude of ways for interfering with spermatogenesis and for developing male contraceptives .
- the problem yet to be solved is how to select from the multitude of hormonal growth regulators one which can sufficiently specifically be targeted to the testis and at the same time provides a system for regulating spermatogenesis without disturbing side-effects and which system does not interfere negatively with the male mating activity.
- mice with human GDNF were capable of mating but not capable of fertilizing the female.
- the solution to the problem is to use glial cell line-derived neurotrophic factor family-related compounds as defined in the claims of the present invention for regulating spermatogenesis and inhibiting or preventing spermatogenesis followed by infertility and as active ingredients for manufacturing compositions useful as male contraceptives, which are both effective and without severe side-effects .
- the first objective of the present invention is to provide an effective male contraceptive without severe, undesired side-effects by using GDNF-family-related compounds as male contraceptives.
- Another objective of the present invention is to use the GDNF-family-related compounds as active ingredients for manufacturing compositions useful as male contraceptives .
- One other objective of the present invention is to provide tools for regulating and studying spermatogenesis. This is facilitated by inhibiting the differentiation of sperm cells.
- a third objective of the present invention is to provide tools, which have an effect on infertility caused by disturbed differentiation of sperm cells.
- the objective of the present invention is also to provide tools for regulating and studying spermatogenesis as well as for inhibiting the differentiation of sperm cells.
- the objective of the present invention is to provide transgenic animals, especially mice, which due to the testis targeted GDNF-family-related compounds provide a useful tool for studying spermatogenesis.
- the characterstics of the present invention are as defined in the claims.
- the present invention describes a method, whereby it is possible to study sterility caused by disturbed differentiation of sperm cells and secondly it provides a system for inhibiting the differentiation of sperm cells in a contraceptive sense, whereby the male (man) is sterile (infertile) .
- the GDNF-transgene carrying transgenic mouse strain, the GDNF t-mouse strain is a model for infertility caused by disturbances in the differentiation of sperm cells.
- GDNF compounds acting like, said GDNF, another cRet receptor, another GDNF-receptor activating compound or a compound, which activates the cRet receptor signal transmitting tubules in spermatogonia
- the GDNF t-mouse model is suitable as an animal model for studying the regulation of spermatogenesis and spermatogenesis.
- Figure la Testicular morphology in wild-type mice at 3 -weeks of age. Scale bar 100 ⁇ m.
- Figure lb Testicular morphology in transgenic mice at 3 -weeks of age. Scale bar 100 ⁇ m. Note the undifferentiated spermatogonia formed cell cluster.
- Figure lc Testicular morphology in wild-type mice at 8 -weeks of age. Scale bar 100 ⁇ m.
- Figure Id Testicular morphology in transgenic mice at 8 -weeks of age. Note in d the remnants of type A spermatogonia clusters that are abundant in the young mice. Scale bar 100 ⁇ m.
- Figure le Testicular morphology in wild-type mice at 6-months of age. Scale bar 100 ⁇ m.
- Figure If Testicular morphology in transgenic mice at 6- months of age. Note the advanced germ cell atrophy and Leydig cell hyperplasia (star) . Scale bar 100 ⁇ m.
- Figure lh Testicular morphology in transgenic epididymal ducts at 8-weeks. Note the lack of sperm. Scale bar 100m.
- FIG. 2a Northern blotting for GDNF RNAs in wild-type mice at various ages . GDNF levels are strongly downregulated after the first or second postnatal week.
- FIG. 2b Northern blotting for Ret mRNAs in wild-type mice testes at various ages. GDNF mRNA levels are strongly downregulated after the first or second postnatal week.
- Figure 2c Northern blotting for GFRcel mRNAs in wild-type mice at various ages. GFR ⁇ l mR ⁇ A levels are strongly downregulated after the first or second postnatal week.
- Figure 2d Northern blotting for GDNF mRNAs in transgenic testes at various ages. The hGDNF levels remain high after the first or second postnatal week.
- Figure 2e Northern blotting for Ret mRNAs in transgenic mouse testis at various ages.
- FIG. 2f Northern blotting for GFR ⁇ l mRNAs in transgenic testes at various ages. GFR ⁇ l RNA levels are high in the transgenic mice at all stages analysed.
- Figure 2g depicts immunoprecipitation of GDNF from wild-type (WT) and transgenic (TG) testes at 3 and 6 weeks of age. In wild-type testes, the GDNF protein is not detectable. The control, 25 ng of reco binant human GDNF protein is shown on the right side.
- FIG. 3a The distribution of GDNF transcripts in testes of wild-type (WT) mice by cRNA in si tu hybridisation. Sense controls did not show grain density above the background. 1- week-old wild-type testis. Scale bar 100 ⁇ m.
- FIG. 3b The distribution of GDNF transcripts in 8 -weeks-old testis of wild-type (WT) mouse by cRNA in si tu hybridisation. Sense controls did not show grain density above the background. Scale bar 100 ⁇ m.
- FIG. 3c The distribution of GDNF transcripts in testes of transgenic mice (TG) by cRNA in si tu hybridisation. Sense controls did not show grain density above the background. 8-weeks-old transgenic testis. Note that the spermatogonia in the transgenic mice continuously express human GDNF at high levels, but the number of these cells is clearly diminishing at 8 weeks of age. Scale bar 100 ⁇ m.
- FIG. 3d The distribution of Ret transcripts in testes of wild-type (WT) mice by cRNA in situ hybridisation. 2-weeks-old wild-type testis. At two weeks of age, Ret expression is localised to some spermatogonia in the basal layer of the seminiferous tubules (arrows) .
- FIG. 3e The distribution of Ret transcripts in testes of wild-type (WT) mice by cRNA in si tu hybridisation. 8 -weeks-old wild-type testis. At 8 weeks of age, Ret is expressed in both some spermatogonia and spermatides.
- FIG. 3f The distribution of Ret transcripts in testes of transgenic mice (TG) by cRNA in si tu hybridisation. 8 -weeks- old transgenic testis. Note that the spermatogonia in the transgenic mice continuously express Ret at high levels, but the number of these cells is clearly diminishing at 8 weeks of age. In transgenic mice, Ret is expressed in the clusters of spermatogonia. Scale bar 100 ⁇ m.
- FIG. 3g The distribution of GFR ⁇ l transcripts in testes of wild-type mice (WT) . 1- to 2-weeks-old wild-type testis. GFR ⁇ l is expressed by some spermatogonia, morphologically similar to those with Ret (arrows) .
- FIG. 3h The distribution of GFR ⁇ l transcripts in testes of wild-type by cRNA in si tu hybridisation. 8 -weeks-old wild- type testis. GFR ⁇ l mRNA distribution follows that of Ret, but is also highly expressed by spermatocytes. Scale bar 100 ⁇ m.
- FIG. 3i The distribution of GFR ⁇ l transcripts in testes of transgenic mice (TG) by cRNA in situ hybridisation. 8-weeks- old transgenic testis. Note that the spermatogonia in the transgenic mice continuously express human GDNF at high levels, but the number of these cells is clearly diminishing at 8 weeks of age. In transgenic mice, GFR ⁇ l is expressed in the clusters of spermatogonia. Scale bar 100 ⁇ m.
- Figure 4a Cell proliferation in wild-type mice testes. In 3 -weeks-old testis, BrdU labelling of S-phase cells is seen only in the periphery of wild-type seminiferous tubules. Note the absence of labelling in some transsections of tubules, reflecting the segmented distribution of S-phase cells during spermatogenesis (stars) . Scale bar 100 ⁇ m.
- Figure 4b Cell proliferation in transgenic mice testes.
- many BrdU-labelled cells are seen in the luminal area of the seminiferous tubules, reflecting the abnormal distribution of spermatogonia (arrow) .
- the segmental distribution of S-phase cells is not seen in the transgenic tubules.
- Scale bar 100 ⁇ m.
- Figure 4c Distribution of cell line proliferation indexes in wild type and transgenic testes. The number of BrdU labeled cells / 100 spermatogonia was counted in 100 transections of wild type and transgenic seminiferous tubules.
- FIG. 4d Apoptosis in wild-type mice testes. TUNEL- labelling for apoptosis in 4-week-old mice testes. Only a few apoptotic cells are seen. Scale bar (c) 100 ⁇ m.
- Figure 4e Apoptosis in transgenic mouse testis. TUNEL-label- ling for apoptosis in 4-week-old testes. Note the increase of labelling in spermatogonia clusters in ( Figure 4e) , as compared to wild-type ( Figure 4d) seminiferous tubules. Scale bar 100 ⁇ m.
- FIG. 4f Live morphology in both a wild-type and transgenic seminiferous tubule. Live preparation of seminiferous tubules from 15-week-old wild-type (WT) and transgenic (TG) mice. Note the reduced diameter of the transgenic tubule and the increase of Leydig cells (arrows) around it. Scale bar 200 ⁇ m.
- FIG. 4g Live morphology in transgenic testes. High magnification of living cells from a transgenic seminiferous tubule in an unfixed squash preparation showing large numbers of dead cells (arrow) , some of which resemble type A spermatogonia, in a Sertoli cell (arrow head) . Scale bar 10 ⁇ m.
- GDNF glial cell line-derived neurotrophic factor family-related compounds
- GDNF-family-related compounds comprises glial cell line-derived neurotrophic factor (GDNF) , neurturin, persephin, artemin and other similar growth factors, but also other compounds which have the same effect as GDNF-like compounds on the GDNF-receptor (s) or co-receptors.
- Said receptor (s) or co-receptor (s) comprises Ret tyrosine kinase and GDNF-family-receptor a : s , respectively.
- glial cell line-derived neurotrophic factor means the glial cell line-derived neurotrophic factor (GDNF) , which is a member of the transforming growth factor- ⁇ family (Lin, L. F., et al . , Science 260, 1130-1132 (1993).
- GDNF maintains the dopaminergic , noradrenergic, cholinergic and motor neurons in the nervous system and also protects peripheral, parasympathetic, ciliary and sensory nerve cells (Lin, L.F., Neural Notes. 2, 3-7 (1996)).
- GDNF is expressed also in other tissues, such as embryonic kidney and testis (Suvanto P, et al .
- GDNF-like factors or GDNF-like compounds mean growth factors which have a similar structure or act like GDNF on its receptor or co-receptors.
- GDNF-like factor or "GDNF-like compound” above all relates to three GDNF-like growth factors, persephin, artemin and neurturin, which are described in (Milbrandt, J., et al . , Neuron 20, 1-20 (1998); Kotzbauer P. T., et al . , Nature 384, 467-470 (1996)).
- GDNF-family All four growth factors of the GDNF-family are structurally similar and cRet receptor tyrosine kinase (Durbec, P., et al., Nature 381, 789-793 (1996); Buj-Bello, A., et al . , Nature. 387, 721-724, (1997)) acts as the common signal transmitting receptor of GDNF, artemin, persephin and neurturin.
- the "compounds which act like said GDNF” means compounds which act as GDNF on the receptor transmitting signals of GDNF or co-receptors thereof.
- receptor means GDNF-family binding substances, i.e. substances or compounds belonging to the GDNF-family-related compounds which are capable of transmitting the signals of GDNF-family-related compounds.
- receptor above all means the GDNF-family signal mediatory receptor is cRet receptor tyrosine kinase.
- co-receptors means receptors, which do not transmit the signals of GDNF-family related compounds, but activate the receptor transmitting the signals of the GDNF-family-related compounds.
- Such compounds are above all the GDNF-family receptor a : s (GFRcn) , which is a new receptor class, the members of which are called GDNF-family-receptor c.s (GFRc . These also belong to the GDNF, persephin, artemin and neur- turin signalling receptor complex.
- GFR ⁇ l-4 Jing, S., et al . , Cell 85, 9-10 (1996), Jing, S. Q., et al., J. Biol.
- Chem. 272, 33111-3311 7 (1997); Treanor, J. J., et al., Nature. 382, 80-83, (1996); Suvanto, P., et al., Human Molecular Genetics. 6, 1267-1273, (1997)) of said family are known. They may transmit signals independently, but they are essential for ligand binding and cRet activation.
- concentration of GDNF-family-related compounds in such amounts that the differentiation of sperm cells is prevented, spermatogenesis can be regulated and infertility caused by disturbed differentiation of sperm cells or that the male is rendered unable to fertilize the female means a concentration of GDNF-family-related compounds, which is approximately 50 - 5000, preferably 50 - 500, most preferably 50 - 100 times higher than the concentration of endogenous GDNF in the testis of said male. Said amount can be calculated from the test results obtained when measuring the GDNF mRNA levels of transgenic mice shown in the Northern blots ( Figures 2) and which indicate an elevated amount of mRNA from which the expressed protein levels can be calculated.
- the amount of GD ⁇ F in transgenic mice is 50-500 times higher than the endogenic level in three to six week old mice, but only a little bit higher than in new born mice, when the GD ⁇ F levels are at their highest and no spermatogenesis is presented. From these results it is possible for those skilled in the art to calculate the amounts needed for obtaining the desired effects in man.
- derivatives means iso ers of GD ⁇ F-family-related compounds . It also includes hybrid molecules constructed based on the structures of the compounds GD ⁇ F, persephin, neurturin and artemin, as well as truncated forms, complexes and fragments of said compounds. The only prerequisite for said compounds is that they still have substantially the same properties and effects as the compounds listed above, determined by the same methods as described in the present inven- tion. Naturally, the term “derivatives” includes derivatives of the compounds listed above in a more conventional sense, e.g.
- amino- and/or hydroxy- groups may be substituted by alkylation, esterification, etherification and amidation with e.g. alkyl groups, including methyl, ethyl, propyl, etc.
- alkyl groups including methyl, ethyl, propyl, etc.
- Glial cell line-derived neurotrophic factor is a potent survival factor for various sets of neuronal cells, it regulates kidney differentiation (Lin, L. F., et al . , Science 260, 1130-1132 (1993); Tomac, A., et al . , Nature 373, 335-339 (1995); Winkler, C, et al . , J. Neurosci . 16, 7206-7215 (1996), Oppenheim, R. W. , et al . , Nature 373, 344-346 (1995), Nguyen, Q. T., et al .
- GDNF Glial cell line-derived neurotrophic factor
- GDNF a distant member of the transforming growth factor- ⁇ (TGF- ⁇ ) superfamily (Lin, L. F., et al . , Science 260, 1130- 1132 (1993)) is a potent survival factor for dopaminergic neurones of the substantia nigra, and some other sets of neurones in the central and peripheral nervous systems (Lin, L. F., et al., Science 260, 1130-1132 (1993); Tomac, A., et al., Nature 373, 335-339 (1995); Winkler, C, et al . , J. Neurosci. 16, 7206-7215 (1996), Oppenheim, R. W., et al .
- TGF- ⁇ transforming growth factor- ⁇
- GNDF also acts as a signalling molecule in ureteric branching during kidney morphogenesis (Schuchardt, A., et al . , Nature 367, 380-383 (1994); Pichel, J. G., et al . , Nature 382, 73-75 (1996)) . It is synthesised by several neuronal and non-neu- ronal embryonic organs including the embryonic and postnatal testis (Trupp, M. , et al . , J. Cell Biol. 130, 137-148 (1995); Hellmich, H. L., et al . , Mech. Dev. 54, 95-105 (1996); Suvanto, P., et al .
- the signalling receptor-complex for GDNF includes Ret receptor tyrosine kinase (Durbec, P., et al . , Nature 381, 789-793 (1996); Trupp, M., et al . , Nature 381, 785-789 (1996)) and a member of the glycosylphosphatidylinositol- linked co-receptors, GDNF-family-receptor a : s (GFRo.1-4) (Jing, S., et al., Cell 85, 9-10 (1996); Jing, S. Q., et al . , J. Biol. Chem.
- mice exhibit remarkably similar phenotypes with absent or hypodysplastic kidneys and severe defects in enteric innervation (Schuchardt, A., et al . , Nature 367, 380-383 (1994); Pichel, J. G., et al., Nature 382, 73-75 (1996)). As they die during the first postnatal day, spermatogenesis could not be analysed in these mutant mice.
- the present inventors studied transgenic mice with targeted overexpression of GDNF in testicular germ cells.
- the transgenic males were infertile and showed an early arrest in the differentiation of spermatogenic cells, the type A spermatogonia. They formed clusters within seminiferous tubules, finally resulting in testicular atrophy.
- the data obtained showed a novel function for GDNF as a paracrine inhibitory signal during early spermatogenesis.
- GDNF is believed to regulate the pool of spermatogonia that is gradually committed to sperm differentiation.
- the activation of the GDNF signalling cascade possibly provides us with means to develop contraceptives for man.
- the effects of GDNF on spermatogenesis should also be taken into consideration, when GDNF-like molecules are used in clinical trials of neurodegenerative disorders, such as Parkinson's disease.
- GDNF targeting to testis provides a new animal model for male infertility.
- the activation of the GDNF signalling cascade by GDNF, Ret agonists or downstream substrates possibly provides us with means whereby sperm production in man could be prevented and male contraceptives developed.
- the effects of GDNF on spermatogenesis should also be taken into consideration, when GDNF-like molecules are designed for and used in clinical trials of neurodegenerative disorders, such as Parkinson's disease .
- transgenic mice overexpressing human GDNF under human translation elongation factor-1 (EF-1) promoter (Mizushima, S. & Nagata, S., Nucleic Acids Res. 18, 5322 (1990)), which targets the transgene expression specifically to testicular germ cells (Furuchi, T., et al . , Development 122, 1703-1709 (1996) ) .
- EF-1 human translation elongation factor-1
- Other promoters targetting the transgene expression specifically to testicular germ cells can be used as well.
- Four independent transgenic founders were analysed, two males CIO, C12 and two females S6, E19 with transgene copy numbers 10, 3, 20, and 4, respectively.
- the male founders CIO and C12 were infertile and further analysis of the male phenotype was done with the offspring of the two female founders S6 and E19.
- the transgenic mice developed normally to adulthood and showed a similar phenotype. The body weight was normal, and they did not have any defects in the weights and morphology of paren- chymal organs (data not shown), except in the testis. After four weeks of age, a reduction in weight of testes was observed in the transgenic mice as compared ' to wild-type controls (Table 1) . All transgenic males in the offspring of the female founders S6 and E19 were infertile.
- transgenic males were caged with wild-type FVB and NMRI females in three to four day intervals.
- the wild-type females mated with transgenic males at normal frequency, as shown by the appearance of vaginal plugs, but none became pregnant.
- 20 transgenic males at breeding age were subjected to wild-type FVB and NMRI females in short term breeding test.
- approximately 200 females from both strains were plugged, but none became pregnant.
- Strain dependence of the infertile phenotype was excluded by crossbreeding the transgenic FVB mice to NMRI background.
- those of the FI generation were also infertile.
- testicular histology of one-week-old transgenic mice was normal . From two to three weeks of age (Fig. la, lb) , they showed clusters of cells morphologically similar to type A spermatogonia, partially detached from the basal lamina of the seminiferous tubules. These cells underwent gradual degeneration during subsequent weeks so that by ten weeks the tubules showed already advanced atrophy with Sertoli cells and only spermatogonia (Fig. lc-lf) . The Leydig cells, the testicular interstitial cells, showed progressive hyperplasia from the fourth postnatal week (Fig. Id, If) .
- GDNF mouse and human GDNF and its receptor mRNAs were elevated in the transgenic mice as determined by Northern blotting in wild-type and transgenic testes at various ages (Fig. 2) .
- GDNF, Ret and GFR ⁇ l transcripts were highly expressed at birth and during the first postnatal week (Fig.2a-c) .
- GDNF mRNA was strongly downregulated shortly before the initiation of puberty (at two weeks of age) (Fig2a) .
- the Ret the Ret
- NTN Neurturin
- GFR ⁇ 2 GFR ⁇ 2
- Both NTN and GFR ⁇ 2 transcripts are expressed by postnatal testes (Widenfalk, J. , et al., J. Neurosci. 17, 8506-8519 (1997)), however preferentially after puberty.
- the third member of GDNF-family, persephin Movator-like substance
- the transgenic testes showed a continuous and high expression of the human GDNF mRNA by type A spermatogonia clusters from two weeks of age to adulthood (Fig. 3c) .
- Ret mRNA was expressed by both spermatogonia and spermatids in wild-type testes (Fig. 3d, 3e) and in the clusters of spermatogonia in transgenic testes (Fig. 3f) .
- GFR ⁇ l mRNA was expressed by spermatogonia, spermatocytes and round spermatids in wild-type testes (Fig. 3g, 3h) and also intensively by the spermatogonia clusters in transgenic testes (Fig. 3i) .
- the signalling receptor complex for GD ⁇ F is present in the spermatogonia of both wild-type and transgenic mice.
- GD ⁇ F is obviously acting as a paracrine Sertoli-cell-derived regulator of spermatogenesis, but in transgenic mice this regulation is disrupted by the continuous autocrine expression of G ⁇ DF by germ cells.
- spermatogonia are the only proliferating germ cells in testis and the mitotic activity is highly segmented in normal semini- ferous tubules (Fig. 4a) .
- the BrdU- labelled spermatogonia showed an abnormal distribution within the seminiferous tubules, reflecting the spermatogonia clusters in the lumina of seminiferous tubules (Fig. 4b) . Because of the highly segmental nature of cell proliferation in wild-type testis, it is difficult to compare the net proliferation rates of wild-type and transgenic testes.
- Sertoli cells Fig. 4f, 4g
- increased apoptosis overriding cell proliferation may explain the gradual testicular atrophy in the GDNF overexpressing mice.
- the accumulation of spermatogonia in young mice seems to be caused by the block of their differentiation and not enhanced cell proliferation.
- mice or rats are sterile in the absence of the vitamin A metabolite, retinoic acid (RA) , as shown by feeding them with vitamin A deficient (VAD) diet.
- VAD vitamin A deficient
- Differentiation of type A spermatogonia is completely blocked by VAD diet and restored by reintroduction of vitamin A or its active metabolites (Kim, K.H., et al., Molec . Endocrinology 4: 1679-1688, 1990).
- Retinoic acid receptor a (RAR ⁇ ) mRNA and protein levels decline upon a VAD diet (Akmal, K. , et al . , Endocrinology 139, 1239-1248, 1998).
- RAR ⁇ -deficient transgenic mice are infertile and develop testicular atrophy (Lufkin, T., et al., Devel . Biol 90, 7225-7229, 1993). Therefore, the expression of RAR proteins was analysed in wild type and GD ⁇ F overexpressing transgenic mice. The RAR expression practically disappeared in the spermatogonial clusters of the transgenic mice, as show by indirect immunoperoxidase staining with antibodies to RAR proteins. The data indicate that one of the mechanisms how GD ⁇ F acts as a negative regulator of spermatogonia is downregulation of RAR expression in Ret expressing spermatogonia .
- transgenic mice overexpressing neurturin in testis To analyse whether the other members of the GD ⁇ F family would act as negative regulators of spermatogenesis, the investigators constructed transgenic mice overexpressing neurturin in testis. Indeed, the transgenic mice expressing neurturin under the translation elongation factor l ⁇ promoter that targets the transgene expression specifically to testis show also infertility during the first four postpubertal weeks as described by mating tests identical to those with the GD ⁇ F overexpressing mice .
- sperm differentiation in mammals can be divided into three distinct phases that are regulated by both local cellular interactions and hormonal control (Russell, L. D., et al . , Histological and histopathological evaluation of the testes. Clearwater, F. L., Cache River, pp. 1-40 (1990)).
- the first phase of spermatogenesis is the mitosis phase, when the spermatogonia give rise to the spermatocytes . Beyond the spermatogonial stage, spermatogenesis proceeds without mitotic cell divisions.
- Spermatogonia are further classified to subtypes, in which the type A spermatogonia subtypes include the spermatogenic stem cells.
- the second phase is the meiosis phase of spermatocytes resulting in haploid spermatids .
- the third phase is the spermiogenic phase, in which the spermatids transform into spermatozoa, the sperm cells, structurally equipped to reach and fertilise the egg.
- type A spermatogonia proliferate and are triggered to differentiate. However, some of them remain undifferentiated and are considered as the reservoir of stem cells (Russell, L. D., et al . , Histological and histopathological evaluation of the testes. Clearwater, F. L., Cache River, pp. 1-40 (1990)).
- Sertoli and germ cells may involve a number of molecules, such as stem cell factor (c-kit ligand) , insulin-like growth factor- I, three members of the TGF- ⁇ family (M ⁇ llerian inhibiting substance, activin, and inhibin) , and transferrin (Pescovitz, 0. H. , et al . , Trends Endocrinol . Metab. 5, 126-131 (1994) ) .
- Sertoli cells secrete all these molecules and germ cells possess their receptors. Conversely, Sertoli cells express receptors for ligands secreted by germ cells.
- Sertoli cells for instance express p75 nerve growth factor receptor and fibroblast growth factor receptors, whose cognate ligands are secreted by testicular germ cells (Pescovitz, 0. H., et al., Trends Endocrinol. Metab. 5, 126-131 (1994); Parvinen, M., et al., J. Cell Biol. 117, 629-41 (1992); Djakiew, D., et al., Biol. Reprod. 51, 214-21 (1994)). Furthermore, Sertoli cells also interact with Leydig cells. Desert hedgehog (Dhh) gene is expressed by Sertoli cells and its putative receptor, patched, predominantly by Leydig cells (Bitgood, M. J.
- the human GDNF cDNA was cloned to the p ⁇ F-BOS vector with the EFl ⁇ -promoter (Migushi a, S. and Nagaca, S., Nucleic Acid Res. 18, 5322 (1990)) by replacing Xbal-Xbal stuffer fragment with entire coding sequence of human GDNF cDNA (GenBank accession number L15306) .
- the correctness of the expression construct was verified by sequencing. Proper expression GDNF protein with the construct was verified by Cos cell transfection followed by i munoprecipitation of GDNF from lysate and culture supernatant.
- Transgenic mice were produced by micro- injecting the 2.7kb Pvul-Hindlll fragment of the GDNF cDNA construct into the pronuclei of newly fertilised FVB inbred mouse eggs as described (Hogan, B., et al . , Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1994) ) .
- the founders were identified by Southern blotting with a human GDNF cDNA and the offspring of the transgene positive mice by PCR from tail DNA.
- Primers for human GDNF were from exon 1 (5' ⁇ TGT CGT GGC TGT CTG CCT GGT GC-3') and exon 2 (5'-AAG GCG ATG GGT CTG CAA CAT GCC-3 ' ) . Histology, cell proliferation and apoptosis. For histology, freshly dissected testes and epididymides were fixed in Bouins fixative or 4% PFA for 2-24 hours depending on the size, processed in paraffin, cut at 7 ⁇ m and stained by hemato- xylin/eosin.
- mice were injected intraperitoneally with a BrdU and 5-fluoro2 -deoxy- uridine (FrdU) cocktail (Amersham) at a concentration of 10 mg/kg in PBS. After 2 hr, the mice were sacrificed by neck dislocation, autopsy was done, testes were removed and fixed in Bouin solution. Testes were processed in paraffin, 7 ⁇ m sections were cut and depara finised. BrdU incorporation was detected by indirect immunofluorescence with monoclonal antibodies to anti-bromodeoxyuridine (Amersham) and rhoda- mine-conjugated goat anti-mouse IgG (Jackson) . Apoptotic cells were detected in deparaffinised sections with ApopTag in situ cell death detection kit (Oncor) according to manuf cturer's instructions .
- RhdU 5-fluoro2 -deoxy- uridine
- RNA of different tissues were isolated with TRIZOL Reagent kit (Life Technology) according to the manufacturer's instructions and 30g of total RNA was used in each lane.
- Northern blotting was carried out according to Maniatis protocol (Sambrook, J. , et al . , T. Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press 1989 pp. 7.43-7.50).
- the cDNA probes (the same as in situ hybridisation, see below) were labelled with [ 32 P]dCTP.
- the filters were exposed in a FujiBAS phospho- i ager . Im unoprecipitation and Western blotting with anti-GDNF antibody.
- Freshly dissected testes were homogenized and lysed in high salt lysis buffer (300 mg protein/ml; high salt lysis buffer: 1M NaCl, 100 mM Tris HCl, pH 8 , 2 % BSA, 4 mM EDTA, 0.2 % Triton X-100, 2 mM PMSF, 1 mM sodium orthovanadate and 1 tabl. /10 ml of protease inhibitor cocktail tablets (Complete, Mini EDTA, Boehringer Mannheim) ) .
- the lysates were immuno- precipitated with a polyclonal antibody to human recombinant GDNF peptide that crossreacts with mouse GDNF (R & D Systems) and Protein A-Sepharose.
- the immunoprecipitates were run on a 20 % SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Life Science) .
- the membranes were blocked with 5 % bovine serum albumin and immunoprecipitated GDNF was detected with a polyclonal anti-GDNF antibody (Santa Cruz Biotechnology) at room temperature for 2 h.
- Detection was accomplished by using anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma) and ECL chemilumine- scence (Amersham) according to the manufacturer's instructions
- In situ hybridization In situ hybridisation was performed as described (Wilkinson, D. & Green, P., In situ hybridization and the three-dimensional reconstruction of serial sections. In: Postimplantation Mammalian Embryos. A practical approach (ed. A. Copp & D. Cockroft) , pp. 155-171. Oxford University Press: London (1990)) .
- Antisense and sense cRNA probes (mouse GDNF 328 bp from exon 3, human GDNF 513 bp from exon 1 and exon 2, mouse Ret from 3 end 714 bp, mouse GFR ⁇ l 777 bp) were synthesized using appropriate RNA polymerases and 35g_]_ a b )e- L-L e ci UTP.
- Hybridisation temperature was 52°C and autoradiography slides were exposed at +4°C for 2-4 weeks. The slides were photographed with CCR camera hooked with computer. In PhotoShop graphics programme, the dark field images were inverted, artificially stained red and combined with the bright field images .
- Example 1 Example 1
- GDNF human GDNF
- the 2.7 kb HindiII -EcoRI fragment isolated from the pEF-BOS vector contains EFl ⁇ (translation elongation factor l ⁇ ) promoter and the 0.7 kb Xba-EcoRI fragment having a ply (A) adenylation signal.
- the size of the entire coding region of human GDNF cDNA is 636 bp .
- Transgenic mice are produced by microinjecting the 2.7 kb PVuI-Hindlll fragment into the pronucleus of fertilized egg cells. The injection is at first made into a FVB/NIH-mice strain and later the transgene positive mice are mated with a FVB/NIH- and NMRI-mice strains.
- Transgenic founder mice are identified by Southern hybridization with a hGDNF-probe and the transgenic offspring is identified by polymerase chain reaction (PCR) from tail DNA (the transgene transferring mouse strain is later called GDNF t-mice) .
- Primers used in PCR are from exon 1 (5'-TGT CGT GGC TGT CTG CCT GGT GC-3') and exon 2 (5'-AAG GCG ATG GGT CTG CAA CAT GCC-3'). The results indicated that GDNF t-mice have a coding region of human GDNF cDNA and they transfer it to their offspring.
- RNA from the testis of a three week old mouse with which the expression levels of endogenic GDNF mRNA and the human transgenic GD ⁇ F R ⁇ A of a mouse is compared.
- RT-PCR reverse transcription polymerase chain reaction
- 1 ⁇ g entire R ⁇ A is copied by reverse transcrition in a random-primed reaction (20 ⁇ l) using AMV reversed copy enzyme (Finnzymes, Helsinki) according to the instructions of the manufacturer. 2 ⁇ l of the reaction product is used as template when copying the endogenic mouse GD ⁇ F cD ⁇ A or the transgenic human GD ⁇ F cD ⁇ A.
- the first primer (5 ' GCTGAG/CCAGTGACTCA/CAATATGCC3 ' ) recognizes both the mouse and human GD ⁇ F alleles and covers the intron region in order to avoid genomic contamination.
- the primers of the other end were selective to endogenic and transgenic GD ⁇ F (GD ⁇ F specific region in mouse: 5 ' TGTTAGCCTTCTACTCCGAGACAG3 ' ) and the transgenic primer (transgenic GD ⁇ F specific region 5 ' CACCAGCCTTCTATTTCTGGATAA3 ' ) .
- primers are used to copy in identical conditions (l ⁇ M primer, lmM, d ⁇ TP and Dynazyme polymerase (Finnzymes, Helsinki) in their own buffer) fragments of 373 bp length from endogenic and transgenic GD ⁇ F cD ⁇ A, which has been reversed copied either from RNA from the testis of a normal or transgenic mouse.
- the cycle is 30s 95°, 30s 62° ja 45s 72°, and it is renewed 20-36 times after a "hot starting" 95°C for 2 minutes.
- the copying is ended after the last cycle for 5 minutes in 72°C.
- the PCR product is run in an agarose gel containing ethidium bromide, is studied under UV-light and is photographed with a Polaroid-camera.
- Northern blot hybridization to identify transgenic hGDNF and endogenic mouse GDNF mRNA expression is carried out with species specific [ ⁇ - 32 P] -labelled GDNF cDNA probes from total-RNA isolated from testis with a TRIzol Reagent-kit (Life Technologies) according to the manufacturer's instructions.
- the probes are: mouse GDNF (328 bp exon 3 (NCB1 Gene Bank, accession number U37459) and human GNDF (536 bp from exon 1 ja 2, (Lin et al .
- Northern blot hybridization is carried out according to Sambrook, J. , et al., (Molecular Cloning: A Laboratory Manual. Second Edi- tion. Cold Spring Harbor Laboratory Press 1989 pp. 7.43-7.50). In each sample the total RNA used is 30 ⁇ g.
- the hybridization filter is kept on a Fuji phosphoimager plate and exposed to light overnight and the image is recorded with a FujiBAS phosphoimager.
- a actin-probe is used as a probe to control even loading of R ⁇ A, which is hybridized on each filter after the hybridization of the G ⁇ DF-probe.
- GDNF-transgene Due to the testis targeting elongation factor l ⁇ promoter the GDNF-transgene is not expressed in the male elsewhere than in testis (Furuchi, T., et al . , Development 122, 1703-1709 (1996) ) , which is studied with Northern blot hybridization from different female and male tissues. Also, cRNA in si tu hybridization is carried out from testis, ovary and kidney according to Wilkinson, D. & Green, P., (Tn situ hybridisation and the three-dimensional reconstruction of serial sections. In: Postimplantation Mammalian Embryos. A practical approach (ed. A. Copp & D. Cockroft) , pp. 155-171. Oxford University Press: London (1990)).
- the probes are: mouse GDNF-probe (328 bp) , human GDNF-probe (536 bp) , GFR ⁇ l-probe (777 bp) and GFR ⁇ 2-probe (approximately 500 bp) .
- mouse GDNF-probe 328 bp
- human GDNF-probe 536 bp
- GFR ⁇ l-probe 777 bp
- GFR ⁇ 2-probe approximately 500 bp
- mice GDNF-receptor Ret is expressed in prepubertal mice spermatogonia and in adult mice also in spermatides, but not any more in differentiated spermatozoa and the co-receptors of GFR ⁇ l and GFR ⁇ 2 are expressed during spermatogenesis also in germ line cells ( Figure 3) .
- transgenic GDNF The morphological results of the expression of transgenic GDNF is followed by histological studies of the testis. Normal and transgenic mouse tissues are fixed with 4 % paraformaldehyde overnight, processed in paraffin, cut in 4-7 ⁇ m sections, deparaffinized and stained with hematoxylineosin- stain.
- GDNF can prevent the formation of sperm cells
- the fertilization ability of male GDNF t-mouse strain was studied by allowing them to mate with fertile, more than 8 week old females. 20 males (more than six weeks old) were kept with females for two weeks. Calculated together they mated with 200 females. Additionally, three males (from the age of eight weeks) were kept half a year with females so that the females were changed with three to four days intervals . They were mating as often as normal mice. After mating the uterus and egg leaders of some females were studied in order to find sperm cells.
- Figure 1 Testicular morphology in wild-type (left panel: a,c,e) and transgenic (right panel: b,d,f) mice at three different ages, and wild-type (g) and transgenic (h) epididymal ducts at 8-weeks. (a and b) 3 -weeks of age. (c and d) 8 weeks, (e and f) 6 months. Note in d the remnants of type A spermatogonia clusters that are abundant in the young mice (d) , and the advanced germ cell atrophy and Leydig cell hyperplasia (star) in f. Epididymal ducts from a wild-type (g) and transgenic (h) mouse show the lack of sperm in the transgenic mice. Scale bar 100 ⁇ m.
- FIG. 1 Northern blotting for GDNF (a,d), Ret (b,e) and GFR ⁇ l (c,f) mRNAs in wild-type (left panel) and transgenic testes (right panel) at various ages.
- Mouse GD ⁇ F cD ⁇ A and human GD ⁇ F cD ⁇ A probes were used in a and d, respectively.
- GD ⁇ F (a) , Ret (b) , and GFR ⁇ l (c) R ⁇ A levels are strongly downregulated after the first or second postnatal week.
- the levels of human GD ⁇ F mR ⁇ A transgene (d) remain high to adulthood.
- Figure 3 The distribution of GD ⁇ F (a,b,c), Ret (d,e,f) and GFR ⁇ l (g,h,i) transcripts in testes of wild-type (WT, left and middle panel) and transgenic mice (TG, right panel) by cR ⁇ A in situ hybridisation.
- (a,b) mouse GD ⁇ F cD ⁇ A template and, in (c) , human GD ⁇ F cD ⁇ A template were used. All other probes were to mouse transcripts. Sense controls did not show grain density above the background.
- (a,d,g) 1- to 2-weeks-old wild-type testes.
- GFR ⁇ l mRNA distribution follows that of Ret, but is also highly expressed by spermatocytes. In transgenic mice, both Ret (f) and GFR ⁇ l (i) are expressed in the clusters of spermatogonia. Scale bar 100 ⁇ m.
- Figure 4. Cell proliferation (a,b,c), apoptosis (d,e), and live morphology (f,g) in wild-type (a,d) and transgenic (b,e,g) testes.
- (f) depicts both a wild-type and transgenic seminiferous tubule
- Testis weights (average + SD) in wild-type and transgenic mice at various ages
- n the number of testes in each group
Abstract
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EEP200100103A EE200100103A (en) | 1998-08-21 | 1999-08-20 | Use of compounds related to the glial cell line neurotrophic factor family for the regulation of spermatogenesis and for the preparation of contraceptives for use in males |
NZ509592A NZ509592A (en) | 1998-08-21 | 1999-08-20 | The use of GLIAL cell line-derived neurotrophic factor familty-related compounds for regulating spermatogenesis and for preparing male contraceptives |
PL99346298A PL346298A1 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
CA002338644A CA2338644A1 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
IL14103799A IL141037A0 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family related compounds for regulating spermatogenesis and for preparing male contraceptives |
EP99939468A EP1105151A1 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
JP2000565914A JP2002523374A (en) | 1998-08-21 | 1999-08-20 | Use of glial cell line-induced neurotrophic factor family related compounds to modulate spermatogenesis and produce male contraceptives |
HU0103419A HUP0103419A3 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
AU53752/99A AU763678B2 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
SK247-2001A SK2472001A3 (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
KR1020017002234A KR20010088793A (en) | 1998-08-21 | 1999-08-20 | The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives |
NO20010859A NO20010859L (en) | 1998-08-21 | 2001-02-20 | The use of glial cell line-derived neurotrophic factor family-related compounds to regulate spermatogenesis and to produce contraceptives for men |
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WO2001062273A1 (en) * | 2000-02-22 | 2001-08-30 | Hannu Sariola | The use of gdnf family-related compounds for manufacturing products for treating testicular tumors |
CZ303358B6 (en) * | 1998-07-06 | 2012-08-15 | Nsgene A/S | Neurotrophic polypeptide, process for its preparation and use, encoding polynucleotide, corresponding vector and host cell, synthetic gene and antibody |
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WO2001062273A1 (en) * | 2000-02-22 | 2001-08-30 | Hannu Sariola | The use of gdnf family-related compounds for manufacturing products for treating testicular tumors |
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