SK2472001A3 - The use of glial cell line-derived neurotrophic factor family-related compounds for regulating spermatogenesis and for preparing male contraceptives - Google Patents

Abstract

The present invention is related to glial cell line-derived neurotrophic factor (GDNF) family-related compounds, such as glial cell line-derived neurotrophic factor (GDNF), GDNF-like factors, such as persephin, artemin and neurturin or other compounds which act like GDNF on its signal transmitting receptor, i.e. cRet receptor tyrosine kinase and/or co-receptors, e.g. GDNF-family-receptor alpha :s (GFR alpha 1-4) and their use for regulating and studying spermatogenesis, for inhibting the differentiation of sperm cells, for developing male contraceptives or as a male contraceptive as well as their use for manufacturing male contraceptives.

Description

Use of compounds related to the glial cell line-derived neurotrophic factor family for the regulation of spermatogenesis and for the preparation of male contraceptives

Technical field

The present invention relates to the use of compounds related to the glial cell line-derived neurotrophic factor (GDNF) family, such as glial cell line-derived neurotrophic factor (GDNF), GDNF-like factors and / or other compounds that act as GDNF at its receptor and / or related receptors, for the regulation and study of spermatogenesis, for inhibiting spermatocyte differentiation, for the development of male contraception or as male contraception, as well as their use in the manufacture of male contraceptives.

BACKGROUND OF THE INVENTION

Spermatogenesis is a complex sequence of events in which both hormonal regulators and local interactions between Sertoli and germinal cells are important (Bellve, AR & Zhang, WJ, Reprod. Fertil. 85, 771-793 (1989); Skinner, MK, Endocr. Rev. 12, 45-72 (1991); Pescovitz, OH, et al., Trends Endocrinol. Metab., 5, 126-131 (1994); Parvinen, M., et al., J. Cell Biol. 629-41 (1992); Djakiew, D., et al., Biol. Reprod. 51, 214-21 (1994); Cancilla, B. & Risbridger, GP, Biol. Reprod. 58, 1138-4.5 (1998) Bitgood, MJ, et al., Curr Biol., 6, 298-304 (1996); Zhao, GQ, et al., Genes Dev. 10, 16571669 (1996); Zhao, GQ, et al., Development 125 , 1103-1112 (1998) Sperm cell differentiation or spermatogenesis can be divided into three phases.In the first phase of spermatogenesis, undifferentiated stem cells from the germ line, spermatogonia, divide mitotically and differentiate into spermatocytes. In the type of injury and upon disruption, spermatogenesis is recovered from these cells. At present, the mechanism by which spermatogonies are stored and the mechanism by which their differentiation into spermatocytes is conserved is unknown. The spermatogonies are located on the periphery of the seminal canals and are in contact with Sertoli cells, which are regulatory cells in the seminal canals and which are classified as somatic cells of the organism.

In the next phase, spermatogonies first differentiate into primary and then secondary spermatocytes, where their chromosomes become hapioid after two meiotic divisions, after which they are referred to as spermatides. In the third phase, the spermatides are converted into spermatozoa or mature sperm cells.

Luteinizing hormone, follicle-stimulating hormone and testosterone are the most important regulators of spermatogenesis. The germline cells in the seminal canals are not direct targets of hormonal regulation, but these are mediated through Sertoli cells. These cells convert hormonal signals to paracrine signals which are signaling 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 β family (transforming growth factor β, activin and inhibin) (Bellve, AR & Zhang, WJ, Reprod. Fertil. 85: 771-793 (1989); Skinner, MK, Endocr. Rev. 12, 45-72 (1991); Pescovitz, OH, et al., Trends Endocrinol Metab., 5, 126-131 (1994)). All of said ligands are expressed by Sertoli cells and their receptors are located on germline cells. Knowledge of the functioning of the regulatory system is poor. Probably the best experimental evidence for paracrine regulation of spermatogenesis was obtained using transgenic mice deficient in the major hedgehog protein (Bitgood, M. J., et al., Curr. Biol. 6, 298-304 (1996)). The major hedgehog protein is a signaling molecule that is expressed on Sertoli cells. Another receptor for this molecule is present on Leydig cells, i. testosterone producing interstitial testicular cells. When the main hedgehog molecule is absent, sperm differentiation ceases, sperm cells do not form, and male mice are all sterile.

Spermatogenic cells can also send signals back to Sertoli cells (Bellve, A.R. & Zhang, W.J., Reprod. Fertil. 85, 771-793 (1989); Skinner, M.

K., Endocr. Rev. 12: 45-72 (1991); Pescovitz, 0H, et al., Trends Endocrinol

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Metab. 5, 126-131 (1994)). This is demonstrated, for example, by the fact that nerve growth factor and fibroblast growth factor-1 are expressed on germline cells and the p75 nerve growth factor receptor and fibroblast growth factor receptor are present on Sertoli cells. There are also indications of autocrine regulation of spermatogenesis. Bone morphogenetic protein 8a and 8b and their receptors are expressed on the same spermatogenic cells (Zhao, GQ, et al., Genes Dev. JO, 1657-1669 (1996); Zhao, GQ, et al., Development 125, 1103-1). 112 (1998)). Functional evidence of autocrine regulation is still lacking.

The glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-β superfamily (Lin, L.F. et al., Science 260, 1130-1132). GDNF maintains dopaminergic, noradrenergic, cholinergic and motor neurons in the nervous system and also protects peripheral parasympathomimetic ciliary and sensory nerve cells (Lin, LF, Neural Notes 2: 3-7 (1996). During mammalian development, GDNF is also expressed in other tissues, such as embryonic kidneys and testes (Suvanto, P. et al., European Journal of Neuroscience 8: 816-822 (1996); Trupp, M. et al., Journal of Cell Biology 130: 137-148 (1996)). The role of GDNF as a regulator of kidney differentiation has been demonstrated by deletion or gene deletion techniques in mice (Pichel. JG et al., Nature 382: 73-75 (1996). Because these mice die at neonatal age, it was not possible to study in the deletion model. in other experimental models, the GDNF function in spermatogenesis, which is initiated much later after birth.

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Studies on other systems have shown that there are many ways to interfere with spermatogenesis and to develop male contraceptives. The methods developed so far have been very coarse with serious adverse effects for men. Therefore, the current problem is to select such a hormonal regulator from many regulators that can sufficiently specifically affect the testes while regulating spermatogenesis without adverse effects and which does not interfere negatively with the male sexual activity.

This problem was solved by the present inventors in the study of transgenic mice incorporating GDNF into their testes. It has been shown that mice with elevated levels of human GDNF have been shown to be they are sexually active but are not able to fertilize females. The solution to the problem is therefore the use of compounds related to the glial cell line-derived neurotrophic factor as defined in the claims for the regulation of spermatogenesis and the inhibition or prevention of spermatogenesis, resulting in infertility, and the use of these compounds as active ingredients for producing compositions useful as male contraceptives that are effective without serious side effects.

SUMMARY OF THE INVENTION

It is a first object of the present invention to provide an effective male contraceptive without serious adverse effects, in which compounds related to the GDNF family are used as male contraceptive compounds.

It is another object of the present invention to use compounds related to the GDNF family as active ingredients for the manufacture of compositions useful as male contraceptives.

Another object of the present invention is to provide a tool for regulating and studying spermatogenesis. This is facilitated by inhibiting the differentiation of sperm cells.

A third object of the present invention is to provide tools that affect infertility caused by a sperm cell differentiation disorder.

It is also an object of the present invention to provide a tool for regulating and studying spermatogenesis as well as inhibiting sperm cell differentiation.

It is a further object of the present invention to provide transgenic animals, particularly mice, which are useful tools for studying spermatogenesis due to compounds related to the testis-targeted GDNF family.

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The features of the present invention are defined in the appended claims. The present invention discloses a method that allows the study of sterility due to impaired sperm cell differentiation and further provides a system for inhibiting sperm cell differentiation in a contraceptive sense, wherein the male is sterile (infertile). The transgenic mouse strain carrying the GDNF transgene, the GDNF t-mouse strain, is a model of infertility caused by a sperm cell differentiation disorder. The invention is characterized in that GDNF, a compound acting as said GDNF, another cRet receptor, another compound activating the GDNF receptor, or a compound activating signal transduction from the cRet receptor in spermatogons, are useful as male contraceptives, but is also characterized in that GDNF t The mouse model is suitable as an animal model for studying the regulation of spermatogenesis and spermatogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. Ia. Testicular morphology in a natural mouse at 3 weeks of age. Scale 100 μπι.

Fig. lb. Testicular morphology in transgenic mice at 3 weeks of age. Scale 100 μπι. Note the undifferentiated spermatogonia forming a cluster of cells.

Fig. c. Testicular morphology in natural mice 8 weeks of age. Scale 100 μπι.

Fig. Id. Testicular morphology in transgenic mice 8 weeks of age. Note the remainder of the group of spermatogonies type A that are abundant in young mice. Scale - 100 μπι.

Fig. le Testicular morphology in natural mice at 6 months of age. Scale - 100 pm.

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Fig. If. Testicular morphology in transgenic mice at 6 months of age. Note the advanced germ cell atrophy and Leydig cell hyperplasia (asterisk). Scale - 100 pm.

Fig. Ig. Testicular morphology in the epididymal channels in a natural mouse at 8 weeks of age. Scale - 100 pm.

Fig. lh. Testicular morphology in epididymal canals in transgenic mice at 8 weeks of age. Note the lack of sperm. Scale - 100 pm.

Fig. 2a. Northern hybridization for GDNF mRNA in normal mice of different ages. GDNF levels are strongly reduced after the first and second weeks after birth.

Fig. 2b. Northern hybridization for Ret mRNA in testes of normal mice of different ages. GDNF levels are strongly reduced after the first and second weeks after birth.

Fig. 2c. Northern hybridization for GFRal mRNA in normal mice of different ages. GFRal levels are strongly reduced after the first and second weeks after birth.

Fig. 2d. Northern hybridization for GDNF mRNA in transgenic testes of mice of different ages. The levels of hGDNF remain high after the first and second weeks after birth.

Fig. 2e. Northern hybridization for Ret mRNA in testes of transgenic mice of different ages.

Fig. 2f. Northern hybridization for GFRa1 mRNA in testes of transgenic mice of different ages. GFRα1 mRNA levels remain high in transgenic mice at all stages analyzed.

Fig. 2g shows immunoprecipitation of GDNF from normal (WT) and transgenic (TG) testes of mice 3 and 6 weeks of age. GDNF protein is not detectable in normal testes. The control, 25 ng of recombinant human GDNF protein, is shown on the right.

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Fig. 3a. Distribution of GDNF transcripts in testes of normal (WT) mice by in situ cRNA hybridization. Sequence controls show no higher density than background. Normal testes from 1 week old mice are tested. The scale is 100 pm.

Fig. 3b. Distribution of GDNF transcripts in testes of normal (WT) mice at 8 weeks of age by cRNA hybridization in situ. Sequence controls show no higher density than background. The scale is 100 pm.

Fig. 3c. Distribution of GDNF transcripts in the testes of transgenic (TG) mice by in situ hybridization cRNA. Sequence controls show no higher density than background. Transgenic testes from 8 weeks old mice are tested. Note that spermatogonia in transgenic mice continuously express human GDNF at high levels, but the number of these cells is clearly lower at 8 weeks of age. The scale is 100 pm.

Fig. 3d Distribution of Ret transcripts in testes of normal (WT) mice by cRNA hybridization in strength. Normal testes from mice 2 weeks of age are tested. At two weeks of age, Ret expression is localized to some spermatogonia in the basal layer of the seminal canals (arrows).

Fig. 3e. Distribution of Ret transcripts in testes of normal (WT) mice by in situ cRNA hybridization. Normal testes from mice 8 weeks of age are tested. At eight weeks of age, Ret is expressed by some spermatogonia and spermatides.

Fig. 3f. Distribution of Ret transcripts in testes of transgenic (TG) mice by in situ cRNA hybridization. Transgenic testes from 8 weeks old mice are tested. Note that spermatogonia in transgenic mice continuously express Ret at high levels, but the number of these cells is clearly lower at 8 weeks of age. In transgenic mice, Ret is expressed in clusters of spermatogonia. The scale is 100 pm.

Fig. 3 g. Distribution of GFRal transcripts in testes of normal (WT) mice. Normal testes from mice aged 1-2 weeks are tested. GFRal is expressed by some spermatogonies, morphologically similar to spermatogonies expressing Ret (arrows).

Fig. 3h. Distribution of GFRa1 transcripts in testes of normal mice as detected by cRNA by in situ hybridization. Normal testes from mice 8 weeks of age are tested. The distribution of GFRal mRNA is the same as the Ret distribution, but significant expression on spermatocytes is also detected. The scale is 100 pm.

Fig. 3i. Distribution of GFRa1 transcripts in the testes of transgenic (TG) mice as detected by cRNA by in situ hybridization. Testes from transgenic mice at 8 weeks of age are tested. Note that spermatogonia in transgenic mice continuously express GDNF at high levels, but the number of these cells is clearly lower at 8 weeks of age. In transgenic mice, GFRa1 is expressed in clusters of spermatogonia. The scale is 100 pm.

Fig. 4a. Cell proliferation in testes of normal mice. In the testes of 3 week old mice, BrdU S-phase cell labeling is detectable only on the periphery of normal seminal canals. Note the absence of labeling in some channel cross-sections, indicating the segmental distribution of S-phase cells during spermatogenesis (asterisks). The scale is 100 pm.

Fig. 4b. Cell proliferation in the testes of transgenic mice. In the testes of transgenic mice at 3 weeks of age, many BrdU-labeled cells are present in the lumen of the seminal canals, reflecting the abnormal distribution of spermatogonia (arrow). The segmental distribution of cells in the S-phase is not evident in the transgenic channels. The scale is 100 pm.

Fig. 4c. Distribution of cell proliferation indices in normal and transgenic testes. The number of cells labeled with BrdU / 100 spermatogonia was counted in 100 sections by normal and transgenic seminal canals.

Fig. 4d. Apoptosis in testes of normal mice. TUNEL labeling for apoptosis was performed on testes from mice 4 weeks of age. Only a few apoptotic cells are seen. Scale (c) 100 pm.

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Fig. 4e. Apoptosis in testes of transgenic mice. TUNEL labeling for apoptosis was performed on testes from mice 4 weeks of age. Note the increase in labeling in the clusters of spermatogonia in FIG. 4e compared to normal (FIG. 4d) seed channels. Scale (c) 100 μιη.

Fig. 4f. Morphological picture of unfixed normal and transgenic seminal canals. Seed channel preparations from normal (WT) and transgenic (TG) mice at 15 weeks of age. Note the decreased diameter of the transgenic duct and the increase in the number of Leydig cells (arrows) around the duct. Scale 200 μπι.

Fig. 4G. Morphological picture of unfixed transgenic testes. Imaging of living cells from the transgenic seminal canal in an unfixed crushed preparation showed a large number of dead cells (arrows), some of which looked like type A spermatogonia, in the Sertoli cell (upper arrow). Scale 10 μπι.

DETAILED DESCRIPTION OF THE INVENTION

definitions

In the present invention, the terms used have their usual meanings in the field of biochemistry, pharmacology, recombinant DNA technology, including the preparation of transgenic animals, but some terms have a slightly different or broader meaning than their usual meaning. Therefore, the following terms are precisely defined.

The term compounds related to the neurotrophic factor family derived from the glial cell line, i. j. GDNF family-related compounds, including glial cell line-derived neurotrophic factor (GDNF), neurturin, persephine, artemin and other similar growth factors, but also compounds that have the same effect on GDNF-like compounds at the GDNF receptor or co10 ·· ·· · 9 99 99 99 99 99 receptors. Said receptor and co-receptor are Ret-tyrosine kinase and the α: s receptor for the GDNF family, respectively.

The term glial cell line neurotrophic factor (GDNF) refers to a glial cell line (GDNF) neurotrophic factor that is a member of the transforming growth factor β family (Lin, LF et al., Science, 260: 1130-1132 (1993)) . GDNF maintains dopaminergic, noradrenergic, cholinergic and motor neurons in the nervous system and also protects peripheral parasympathomimetic ciliary and sensory nerve cells (Lin, LF, Neural Notes 2: 3-7 (1996). During mammalian development, GDNF is also expressed in other tissues, such as embryonic kidneys and testes (Suvanto, P. et al., European Journal of Neuroscience 8: 816-822 (1996); Trupp, M. et al., Journal of Cell Biology 130: 137-148 (1996)). The properties, structure and amino acid sequence are described in (Lin, LF et al., Science, 260: 1130-1132 (1993)) The protein is encoded by a gene characterized by the GDNF cDNA sequence deposited with GenBank under accession number L15306.

The terms GDNF-like factors or GDNF-like compounds refer to growth factors having a similar structure or acting as GDNF at its receptors or co-receptors. The terms GDNF-like factors or GDNF-like compounds include all three GDNF-like growth factors, neurturin, persephine, artemin, as described in Milbrandt, J. et al., Neuron 20: 1-20 (1998); Kotzbauer, P.T. et al., Nature 384; 467-470 (1996).

All four growth factors of the GDNF family are structurally similar and the 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 a common receptor for signal transduction from GDNF, artemin, persephine and neurturin.

The term compounds acting as said GDNF refers to compounds that act as GDNF at a receptor transmitting signals from GDNF or its co-receptors.

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The term receptor refers to a substance binding elements of the GDNF family, i. j. substances or compounds belonging to compounds related to the GDNF family, which can transmit signals from compounds related to the GDNF family. The term receptor refers to a receptor mediating signal from the GDNF family and is a cRet receptor tyrosine kinase.

The term "co-receptors" refers to receptors that do not transmit signals from GDNF family-related compounds, but which activate the signal transduction receptor from GDNF family-related compounds. Such receptors are, in particular, the α: s receptor for the GDNF family (GFRα), which belongs to a new class of receptors whose members are also referred to as α-s receptors for the GDNF family (GFRα). Also included are GDNF, persephine, artemin, and neurturin signaling receptor complexes. Currently, four members of the family are known, (GFRal-4) (Jing, S. et al., Cell 85: 9-10 (1996); Jing, SQ et al., J. Biol. Chem. 272: 3311 1). -33117 (1997); Treanor, JJ et al., Nature, 382: 80-83 (1996); Suvanto, P. et al., Human Molecular Genetics, 6: 1267-1273 (1997)). These receptors can transmit the signal independently, but are essential for ligand binding and activation of cRet.

The term concentration of GDNF family-related compounds in amounts such that spermatogenesis is prevented, that spermatogenesis can be regulated and that infertility due to impaired sperm cell differentiation can be caused, or that a man is rendered unable to fertilize a woman indicates a GDNF-like compound concentration of approximately 50 -5000, preferably 50-500, preferably 50-100 times the concentration of endogenous GDNF in the testes of the male. Said amount can be determined from the results obtained by measuring the amount of GDNF mRNA in transgenic mice by Northern hybridization (Fig. 2), from which the amount of expressed protein can be calculated at higher mRNA. From these results, it can be deduced that the amount of GDNF in transgenic mice is 50500 times higher than the endogenous amounts in mice 3-6 weeks of age, but only slightly higher than in newborn mice when they reach GDNF concentrations and are not no spermatogenesis present. From these results 9 9 9 9 99 99

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9999 99 99 9 999, those skilled in the art can calculate the amounts necessary to achieve the desired effects in humans.

The term derivative refers to isomers of compounds related to the GDNF family. It also includes hybrid molecules constructed according to the structure of GDNF, persephine, neurturin, and artemin compounds, as well as truncated forms, complexes, and fragments thereof. The only condition for said compounds is that they have substantially the same properties and effects as the above compounds, determined by the same methods of the present invention. Naturally, the term derivative includes derivatives of the compounds mentioned above in a more common sense, for example free groups in said compounds, such as carboxyl, amino and / or hydroxyl groups, may be substituted by alkylation, esterification, etherification and amidation, for example with alkyl groups such as is methyl, ethyl, propyl, etc. The only condition in this case is that the substitutions do not significantly alter the properties or potency of the molecules or compounds mentioned above in this paragraph.

Glial cell line-derived neurotrophic factor (GDNF) is a potent factor supporting the survival of various neuronal cells, regulating renal differentiation (Lin, LF, 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, RW, et al., Nature 373, 344-346 (1995); Nguyen, QT, et al., Science 279, 1725-1729 (1998); Trupp, M., et al., J. Cell Biol. 130, 137-148 (1995); Hellmich, HL, et al., Mech Dev. 54, 95-105 (1996); Schuchardt, A., et al., Nature 367, 380-383 (1994); Pichel, JG, et al., Nature 382, 73-75 (1996), and is expressed in the testes (Trupp, M., et al., J. Cell Biol. 130, 137-148 (1995); Hellmich, HL et al., Mech. Dev. 54, 95-105 (1996); Suvanto , P., et al., European Journal of Neuroscience 8, 816-822 (1996), but its function is still unknown.

GDNF, a distinct member of the transforming growth factor β (TGF-β) superfamily (Lin, LF, et al., Science 260, 1130-1132 (1993)), is an effective survival promoting factor for dopaminergic neurons in the substantia nigra, and for some other groups of neurons in the central and peripheral nervous systems13 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Lin, LF, et al., Science 260: 1130-1132 (1993); Tomac, A., et al., Nature 373, 335-339 (1993); Winkler, C., et al., J. Neurosci., 16, 7206-7215 (1996), Oppenheim, RW, et al., Nature 373, 344-346 (1995), Nguyen, QT, et al. Science 279, 1725-1729 (1998), Trupp, M., et al., J. Cell Biol. 130, 137-148 (1995); Hellmich, HL, et al., Mech. Dev. 105 (1996); Schuchardt, A., et al., Nature 367, 380-383 (1994); PicheI, JG, et al., Nature 382, 73-75 (1996)). GNDF also acts as a signaling molecule in urethral branching during kidney morphogenesis (Schuchardt, A., et al., Nature 367, 380-383 (1994); PicheI, JG, et al., Nature 382, 73-75 (1996)) . It is synthesized by several neuronal and non-neuronal embryonic organs, including embryonic and postnatal testes (Trupp, M., et al., J. Cell Biol. 130, 137-148 (1995); Hellmich, HL, et al., Mech. Dev. 54, 95-105 (1996), Suvanto, P., et al., European Journal of Neuroscience 8, 816-822 (1996)). Signaling receptor complexes for GDNF include Ret receptor tyrosine kinase (Durbec, P., et al., Nature 381, 789-793 (1996); Trupp, M., et al., Nature 38J., 785-789 (1996) ) and a glycosylphosphatidylinositol-linked co-receptor, the?: GDNF family receptor (GFRal-4) (Jing, S., et al., Cell 85, 9-10 (1996); Jing, SQ, et al., J. Biol Chem 272: 33111-33117 (1997), Enokido Y, et al., Curr Biol 18, 1019-1022 (1998)).

GDNF-, GFRal, and Ret-null mice show markedly similar phenotypes with the absence or hypoplasia of the kidneys and severe enteric innervation defects (Schuchardt, A., et al., Nature 367, 380-383 (1994); PicheI, JG, et al. Nature 382, 73-75 (1996)). Since these mice die within a few days of birth, spermatogenesis cannot be analyzed in these mutant mice.

To investigate the role of GDNF in spermatogenesis, the inventors studied transgenic mice targeting GDNF overexpression in testicular germ cells. The transgenic males were infertile and were found to stop early differentiation of spermatogenic cells, spermatogonies of type A. These cells formed clumps in the seminal canals, resulting in testicular atrophy. The data obtained show a new function of GDNF as a paracrine inhibitory signal during the early phase of spermatogenesis.

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In normal mice, GDNF is thought to regulate the spermatogonia stock so that they are progressively differentiated into spermatozoa. These infertile transgenic mice serve as a model for male infertility with spermatogenic deficiency. Activating the GDNF signaling cascade provides us with the means to develop contraception for men. The effects of GDNF on spermatogenesis should be considered when GDNF-like molecules are used in clinical studies to treat neurodegenerative diseases such as Parkinson's disease.

In the study of transgenic mice overexpressing human GDNF under the control of the human translational elongation factor 1 (EF-1) promoter (Mizushima, S and Nagata, S., Nucleic Acids Res. 18: 5322 (1990)), which localizes transgene expression specifically into testicular germ cells (Furuchi, T. et al., Development 122: 1703-1709 (1996)), a new function for GDNF was found. Impaired spermatogenesis in transgenic testes, caused by overexpression of GDNF, suggests that GDNF acts as a local inhibitory signal in spermatogonia differentiation. Progressive germ cell degeneration and Leydig cell hyperplasia in transgenic mice are clearly secondary to spermatogenesis deficiency, since Sertoli cells eliminate poorly differentiated and abnormal germ cells, and cooperation between different cell types is disrupted. Therefore, targeted expression of GDNF in testes provides a new animal model for male infertility. Activation of the GDNF signaling cascade by GDNF, Ret agonists or other substrates may be a means of preventing sperm production in men and may be the basis for the development of male contraception. The effects of GDNF on spermatogenesis should be considered when GDNF-like molecules are designed and used in clinical trials to treat neurodegenerative diseases such as Parkinson's disease.

To study the function of GDNF in the testes, the inventors prepared transgenic mice overexpressing human GDNF under the control of the human translational elongation factor 1 (EF-1) promoter (Mizushima, S and Nagata, S., Nucleic Acids Res. 18: 5322 (1990)) , which localizes the expression of the transgene specifically to testicular germ cells (Furuchi, T. et al., Development 122: 1703-1709 (1996)). Other localized promoters may also be used15

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for expression of the transgene specifically into testicular germ cells. Four independent animals, two males (C10, C12) and two females (S6, E19), with a copy number of transgene of 10, 3, 20 and 4, were analyzed, respectively. Male C10 and C12 were infertile and further male phenotype analysis was performed on the progeny of two female S6 and E19. Transgenic mice developed normally into adulthood and had a similar phenotype. Body weight was normal and had no defects in parenchymal organ weight and morphology (data not shown) except for the testes. After reaching 4 weeks of age, a decrease in testicular weight was observed in transgenic mice compared to normal controls (Table 1). All transgenic males in the offspring of S6 and E19 females were infertile. During a six-month continuous crossover, transgenic males were placed in a box with normal FVB and NMRI females at 3-4 daily intervals. Normal females copulated with transgenic males at normal frequency, as evidenced by the presence of vaginal bays, but none became pregnant. A further 20 transgenic male mice of fertile age were tested in a short crossover test with normal FVB and NMRI females. During 2 weeks of continuous cross-linking, approximately 200 females from both strains were coupled, but none became pregnant. Strain dependence of the infertile phenotype was eliminated by backcrossing transgenic FVB mice with NMRI background. Like transgenic male FVB strains, male F1 generations were also infertile.

Histological findings in testes from transgenic mice at 1 week of age were shown to be normal. From 2-3 weeks of age (Figs. 1a, 1b), clusters of spermatogonies of type A were observed, which were partially separated from the basement membrane of the seminal canals. These cells were undergoing gradual degeneration over the following weeks, so that by the tenth week already advanced atrophy was present with the presence of Sertoli cells and only spermatogonia (Fig. Ic-1f). Leydig cells, testicular interstitial cells, showed progressive hyperplasia from 4 postnatal week (Fig. 1d, If). Occasionally, germ cells outside spermatogonia clusters developed further into spermatocytes and rarely spermatides, but their development stopped after meiosis and the cells degenerated. Mature semen was not observed in the seminal canal or epididymal canal, although no obstruction was observed in the duc16, but no obstruction was observed in the duc16. · ··· · ····· · · ··· · ··· · · ··· · · · ··· · · · · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · Ig, 1h).

Concentrations of mouse and human GDNF and its receptor mRNA were also shown to be increased in transgenic mice as determined by Northern hybridization in normal and transgenic testes from mice of different ages (Fig. 2). In normal testes, GDNF, Ret and GFRa1 transcripts are expressed in significant amounts at the time of birth and during the first week after birth (Fig. 2a-c). Expression of GDNF mRNA was significantly reduced before the onset of puberty (at 2 weeks of age) (Fig. 2a). At the same time, the levels of Ret (Fig. 2b) and GFRa1 (Fig. 2c) mRNA decreased, but were still detectable in the testes of adult mice. Artemin (ART) binds to GFRα3 and also signals through Ret.

Neurturin (NTN) is a GDNF-related molecule that uses the same multi-component receptor complex as GDNF, but prefers GFRα2 (Klein, RD et al., Nature 387: 717-721 (1997) as the co-receptor, which is a homolog to GFRα. Both NTN and GFRα2 transcripts are expressed in the testes after birth (Widenfalk, J et al., J. Neurosci. 17: 8506-8519 (1997)), but preferably after puberty.Third member of the GDNF family, persephin (Milbrandt, J. et. al., Neuron 20: 1-20 (1998)), binds to chicken GFRα4 (Enokido, Y. et al., Curr. Biol. 18: 1019-1022 (1998)), whose homologue has not yet been discovered in mammals.

Northern hybridization with a probe specific for human GDNF showed that the transgene is expressed only in the testes and not in other organs in both sexes, such as the ovary, penis, brain, liver, kidney, lung and skin (data not shown). In contrast to the decrease in GDNF mRNA levels in normal mice, transgenic mice remain GDNF mRNA expression in testes high from neonatal to adulthood (Fig. 2d). Also, Ret and GFRα1 mRNA levels remain high at each age analyzed (Fig. 2e, 2f). Immunoprecipitation of GDNF from the testes from normal and transgenic mice at 3 and 6 weeks of age confirmed that GDNF protein levels are significantly higher in the transgenic testes compared to normal testes (Fig. 2g).

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It has previously been shown that embryonic Sertoli cells and Sertoli cell lines express GDNF (Trupp.M., Et al., J. Cell Biol. 130, 137148 (1995); Hellmich, HL et al., Mech. Dev. 54, 95-105 (1996)). Using in situ hybridization, GDNF mRNA was detectable in normal seminal canals during the first week after birth, but not later (Fig. 3a, 3b), and the probe for GDNF mRNA was detectable on Sertoli cells. Transgenic testes showed continuous and high expression of human GDNF mRNA on spermatogonies of type A from two weeks of age to adulthood (Fig. 3c). Ret mRNA was expressed by both spermatogonia and spermatides in normal testes (Figs. 3d, 3e) and in spermatogonia clusters in transgenic testes (Fig. 3f). GFRα1 mRNA was expressed by spermatogonies, spermatocytes and spermatides in normal testes (Figs. 3g, 3h), and also intensively in spermatogonia clusters in transgenic testes (Fig. 31). Thus, the GDNF signaling receptor complex is present in the spermatogony of both normal and transgenic mice. In normal mice, GDNF acts as a paracrine regulator of spermatogenesis from Sertoli cells, but in transgenic mice this regulation is disrupted by the continuous autocrine expression of GDNF by germ cells.

Spermatogonies are the only proliferating germ cells in the testes and mitotic activity is extensively segmented in normal seminal canals (Fig. 4a). In transgenic mice, BrdU-labeled spermatogonies exhibit an abnormal distribution in the seminal canals due to the aggregation of spermatogonia in the lumen of the seminal canals (Fig. 4b). Due to the highly segmental nature of cell proliferation in normal testes, it is difficult to compare degrees of proliferation in normal and transgenic testes. However, the peak proliferation index of spermatogonia (nuclei labeled BrdU / 100 spermatogonia) was clearly lower in transgenic mice compared to normal: 0.26 ± 0.11 (n = 714) and 0.72 ± 0.14 (n = 436 cells). , calculated in the proliferative segments of the seminal canals), respectively (Fig. 4c).

Apoptosis was pronounced in clusters of spermatogonies and other poorly differentiated germ cells from the 2nd week after birth, as shown. · · • · · · · · · · · · · · · · · · · · · · · ·

TUNEL marking (Fig. 4d, 4e). A large number of dead cells, some of which were similar to spermatogonies of type A, were captured and phagocytically removed by Sertoli cells (Figs. 4f, 4g). Thus, increased apoptosis higher than cell proliferation may explain gradual testicular atrophy in mice overexpressing GDNF. On the other hand, the accumulation of spermatogonia in young mice appears to be due to a block of their differentiation and not a higher proliferation of these cells.

Three GDNF-overexpressed males were observed for 5 months and neither macroscopic testicular tumors nor microinvasive tumors were observed during this time. Furthermore, we did not observe testicular malignancies in any biopsy from young male mice (n = 103), suggesting that a defect in spermatogonia differentiation in GDNF overexpressed mice does not lead to germ cell malignancies but to testicular atrophy. However, mice should be observed for an extended period of time to avoid any increase in the risk of testicular carcinomas.

Male or rat male animals are sterile in the absence of vitamin A metabolite, retinoic acid (RA), as demonstrated by administration of a vitamin A-free diet (VAD). The differentiation of type A spermatogonias is completely blocked by the VAD diet and is restored by the administration of vitamin A or its active metabolites (Kim, K. H. et al., Molec. Endocrinology 4: 1679-1688, 1990). Retinoic acid receptor (RARα) and protein mRNA levels decrease in the VAD diet (Akmal, K. et al., Endocrinology 139: 1239-1248, 1998). Accordingly, RARα-deficient transgenic mice are infertile and develop testicular atrophy (Lufkin, T. et al., Devel. Biol. 90: 7225-7229, 1993). Therefore, RAR protein expression was analyzed in normal mice and in GDNF overexpressed transgenic mice. Expression of RAR virtually disappears in clusters of spermatogonies of transgenic mice as demonstrated by indirect immunoperoxidase staining with antibodies to the RAR protein. Data show that one mechanism by which GDNF acts as a negative regulator of spermatogonia is to reduce RAR expression in spermatogonies expressing Ret.

To analyze whether other GDNF family members can act as negative regulators of spermatogenesis, the inventors prepared transgenic mice

Φ · · · · · · Φ With overexpression of neurturin in the testes. Indeed, the transgenic mice expressing neurturin under the control of the translational elongation factor Ia promoter, which locates expression of the transgene specifically in the testes, were also infertile during the first 4 weeks after puberty, as found in fertility tests identical to those performed in mice overexpressing GDNF.

Sperm differentiation in mammals can be divided into three distinct phases that are regulated both by local cellular interactions and hormonal (Russell, LD: et al., Clearwater, FL, Cache River, pp. 1- 40 (1990)). The first phase of spermatogenesis is mitosis, in which the spermatogonies result in spermatocytes. After the spermatogonial stage, spermatogenesis continues without mitotic division. Spermatogonies are further subdivided into subtypes where spermatogonies of type A include spermatogenic stem cells. The second phase is spermatocyte meiosis, which leads to the formation of haploid spermatids. The third phase is a spermiogenic phase in which spermatides turn into spermatozoa, sperms that are structurally equipped to reach the egg and are capable of fertilization. During meiosis, spermatogonies of type A proliferate and begin to differentiate. However, some of them remain undifferentiated and are considered to be a stem cell reservoir (Russell, L. D. et al., Clearwater, F.L., Cache River, pp. 1-40 (1990)). The exact mechanism by which stem cells are maintained or transformed into differentiated spermatogonia is unknown. Our Northern hybridizations have shown that the reduction of GDNF expression coincides with the initiation of sperm differentiation in normal mice and overexpression of GDNF blocks spermatogonia differentiation. Therefore, local GDNF signaling may be essential to maintain spermatogonies in an undifferentiated state.

There is evidence that each stage of spermatogenesis requires paracrine interactions between somatic Sertoli cells and germ cells and that these interactions may be mediated by growth factors and their mediators (Bellvé, A. R & Zhang, WJ, Reprod. Fertil. 85, 771-793 ( Skinner, M. (1989);

K., Endocr. Rev. 12: 45-72 (1991); Pescovitz, A. H., et al., Trends Endocrinol.

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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. & Risbridger, G. P., Biol. Reprod. 58, 1138-4.5 (1998); Bitgood, M.J., et al., Curr. Biol. 6, 298-304 (1996); Zhao, G. Q., et al., Genes Dev. J. O., 1657-1669 (1996); Zhao, G. Q., et al., Development 125: 1103-1112 (1998). However, direct data on intercellular communication between testicular somatic cells and germ cells is still weak. Communication between Sertoli cells and germ cells can include many molecules such as stem cell factor (c-kit ligand), insulin-like growth factor-1, three members of the transforming growth factor β family (transforming growth factor β, activin and inhibin) and transferrin (Pescovitz, A. H., et al., Trends Endocrinol. Metab. 5, 126131 (1994)). Sertoli cells secrete all of these molecules and the germ cells carry receptors for these molecules. In contrast, Sertoli cells express germ cell secreted receptors. For example, Sertoli cells express the p75 nerve growth factor receptor and fibroblast growth factor receptors whose respective ligands are secreted by testicular germ cells (Pescovitz, OH, 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)). Further, Sertoli cells also interact with Leydig cells. The major hedgehog (Dhh) gene is expressed by Sertoli cells and its putative receptor mainly by Leydig cells (Bitgood, M. J., et al., Curr. Biol. 6, 298-304 (1996)). Indeed, Dhh null mice show severe differentiation defects in spermatogenesis (Bitgood, M. J., et al., Curr. Biol. 6, 298-304 (1996)). Recently, bone morphogenetic proteins 8a and 8b, together with their putative receptors, have been discovered on germ cells and inactivation of these ligands has led to abnormal spermatogenesis and male infertility (Zhao, GQ, et al., Genes Dev. JO, 1657-1669 (1996); Zhao) , GQ, et al., Development 125: 1103-1112 (1998)). The simultaneous presence of these growth factors and their germ cell receptors indicates autocrine regulation of germ cells.

Our experiments show a new GDNF function. Impaired spermatogenesis in transgenic testes, caused by overexpression of GDNF, suggests that GDNF acts as a local inhibitory signal in spermatogonia differentiation.

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Progressive testicular atrophy and hyperplasia of Leydig cells in transgenic mice are clearly secondary to spermatogenesis deficiency. Therefore, targeted insertion of GDNF into the testes provides a new animal model for male infertility. Activation of the GDNF signaling cascade by GDNF, Ret agonists or other substrates may be a means of preventing sperm production in men and may be the basis for the development of male contraception. The effects of GDNF on spermatogenesis should be considered when GDNF-like molecules are designed and used in clinical trials to treat neurodegenerative diseases such as Parkinson's disease.

The invention is described in more detail in the following examples which describe the materials and methods used to prepare transgenic mice and the experiments used to demonstrate the effect of GDNF in an animal model. The examples are not intended to limit the scope of the present invention. Various other embodiments will be apparent to those skilled in the art from the results of the following examples.

DETAILED DESCRIPTION OF THE INVENTION

techniques

Construction of GDNF Transgene and Preparation of Transgenic Mice

Human GDNF cDNA was cloned into pEF-BOS vector with EFlapromotor (Migushima, S. and Nagata, S. Nucleic Acids Res. 18: 5322 (1990)) by replacing the XbaI-XbaI original fragment with the entire coding sequence of human GDNF cDNA (GenBank accession number L15306). The correctness of the expression construct was confirmed by sequencing. Correct expression of the GDNF protein by the construct was confirmed by transfection of Cos cells followed by immunoprecipitation of GDNF from the lysate and culture supernatant. Transgenic mice were prepared by microinjection of a 2.7 kb Pvul-HindIII fragment of the GDNF cDNA construct into a projector of newly fertilized FVB inbred mouse eggs, using the method described earlier (Hogan, B. et al., Manipulating the Mouse Embryo, Cold Spring Harbor, NY). Cold Spring Harbor Laboratory Press (1994). Transgenic mice have been identified by Southern Hybris22. 9 9 9 9 9 10 11 12 13 14 15 16 17 18 19 20 99 99 999 by design with human GDNF cDNA and progeny transgenic mouse PCR from terminal DNA. Primers for human GDNF were from exon 1 (5'-TGT CGT GGC TGT CTG CCT GGT GC-3 ¹ ) and exon 2 (5'-AAG GCG ATG GGT CTG CAA CAT GCC-3 ¹ ).

Histology, cell proliferation and apoptosis. For histological examination, freshly removed testes and epididymis were fixed in Bouin's fixative or 4% PFA for 2-24 hours, depending on size, paraffin treated, 7 µm sections prepared and stained with hematoxylin / eosin. For the cell proliferation assay, mice were injected intraperitoneally with a mixture of BrdU and 5-fluoro-2-deoxyuridine (FrdU) (Amersham) at a concentration of 10 mg / kg in PBS. After 2 hours, the mice were sacrificed by breaking the ligament, necropsied, the testes were removed and fixed in Bouin's fixative. The testes were paraffin treated, 7 µm sections were prepared and deparaffinized. BrdU incorporation was detected by indirect immunofluorescence with monoclonal antibodies to bromodeoxyuridinium (Amersham) and goat anti-mouse IgG antibody conjugated to rhodamine (Jackson). Apoptotic cells were detected in deparaffinized sections using the Apoptag in situ cell death detection kit (Oncor) according to the manufacturer's instructions.

Living cells. Seed channels were sectioned and crushed on slides with PBS buffer. Computer analysis of the cells in the seminal canals was performed according to the method of Parvinen, M. et al., Histochem. and Biol. 108: 77-81 (1997)).

Northern hybridization. Total RNA from various tissues was isolated using the TRIZOL Reagent Kit (Life Technology) according to the manufacturer's instructions, and 30 g of total RNA was used in each lane. Northern transfer was performed according to the Maniatis procedure (Sambrook, J. et al., T. Molecular Cloning: A).

Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press 1989, p.

7:43 to 7:50). cDNA probes (same as in situ hybridization, see below) were labeled with [ ³² P] dCTP. The filters were exposed to a FujiBAS phosphoimager.

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Immunoprecipitation and Western blotting with anti-GDNF antibody. Freshly collected testes were homogenized and the cells were lysed in high salt lysis buffer (300 mg protein / ml; high salt lysis buffer: IM 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 tablet / ml protease inhibitor mixture (Complete, Mini EDTA, Boehringer Mannheim) The lysates were immunoprecipitated with a polyclonal antibody to human recombinant GDNF peptide that crosses reacts with mouse GDNF (R and D Systems) and protein A sepharose .. Immunoprecipitates were processed on 20% SDSPAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham Life Scieces) .Membranes blocked with 5% bovine serum albumin and immunoprecipitated GDNF with anti-GDNF antibody (Santana Cruz Biotechnology) at ambient temperature for 2 hours Detection was performed using an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma) and ECL chemiluminescence (Amersham), according to the manufacturer's instructions.

In situ hybridization. In situ hybridization was performed as described (Wilkinson, D. and Green, P. In situ hybridization and threedimensional reconstruction of serial sections. In: Postimplantation Mammalian Embryos. A Practical Approach (ed. A. Copp and D. Cocroft), p. 155-171, Oxford University Press, London (1990)). Upstream and upstream cRNA probes (mouse GDNF 328 bp from exon 3, human GDNF 513 bp from exon 1 and exon 2, mouse Ret 714 bp from 3 end, mouse GFRal 777 bp) were synthesized using the appropriate RNA polymerases and ³⁵ S-labeled UTP. The hybridization temperature was 52 ° C and the autoradiographic membranes were exposed at 4 ° C for 2-4 weeks. The membranes were photographed with a CCR camera connected to a computer. In the PhotoShop graphics program, dark areas of images were inverted, artificially colored red, and combined with light areas of images.

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Example 1

Preparation of transgenic mice

In the example, localized expression of GDNF under the control of the translational elongation factor 1 promoter in the testes and the role of GDNF in the testes are studied. The entire coding region of the human GDNF (hGDNF) cDNA was cloned into the pEF-BOS vector by replacing the XbaI-XbaI fragment of the hGDNF cDNA (Migushima, S. and Nagata, S. Nucleic Acids Res. 18: 5322 (1990)).

The 2.7 kb HindIII-EcoRI fragment was isolated from a pEF-BOS vector containing the EF1a (translational elongation factor 1a) promoter and a 0.7 kb Xba-EcoRI fragment containing the poly (A) adenylation signal. The size of the entire coding region of human GDNF cDNA was 636 bp. Transgenic mice were prepared by microinjection of a 2.7 kb Pvul-HindIII fragment into a fertilized mouse egg projector. The injection was first administered in the FVB / NIH strain and then the transgene positive mice were crossed with the FVB / NIH- and NMRI-strains of mice. Transgenic mice were identified by Southern hybridization with human GDNF cDNA and progeny of transgenic mice PCR from terminal DNA (the transgene carrying the mouse strain is hereinafter referred to as GDNF t-mice). The 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'). The results show that GDNF t-mice carry the coding region of human GDNF cDNA and transmit it to their progeny.

Example 2

Transgene expression studied by Northern hybridization and RT-PCR

Transgene expression in mice of different ages (0-13 weeks) was studied by Northern hybridization, which detected concentrations of endogenous and transgenic GDNF mRNA mice of different ages, and reverse transcription - polymerase chain reaction (RT-PCR) from RNA from mouse testes, comparing the expression levels of endogenous GDNF mRNA and human transgenic GDNF mRNA in mice. For reverse transcription PCR ·· · 1 µg of total RNA was replicated by reverse transcription in a randomly initiated reaction (20 μΐ) using AMV reverse copying enzyme (Finnzymes, Helsinki) according to the manufacturer's instructions. 2 μΐ of the reaction product was used as a template to copy endogenous mouse GDNF cDNA or transgenic human GDNF cDNA.

The first primer (5'GCTGAG / CCAGTGACTCA / CAATATGCC-3 *) recognizes both mouse and human GDNF alleles and covers the intron region to exclude genomic contamination. Second end primers were selective for endogenous and transgenic GDNF (specific mouse GDNF region: 5'-TGTTAGCCTTCTACTCCGAGACAG-3 ') and transgenic primer (transgenic GDNF specific region 5'-CACCAGCCTTCTATTTCTGGATAA-3'). These primers were used under identical conditions (1 μΜ primer, 1 mM dNTP and Dynazyme polymerase (Finnzymes, Helsinki) in their own buffer) to copy 373 bp fragments from endogenous and transgenic GDNF cDNAs that were reverse-copyed from RNA from normal testes or transgenic mice. The cycle was 30 seconds at 90 ° C, 30 seconds at 62 ° C and 45 seconds at 72 ° C, and was repeated 20-36 times after a hot start at 95 ° C for 2 minutes. Copying was completed after the last cycle at 72 ° C for 5 minutes. The PCR product was introduced into an agarose gel containing ethidium bromide, analyzed under UV light and photographed with a Polaroid camera. Northern blotting to identify expression of transgenic hGDNF and endogenous mouse GDNF mRNA was performed using [α- ³² P] -labeled species-specific GDNF cDNA probes and total RNA isolated from the testes using the TRIzol Reagent Kit (Life Technologies) according to the manufacturer's instructions. The probes were: mouse GDNF (328 bp from exon 3 (NCB1 GenBank accession no. U37459)) and human GDNF (536 bp from exon 1 and 2 (Lin et al., Science 260: 1130-1132 (1993)). performed according to Sambrook, J. et al (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press 1989, pp. 7.43-7.50), 30 µg of total RNA was used in each sample. The Fuji phosphoimager plate was exposed and exposed to light overnight and the image was captured with a FujiBAS phosphoimager.The actin probe was used as an RNA control probe and hybridized to each filter after hybridization of the GDNF probe The results showed strong expression of GDNF ·· ·· ·· 99 99 99 99 99 99 99

9

9999 99 99 999 9 mRNA in the testes of mice at the time of birth (Fig. 2a), where this expression weakens (decreases) during the first weeks after birth, especially when the mice reach puberty, and is difficult to detect in adult mice. According to Northern hybridization, it can be determined that in the testes of GDNF t-mice at 3 weeks of age (Fig. 2d), the transgenic human GDNF mRNA is 50-500 fold more abundant than the endogenous GDNF mRNA (Fig. 2a).

Example 3

Localization of GDNF into the testes by Northern hybridization and in situ hybridization

Due to the presence of the elongation factor 1a promoter, the GDNF transgene is not expressed in males other than the testes (Furuchi, T. et al., Development 122: 1703-1709 (1996)) as analyzed by Northern hybridization of various male and female tissues. In situ hybridization of the cDNA was also performed on the testes, ovaries and kidneys, according to the method of Wilkinson, D. and Green, P. In situ hybridization and the three-dimensional reconstruction of the serial sections. In: Postimplantation Mammalian Embryos. A Practical Approach (edited by A. Copp and D. Cocroft), p. 155-171, Oxford University Press, London (1990). The probes were: mouse GDNF probe (328 bp), human GDNF probe (536 bp), GFRa1 probe (777 bp) and GFRa2 probe (approximately 500 bp). For in situ hybridization, the temperature for all probes was 52 ° C and the exposure time at 4 ° C was 1.5-4 weeks. At least 3 different exposures were performed for each probe.

The results obtained with Northern hybridization and in situ hybridization cRNA show that the human GDNF probe hybridizes only to testicular RNA, where the in situ hybridization signal is localized to spermatogonia and some early spermatocytes (Fig. 3).

It has also been shown that, in both transgenic GDNF t-mice and normal mice, the GDNF Ret receptor is expressed in prebubertal mice by spermatogonia and in adult mice also by spermatides but not differentiated by spermatozoa and that the GFRα and GFRα2 co-receptors are also expressed by germ cells during spermatogenesis (Fig. 3).

Example 4

Expression morphology detected histologically

Morphological results of transgenic GDNF expression were determined by histological examination of the testes. Tissues of normal and transgenic mice were fixed in 4% formaldehyde overnight, paraffin treated, 7 µm sections were prepared and these sections were deparaffinized and stained with hematoxylin / eosin.

Histological examinations of testes from mice aged 1-4 weeks revealed a significant elevation in the amount of spermatogonia in male GDNF mice, related to the fact that there was no differentiation into spermatides and spermatozoa. According to the histological structure, cell clusters in the seminal canals could be identified as type A spermatogonia (Fig. 1b). After four weeks, the number of said spermatogonia clusters in the seminal canals decreased and only a few spermatogonia were present after 11 weeks. Mature and free spermatozoa were not formed at any stage and sperms were not detected in the epididymal canals.

Example 5

Demonstration of GDNF's ability to cause infertility

Because GDNF may inhibit sperm production, the fertility of GDNF-t mice was studied by mating with fertile females over 8 weeks of age. 20 males (over 6 weeks of age) were kept with females for 2 weeks. Altogether there were 200 matings. In addition, three males (8 weeks of age) and females were treated for 1/2 year with the females changing at 2-3 daily intervals. The mating was the same as in normal mice. After mating, the uterus and oviducts of some females were examined for sperm.

The results showed that neither female mating with the transgenic male of the GDNF t-mouse strain became pregnant and no sperm was detected in their uterus or oviducts. Males carrying the transgene from the GDNF strain of t-mice are completely infertile and cannot produce progeny.

The results are also shown in the accompanying drawings, where:

Fig. 1 shows the testicular morphology of normal (left panel, a, c, e) and transgenic (right panel, b, d, f) mice at three different stages, and the normal (g) and transgenic (h) channel of the epididymis in mice aged 8 weeks, (a and b) age 3 weeks, (c and d) age 8 weeks, (eaf) age 6 months. In FIG. d note the remnants of type A spermatogonia clusters that are abundant in young mice (d) and advanced germ cell atrophy and Leydig cell hyperplasia (asterisk) in (f). Adrenal channels from normal (g) and transgenic (h) mice show sperm deficiency in transgenic mice. The scale is 100 μιη.

Fig. 2. Northern hybridization for GDNF (a, d), Ret (b, e) and GFRa1 (c, f) mRNA in normal (left panel) and transgenic mice (right panel) of different ages. Probes for murine GDNF cDNA and human GDNF cDNA were used in a and d, respectively. GDNF (a), Ret (b) and GFRa1 (c) mRNA levels are strongly reduced after the first and second weeks after birth. In contrast to decreasing levels of endogenous GDNF mRNA in normal mice, levels of transgenic GDNF mRNA (d) remain high until maturity. Also, the levels of Ret (e) and GFRal (f) mRNA are high in transgenic mice at all stages analyzed, (g). Immunoprecipitation of GDNF from normal (WT) and transgenic (TG) testes from mice 3 and 6.5 weeks of age. GDNF protein is not detectable in normal testes. On the right is 25 ng of recombinant human GDNF protein as a control.

Fig. 3. Distribution of GDNF (a, b, c), Ret (d, e, f) and GFRa1 (g, h, i) transcripts in the testes of normal (WT, left and middle panel) and transgenic mice (TG, right panel) by in situ hybridization of cRNA. In (a, b) the mouse GDNF • cDNA template was used and in (c) the human GDNF cDNA template was used. All other probes were mouse transcripts. Sequence controls showed no higher background density, (a, d, g) testes of normal 1- to 2-week-old mice, (b, e, h) testes of normal 8-week-old mice, (c, f, i) testes 8- weekly transgenic mice. Note that spermatogonia in transgenic mice continuously express human GDNF at high levels, but the number of these cells is clearly lower at 8 weeks of age, (d) - at 2 weeks of age, Ret expression is localized to some spermatogonia in the basal layer of seminal canals (g) - also GFRα1 is expressed by some spermatogonia, morphologically similar to spermatogonia expressing Ret (arrows), (e) - at 8 weeks of age, Ret is expressed by some spermatogonia and spermatides. (h) - The distribution of GFRa1 mRNA is similar to that of Ret but is also expressed in large quantities on spermatocytes. In transgenic mice, both Ret ((f) and GFRa1 (i) are expressed on clusters of spermatogonia at 100 µm.

Fig. 4. Cell proliferation (a, b, c), apoptosis (d, e) and living cell morphology (f, g) in testes of normal (a, d) and transgenic (b, e, g) mice, (f) shows both normal and transgenic seminal canals, (a) - in the testes of mice of 3 weeks of age, BrdU S-phase cell labeling is detectable only on the periphery of normal seminal canals. Note the absence of labeling in some cross-sections through the channels, indicating the segmental distribution of S-phase cells during spermatogenesis. (b) - In the testes of transgenic mice at 3 weeks of age, many BrdU-labeled cells are present in the lumen of the seminal canals, reflecting the abnormal distribution of spermatogonia (arrow). The segmental distribution of S-phase cells is not apparent in the transgenic channels. (C) Distribution of cell proliferation indices in normal and transgenic testes. The number of cells labeled with BrdU / 100 spermatogonia was counted in 100 sections by normal and transgenic seminal canals. The normal segmental distribution of BrdU-labeled cells is disrupted in transgenic testes. (d and e) - TUNEL labeling for apoptosis in 4-week-old mice. Note the increase in labeling in clusters of spermatogonia (arrow) (e) compared to normal (d) seminal canals. (f) - Preparation of non-fixed seminal canals from normal (WT) and transgenic (TG) mice. Note the altered diameter of the transgenic duct and the increase in the number of Leydig cells (arrows) around the duct. (g) - microscopic examination of living cells from the transgenic seminal canal in an unfixed crushed preparation at high magnification showed a large number of dead cells (arrows), some of which looked like spermatogonia of type A, in the Sertoli cell (upper arrow). Scale (a-d) 100 μιη, (e) 200 μιη, (f) 10 μιη.

Table 1: Testicular weights (mean ± SD) from normal and transgenic mice of different ages

Age (weeks) Normal (mg) n Transgenic (mg) n 1 5.5 ± 2.6 20 4.8 ± 2.5 20 2 27.8 ± 6.0 20 22.7 ± 12 20 3 50.2 ± 6.5 20 58.7 ± 8.6 20 4 68.8 ± 8.0 10 40.3 ± 10.0 10 5 79.9 ± 7.3 10 30.5 ± 9.3 10 8 102.2 ± 4.6 10 34.7 ± 8.0 8 13 100.9 ± 6.0 10 38.3 ± 8.3 6 24 90.0 ± 4.6 10 43.2 ± 8.9 6

n = number of testes in each group

Claims (16)

PATENT CLAIMS Use of compounds related to the glial cell line-derived neurotrophic factor (GDNF) family, which are glial cell line-derived neurotrophic factor (GDNF), GDNF-like factors or other compounds and derivatives which act as said GDNF on its signal transduction receptor from GDNF, GDNF-like factors or compounds or co-receptors thereof, for the manufacture of a composition for men comprising GDNF, GDNF-related compounds or compounds acting as GDNF on GDNF receptors or co-receptors in an amount sufficient to prevent sperm differentiation, regulation spermatogenesis and infertility due to impaired sperm differentiation. Use according to claim 1 for the manufacture of compositions effective as male contraceptives comprising GDNF, GDNF-related compounds or compounds acting as GDNF at GDNF receptors or co-receptors in an amount sufficient to render the male unable to fertilize the female. Use of GDNF-related compounds for the manufacture of a composition effective as a male contraceptive according to claim 2 comprising an amount of GDNF, a GDNF-related compound or a compound acting as GDNF at GDNF receptors or co-receptors such that the concentration of said compound in man is 50-5000, preferably 50-500, and preferably 50-100 times higher than the concentration of endogenous GDNF in the male testes. 4. A male contraceptive comprising glial cellular neurotrophic factor-related compounds · · • · • • • · • ··· • · · · • • • · ···· * · • · • • • · • • • • • • · • • • · · · · • • · · · lines (GDNF), capable of preventing sperm differentiation, regulating spermatogenesis and inducing infertility due to impaired sperm differentiation. The male contraceptive of claim 4, wherein the GDNF family-related compound is GDNF, a GDNF-like factor or derivative thereof with the same effect on GDNF receptors or co-receptors as GDNF or GDNF-like compounds. A male contraceptive according to claim 5, characterized in that the GDNF-like factor is selected from the group consisting of persephine, neurturin and artemin or derivatives thereof acting on GDNF signaling receptors or GDNF-like factors or co-receptors thereof as well as GDNF or similar factors GDNF. The male contraceptive of claim 4, wherein the GDNF family-related compounds are GDNF and derivatives thereof. The male contraceptive of claim 4, wherein the GDNF family-related compounds are persephine and derivatives thereof. The male contraceptive of claim 4, wherein the GDNF family-related compounds are neurturin and derivatives thereof. The male contraceptive of claim 4, wherein the GDNF family-related compounds are artemin and derivatives thereof. ··· ·· ·· ········································· · ···· ·· ·· · ·· The male contraceptive of claim 5, wherein the signal transducer receptor of compounds related to the GDNF family is a cRet receptor tyrosine kinase. The male contraceptive according to claim 5, wherein the receptor-activating co-receptors transmitting signals from compounds related to the GDNF family are receptors α: for the GDNF family (GFRα). 13. A composition for men comprising a compound related to the glial cell line (GDNF) family of neurotrophic factor (GDNF) in an amount sufficient to prevent sperm differentiation, control of spermatogenesis, and cause infertility due to sperm differentiation disorder in combination with pharmaceutically acceptable carriers or excipients useful for the particular mode of administration. The composition of claim 13, wherein the amount of glial cell line-derived neurotrophic factor (GDNF) family related compounds in a composition causing male inability to fertilize a female is such that the concentration of the compound administered in the male is 50-5000 times the concentration endogenous GDNF in male testes. The composition of claim 13, wherein the amount of glial cell line-derived neurotrophic factor (GDNF) family related compounds in a composition causing male inability to fertilize a female is such that the concentration of the compound administered in the male is 50-500 times the concentration endogenous GDNF in male testes. ·· • · • • · · • · · · • · · · • · • · • · · • • • · ···· ·· · • · • · • · · • · • • ···· • · · · • · · · · · The composition of claim 13, wherein the amount of glial cell line-derived neurotrophic factor (GDNF) family related compounds in a composition causing male inability to fertilize a female is such that the concentration of male compound administered is 50-100 times higher than the concentration endogenous GDNF in male testes.