DE102005028453A1 - Method for determining and influencing the fertility of male mammals and humans - Google Patents

Abstract

The present invention relates to a method for determining the fertility of male mammals and humans, a method of contraception and a method for increasing the fertility of male mammals and humans. Further, the present invention relates to the use of an inhibitor of the T1R3 receptor for contraceptive use in mammals and humans and to the use of a potentiating ligand for the T1R3 receptor to increase the fertility of male and human mammals.

Description

The The present invention relates to a method for the determination of fertility male mammals and humans, a method of contraception and a procedure for increase of fertility male mammals and of man.

fertility or fertility is one of the most important prerequisites for the development of organisms and can always reproduce again. For this reason, fertility determinations are more male mammal and the increase their fertility from central to the process of reproduction.

to Determination of fertility male mammals and man are aware of a number of standard methods that often on a recording of the morphology of the sperm cells of the examined Individual, the collection of sperm concentration and / or the Capture of sperm motility based. However, the evaluation criteria of some of these Tests are relatively inaccurate. Moreover, part of them can only be found in the preovulatory Phase performed become. At the Sims-Huhner 'test For example, 2 to 6 hours after cohabitation will be mucus from the cervical canal taken, and on the presence and the number of movable Sperm examined. There are plenty of mobile cervical mucus normal sperm, the test is considered positive, i. the sperm of the man is likely to be considered fertile, and it lies no functional cervical disorder in front.

task The present invention is therefore a method of determination fertility male mammals and humans available to provide, which is easy to perform and conclusions on the fertility of the examined individual. It is also desirable to have a method for increasing male fertility mammals and a method of contraception in mammals. All procedures should also the biological and hormonal household of an individual preferably little influence.

According to the invention this Task by the characterizing part of claims 1, 14 and 20 and by the requirements 19 and 25 solved.

in the The scope of the present invention has been found and demonstrated that the taste receptor subtype T1R3 is different in the acrosome area Mammalian sperm, i.e. in the vesicular Cap in the head area of the sperm, which has an important function regarding the Preparation for the actual fusion of the sperm with the egg comes to occur.

This Receptor plays after initial knowledge an important role the pathfinding of sperm to fertilizable oocytes and initiation the fusion process between sperm and egg cells. After the until now obtained results of the present invention is the T1R3 receptor is therefore an indicator of male fertility mammals and of man. This was particularly unexpected because the T1R3 receptor has so far only been characterized as a taste receptor.

The amino acid sequences in different mammalian species are expressed T1R3 receptor known and for example in WO 03/001 876 A2 and US 2004/0 219 632 A1. From the amino acid sequences can be suggest that the T1R3 receptor is approximately 850 amino acids in length Protein with seven transmembrane helices and a long N-terminal Domain is. From US 2004/0 185 469 A1, US 2004/0 175 793 A1, US 2004/0 191 862 A1 and WO 03/001 876 A2 it is known that the T1R3 receptor in conjunction with the T1R2 receptor, a sweet carbohydrate taste receptor, artificial sweeteners as well as sweet proteins whereas the T1R3 receptor in conjunction with the T1R1 receptor, a receptor for umami taste (glutamate) results. In addition, in US 2004/0 219 632 A1, the expression of the T1R3 receptor detected in taste buds comprehensive tongue tissue, whereas tongue tissues without taste buds have this receptor not expressed. In addition, the transcription of the T1R3 receptor gene was observed in testes, thymus and brain, with the corresponding Transcripts, however, shorter were than those tongue tissues containing taste buds. Furthermore, the nucleotide sequence of the human T1R3 receptor in the US 2004/01 320 775 A1 and US 2004/0 219 632 A1. A Heterologous Expression Method for the Human T1R3 Receptor is known for example from US 2004/0 191 862 A1.

According to the invention there is now provided a method for determining the fertility of male mammals and humans, wherein the content of T1R3 receptors in sperm of the subject to be examined is determined in vitro. Since the method according to the invention can easily be standardized, it can be used both in human medicine, in particular in the monitoring of in vitro fertilization or in fertilization diagnoses and Fertili be used in the context of modern livestock breeding.

Preferably quantification of T1R3 receptor content in sperm, by comparing determined measured data with a reference value. That way you can reliable Conclusions on the fertility of the examined individuals. As a reference value is usually a normal T1R3 receptor level in sperm. This one will For example, determined from comparison curves or comparison tables or in the form of comparative values. Preferably determine it parallel to the analysis of the sample of the examined Individuals by analysis of a reference sample, the one defined content of recombinantly expressed or from HEK cells has recovered T1R3 receptor. In the latter case, systematic Error in execution be excluded from the provision. Is the content of T1R3 receptor the sample below the value of the reference sample, this is an indication on lack of fertility the sperm.

According to one first preferred embodiment The present invention determines the T1R3 receptor content by detection of the T1R3 receptor transcript in the sperm by polymerase chain reaction (PCR). in principle can PCR with chromosomal DNA isolated from testes as template DNA and suitable primers become. The latter are at an amplification of at least one Part of the gene region coding for the T1R3 receptor in the DNA send comprehensive nucleic acid sequence customized. Following the PCR, the PCR product, for example by sequencing, on agreement with a functional one T1R3 receptor coding sequence analyzed.

Around but more reliable information about one actually transcriptional transcription of the T1R3 receptor gene and thus via the Formation and content of T1R3 receptor in the sperm, an RT-PCR is performed, wherein first Total RNA or mRNA from testes of the individual to be examined is isolated. This is then followed by reverse transcriptase transcribed into cDNA and the latter of a PCR with suitable primers subjected.

Preferably For example, RT-PCR employs two or more primers attached thereto adapted to at least a portion of the T1R3 receptor coding gene region in the DNA comprising nucleic acid sequence to amplify

Reliable results are obtained, in particular, when a quantitative PCR or quantitative RT-PCR is carried out, with a real-time PCR using fluorescence-labeled probes or using fluorescent substances intercalating into the PCR products formed being particularly suitable Has. Suitable systems for this include the Light Cycler ^® real-time PCR system and the Taqman ^® system from Roche. Of course, the quantitative PCR can also be carried out with any other method known to the skilled person for this purpose.

According to one second preferred embodiment The present invention determines the T1R3 receptor content in sperm using immunological methods that target proteins are directed. In particular, immunocytochemical methods, a Determination by radioimmunoassay (RIA) or a determination by an "enzyme linked immunosorbent assay "(ELISA) have proven to be particularly suitable.

at The latter method is usually the first to examining sample on a surface of, for example, a microtiter plate, a Immobilized polyvinyl chloride plate or a suitable tissue plate, before a for the protein of specific antibodies to be identified, here a specific antibody for the in the acrosome area of the sperm present T1R3 receptor, abandoned becomes. If the sample contains the T1R3 receptor, the antibody binds the on the surface immobilized sample. After a washing step, the surface is finally with a second secondary antibody detecting the T1R3 receptor antibody (commercial available), with an enzyme such as an alkaline phosphatase or peroxidase is detected. After a renewed Washing is finally a colorless or non-fluorescent Substrate for that on the second antibody added bound enzyme. This enzyme turns the substrate into a colored or fluorescent Product whose concentration is determined by a suitable detector, For example, a photometer can be determined. With the ELISA method Even small amounts of the T1R3 receptor can be released quickly and easily reliable prove. Another advantage of this method is that it is in use ELISA devices and commercially available Microtiter plates in 96, 256 or even 1024 format is operable, which is why a big one Number of analyzes at the same time.

In the RIA method, in principle, the same steps as in the ELISA method take place, except that instead of a conjugated with a suitable enzyme second antibody radiolabelled second antibody is used. The quantitative detection takes place in this case, for example by means of a scintillation counter.

In the immunocytochemical method, the spermatozoa of the individual to be examined are preferably fixed on a suitable surface and, after subsequent blocking of the remaining surface sites, incubated with a T1R3-receptor-specific antibody. Thereafter, the bound first antibody is treated, for example, with a commercially available FITC-conjugated anti-rabbit IgG, and visualized for fluorescence microscopy, before being allowed to fluoresce, as in 2 seen, recorded by fluorescence microscopy. Not only the amount of T1R3 receptor but also its exact location in the sperm of the individual to be examined can be determined with this method.

apart Of the aforementioned methods, the determination of the T1R3 receptor content in sperm also with other immunological known to those skilled in the art Methods are quantified, with only for example, the "Western Blot" method or the Called dot blot method.

According to one third preferred embodiment The present invention determines the T1R3 receptor content in the sperm or their reactivity by from the to be examined Individual isolated sperm with one or more flavors, speak the natural Binding partners of the T1R3 receptor, contacted and stimulated become. These include sweeteners like the course sugar, sucrose, glucose, maltose, galactose, lactose and fructose and the synthetically produced compounds acesulfame K, saccharin. Aspartame, dulcine, cyclamate and SC-45647. To the sweeteners also belong Guanidine-1 Esssigsäure (GA-1), guanidine-2-acetic acid (GA-2), glycyrrhizic acid (GA), Neohesperidin Dihydrochalcone and Sweeteners proteinaceous, preferably monellin, thaumatin, mabinlin, Pentadine, Brazzein, Curculin and Miraculin. Finally, it is possible at least one amino acid, preferably one of glutamate, L-alanine, L-proline, L-serine, L-aspartate, L-asparagine, L-arginine and combinations thereof Group selected amino acid or their corresponding salt, in particular alkali or alkaline earth metal salt as a flavor and stimulant for the T1R3 receptor in sperm deploy. After contacting, so after binding of the flavor to one for that provided binding site on the T1R3 receptor, the cytosolic calcium concentration in the sperm with the help of membrane-permeable calcium-sensitive dyes, for example Fura2 or Fluo determined.

According to one fourth preferred embodiment The determination of the content is made in accordance with the present invention intact T1R3 receptor in the sperm by determining the taste profile from male mammals or preferably by men for example, with the help of the "two bottle preference test "or other taste tests. These methods for determining thresholds for certain Flavors (creation of a taste profile) are those skilled in the art (Feigin et al., Neurosci Biobehav Rev.11, pp. 231-240, 1987; Ruiz et al., Chemical Senses, 28, pp. 573-579, 2003) and are routinely administered to mammals and People used.

Preferably is in the embodiments three and four the threshold of one or more flavorings of the individual to be examined. The inventive method Can be used for both medical and non-medical purposes respectively.

One Another object of the present invention is a method for contraception mammals and humans, where a male mammal or at least one inhibitor for the T1R3 receptor is administered to a human.

inhibitors according to the invention are substances that interact with the T1R3 receptor and aggravate an attachment of flavors to it or Completely prevent (competitive and non-competitive antagonists). Especially with a mixture of lactisol, gurmarine, cyclohexylacetic acid, indoleacetic acid and Combinations of this existing group selected compound become special get good results. Also derivatives of these compounds are among the Inhibitors. In the same way, one or more representatives of one Administered a group of compounds that are able to become firmly attached to receptors for the sweet taste to bind.

Everyone Inhibitor may be used separately, in combination with one or more others Inhibitors and / or together with pharmaceutically acceptable excipients, speak to be administered as a pharmaceutically acceptable composition, the ratio from inhibitor to carrier by the solubility or chemical nature of the inhibitor, by the selected route of administration and / or determined by common practice in the pharmaceutical practice becomes.

Pharmaceutically acceptable compositions may also contain the inhibitor (s) in the form of a salt, ester, amide or as a "prodrug", that is to say as an inactive inhibitor precursor For the local administration of the inhibitor they comprise ointments, Powders, sprays or inhalants. Said compositions are prepared by mixing the inhibitor under sterile conditions, with a physiologically acceptable carrier and possible colors and flavors, buffers or propellants, as needed.

The Administration of the inhibitor or its pharmaceutically acceptable Composition is preferably oral, sublingual, rectal, parenteral, intracisternal, intrathecal or intravesical (by instillation in the bladder).

Around to prevent digestion of the inhibitor in the gastrointestinal tract he or his pharmaceutically acceptable composition is particularly preferred fed parenterally. Possible Forms of parenteral administration are an intravascular, for example intravenous Administration (with a syringe into a vein), an intramuscular injection (into a skeletal muscle), a subcutaneous injection (under the skin), a transdermal (e.g., with a "patch"), intragenital administration into the genital organ, an intrapulmonary dose (as aerosol or Injection), an intraperitoneal injection (into the abdominal cavity), one intracardiac injection (into the heart) or an application to the mucous membranes (e.g. the genital mucosa, the nasal mucosa or locally on the Conjunctiva), or locally as powder, ointment, drops or in spray form (Aerosol) preferably in the genital area. Parenterals include injections, Infusions, powders for solution, concentrates and implants.

Around to achieve the same effect as with parenterally administered amounts, have to if administered orally, larger quantities be used on inhibitor.

With the method according to the invention can Pregnancies can be prevented easily and reliably without that side effects in the treated individuals are to be feared. Across from the conventional one Contraception methods like hormonal contraception for women, for example, estrogens or gestagens are used, or hormonal contraception for men the base of testosterone, progestogen and etonogestrel, respectively associated with significant side effects, provides the inventive contraceptive method considerable advantages. It is compared to the known birth control methods cost-effective, shows almost no side effects and is very reliable. The inventive method Can be used for both medical and non-medical purposes respectively

It also concerns the present invention the use of an inhibitor for the T1R3 receptor for contraception Mammals and People. As one skilled in the art realizes, the use of both can be medical as well as non-medical purposes.

One Another object of the present invention is a method to increase fertility male mammals or human, in which a male mammal or human at least a ligand potentiating the recognition of the T1R3 receptor, in particular a positive allosteric modulator is administered.

potentiating Ligands according to the invention are molecules that bind to a binding site bind to the T1R3 receptor spaced from the binding site for flavorings or inhibitors is arranged. Interacting with the receptor potentiating ligands cause this to have an even greater affinity for flavorings or inhibitors and / or binds them more tightly. On off of monophosphates, in particular of inosine monophosphate (IMP) and guanosine monophosphate (GMP) and combinations thereof Group selected Ligand gives particularly good results. Also derivatives of these monophosphates counting to the potentiating ligands.

Everyone potentiating ligand settles separately, in combination with one or more other ligands and / or together with pharmaceutically acceptable excipients, speak to administer as a pharmaceutically acceptable composition, wherein The relationship from ligand to carrier by the solubility or chemical nature of the ligand, by the selected route of administration and / or by procedures commonly used in the pharmaceutical practice is determined.

pharmaceutical acceptable compositions can the ligand (s) also in the form of a salt, ester, amide or as a "prodrug", ie as inactive Ligand precursor. For the local administration of the ligand, they include ointments, powders, sprays or inhalants. Received named composition one, by the ligand (s) under sterile conditions with a physiologically acceptable carrier and possible Colorants and flavorings, buffers or propellants, as appropriate, is mixed.

The Administration of the ligand or the pharmaceutically acceptable composition is preferably oral, sublingual, rectal, parenteral, intracisternal, intrathecal or intravesical (by instillation into the bladder).

To prevent digestion of the ligand in the gastrointestinal tract, he or his pharmaceutically acceptable composition besondes preferably fed parenterally. Possible forms of parenteral administration are: an intravascular, for example intravenous administration (with a syringe into a vein), an intramuscular injection (into a skeletal muscle), a subcutaneous injection (under the skin), a transdermal administration (eg with a "plaster") ), an intragenital administration into the genital organ, an intrapulmonary administration (as aerosol or injection), an intraperitoneal injection (into the abdominal cavity), an intracardiac injection (into the heart), or an application to the mucous membranes (eg to the genital mucosa, the Nasal mucosa or locally on the conjunctiva), or locally as a powder, ointment or drop or in spray form (aerosol) preferably in the genital area. Parenterals: include injections, infusions, powder for solution, concentrates and implants.

Around to achieve the same effect as with parenterally administered amounts, have to if administered orally, larger quantities be used on ligand. The inven tion proper method can both medical as well as non-medical purposes.

Further The present invention relates to the use of a recognition of the T1R3 receptor potentiating ligand, especially a positive one allosteric modulator for increasing male fertility mammals and people. As one skilled in the art realizes, the use of both for medical as well as non-medical purposes.

Further Features, details and advantages of the invention will become apparent the wording of the claims and the following description of exemplary embodiments with reference to FIG Drawings. Show it:

1 an agarose gel with the PCR products obtained in Example 1 and

2 an immunocytochemical detection of the T1R3 receptor in isolated sperm of the mouse according to Example 2.

example 1

To the Detection of expression of the T1R3 receptor in testis became a RT-PCR with mouse testis tissue and tissue circumvallater taste papillae carried out as a positive control (CV).

For this purpose, total RNA from mouse testicular cells and total RNA from circumvallated taste buds of a mouse were first isolated using a standard molecular biological method. Subsequently, the RNA of both samples was separated into cDNA by hybridizing the samples with an oligo-dT primer or "random" hexamer primer and incubated with reverse transcriptase after addition of a deoxynucleotide mixture, after which a subset of the thus obtained PCR-amplified samples with a primer pair specific for the T1R3 receptor before each of a subset of the resulting amplification products was spiked with DNA sample buffer and electrophoresed on an agarose gel 1 played.

As T1R3 receptor specific primer pair a mouse specific T1R3 primer pair was used with the sequence 5 'TGA GCT GGG CAA ACT GGC TA 3' as coding or sense primer and the sequence 5 'TCT TGG CAT TCC TTC CCA GG 3' as not coding or antisense primer. The PCR reaction was carried out for one minute at an annealing temperature of 60 ° C in a total volume of 50 μl. This contained 20-100 ng mouse cDNA, 0.4 μM of each primer, 0.4 mM dNTP, 1.5 mM MgCl ₂ and 2.5 U Taq DNA polymerase (Master Taq Kit, Eppendorf, Hamburg). To determine the size of the amplification products by molecular weight, a standard molecular weight marker was placed on the far left of the agarose gel.

Like from the 1 As can be seen, PCR products of the T1R3 receptor with an expected size of 510 base pairs (bp) were obtained both with the tissue from the mouse testes and the tissue from the taste papillae. This shows that the T1R3 receptor, which has hitherto only been characterized as a taste receptor, is expressed not only in taste buds but also in mammalian testes.

Example 2

Around to locate the T1R3 receptor expressed in the mouse testis, Sperm were isolated from the epididymis (epididymis) of the mouse and examined immunocytochemically.

For this The sperm were fixed with ice-cold methanol on a microscope slide and after that Blocking with PBS / 10% FCS with a T1R3 receptor specific antibody at 4 ° C overnight incubated. The T1R3-specific antibody is against the amino acid sequence 239-254 of the T1R3 mouse receptor (Damek et al., Science, 301, pp. 850-853, 2003). The used T1R3-specific antibodies against the human receptor recognizes the amino acid sequence 829-843 (Damek et al., Science, 301, pp. 850-853, 2003).

After that became the bound antibody with a commercially available FITC-conjugated anti-rabbit IgG treated the former for the Fluorescence microscopy was visualized before being so prepared slides under the microscope.

The respective frames of the 2A and B show at the top left a nuclear staining with propidium iodide, the picture next to it shows the corresponding transmitted light image; below this, the labeling of the T1R3 receptor can be seen by detection with the FITC-labeled antibody. The illustration at the bottom right shows the superimposition of all three single shots. It can be seen that the T1R3 receptor is located exclusively in the acrosome area of the sperm.

Claims (25)

Method for determining the fertility of male mammals and humans, characterized in that the content of T1R3 receptors in sperm of the individual to be examined is determined in vitro. Method according to claim 1, characterized in that that the determined T1R3 receptor content with a reference value is compared. Method according to claim 1 or 2, characterized that the determination of T1R3 receptor content in the sperm by Polymerase chain reaction (PCR) and preferably by quantitative PCR takes place. Method according to claim 3, characterized that in the PCR as a DNA template cDNA from testes of the examined Individual is used. Method according to claim 3 or 4, characterized that two or more primer pairs are used in the PCR, which are adapted to at least part of the T1R3 receptor coding gene region in the DNA comprising nucleic acid sequence to amplify. Method according to claim 1 or 2, characterized that the determination of the T1R3 receptor content in the sperm by means of an immunological method. Method according to Claim 6, characterized that the determination of T1R3 receptor content in the sperm immunocytochemical, by a radioimmunoassay (RIA) or an enzyme-linked immunosorbent Assay (ELISA) takes place. Method according to claim 6 or 7, characterized that in the immunological method a specific for the T1R3 receptor antibody is used. Method according to claim 1 or 2, characterized that the determination of T1R3 receptor content in the sperm by Contact the sperm of the individual to be examined with to the T1R3 receptor binding substances, preferably with the T1R3 receptor-binding flavors, and subsequent measurement the cytosolic calcium concentration in the sperm by means of membrane permeable calcium-sensitive dyes is determined. Method according to claim 9, characterized in that that at least one sweetener is used as the flavoring agent. Method according to claim 9 or 10, characterized that one from sucrose, glucose, maltose, galactose, lactose, Fructose, acesulfame K, saccharin. Aspartame, dulcin, cyclamate, SC-45647, Guanidine-1 Esssigsäure (GA-1), guanidine-2-acetic acid (GA-2), glycyrrhizic acid (GA), neohesperidin dihydrochalcone and combinations thereof Group selected Flavor is used. Method according to claim 9 or 10, characterized characterized as sweetener at least one sweetener with Protein character, preferably one of monellin, thaumatin, Mabinlin, pentadine, brazzein, curculin and miraculin and combinations sweet protein selected from the group consisting of flavor is used. Method according to claim 9, characterized in that that at least one amino acid, preferably one of glutamate, L-alanine, L-proline, L-serine, L-aspartate, L-asparagine, L-arginine and combinations thereof existing group selected amino acid or their corresponding salt, in particular alkali metal or alkaline earth metal salt is used as a flavor. Method of contraception in mammals and humans, thereby marked that a male mammal or administering to a human at least one inhibitor of the T1R3 receptor becomes. Method according to claim 14, characterized in that the existence from the lactisol, gurmarine, cyclohexylacetic acid, indoleacetic acid and Combinations thereof existing group selected inhibitor used becomes. The method of claim 14 or 15, since characterized in that the inhibitor is administered as a pharmaceutically acceptable composition containing the inhibitor, for example in the form of a salt, ester, amide or as a "prodrug". Method according to one of claims 14 to 16, characterized that the inhibitor is used orally, sublingually or rectally. Method according to one of claims 14 to 16, characterized that the inhibitor parenterally, intravascularly, for example, intravenously, subcutaneously or transdermal, intragenital, local as powder, ointment or drops or supplied in the form of an aerosol or by order on the mucous membranes. Use of an inhibitor of the T1R3 receptor for contraception mammals and people. Method for increasing male fertility mammals or man, characterized in that a male Mammal or the human being at least one potentiation of the recognition of the T1R3 receptor Ligand, in particular a positive allosteric modulator administered becomes. Method according to claim 20, characterized in that that one from monophosphates, in particular from inosine monophosphate (IMP) and guanosine monophosphate (GMP) and combinations thereof Group selected Ligand is used. Method according to claim 20 or 21, characterized that the ligand is administered as pharmaceutically acceptable compositions containing the ligand, for example in the form of a salt, ester, Amids or as a "prodrug" includes. Method according to one of claims 20 to 22, characterized that the ligand is used orally, sublingually or rectally. Method according to one of claims 20 to 22, characterized that the ligand is parenteral, intravascular, for example intravenous, subcutaneous or transdermal, intragenital, local as powder, ointment or drops or supplied in the form of an aerosol or by order on the mucous membranes. Use of a recognition of the T1R3 receptor potentiating ligands, in particular a positive allosteric Modulators to increase of fertility from male mammals and people.