KR20110002407A - Novel biomarkers for predicting fertility of sperm - Google Patents

Abstract

PURPOSE: A marker for predicting fertility of animal is provided to predict fertility and to stably produce animal of high quality. CONSTITUTION: A marker for predicting animal fertility contains porin, ropporin, enolase 1A, or actin-related protein T. A method for obtaining the marker for predicting fertility of animal comprise; a step of classifying sperms which have high fertility and low fertility; a step of treating the sperms with heparin; a step of isolating protein form the sperms; and a step of comparing expression level of the isolated protein to select protein of which expression is increased or decreased.

Description

Marker for predicting fertility of animal sperm {Novel biomarkers for predicting fertility of sperm}

The present invention relates to markers and methods for predicting the fertility and modality of animal sperm acquisition. More specifically, to identify mRNA or protein that is expressed differently according to the animal's fertilization ability, including a marker for predicting fertilization ability of at least one animal sperm selected from the group consisting of the mRNA or proteins and antibodies specific for the marker It relates to a method for predicting the fertilization ability of the animal sperm using a kit comprising a composition, a primer specific for the gene encoding the marker, and the like.

Artificial insemination (AI) has been used to improve the rapid supply of valuable genes and the genetic quality of animal lineage. In case of cows, cow frozen semen is used mainly for artificial fertilization, which plays an important role in genetically improving cows' production capacity through the spread of specific genes.

However, artificial seeds have only about 50% success rate. Since a single bull frozen semen is used to conceive thousands of cows, the production of bulls with high fertility has a very high economic value in increasing pregnancy rates. Sperm counts, motility, morphological aspects (Bonde et al., 1998), sperm membrane damage (Thundathil et al., 1990), cervical semen or oocyte penetration (Sharara et al., 1995) Although the back has been studied to determine the fertility of the animal, it has not been proved that the above factors also affect the fertility of cattle. Moreover, bulls that have proven to have high fertility in vivo may have different fertility in vitro (Palma and Brem, 1994).

For these reasons, research on an efficient method for predicting the fertility of an animal is necessary, and in particular, it is urgent to obtain markers related to fertility in cattle.

Mammalian sperm require some time to develop physiological changes and acquire fertilization. This change is called capacitaion (Austin, 1952). Fertility gains include tyrosine kinase by cyclic AMP, lipids in the sperm membrane, protein changes, ion migration, sperm motility, protein transport, protein phosphorylation (Zaneveld et al., 1991; Chang, 1995) has been reported to be relevant. Since capacitation and fertility are deeply related, fertility acquisition status is very important in determining fertility. Fraser (1995) et al. Proposed that the use of the fluorescent antibiotic chlorotetracycline (CTC) in the presence of calcium can determine whether to obtain fertility. Many researchers have demonstrated the link between fertility status and fertility with CTC, but the molecular mechanisms underlying sperm gain are unknown.

Proteome analysis provides a good way to understand the comprehensive process of molecular function of sperm related to fertility, so previous studies have used proteomic analysis of molecular markers of proteins involved in fertility and acquisition of fertility. Roncoletta et al. (2006) define many proteins for bull fertility through 2-dimensional electrophoresis (2-DE). Peddinti et al. (2008) argued that sperm from bulls with high fertility showed expression of proteins involved in energy metabolism, intercellular communication, spermatogenesis, and cell movement. In particular, cytoskeleton proteins and tyrosine phosphoproteins are related to the acquisition of fertility, and the above mentioned groups of proteins are also highly associated with the process of fertility acquisition. In many types of proteome studies, mass spectrometry (MS) is widely used in this field to identify proteins through 2-DE, peptide sequencing to separate proteins.

As described above, in order to obtain sperm with excellent fertility, it is urgently necessary to find molecular mechanisms and markers affecting fertility and acquisition of fertility.

Accordingly, the present inventors have made an effort to confirm that, in the case of a biomarker using a protein having a difference in expression according to an animal, in particular, a cow's fertilization ability, it is possible to select an animal having a high fertilization ability, thereby completing the present invention.

It is therefore an object of the present invention to provide markers for predicting fertility of animal sperm.

Another object of the present invention is to provide a method for obtaining a marker for predicting fertilization ability of animal sperm.

Still another object of the present invention is to provide a composition for predicting fertilization ability of sperm comprising an antibody of a marker for predicting fertility of an animal sperm and a primer specific for a gene encoding the marker.

Still another object of the present invention is to provide a method for predicting the fertilization ability of the animal sperm and the acquisition mode of fertilization.

The present invention relates to bovine mitochondrial F1-ATPase (Bovine) in combination with porin, ropororin, enolase 1, actin-related protein T2 and aurovertin B. mitochondrial F1-ATPase complexed with Aurovertin B) at least one selected from the group consisting of markers for predicting fertility of the animal, characterized in that increased expression in the semen of the animal.

As used herein, the term marker for predicting fertility of an animal refers to an organic biomolecule that is expressed differently according to sperm fertilization ability. Organic biomolecules include, but are not limited to, for example, polypeptides, proteins, nucleic acids, lipids, glycolipids, glycoproteins, sugars, and the like. The marker for predicting fertility in the present invention may be a protein that is overexpressed in other sperm or a nucleic acid encoding the same.

In addition, the present invention comprises the steps of (1) classifying sperm collected from the animal into sperm with high fertilization ability and low fertilization ability; (2) treating each of the classified sperm with heparin to induce fertility gain; (3) separating the fertilization ability of step (2) from the obtained high fertilization sperm and low fertilization sperm; And (4) comparing the expression amounts of the proteins separated from the sperm with high fertilization ability and sperm with low fertilization ability, respectively, and selecting the protein whose expression is increased or decreased. It is about a method.

The cow bound to Porin, Roporin, Enolase 1, Actin-related protein T2, and aurovertin B disclosed in the present invention through the method of obtaining the marker. Except for mitochondrial F1-ATPase complexed with Aurovertin B, markers for predicting fertility of another animal may be obtained.

In the present invention, the animal is preferably characterized by a bovine.

In the above method of the present invention, the classification step of (1) may be performed by a dual dyeing method.

The dual staining method comprises the steps of: (1) adding sperm to the H33258 solution, culturing in a light-blocking state; (2) suspending the pellets by centrifugation after removing excess staining; (3) observing the suspension under epifluoresce illumination using UV and BP filters.

The suspension may comprise PBS, which may consist of 750 μΜ CTC, 20 μΜ Tris, 130 μΜ NaCl and 5 μΜ cystein.

In the above method of the present invention, classification of sperm fertilization ability includes classifying according to the sperm film pattern. According to the sperm's membrane pattern, it can be classified into F (when living and incapacitating ability), B (when living and incapable of acquiring ability), and AR (when living and acrosome responds).

In the method of the present invention, the step of selecting the protein of (4) can be analyzed the concentration of the spot using PD Quest.

According to the embodiment of the present invention, the protein expressed before the fertility can be obtained, 287 protein spots in the sperm with low fertilization ability, 301 protein spots in the sperm with high fertilization ability can be found. According to the embodiment of the present invention in which the concentration of the spot was analyzed using PD-Quest, 12 proteins were highly expressed in sperm having low fertility, and 1 protein was highly expressed in sperm having high fertility. As a result of analyzing these 13 protein spots using LC / MS-MS, highly expressed porin was obtained at low fertilization ability.

Furthermore, according to an embodiment of the present invention, after inducing fertilization gain through heparin treatment, the protein levels of sperm and sperm with low fertility were analyzed and 592 and 394 protein spots were expressed in each sperm. .

In the embodiment of the present invention as a result of analyzing the concentration of the spot using PD-Quest, a total of eight protein spots showed different expression patterns according to the ability to modify. Four of them were highly expressed in sperm with low fertility, and four of them were highly expressed in sperm with high fertility. Analysis of these 8 protein spots using LC / MS-MS revealed that ropoorin inhibits motility in individuals with low fertility and enolase 1 involved in the production of abnormal and immature sperm. Was highly expressed. On the other hand, actin-related T2, which is involved in germ cell division after fertilization, acrosome reaction and fertilization, was highly expressed in individuals with high fertility. In addition, the bovine mitochondrial F1-ATPase complexed with Aurovertin B protein coupled with aurovertin B, which was newly discovered in the present invention, was highly expressed in individuals with high fertilization ability.

The porin is NCBI gi No. A protein represented by 190201, ropporin is a protein represented by NCBI gi No.74002908, and enolase 1 is represented by NCBI gi No. The protein represented by 87196501, actin-related T2 (actin-related T2) is a bovine mitochondrial F1-ATPase complexed with Aurovertin bound to a protein represented by NCBI gi No.84000199, aurovertin B. B) is a protein represented by NCBI gi No.1827812.

Another aspect of the present invention provides a composition for predicting fertility of an animal comprising an antibody specific for a protein showing a difference in expression according to the fertility of the animal.

 The present invention also provides a composition for predicting fertility of an animal comprising a primer or probe specifically binding to a nucleic acid encoding a protein showing a difference in expression amount according to the fertility of the animal.

Proteins showing a difference in expression levels include porin, roporin, enolase 1, actin-related protein T2 and auromitin B in combination with Chodrial F1-ATPase (Bovine mitochondrial F1-ATPase complexed with Aurovertin B).

Preferably, the predictive composition of the present invention may be provided in the form of kits, microarrays and DNA and protein chips.

Compositions for predicting fertility in animals provided herein may require specific additional equipment, such as PCR cycles, microarray readers, or FACS devices.

One aspect of the compositions of the present invention is that the expression of Porin, Ropoorin and Enolase 1 is upregulated in the sperm of animals with low fertility, and therefore, Porin, Ropoorin And a primer or probe that specifically binds to an antibody or encoding nucleic acid for enolase 1, and may be a composition that analyzes whether the fertilization ability is increased compared to protein level or mRNA level of sperm. .

Another aspect of the compositions of the present invention is that the expression of actin-related protein T2 and bovine mitochondrial F1-ATPase complexed with Aurovertin B combined with aurovertin B is capable of modifying. Antibodies to bovine mitochondrial F1-ATPase complexed with Aurovertin B combined with actin-related protein T2 and aurovertin B because they are upregulated in the sperm of these high animals Or a primer or probe that specifically binds to the encoding nucleic acid and analyzes whether the fertilization ability is increased compared to the protein level or mRNA level of low sperm.

The antibodies can be prepared using techniques well known in the art. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fν molecules. , Chimeric antibodies and the like.

Since the marker proteins of the present invention are known proteins, the antibodies used in the present invention can be prepared by conventional methods well known in the field of immunology using the known proteins as antigens.

Proteins used as antigens of the antibodies according to the invention can be extracted or synthesized in nature and can be prepared by recombinant methods based on DNA sequences. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.

For example, polyclonal antibodies can be produced by the method of injecting a protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

The diagnostic composition of the present invention may further comprise reagents known in the art for use in immunological assays in addition to antibodies specific for the protein.

Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

On the other hand, the diagnostic composition of the present invention can be provided in a fixed state using a variety of methods known on a suitable carrier or support to increase the speed and convenience of diagnosis (Antibodies: A Labotory Manual, Harlow & Lane Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography to detect a specific protein in a sample. The lateral flow assay kit includes a sample pad applied to the sample, a release pad coated with a detection antibody, a developing membrane (e.g., nitro) in which the sample is moved and separated and an antigen-antibody reaction occurs. Cellulose) or strip, and an absorption pad.

The microarray is generally intended to attach an antibody onto a slide glass surface treated with a specific reagent to detect a protein that specifically attaches to the antibody by an antigen-antibody reaction.

Those skilled in the art can easily determine the combination of various primer pairs having high specificity and sensitivity without causing non-specific amplification by considering variables such as the degree of hybridization between sequences in target genes based on the gene sequences of known markers. As used herein, the term "primer" refers to a short nucleic acid sequence having a short free hydroxyl group, which can form a base pair with a complementary template, and serves as a starting point for template strand copying. "Non-specific amplification" refers to nucleic acid amplification other than the target sequence that occurs by primers hybridizing to nucleic acid sequences other than the target sequence and acting as a substrate for primer extension.

Another example of an agent for measuring mRNA levels, "probe" refers to a nucleic acid fragment, such as RNA or DNA, which is short or several hundred bases, capable of specific binding with mRNA, and is labeled. The presence or absence of a specific mRNA can be confirmed. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like.

Primers or probes of the invention may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. It can also be modified by methylation, capping, or the like by known methods.

The composition for predicting fertility of the present invention may further include a reagent generally used in a method for detecting a nucleic acid. For example, metal ion salts such as deoxynulceotide triphosphate (dNTP), a heat resistant polymerase, and magnesium chloride required for a PCR reaction may be included, and include dNTP, a sequencease, etc. required for sequencing. Can be.

In addition, fertility prediction using the marker for predicting fertility of the animal sperm according to the present invention can be carried out by confirming the presence of a marker protein or nucleic acid of the present invention in a sample.

The present invention relates to (1) bovine mitochondrial in combination with porin, roporin, enolase 1, actin-related protein T2 and aurovertin B in a sample. Measuring mRNA or protein levels of a fertility predictor marker of at least one animal selected from the group consisting of F1-ATPase (Bovine mitochondrial F1-ATPase complexed with Aurovertin B); And (2) analyzing the increase or decrease in the amount of mRNA or protein expression.

The sample is characterized in that bovine sperm.

If the expression of porin (Porin), Ropoorin (Ropporin) and Enolase 1 (enolase 1) increases in the sample is characterized in that it further comprises the step of predicting the low fertility of the animal.

An increase in the expression of actin-related protein T2 and bovine mitochondrial F1-ATPase complexed with Aurovertin B in combination with aurovertin B resulted in an increase in animal fertility. The method is characterized by further comprising the step of predicting the high.

 According to the fertility of the present invention, the composition and method for predicting fertilization ability of an animal sperm provided through analysis of a protein expressed differently predict a fertilization ability and a fertility acquisition aspect of an animal sperm and have a high fertilization ability. It is very useful for easy selection and stable production of excellent animals.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art to which the present invention pertains. Will be self-evident.

Example 1 Material Preparation

1-1. Classification of semen source and fertility

Hanwoo frozen semen, which had normal fertility at the insemination (AI) stage, was obtained from Hanwoo improved in NACF. NRR (Non-Return Rate) values of Hanwoo frozen semen were obtained from the conception data of each farm. NRR was determined as the ratio of the number of cows that did not show estrus until later conception up to 60 days of the total number of cows. Fertility was expressed as NRR up to 60 days in bulls, ranging from 47.78 to 88.3% (p <0.05, Table 1).

[Table 1] Categorization of cattle by reproductive performance

Fertility ID No. of inseminated cows NRR (%) height 537 25 88.3 ± 1.84 641 15 80.56 ± 5.78 642 45 88.56 ± 2.73 Average 85 85.80 ^a lowness 550 90 51.31 ± 7.09 606 183 36.24 ± 4.38 622 79 56.80 ± 6.68 Average 352 48.12 ^b

1-2.Preparation of medium

100 mM NaCl, 3.1 mM KCl, 2.0 mM CaCl 2 H ₂ O, 0.4 mM MgCl 6H ₂ O, 0.3 mM mM Na ₂ HPO ₄ , 12H ₂ O, 21.6 mM sodium lactate, 25 mM NaHCO ₃ TALP medium containing 1.0 mM sodium pyruvate was used as a medium for the base.

The medium was incubated at a temperature of 39 ° C. with 5% CO _{2 in} air for the night before the day of the experiment. The acidity and osmoticity of the medium were maintained at 7.5 and 300 mOsmo / kg, respectively.

TALP medium to which 10 μg / ml of heparin (Sigma Chemical Co., St. Louis MO, USA) was added was used as a capacitation medium.

1-3.Sperm Preparation and Processing

Six cattle straws were frozen in two groups: sperm showing high fertilization and low fertilizing sperm in 39 ° C. water for 20 seconds. Frozen sperm were washed with a discontinuous Percoll density gradient.

Sperm layered in 45% and 90% Percoll were diluted with phosphate buffered saline (PBS). After centrifugation at 400 Xg for 20 minutes at room temperature, the supernatant was removed and the pellet was again suspended with 300 μl of TALP. The supernatant was then discarded after centrifugation for 5 min at 400 Xg (group without fertility; NC).

Sperm induced fertility gain at 400 μL CM, 5% CO ₂ , 39 ° C. for 20 min. After 20 minutes, the obtained sperm was centrifuged at 20,000 Xg for 2 minutes, and the supernatant was removed.

Example 2 Combined Hoechst 33258 / chlortetracycline Fluorescence Analysis (H33258 / CTC)

The fertility status is based on the addition of several substances to Perez et al. It was determined by the Dual staining method devised by (1996). In brief, 100 μl of sperm were added to 15 μl of H33258 solution and incubated at 39 ° C. for 5 minutes in a light-blocked water bottle. Excess staining was removed by layer separation of the mixture using 250 μl of 2% poly vinylpyrrolidone in PBS. After centrifugation at 400 Xg for 5 minutes, the supernatant was discarded and the pellet suspended in 100 μl of PBS. PBS is 100 μl of freshly prepared CTC solution (750 μM CTC in 5 mL buffer, 20 μM Tris, 130 μM NaCl, 5 μM cysteine).

Samples were observed with Nikon microphot-FXA under epifluorescence illumination using UV BP 340-380 / LP 425 and BP 450-490 / LP 515 excitation / emission filters for H33258 and CTC, respectively.

As shown in Table 2, sperm were classified as follows.

-Dead: Dead (Type D, with bright blue fluorescence on sperm head)

Live and capacitive: live non capacitated (F type, with bright green fluorescence evenly over the entire sperm head with strong or no fluorescence in the center)

Live capacitated (type B, dark fluorescence on the acrosome region and dark back of the acrosome)

Live acrosome reacted (AR type, green fluorescence on the sperm head or green fluorescence on the back of the acrosome or no fluorescence on the head)

All sperm had green fluorescence in the middle. Two slides for each sample were estimated to have at least 400 sperm per slide.

[Table 2] Definition of sperm film pattern by CTC staining

pattern describe F When the assessed sperm is fully fluorescent: characteristics of the sperm that do not acquire fertility B When expressed in band form or fluoresce only in the acrosome: fertility features AR Typical acrosome reaction sperm

Example 3 2-D Electrophoresis

Sperm samples were 7M urea, 2M thiourea, 4% CHAPS, 0.05% Triton X-100, 1% octyl beta-di-glucopyroside, 24 μM Solubilized at room temperature for 1 hour in 1 ml of rehydrated buffer containing phenylmethane sulfonyl fluoride, 0.1% DTT, 0.5% IPG buffer and 0.005% bromophenol blue.

Insoluble materials were removed by centrifugation at 20,000 Xg for 10 minutes and the supernatants collected. Protein amount was maintained in a 450 ul equilibrium sample solution. This solution was added to the IPG box kit and then placed on an IPG strip (24 cm, pH 3-11, non linear). This state was maintained for 24 hours at room temperature in consideration of the hydration time. The hydrated portion was then placed in a strip holder manifold (top of the gel) covered with mineral oil. Application time is designed as 100V (1 hour), 200V (1 hour), 500V (1 hour), 1000V (1 hour), 5000V (1.5 hours), 8000V (1.5 hours), and 40000Vhr as voltage gradient It was set as.

The focused strips were 10 ml of equilibration buffer [0.67 ml of 1.5 M Tris-cl (pH 8.8) / ml, 6 M urea, 30% in each strip containing 65 mM DTT for 15 minutes. (v / v) glycerol, 2% (w / v) sodium dodecyl sulfate and 0.005% (w / v) bromophenol blue] It was equilibrated with 135 mM iodine acetamide in 10 ml of equilibration buffer for 15 minutes.

Silver staining of the gel was proposed by Roncoletta et al. (1996) and proceeded with the addition of several compounds. Staining is divided into five stages: fixing, sensitizing, staining, developing and stopping.

Each gel was fixed in 5% (v / v) acetic acid and 50% (v / v) ethanol for 20 minutes and rinsed twice with distilled water (DW) for 20 minutes. Rinse twice with distilled water for 2 minutes for sensitizing in 0.02% (w / v) sodium thiosulfate. After staining the gel with 0.1% (w / v) silver nitrate, each was rinsed with distilled water for 1 minute. Protein spots were developed with 4% (w / v) sodium carbonate containing 0.1% (v / v) formaldehyde and each solution was used as clean. The developed gel stopped moving to 1% (v / v) acetic acid.

Example 4 Gel Analysis

A total of 12 gels were scanned at high resolution of GS-800 (Bio-rad). Protein spots of each gel were found with PDQuest (Bio-rad) and spotted while warping and matching all gel sets. Background substraction was used with 45 margins, and the actual number of protein spots was directly drawn from the gel. With a benchmark mark as the reference, gel matching was performed by automatically estimating the corresponding pair of spots. Twelve matched sets were found and protein amount reporters were generated.

In these experiments, the marked difference in spot volume was more than three times that of the software device. The amount of spots was read into the 'Spot parameters' window. The results were derived using the 'Comparisions window', and the numbers represented differently were compared with known volume ratios.

Example 5 Protein Degradation

The dry protein portions were removed by dissolving with a solution containing urea (8M), EDTA (5 mM), TCEP (10 mM) (10 μl) and incubated at room temperature for about 2 hours. In order to avoid the problem of excess alkylation, the space of 15 cysteines was not capped and continued to maintain safe TCEP to prevent the re-formation of disulfides. The removed proteins were diluted to 40 μl with 50 mM ammonium bicarbonate at pH 7.8 to provide the final 2M urea. Sequenced trypsin (20 pmol) (V5111, Pomega, Madison, Wis.) Was added to the protein solution and cleaved overnight at 37 ° C.

Example 6 LC-MS / MS Analysis

Cleaved trypsin peptides of the secreted proteins were analyzed using nanoflow electrospray and liquid chromatography on a QTOFII (Micromass, UK) mass spectrometer. Peptides were isolated using a two-way solvent system in a C18 reverse phase column (0.15 mm × 150 mm, VC-10-C18-150; Micro-Tech Scientific, Vista, CA, USA). The binary solvent system consisted of solvent A consisting of 98.9% water, 1% acetonitrile, 0.2% formic acid, and solvent B consisting of 94.8% acetonitrile, 5% water, 0.2% formic acid. Peptides were eluted from the column with a linear gradient program that changed the composition of the solvent from 5% B to 90% B for 100 minutes at a constant rate of 1 i L / min. Data of the eluted peptides were obtained using Water MassLynx 4.0 software. The first 40 minutes of MS / MS analysis (1 μl injected into each site) was performed to confirm the quality and concentration of the secretome. The final data is 180 min analyses in the data-dependant mode where each full MS scan was performed on the MS / MS of the four most intense ions (injecting approximately 2 μg protein based on the intensity of the initial operation). Obtained using To optimize peptide coverage, a mass / ion exclusion list was maintained so that the same peptide was not selected in MS / MS within 1 minute. The analysis of two samples from each cell line was replicated to confirm the reliability of the MS results.

Example 7 Statistical Analysis

Statistical analysis was performed using the statistical software program SPSS version 12.0 (USA). T-tests were calculated to determine significant differences in fertility gain status and in vivo fertility (NRR). P <0.05 is statistically significant.

The results of protein expression related to fertility gain and relative fertility in bovine frozen semen of the present invention were as follows.

7-1. Protein Expression According to Fertility

To illustrate the results obtained by performing 2-DE on bovine semen, gel analysis was performed using the PDQest program in three pairs of experiments, the results of which are shown in Table 3, FIG. 1 and FIGS. 2A-2E. .

TABLE 3 Evaluation of sperm proteome through gel analysis

Experimental comparison Total number of spots detected in the reference gel Number of spots with increased expression compared to other sperm Number of spots with reduced expression compared to other sperm Low fertility 287 ± 20.07 12 One High fertility 301 ± 25.33 One 12

In Figure 1, H-1, H-2, H-3 is a high sperm sperm, L-1, L-2, L-3 is a low sperm for sperm. Each gel was loaded with 200 μg of protein. The apparent molecular weight is indicated on the vertical axis and the pH range is indicated on the horizontal axis (pH range is indicated so that the tip of the gel is low). All experiments were repeated three times, and the spots shown included increased / decreased or missing protein expression in small or large size gels. The other points are marked with arrows.

We identified 287 ± 20.07 and 301 ± 25.33 spots in the groups classified according to fertility (high and low fertility), respectively. The mean error in the number of protein spots for each group was not so large that we thought to minimize all possible changes. Thirteen protein expression spots showed significant differences in fertility and the number of increased or decreased spots (more than threefold in intensity) was also detected. In sperm with low fertility, 12 spots were expressed higher than in sperm with high fertility.

In other words, only one spot was highly expressed in the fertile sperm. Since only 13 spots identified by at least three peptides were expressed differently, we confirmed that 13 proteins were expressed differently in semen with different fertility, through 2-DE and LC / MS-MS mass spectrometry analysis. Could.

Analysis of the 13 protein spots using LC / MS-MS revealed that only porin protein, which is highly expressed in sperm with low fertility, explains the mechanism of sperm motility and apoptosis. The mechanism and name are not yet known.

2A to 2E show analysis of proteins expressed differently in sperm according to differences in fertility. To illustrate this, three pairs of experiments were performed using a detailed gel analysis using PDQest software. The upper spots and white bars are the amount of protein expression in sperm with high fertility, and the lower spots and black bars are the amount of protein expression in sperm with low fertility.

7-2. Identify proteins related to acquiring fertility in sperm with different fertility

To identify the proteins involved in fertility gain, sperm with different fertility abilities derived from bulls were fertilized (20 min CM) and fertility free (NCM, not cultured).

First, fertility gain status was determined by CTC / H33258 staining.

Figure 3 shows the analysis using a chlorotetracycline (CTC) fluorescence assay after treatment with low-fertilizing sperm and high sperm to give a fertility obtained by treatment with 10 ㎍ / ml heparin for 20 minutes will be. The data in FIG. 3 are expressed in% cells (mean ± SEM), labeled F, B, AR.

In FIG. 3, the other superscripts of a and b showed a significantly different pattern compared to the control group and the F pattern group by T-test (P <0.05), and the other superscripts of A and B were T -test showed significantly different patterns compared to the control group and the B pattern group (P <0.05).

Since the B pattern is related to the acquisition of sperm power, the sperm acquired by fertility have high fertilization ability, and the sperm that do not acquire fertility has low fertility ability.

According to FIG. 3, sperm (B pattern) obtained with fertility were significantly increased in CM as compared to NCM.

Second, 2-DE confirmed the protein expressed in the sperm according to the acquisition of fertility. Fertility Obtained proteins and proteins not identified were identified. Gels were analyzed using PDQest software for three pairs of experiments and the results are shown in Tables 4, 4A-4C, 5A-5D, FIG. 6.

529 ± 94.64 protein spots were expressed in sperm showing low fertility gain and 394 ± 41.41 protein spots in all sperm showing high fertility gain. The error in the number of protein spots for each group is not so large that we have thought to minimize all possible changes. Eight spots among these proteins showed marked differences in protein expression according to fertility (more than three-fold differences in intensity) (see Table 4 and FIGS. 4A-4C).

[Table 4] Description of fertilization obtained sperm protein by automated gel analysis

Experimental comparison Total number of spots found on the reference gel Number of spots highly expressed on one gel (percent of total spots) compared to other gels  The number of spots that are low on one gel compared to other gels (percent of total spots) Capacitated protein in low fertility 529 ± 94.64 4 4 Capacitated protein in high fertility 394 ± 41.41 4 4

After acquiring fertility, high fertility capacitation sperm showed high expression at four spots as compared to low fertility capacitation sperm (see FIGS. 5A-5B). ). On the other hand, the four spots after acquiring fertilization showed high expression in sperm having low fertility as compared to sperm having high fertility (see FIGS. 5C to 5D). These proteins were identified by LC / MS-MS mass spectrometry (see Table 5). That is, ropolinin, which inhibits motility, and enolase 1, which is involved in the generation of abnormal sperm and immature sperm, were highly expressed in individuals with low fertility. On the other hand, actin-related T2 related to germ cell division after fertility gain, acrosome reaction and fertilization were highly expressed in individuals with high fertility. Bovine mitochondrial F1-ATPase complexed with Aurovertin B was also highly expressed in high fertility individuals. It can be inferred that this would be a protein that affects fertility gain.

Table 5 Differently Expressed Proteins

function Spot No. NCBI gi No. Protein name Peptide Motility 11 190201 Porin K.LTFDTTFSPNTGK.K 1111 74002908 Prediction: similar to ropporin, rhophilin associated protein 1 R.FTEEIEWLK.F Fertility gain and acrosome reaction 2404 84000199 Actin-related protein T2 K.AGLSGEIGPR.H Involved in glycolysis / premature sperm production 5508 87196501 Enolase 1 K.SCNCLLLK.V + 2 Carbamidomethyl (C) Feature not known 1505 1827812 Chain D, bovine mitochondrial F1-ATPase complexed with Aurovertin B K.VLDSGAPIR.I Other proteins One 189054178 Unnamed protein product K.YEELQITAGR.H 2 189054178 Unnamed protein product R.DYQELMNTK.L 3 28317 Unnamed protein product K.VTMQNLNDR.L 4 28317 Unnamed protein product K.DAEAWFNEK.S 5 12859782 Unnamed protein product R.TNAENEFVTIK.K 6 12859782 Unnamed protein product R.FLEQQNQVLQTK.W 7 189054178 Unnamed protein product R.DYQELMNTK.L 8 119912330 Expected: hypothetical protein K.QAMQNLNDR.L 9 189054178 Unnamed protein product R.SLVNLGGSK.S 10 189054178 Unnamed protein product K.YEELQITAGR.H 12 12859782 Unnamed protein product R.TNAENEFVTIK.K  13 126308132 Expected: hypothetical protein R.LENEIETYR.R 312 189054178 Unnamed protein product K.AEAESLYQSK.Y 1525 56242 Unnamed protein product K.LVDENLLFHGEASEQLR.T 2419 189054178 Unnamed protein product R.DYQELMNTK.L 3411 28317 Unnamed protein product R.VLDELTLTK.A

1 shows 2-D electrophoretic separation of sperm proteins in high and low fertility sperm.

2A-2E show the analysis of sperm proteins in sperm of high and low fertility.

Figure 3 shows the high and low fertilization ability was analyzed by chlorotetracycline (CTC) fluorescence assay after treatment with sperm for 10 minutes to 10 μg / ml heparin to cause fertility acquisition.

4a to 4c show the results of 2-D gel electrophoresis of sperm cells (left) that did not acquire fertility and sperm cells that obtained fertility (right) according to the difference in fertility.

5a to 5d show the strength of capacitated sperm proteins in sperm having different fertility.

Claims (12)

Bovine mitochondrial F1- combined with Porin, Ropporin, Enolase 1, actin-related protein T2 and aurovertin B ATPase complexed with Aurovertin B) A marker for predicting fertility of an animal, characterized in that the expression is increased in semen of the animal, at least one selected from the group consisting of. (1) classifying sperm collected from the animal into sperm having high fertilization ability and sperm having low fertilization ability; (2) treating each of the classified sperm with heparin to induce fertility gain; (3) separating the fertilization ability of step (2) from the obtained high fertilization sperm and low fertilization sperm; And (4) selecting proteins with increased or decreased expression by comparing expression amounts of proteins isolated from sperm with high fertility and sperm with low fertility; Method of obtaining a marker for fertility prediction of the animal comprising a. The method of claim 2, wherein said animal is a cow. The method of claim 2, wherein the classification step (1) is performed by a dual dyeing method. The method of claim 2, wherein the step of selecting the protein of (4) is characterized in that for analyzing the concentration of the spot using PD Quest. Bovine mitochondrial F1- combined with Porin, Ropporin, Enolase 1, actin-related protein T2 and aurovertin B ATPase complexed with Aurovertin B) A composition for predicting fertility of an animal comprising an antibody that specifically binds to at least one marker selected from the group consisting of. Bovine mitochondrial F1- combined with Porin, Ropporin, Enolase 1, actin-related protein T2 and aurovertin B ATPase complexed with Aurovertin B) A composition for predicting fertility of an animal comprising a primer or probe specifically binding to a gene encoding one or more markers selected from the group consisting of. 8. The composition of claim 6 or 7, wherein the predictive composition is used in a kit, a microarray, and a DNA chip. (1) Bovine mitochondrial F1-ATPase bound to porin, ropororin, enolase 1, actin-related protein T2 and aurovertin B in the sample. Measuring mRNA or protein levels of the fertility predictor marker of at least one animal selected from the group consisting of (Bovine mitochondrial F1-ATPase complexed with Aurovertin B); And (2) analyzing the increase or decrease in the amount of mRNA or protein expression. 10. The method of claim 9, wherein the sample is bovine sperm. 10. The method of claim 9, further comprising the step of predicting that the fertility (Porin), Roporin (Ropporin) and Enolase 1 (enolase 1) in the sample increases the fertility of the animal is low How to. 10. The method according to claim 9, wherein the expression amount of actin-related protein T2 and bovine mitochondrial F1-ATPase complexed with Aurovertin B in combination with aurovertin B in the sample Further increasing the fertility of the animal.

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