CN103361336A - Animal model with male reproductive disorders, as well as preparation method and application thereof - Google Patents

Abstract

The invention provides an animal model with male reproductive disorders of a non-human mammalian animal, as well as a preparation method and application of the animal model, and further identifies the functions of a Prss37 gene and a protein. The Prss37 gene of the animal model is inactivated, thereby affecting the appearance of ADAM3 in mature sperms produced by the animal model, indirectly affecting the combination of the sperms-zona pellucida and the migration of the sperms from the uterus to the oviduct and resulting in the serious male reproductive disorders. The model, the Prss37 gene and the protein thereof can be used for screening ADAM3 ripening accelerators, as well as sterility infertility treatment agents or contraceptive medicaments.

Description

Male fertility obstacle animal model and method for making thereof and purposes

Technical field

The present invention relates to biological technical field.More particularly, the present invention relates to the establishment method and uses thereof of the male fertility obstacle animal model of non-human mammal.

Background technology

Human reproduction relates to the quality of population, and in the modern life, because operating pressure and various environmental factors cause people's fertility quality to descend year by year, therefore, human reproduction's research has been become the research " focus " of researcher.

Male fertility is the process of a complexity, is subject to the impact of a lot of different factors, such as sperm can normally produce, whether good, sperm quantity form and motility whether enough, sperm normal for semen quality, etc. [1].It is reported, in western countries, there is the fertility difficulty in 1/6 man and wife.Wherein half is because sterile (MFI) that male factor causes.1/3 MFI is relevant with chromosome abnormalty; 2/3 remaining reason unknown [2,3].

The generation of sperm occurs in the male testis, and during spermatogenesis is called spermatogenesis, can be divided into three different periods: mitotic division (mitosis), the i.e. self of spermatogonium and propagation; Reduction division (meiosis), namely chromosome number becomes monoploid from diploid; Sperm forms (spermiogenesis), and namely spermatid is divided into mature sperm [4].Wherein arbitrary period is physically different, all will cause sperm normally not produce.

Different period from spermatogenetic the first two, violent form is occuring in the sperm forming process to be changed, Round spermatid is transformed into mature sperm prolongation, the tadpole shape, and size also is reduced to about 1/5 original [5,6].The complicated restructuring procedure of sperm formation stages comprises the formation of acrosome and sperm tail, DNA is by dense packing, plastosome is arranged along the neck of sperm and the stage casing of tail, be combined with zona pellucida and sperm surface and the cross-film structure of signal transduction are synthesized the removal of unnecessary endochylema and the formation of motility of sperm.Regulating effect is brought into play in the expression of the cell reconstitution process need sperm specificity gene that these are unique.600-1 is arranged by inference, and 000 specific gene of sexual cell participates in sperm forming process [7].In in the past 15 years, gene knockout research has identified that more than 20 the specific gene of male sex-cell bring into play keying action [8] in the sperm forming process.These genes participate in regulating acrosomal formations (Hrb, Gopc and Csnk2a2), tail and form (Tektin-t, Vdac 3, Sepp1, Akap4 and Spag6), Chromosome packing (Prm1, Prm2, Tnp1, Tnp2 and Hlt2), surface molecular (the Adams 1-3 relevant with signal transduction with the zona pellucida combination, Tenr, Apob, Clgn, Catsper1 and Catsper2) and energy metabolism (Gapds and sAc).

After sperm formed, sperm was released from the seminiferous tubule top, enters tube chamber, and experience is further ripe in the epididymis transport process, the ability that obtains motility and identification and merge with ovum.

Mammiferous fertilization process is the event of continuous a, multi-step, the sperm that penetrates need to pass female reproductive tract, and capacitation in female reproductive tract, finish from the uterus to oviducal migration, could arrive the ovum that is positioned at ampulla of uterine tube, then comprise the adhesion of sperm and zona pellucida and just can finish fertilization in conjunction with the processes such as film fusion between, sperm and the ovum.

Studies show that multiple protein enzyme and their inhibitor participate in the expression regulation [9] of testicualr development and testis genes involved.No matter testis still at adult all is a highly dynamic organ in embryonic stage, and the event of reinventing of a series of essence is occuring constantly.The supressor of proteolytic enzyme and homology thereof is being set up and is being kept and all brought into play important biological function in testis structure, spermatogeny and the fertilization process.

Serine protease is a main class in the known proteolytic enzyme, and the known proteolytic enzyme more than 1/3 is serine protease, and it is widely distributed at occurring in nature, has found to have [10] in all cell life entities and multiple viral genome.Serine protease is because there being the serine residue of individual nucleophilic to gain the name on the avtive spot of its enzyme; it attacks the carbonyl group in the substrate peptide bond; form acyl group-enzyme intermediate product, then the saponification that water/hydroxyl induces occurs make enzyme regeneration, thus catalytic substrate hydrolysis [11].

In the eukaryotic protein group, serine protease superfamily is one of wherein maximum, the most various enzyme family, comprises approximately the human encoding gene [12] of 200 enzymes or 1%.Serine protease has participated in the various physiological processes such as fetal development, immunne response and coagulation process, with human health closely related [13-17].Proteolytic enzyme express and space-time unusually will cause multiple pathological phenomenon, such as cancer, inflammation, haemophilia, osteoporosis, nerve degenerative diseases and cardiovascular disorder etc.

Show on evidence, serine protease has been brought into play vital role in fertilization process, participate in capacitation, the physiological processs such as essence ovum combination, a member of serine protease subtilisin sample such as PCSK4, high expression level is in male sex-cell, the mouse sperm genesis and development that lacks this gene is normal, but smart ovum binding ability descends, fertilization process can't be finished, infer that it may participate in some synthetic precursor protein of male sex-cell, such as proenkephalin, pronerve growth factor, propituitary adenylate cyclase-activating peptide (proPACAP), the IGF-1 acceptor, the enzymolysis processing of hepatocyte growth factor receptor and ADAM family protein etc., related mechanism is still waiting further research [18]; Prss21 and Acrosin are the serine proteases that two other sperm of having reported is expressed, in vitro study finds that it has participated in smart ovum combination, yet single-gene is rejected and but not to be affected arrenotoky [19], only has the fecundity of hero mouse when dual-gene rejecting just can descend [20].

In the prior art, the gene of Unknown Function has many methods in the research body, and its small mouse gene knochout technique provides strong means for the research Human genome in the function of whole animal level.Change the function information that just can obtain this gene by analyzing the phenotype that obtains the genetic modification mouse.Another advantage of this research method be gene function and human diseases can be carried out related, thereby when obtaining gene function, also can obtain disease information and the disease animal model that this gene can be treated as potential drug or drug target.

Therefore, this area be in the urgent need to studying the function with buck vegetative phase correlation gene, and then is used for the gene knockout animal model of related drugs exploitation.

Summary of the invention

The male fertility obstacle animal model that the purpose of this invention is to provide the non-human mammal of Prss37 gene inactivation.

Another object of the present invention provides preparation method and the purposes of described animal model.

Another object of the present invention is to identify the function of Prss37 gene and albumen thereof.

In first aspect, the invention provides a kind of male fertility obstacle animal model of non-human mammal, the Prss37 gene inactivation of described animal model.

In second aspect, the invention provides the preparation method of the male fertility obstacle animal model of described non-human mammal, described method comprises step: the cell that non-human mammal is provided, with Prss37 gene inactivation in the described cell, and the cell regeneration of described Prss37 gene inactivation formed male fertility obstacle animal model.

In another preference, described inactivation is to insert by gene knockout, gene interruption or gene to cause the Prss37 gene inactivation.

In further preference, described inactivation comprises that the Prss37 gene do not express, or expresses and do not have activated Prss37 albumen.

In another preference, described non-human mammal comprises rodent or primate, preferably comprises mouse, rat, monkey.

In another preference, described method comprises: utilize the ET clone, reject exon3 and the exon4 of Prss37 gene in the ES cell by homologous recombination, thereby obtain positive ES cell clone; Make chimeric rodent with described positive ES cell clone, comprise male mosaic rodent and female rodent; Described male mosaic rodent and female rodent are carried out mating, thereby obtain the heterozygote rodent, comprise the male rodent of heterozygote and the female rodent of heterozygote; With with the male rodent of described heterozygote and the female rodent mating of heterozygote, thereby obtain the rodent of isozygotying of Prss37 gene complete deactivation.

In further preference, comprise that also the rodent to obtaining in each step carries out the genotype checking at DNA, RNA and protein level.

In the third aspect, the invention provides the purposes with male fertility obstacle animal model preparation, non-human mammal of method described in the second aspect, the material that it is used for the screening treatment or improves the male fertility obstacle; Or for the ripe promotor of screening ADAM3.

In fourth aspect, the invention provides the purposes of Prss37 gene or albumen, the material that it is used for the screening treatment or improves the male fertility obstacle; Be used for the material that the screening Inhibit sperm is combined with ovum.

In a preference, described sperm is combined with ovum and is referred to that sperm is incorporated into the ovum with zona pellucida.

Aspect the 5th, the invention provides the method that screening affects the candidate substances that sperm is combined with ovum, described method comprises step: the non-human mammal that test substance is applied to test group; Detect the expression of Prss37 gene or the activity of albumen; With compare with control group, if wherein described test substance causes the rise of Prss37 genetic expression or protein-active, represent that then test substance is the candidate substances that promotes that sperm is combined with ovum; If described test substance causes the downward modulation of Prss37 genetic expression or protein-active, represent that then test substance is the candidate substances that Inhibit sperm is combined with ovum.

In a preference, described use be oral, through skin or spray application.

Can in another preference, described method also comprises step: to the candidate substances that the promotion sperm that obtains is combined with ovum, further test it and treat or improve the male fertility obstacle; And/or the candidate substances that the Inhibit sperm that obtains is combined with ovum, can further test it as contraceptive.

In another preference, the invention provides the promotor of Prss37 gene or albumen or the purposes of inhibitor, it is for the preparation of the conditioning agent that affects sperm and be combined with ovum.

In further preference, described promotor is for the preparation of the medicine for the treatment of or improve the male fertility obstacle; And/or described inhibitor is for the preparation of contraceptive.

In another preference, described Prss37 gene and albumen comprise wild-type or its variant.

In another preference, described Prss37 gene or dietary protein origin are in mouse, rat or people.

In further preference, promotor or the inhibitor of described Prss37 gene or albumen comprise: the sense-rna of Prss37 gene; The antibody of anti-Prss37 albumen.

In further preference, described antibody comprises polyclonal antibody, monoclonal antibody or antiserum(antisera).

Aspect the 6th, the invention provides the method for the ripe promotor of screening ADAM3, described method comprises step: the animal model that test substance is applied to the preparation of method described in the second aspect; Content with ripe ADAM3 in the mature sperm albumen extract that detects described animal model; If compared with the control, the content of described ripe ADAM3 raises, and then described test substance is the ripe promotor of ADAM3.

Aspect the 7th, the invention provides with the described method of second aspect male fertility obstacle animal model preparation, non-human mammal, described animal model has following characteristic: (a) produce ripe sperm, but described sperm can not be combined with the ovum with zona pellucida effectively; (b) in testis tissue, produce the ADAM3 precursor, but do not produce ripe ADAM3.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.

Description of drawings

Fig. 1. reverse transcription PCR and real-time quantitative PCR disclose the tissue specific expression of Prss37mRNA.Extracting RNA from 16 tissues shown in the figure, and become cDNA with the reverse transcription of MMLV-RT ThermoScript II.Take these cDNA as template, the specific region in the amplification Prss37 encoding sequence.Take β-actin as confidential reference items.

Fig. 2. shown that Prss37mRNA and albumen are testes specificities, in epididymis, can't detect mRNA or albumen, and lifetime interval between Prss37 genetic transcription and the mRNA translation.Extracting RNA and albumen from the mouse testis of the rear different ages of being born and epididymis carry out RT-PCR (A and B are respectively real-time quantitative PCR and sxemiquantitative PCR) and Western blot (C and D) and analyze.Prss37mRNA shows that from the rear 23 days transcriptional starts of being born transcribing of it occurs in the initial period that sperm forms.The sperm stretch-out phase of Prss37 albumen after 4-5 days produces.β-actin and GAPDH contrast as applied sample amount.

The immunohistochemical methods location of Fig. 3 .Prss37 albumen in the adult mice testis.Immunohistochemical analysis is carried out in testis section to the wild-type adult mice.The different times of cycle of seminiferous epithelium and its corresponding sperm development step are marked on the top of every pictures.Image Display Prss37 albumen with a kind of period specific mode express, show that the expression of Prss37 is confined to prolong the acrosome stage of sperm, i.e. the 9th to 14 step of forming of sperm.

The target of Fig. 4 .Prss37 gene is rejected.(A) the allelic complete structure of wild-type mice Prss37.Exon and intron represent with square frame and sea line respectively, and the coding region is shown as black.Reject mouse Prss37 allelotrope for target, targeting vector comprises the 5 ' homology arm of 4756bp and the 3 ' homology arm of 3543bp.Homologous recombination causes exon 3 and 4 to be replaced by the Neo gene.Introduce the hsv-TK gene in the targeting vector, as negative control.(B, C) positive ES cell clone and mouse tail DNA carry out pcr amplification by the primer that indicates among the figure and come the identified gene type.(D) RT-PCR of the total RNA of testis analyzes.The specific region of primer amplification Prss37 encoding sequence can obtain the fragment of 403bp in wild-type (wt) and heterozygote (+/-) mouse.M, dna molecular amount mark.(E, F) wt ,+/-and-/-western blot and the immunohistochemical analysis of mouse testis tissue.

Fig. 5 .Prss37-/-form and the histologic analysis of mouse testis, cauda epididymidis and sperm.(A) wt ,+/-and-/-Testicular Size of mouse recently estimates with the percentage of testicular weight percentage of liveweight.(B) wt ,+/-and-/-Hematorylin of mouse testis and cauda epididymidis section/Yihong dyeing.Coloration result shows that all during spermatogenesis is sound.Illustration is the high magnification map of cauda epididymidis sperm.(C) Hematorylin of sperm smear/Yihong dyeing.From wt and-/-cauda epididymidis of mouse separated sperm, be coated on the slide glass.It is unusual that the opticmicroscope analysis has no obvious sperm morphology.(D) the Ultrastructural TEM (transmission electron microscope) analysis of sperm.

Fig. 6. shown since sperm to oviducal migration obstacle, Prss37-/-the internal fertilization rate of male mouse descends.(A, B) obtains two protokaryon phases or two cell stage embryos from the uterine tube of the rear 27h of hCG injection or 45h.Two pronuclear-stage embryos are with Hoechst33258 dyeing and at the fluorescence microscopy Microscopic observation.Two cell stage embryos are at optical microphotograph Microscopic observation and counting.Rate of fertilization is calculated by the ratio of two cell stage embryo numbers/total embryo number.The total embryo number that obtains is shown in (n=3) in the round bracket.Wt and Prss37-/-there are significant difference (P＜0.01) in two protokaryon phases or the two cell stage embryo numbers of mouse.(C) Prss37-/-there is defective in sperm to oviducal migration.With wild-type or Prss37-/-the female mouse of male mouse mating, fix its uterus and uterine tube at post-coitum 2h, carry out serial section behind the paraffin embedding, with Hematorylin/Yihong dyeing.Sperm is analyzed by the section that observation contains Utero-uterine tube connection (UTJ) from the uterus to oviducal migration.

Fig. 7 .Prss37 is on the interactional impact of essence-ovum.The ovum that zona pellucida is complete and the ovum of zona-free respectively with from wild-type and Prss37-/-sperm of mouse epididymis tail hatches jointly.A large amount of wild-type sperms is attached on the complete ovum of zona pellucida, and Prss37-/-sperm only has several or do not have combination.But, wild-type and Prss37-/-quantity that sperm is attached on the zona-free ovum do not have significant difference.

Fig. 8 .Prss37 is on the impact of sperm ADAM3.The disappearance of Prss37 causes the disappearance of the ripe ADAM3 of sperm surface, but the ADAM3 precursor in the testis is unaffected.Wild-type and Prss37-/-testis and the Epididymal sperm protein extract of mouse carry out SDS-PAGE under reductive condition separates, and carry out western blot with ADAM3 antibody and analyze.

Embodiment

The contriver is through extensive and deep research, find that unexpectedly the Prss37 specific gene expression is at the sperm formation stages of testis tissue, by affecting the appearance of ADAM3 in mature sperm, the combination of remote effect sperm and zona pellucida and sperm be from the uterus to oviducal migration, thereby cause serious male fertility obstacle.This male fertility Disorder Model will provide a new direction for infertile and diagnosis and treatment reproductive medicine, for Prss37 provides experiment support as the application prospect of possible contraceptive or infertile therapeutical agent target spot, has important using value simultaneously.Finished on this basis the present invention.

Prss37 gene and albumen thereof

Prss37 is a function without the gene of identifying, the albumen of its expression may be pancreatin sample serine protease.The mouse Prss37 assignment of genes gene mapping is made of coding 237aa in No. 6 karyomit(e) B district 2 bands 6 exon; The people Prss37 assignment of genes gene mapping is comprised of 5 exon in No. 7 long-armed 3 districts of karyomit(e) 4 bands, present known two transcripts that have, and 235aa and 234aa encode respectively.The difference of people Prss37 albumen and its variant only has been more than the exon3 3 Nucleotide.Prss37 albumen is relatively conservative on evolving, in species camber homologies such as people (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), ox (Bos Taurus), domesticated dog (Canis lupus familiaris), rhesus monkey (Macaca mulatta), chimpanzees (Pan troglodytes).Wherein, the homology degree of people and mouse is up to 91%.But the physiological function of Prss37 albumen is not also determined.

The cDNA sequence of mouse Prss37 shown in SEQ ID NO:1, total length 1,049bp, wherein CDS is positioned at the 229-942 position.

agtgaccatt gggaggttca gagcaaacca ttaagggcat gctctccctg agccaaggta 60

gagaatcctg ttcctcacag ctctgtgctc agaaactctg caagaaagag cccttaccta 120

tccaaacctg ctggctccgg tagacatccc ccaggagaca agtgagcaga agattccggc 180

ctctgctgat cttctttcta tacctcgaaa ggaaagagct ggaccatcat gaaacttatt 240

atctatctca ccatcctagc tggaacagct ttagtcactc actcatctgt gcagaaagag 300

gaccatgctc cctacttagc atacctcaag tctaacttca atccctgcgt gggtgtcctc 360

atcaaagcca gctgggtgct ggccccatct cactgctatt taccaaatct gagggtgatg 420

ttgggaaatt tcaagagcag agtcagagat gggacagagc agacaattta tcccatccag 480

attatccgct actggaacta cagccacaca gccccacagg atgacctcat gcttattaaa 540

ctagctaagc ctgccacctt caaccacaaa gtccaggttc ttcccatagc caccactaat 600

gtccggccag gcactgtctg tacactttca ggtttggact ggagtcaaga aaacaatggc 660

aggcaccctg acttacggca gaacctggaa gctcccgtga tgacggacaa agactgccag 720

aaaactcaac aaggaagcag ccacagaaac tccttatgtg tcagatttgt gaaagtattc 780

agccgaattt ttggggaggt tgctgtagct actgtcattt gtaaaaacaa gctccaggga 840

attgaagttg gacacttcat gggaggggac gttggcatct ataccaacat ttactcatat 900

gtaccatgga ttgagaaaac cactaaggag aagatgacat gatgccctgc ttttccctcc 960

gcatctcatt gcctctgcca ttgactgaag gagcttacat ggtccctaaa ctcaaataaa 1020

ctctccaaac agaaagtttg gaatgcagc 1049

(SEQ ID N0.：1)

The cDNA sequence 1 of people Prss37 shown in SEQ ID NO:2, total length 1,197bp, wherein CDS is positioned at the 373-1080 position.

agaaactctg tggggaagat aagtcctgag cctagagctg ccatttccac agacaactga 60

actaccttca aacagatgtg tttttagggg cagcagatag aaggagctga atccaggagt 120

ttccttccct gaaatgcccc aacagaggca cagtcctcca ccttccctgg aacagagggc 180

tttcatctgt ctctctgaag ccatctccct cccctgttta tgcccagagc tgctttccaa 240

tcccatggag tgcctccgta tctaaccata gccactgagt ctaccaggtg ttcctgagaa 300

gacaactaag ccaaccgctc cacactctgc tgattttctt ctacatctca cagggggaag 360

agctggatca ccatgaaata tgtcttctat ttgggtgtcc tcgctgggac atttttcttt 420

gctgactcat ctgttcagaa agaagaccct gctccctatt tggtgtacct caagtctcac 480

ttcaacccct gtgtgggcgt cctcatcaaa cccagctggg tgctggcccc agctcactgc 540

tatttaccaa atctgaaagt gatgctggga aatttcaaga gcagagtcag agacggtact 600

gaacagacaa ttaaccccat tcagatcgtc cgctactgga actacagtca tagcgcccca 660

caggatgacc tcatgctcat caagctggct aagcctgcca tgctcaatcc caaagtccag 720

ccccttaccc tcgccaccac caatgtcagg ccaggcactg tctgtctact ctcaggtttg 780

gactggagcc aagaaaacag tggccgacac cctgacttgc ggcagaacct ggaggccccc 840

gtgatgtctg atcgagaatg ccaaaaaaca gaacaaggaa aaagccacag gaattcctta 900

tgtgtgaaat ttgtgaaagt attcagccga atttttgggg aggtggccgt tgctactgtc 960

atctgcaaag acaagctcca gggaatcgag gtggggcact tcatgggagg ggacgtcggc 1020

atctacacca atgtttacaa atatgtatcc tggattgaga acactgctaa ggacaagtga 1080

gaccctactt ctccctctgc attccactgg ctctgccatg gactatacaa gcagataatt 1140

ttccctctat tcaaaataaa atctccaaat gaaaatttgg gaatgtagca tactagt 1197

(SEQ ID N0.：2)

The cDNA sequence 2 of people Prss37 shown in SEQ ID NO:3, total length 1,194bp, CDS:373-1077bp wherein, it is almost completely consistent with sequence 1, has only lacked three bases of 799-801 position among the SEQ ID NO:2.

Mouse Prss37 albumen (237aa, SEQ ID NO:4):

MKLIIYLTILAGTALVTHSSVQKEDHAPYLAYLKSNFNPCVGVLIKASWVLAPSHCYLPNLRVMLGNFKSRVRDGTEQTIYPIQIIRYWNYSHTAPQDDLMLIKLAKPATFNHKVQVLPIATTNVRPGTVCTLSGLDWSQENNGRHPDLRQNLEAPVMTDKDCQKTQQGSSHRNSLCVRFVKVFSRIFGEVAVATVICKNKLQGIEVGHFMGGDVGIYTNIYSYVPWIEKTTKEKMT

People Prss37 albumen (235aa, SEQ ID NO:5):

MKYVFYLGVLAGTFFFADSSVQKEDPAPYLVYLKSHFNPCVGVLIKPSWVLAPAHCYLPNLKVMLGNFKSRVRDGTEQTINPIQIVRYWNYSHSAPQDDLMLIKLAKPAMLNPKVQPLTLATTNVRPGTVCLLSGLDWSQENSGRHPDLRQNLEAPVMSDRECQKTEQGKSHRNSLCVKFVKVFSRIFGEVAVATVICKDKLQGIEVGHFMGGDVGIYTNVYKYVSWIENTAKDK

People Prss37 protein variant (234aa, SEQ ID NO:6):

MKYVFYLGVLAGTFFFADSSVQKEDPAPYLVYLKSHFNPCVGVLIKPSWVLAPAHCYLPNLKVMLGNFKSRVRDGTEQTINPIQIVRYWNYSHSAPQDDLMLIKLAKPAMLNPKVQPLTLATTNVRPGTVCLLSGLDWSQENSRHPDLRQNLEAPVMSDRECQKTEQGKSHRNSLCVKFVKVFSRIFGEVAVATVICKDKLQGIEVGHFMGGDVGIYTNVYKYVSWIENTAKDK

Materials and methods:

The mouse source: C57BL/6J Strains of Mouse and Prss37 reject mouse model by the SPF level Animal House conservation breeding of model animals research centre, south, Shanghai.

Reverse transcription PCR and real-time quantitative PCR detect the mRNA abundance

The relative expression level of Prss37 in mouse tissue detects by the cDNA sample from 16 mouse tissues, and these tissues comprise brain, cerebellum, thymus gland, the heart, lung, liver,spleen,kidney, stomach, intestines, bladder, skeletal muscle, testis, epididymis, ovary and uterus.RNA operates to specifications with Trizol reagent (Invitrogen) extracting.The total RNA of 2 μ g becomes cDNA with MMTV ThermoScript II (Promega, Madison, WI) reverse transcription.CDNA increases with specific primer pair.The primer of beta-actin and Prss37 is in the reverse transcription PCR: and beta-actin (forward: 5 '-GCCTTCCTTCTTGGGTATG-3 ' SEQ ID NO:7 and reverse: 5 '-ACGCAGCTCAGTAACAGTCC-3 ' SEQ ID NO:8), Prss37 (forward: 5 '-AATCCCTGCGTGGGTGTC-3 ' SEQ ID NO:9 and reverse: 5 '-GGCTGCTTCCTTGTTGAGT-3 ' SEQID NO:10).Amplified production separates by agarose gel electrophoresis and EB dyeing is observed.Real-time quantitative PCR exists with the special fluorescein SYBR Green of double-stranded DNA

Carry out on the ep realplex (eppendorf).The primer sequence of beta-actin and Prss37 is in the real-time quantitative PCR experiment: and beta-actin (forward: 5 '-TACCCAGGCATTGCTGACAGG-3 ' SEQ ID NO:11 and reverse: 5 '-ACTTGCGGTGCACGATGGA-3 ' SEQ ID NO:12), Prss37 (forward: 5 '-CCTGCCACCTTCAACCACAA-3 ' SEQ ID NO:13 and reverse: 5 '-GGCTGCTTCCTTGTTGAGT-3 ' SEQ ID NO:14).The differentiation of target product and non-specific amplification product realizes by the melt curve analysis analysis.The expression level of Prss37 carries out standardization according to the content of beta-actin.

Western blot analyzes

Be organized in homogenate in the RIPA damping fluid that contains proteinase inhibitor (final concentration is pepstatin 1 μ g/ml, Trypsin inhibitor,Trasylol 1 μ g/ml, bright ampeptide elemente 1 μ g/ml, PMSF 1 μ M).Under 4 ℃, with the centrifugal 30min of 12,000g.Protein concentration is measured with the BCA method.The total protein of equivalent boils the 5min sex change in 1 * sample-loading buffer (loading buffer), carry out SDS-PAGE and separate then that electricity forwards on the NC film.With after the 5% skimmed milk sealing, film spends the night in incubated at room 2h or 4 ℃ with primary antibodie.After washing with TBST, film is resisted in incubated at room 1h with anti-rabbit or anti-mouse two.Again wash film, with Odyssey Infrared Imager (LI-COR) observing response band.'beta '-tubulin or GAPDH contrast as applied sample amount.

Immunohistochemical analysis

Separating testis or epididymis is fixed among Bouin ' the s buffer (Glacial acetic acid 5ml, mixing is used front preparation for saturated picric acid 75ml, 37-40% formaldehyde 25ml).After fixing, tissue dewatering and paraffin embedding.Tissue slice seals 1h with 5% normal sheep serum room temperature of PBS dilution, use to resist-the Prss37 rabbit anti-serum (1: 100, sigma) 4 ℃ of night incubation.Wash three times each 5min, section VECTASTAIN with PBS

ABCKIT detects, and operates to specifications.

The Prss37 targeting vector makes up

Make up the Prss37 targeting vector by the ET cloning process.Be briefly described as follows: from the BAC (bacterial artificial chromosome (Bacterial Artificial Chromosome) that contains the Prss37 genome sequence; utilize intestinal bacteria F plasmid or P1 phage for fundamental construction be used for clone amplification A, B, C, D homology arm in the large fragment DNA (100～300kb) carrier is usually used in the analysis of genome structure) intestinal bacteria.Primer is respectively: A1:AATAAGCTTTTTAGATAGCCCTGTCCT (SEQ ID NO:15), and A2:TTCCTCGAGCATTTACTGCCTCTTTCAT (SEQ ID NO:16); B 1:CGCCTCGAGTGGCATAGAGCAGGAGAC (SEQ ID NO:17), and B2:CGCGGTACCCAACATTGGACCAGAAGC (SEQ ID NO:18); C1:AATCCGCGGTCAAGCGACAATTATGAAC (SEQ ID NO:19), and C2:AATGGATCCTCCCAGCAGTAAAGAAGTA (SEQ ID NO:20); D1:ATAAAGCTTGGTGGGATGGAGTAGAAA (SEQ ID NO:21), and D2:ATAGTCGACCACCTCCTTAATGTAAATGG (SEQ ID NO:22).PCR product A and B cut with HindIII/Xho I and Xho I/Kpn I enzyme respectively, being inserted into pBR322-MK-MCS (containing the negative selectors of tk) (is reconstructed as the basis take the pBR322 carrier by southern model animals research centre, increased the negative screening-gene of MC1-hsvTK and multiple clone site MCS) HindIII/Kpn I site, obtain to extract carrier pBR322-MK-MCS-AB.After Xho I linearizing, extract carrier and can from BAC, extract the genomic dna that contains Prss37.Equally, PCR product C and D cut with Sac II/BamH I and HindIII/SalI enzyme respectively, are connected to SacII/BamHI and the HindIII/SalI site of the PL451 carrier that contains the PGK-neo element.With primer C1 and the D2 C-neo-D fragment that from carrier, increases, knock in the pBR322-MK-MCS-AB carrier and obtain targeting vector.This targeting vector contains the homology arm of 4756bp (5 ') and 3543bp (3 ').

Prss37 rejects the preparation of mouse

Targeting vector with the NotI linearizing after electricity forward in the SCR012ES clone.With G418 and ganciclovir screening resistant cell.From 128 clones, extract DNA and identify positive colony with targeting vector generation homologous recombination with PCR.The primers designed of 5 ' and 3 ' homology arm be P1 (5 '-CGAGCATCCTGGCTTATC-3 ', SEQID NO:23), P2 (5 '-CCACTCCCACTGTCCTTT-3 ', SEQ ID NO:24) and P3 (5 '-TTTATTAGGAAAGGACAGTGGGAGT-3 ', SEQ ID NO:25), P4 (5 '-TCTGTGAAGTAGGGATGGGTTGT-3 ', SEQ ID NO:26), the PCR product is respectively 5520bp and 4459bp.With two independently ES clone carry out C57BL/6J blastaea injection, transfer to subsequently in the female mouse of false pregnancy and obtain chimaera progeny.The male mouse of mosaic and the female mouse mating of C57BL/6J produce the heterozygote mouse.All mouse are supported in the controlled cage tool of temperature, and room illumination keeps 12h daytime/cycle at 12h night.Mouse freely ingests and drinks water.Relate to zooperal research approach and obtain mechanism's the care of animal and the use council (IACUC) license.

The genotype identification of mutant mice

The genotype of mutant mice is identified by PCR: extracting mouse coda gene group DNA, carry out pcr analysis with specific primer, the primer of wild-type allele is P5,5 '-ACTAGTTATAGCTCTTGATGCC-3 ' (SEQID NO:27) and P6,5 '-CGAGTTTTAGGTGAAGACATC-3 ' (SEQ ID NO:28), allelic primer is P5 and P7 after practicing shooting, 5 '-TGTCATCTCACCTTGCTCCT-3 ' (SEQ ID NO:29).Article three, primer is used for the multi-PRC reaction system, and amplification condition is: 95 ℃ of 4min denaturations, and 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 1min 20s move 35 cycles, hatch 10min for last 72 ℃.The PCR product separates through 1% sepharose.Wild-type and the rear allelic length of practicing shooting are respectively 642bp and 1138bp.

Fertility is analyzed

Three kinds of genotype (wt ,+/-,-/-, each 5-6 is male mouse only) the single cage of male mouse in 8-9 age in week/only raise, match with the female mouse of C57BL/6J wild-type with 1: 2 ratio.Every morning checks whether female mouse cloudy bolt occurs.Take away and see the female mouse of bolt and put into new female mouse.Provide two new female mouse for male mouse weekly.Undertaken 30 days by this rule.The female mouse of bolt that sees that can produce the offspring after three weeks is identified as pregnant mouse.If see behind the bolt and not produce the offspring in 22 days, then dissect female mouse and confirm that it does not have pregnancy.Count total female mouse quantity, see the female mouse quantity of bolt, a few nest mouse of birth and birth offspring's quantity calculates and sees bolt rate (FCP) and pregnancy rate (FC).

Histologic analysis

Testis and epididymis are fixed and paraffin embedding as stated above.Tissue slice dyes with Hematorylin/Yihong, and the reference standard method operates.Separate wt ,+/-and-/-testis of mouse and weighing.From wt and-/-epididymis of mouse separated sperm and make the sperm smear.

TEM (transmission electron microscope) analysis

TEM (transmission electron microscope) analysis is carried out at medical college of university of communications Electron Microscope Laboratory, according to standard operating instructions.Be briefly described as follows: from wild-type and Prss37-/-the mouse epididymis tail separated sperm, be fixed in glutaraldehyde and the osmic acid, 10% gelatin infiltrates, sucrose dehydration, subsequently liquid nitrogen freezing.The freeze-drying section (LeicaULTRACUT UCT/Leica EMFCS) that preparation 50nm is thick with uranyl acetate and methylcellulose gum dyeing, is observed under transmission electron microscope.

Area of computer aided semen analysis (CASA)

The sperm motility force parameter carries out quantitatively (Hamilton-Thorne Biosciences) by the CASA that has integrated IVOS software.Be briefly described below: the cauda epididymidis sperm at 2-3 monthly age is at 37 ℃ of HTF nutrient solutions (101.6mmol/L NaCl, 4.7mmol/L KCl, 0.2mmol/L MgSO ₄, 0.37mmol/L KH ₂PO ₄, 2mmol/L CaCl ₂, 25mmol/L NaHCO ₃, 2.8mmol/L glucose, 0.33mmol/L Sodium.alpha.-ketopropionate and 4mg/mL bovine serum albumin) in hatch capacitation.Transfer part capacitated sperm suspension is counted in the counting lattice, and each sample is analyzed 1000 sperms at least.

The post-coitum embryo obtains

B6 * female mouse of CBA F1 generation (4-5 age in week) surpasses row by abdominal injection 5IU PMSG and interval 48h injection 5IU hCG.Female mouse mates with male mouse behind injection hCG immediately.27h or 45h separate uterine tube from see the female mouse of bolt after the hCG injection, obtain two protokaryon phases or two cell stage embryos.Two pronuclear-stage embryos (contain 0.01mmol/L EDTA2Na, 95mmol/L NaCl, 2.5mmol/L KCl, 0.35mmol/L KH at the KSOM nutrient solution ₂PO ₄, 0.2mmol/L MgSO ₄, 0.2mmol/L glucose, 25mmol/LNaHCO ₃, 2.25mmol/L CaCl ₂) in dye 1h with Hoechst 33258, through several times the cleaning after, at the fluorescence microscopy Microscopic observation.Two cell stage embryos count at the optical microphotograph Microscopic observation.

Rate of fertilization=two cell stages embryo number/ovum sum.

The sperm migration analysis

B6 * female mouse of CBA F1 generation (4-5 age in week) surpasses row by aforesaid method.The male mouse of the super female mouse of row and detection mates 2h (9-11h after the hCG injection), checks whether cloudy bolt forms.At post-coitum 2h, separation and wild-type or Prss37 ^-/-The uterus of seeing the female mouse of bolt and the uterine tube of male mouse mating also is fixed and paraffin embedding, dyes with Hematorylin/Yihong after the serial section.The section that contains Utero-uterine tube connection (UTJ) by observation is analyzed sperm from the uterus to oviducal migration.

The sperm binding analysis

After the hCG injection, collect ovum the female mouse uterine tube of super row of 14h, and the drop that places the HTF nutrient solution to form.Process ovum with the removal granulosa cell with 0.01% (w/v) Unidasa, and clean.Half degranulation cell ovum is processed 30s to remove zona pellucida with acid tyrode's solution (acidic Tyrode ' s solution, Sigma), then cleans with the HTF nutrient solution.The ovum that zona pellucida is complete and the ovum of zona-free are transferred in the 200ul HTF drop that covers with mineral oil.Separation mixes with ovum behind the In-vitro Capacitation 1h in the HTF nutrient solution from the sperm of cauda epididymidis.After hatching 15min altogether, clean with HTF is gentle, observe the sperm that is attached on zona pellucida or the dotter haut.

Statistical study

All data all are expressed as mean+/-standard error.(n 〉=3) are unless propose in addition.Use Student ' st-test to carry out statistical analysis, P＜0.05 has been considered to significant difference.

Major advantage of the present invention

1. the specified phase that forms at sperm owing to the Prss37 specifically expressing, its rejecting does not affect normal development and the during spermatogenesis of testis, thereby does not affect male hormonal readiness and normal mating behavior yet;

2.Prss37 the rejecting of gene does not cause serious sperm morphology unusual, Prss37-/-sperm can normally produce, and sperm number, form and motility are also unaffected, thus this model-specific and accuracy are higher;

3. although internal fertilization ability is badly damaged, experiment in vitro fertilization shows, although Prss37-/-sperm almost can not or seldom identify the complete ovum of zona pellucida, and sperm still can make ovum fertilization, even grows to blastula stage;

4.Prss37 gene may can be used as new contraceptive and the target spot of infertile therapeutical agent, by suppressing this albumen or promoting the effect of this albumen, reach the effect of normal contraception or raising conceptual quotient, meanwhile can not affect the normal generating process of testicualr development and sperm.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber are weight percent and parts by weight.

Embodiment 1

The tissue expression pattern of Prss37 gene and the special station-keeping mode of cell type

Extracting RNA prepares the cDNA sample from 16 tissues of mouse, and these tissues comprise brain, cerebellum, thymus gland, the heart, lung, liver,spleen,kidney, stomach, small intestine, bladder, skeletal muscle, testis, epididymis, ovary and uterus.Carry out reverse transcription PCR and real-time PCR with special primer and detect, find that the special and high expression level of the Prss37 transcript of a uniqueness is in testis tissue (Fig. 1).By research birth rear 7,14,18,21,23,28,42 days male mouse testis and epididymis tissue, find that further the Prss37 genetic transcription starts from the rear 23 days male mouse testis of being born, and began to detect Prss37 albumen in rear 28 days in birth.Both do not had Prss37mRNA in the epididymis tissue, do not had Prss37 albumen yet, shown that Prss37 albumen was present in the interior sperm of epididymis (Fig. 2) hardly.Carry out immunohistochemical analysis by the paraffin section to testis tissue, find the special 9-14 step that appears at sperm formation of Prss37 immunity signal, corresponding to the acrosomal formation period (Fig. 3) that prolongs sperm.

Embodiment 2

Preparation and the evaluation of Prss37 gene knockout mice model

The gene targeting strategy is shown in Fig. 4 A, and we reject exon3 and the exon4 of Prss37 gene with the mode of ET clone and ES cell homologous recombination, rejects part with the replacement of Neo resistant gene, 5 ' homology brachium 4768bp wherein, 3 ' homology brachium 3543bp.8 positive ES cell clones (Fig. 4 B) have been identified with PCR method.Wherein two independently the ES cell clone be used for blastaea and inject to obtain allophenic mice.The female mouse mating of male mosaic and C57BL/6J obtains the heterozygote of blending inheritance background (129-C57).Mating obtains to meet three kinds of mouse genotypes of mendelian inheritance between the male mouse of heterozygote and the female mouse of heterozygote.And strictly carry out the genotype checking from DNA (Fig. 4 C), RNA (Fig. 4 D) and albumen (Fig. 4 E-F) level.

Embodiment 3

Prss37-/-the male fertility obstacle of mouse identifies

Prss37-/-mouse can experience normal fetal development, birth, ontogeny and maturation, not have obvious heteroplasia.Because testis specific expression and the location of Prss37 gene, we carried out Prss37-/-the Fertility assessment of male mouse.With three kinds of genotype (wt ,+/-,-/-) Female odor of the same age (8-9 week age) 1: 2 mate with the female mouse of wild-type, every morning checks that female mouse sees the bolt situation, takes away and sees the female mouse of bolt and put into new female mouse, the female mouse that more renews weekly simultaneously.Observe and continue 30 days.Statistics is seen the female mouse ratio of bolt and the female mouse ratio (table 1) of becoming pregnant afterwards.As can be seen from Table 1, the male mouse of three kinds of genotype is seeing do not have marked difference aspect the bolt ratio.But aspect conceptual quotient, Prss37-/-male mouse significantly is lower than the male mouse of wild-type, is respectively 7.4% and 82.8%.More than 90% with Prss37-/-male mouse mating and see that the female mouse of bolt does not all have pregnancy.Above data declaration, Prss37-/-there is serious growing barrier in male mouse.

Table 1. target is rejected Prss37 and is caused male mouse growing barrier.The male mouse in 8-9 age in week of different genotype, every is all carried out continuous mating with the female mouse of wild-type.Can find cloudy bolt to show the mating success.See that bolt rate (FCP) is expressed as the ratio of the quantity of all female mouse that the quantity of seeing the female mouse of bolt and every male mouse touch.Conceptual quotient (FC) is expressed as the ratio of the female mouse quantity of the female mouse quantity that bears the offspring and successful mating.

Embodiment 4

Identify Prss37-/-whether sperm generation, sperm number, form and the motility of male mouse influenced

After male mouse sexual maturity, we have dissected and have taken by weighing the testicular weight of three kinds of male mouse of genotype, and analyze the per-cent of its percentage of liveweight.Do not find testicular atrophy or weight decline (Fig. 5 A).Carry out specimens paraffin embedding slices after testis, cauda epididymidis is fixing, the pathological analysis that is undertaken by HE dyeing does not observe that sperm produces and the notable difference (Fig. 5 B) of sperm number aspect.Separate the cauda epididymidis sperm, carry out the sperm smear and observe, also do not find unusual (Fig. 5 C) of sperm morphology aspect.Carry out the analysis of Ultrastructure of sperm with transmission electron microscope, the acrosome structure of sperm is compared Non Apparent Abnormality (Fig. 5 D) with wild-type.With the area of computer aided sperm analyze (CASA) estimate Prss37-/-motility of sperm, find to comprise Progressive motility (propulsion ability), Rapid motility (rapid movement ability), Path velocity (path velocity), the indexs such as Progressive velocity (forward speed), Beat frequency (jumping frequency rate) all similar to wild-type (table 2).Therefore, Prss37-/-male mouse can normally produce sperm, and sperm number, form and motility are all unaffected.

Table 2. wild-type and Prss37-/-motility of mouse epididymis tail capacitated sperm.The cauda epididymidis sperm is hatched and capacitation in the HTF nutrient solution, then carries out the area of computer aided sperm and analyzes (CASA).From the sperm sample (n=5) of five different mouse, each sample is analyzed 1000 sperms at least.The data of sperm analysis are expressed as mean value ± standard error.Wild-type and Prss37-/-all parameters of sperm all do not have significant difference (P＞0.05).

Embodiment 5

Identify Prss37-/-the internal fertilization rate of male mouse and Prss37-/-sperm is from the uterus to oviducal migration

For analyze Prss37-/-reason of male mouse growing barrier, we with the male mouse of wild-type or Prss37-/-separate zygote in the uterine tube of the female mouse of the normal mating of male mouse, analyze the zygote ratio of two protokaryon phases and two cell stages, estimate the rate of fertilization of male mouse with this.Shown in Fig. 6 B, apparent two cell stage zygotes are present in the wild-type group in a large number under two protokaryon phase zygotes of Hoechst33258 dyeing and the opticmicroscope, Prss37-/-group only has indivedual zygotes to be in pair protokaryon phases or two cell stages.The rate of fertilization analytical results shows, the wild-type group be 67%, Prss37-/-group only is 2.3%.These results further confirmed Prss37-/-growing barrier of male mouse, illustrate simultaneously Prss37-/-growing barrier of male mouse is not because the development of fertilized ova problem, but the problem of fertilization process itself.

Mammiferous fertilization process is the event of continuous a, multi-step, at first requires sperm to arrive around the ovum by female reproductive tract.For research Prss37-/-whether the growing barrier of male mouse relevant to oviducal transfer ability from the uterus with sperm, we separated with the male mouse of wild-type or Prss37-/-the female mouse Utero-uterine tube of male mouse post-coitum 2h, carry out paraffin embedding after fixing, and carry out serial section in Utero-uterine tube junction (UTJ), to observe the sperm transfer ability at UTJ place.Shown in Fig. 6 C, with the female mouse of the male mouse mating of wild-type, can observe obvious sperm in its oviducal initial section and distribute; And with Prss37-/-the female mouse of male mouse mating, almost can't see the sperm migration at the UTJ place and enter uterine tube.Therefore, Prss37-/-growing barrier of male mouse may be because Prss37-/-sperm can not move and enter uterine tube.

In sum, Prss37-/-the internal fertilization rate of male mouse descends, Prss37-/-there is obstacle in sperm from the uterus to oviducal migration.

Embodiment 6

Identify that Prss37 is on the interactional impact of essence-ovum

As shown in Figure 7, Prss37-/-sperm seldom or almost can not be in conjunction with the complete ovum of zona pellucida, but the binding ability of itself and zona-free ovum compares with the wild-type sperm, do not have significant difference, illustrates that the disappearance of Prss37 has affected the combination of sperm and zona pellucida.In experiment in vitro fertilization, we observe Prss37-/-sperm can normal dispersion granulosa cell around the ovum, although Prss37-seldom/-sperm centers on and the promotion ovum rotates, but the zygote ratio of two protokaryon phases, two cell stages and blastula stage is with wild-type group suitable (data do not show).

The present embodiment proves, the Prss37 disappearance affects the combination of external sperm-zona pellucida, but Prss37-/-sperm can be combined with the ovum of zona-free and finish fertilization external, and zygote can ectogenesis to blastula stage

Embodiment 7

Identify that Prss37 is on the impact of sperm ADAM3

Owing in the western blot that fails at epididymis or the mature sperm albumen extract experiment, detecting Prss37 protein expression signal, we think may not contain Prss37 albumen in the mature sperm of mouse.Prss37 is probably by destroy participating in the key factor of sperm-zone binding so, indirectly cause Prss37-/-sperm and zona pellucida in conjunction with obstacle.Previous research report prompting ADAM3 is the factor that is closely related most that participates in sperm-zone binding, therefore, we with western blot methods analyst Prss37-/-testis of mouse and sperm protein extract in the expression of ADAM3.Consistent with expected results, the disappearance of Prss37 causes the disappearance of the ADAM3 in the mature sperm albumen extract, but its testis precursor protein is compared with wild-type and do not had difference (Fig. 8).

The present embodiment proves, the disappearance of Prss37 causes that ADAM3 disappears in the mature sperm.

Conclusion

The present invention has obtained the clear and definite arrenotoky disorder disease model of a kind of phenotype---Prss37 gene knockout mice model.The Prss37 gene specific is expressed the sperm formation stages at testis tissue, and by affecting the appearance of ADAM3 in mature sperm, the combination of remote effect sperm-zona pellucida and sperm cause serious male fertility obstacle from the uterus to oviducal migration.Simultaneously because the high homology of mouse Prss37 and people Prss37 albumen, this male fertility Disorder Model will provide a new direction for infertile and diagnosis and treatment reproductive medicine, for Prss37 provides experiment support as the application prospect of possible contraceptive target spot, has important using value simultaneously.

All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

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Claims (11)

1. the preparation method of the male fertility obstacle animal model of a non-human mammal is characterized in that, comprises step:

The cell of non-human mammal is provided, with Prss37 gene inactivation in the described cell, and

The cell regeneration of described Prss37 gene inactivation is formed male fertility obstacle animal model.

2. the method for claim 1 is characterized in that, described inactivation is to insert by gene knockout, gene interruption or gene to cause the Prss37 gene inactivation.

3. the method for claim 1 is characterized in that, described non-human mammal comprises rodent or primate, preferably comprises mouse, rat, monkey.

4. the method for claim 1 is characterized in that, described method comprises:

Utilize the ET clone, reject exon3 and the exon4 of Prss37 gene in the ES cell by homologous recombination, thereby obtain positive ES cell clone;

Make chimeric rodent with described positive ES cell clone, comprise male mosaic rodent and female rodent;

Described male mosaic rodent and female rodent are carried out mating, thereby obtain the heterozygote rodent, comprise the male rodent of heterozygote and the female rodent of heterozygote;

With the male rodent of described heterozygote and the female rodent mating of heterozygote, thus the rodent of isozygotying of acquisition Prss37 gene complete deactivation.

5. the purposes with the described method of claim 1 male fertility obstacle animal model preparation, non-human mammal is characterized in that, is used for the material that the male fertility obstacle was treated or improved in screening; Or for the ripe promotor of screening ADAM3.

6. the purposes of a Prss37 gene or albumen is characterized in that, is used for the screening treatment or improves the material of male fertility obstacle; Be used for the material that the screening Inhibit sperm is combined with ovum.

7. one kind is screened the method that affects the candidate substances that sperm is combined with ovum, it is characterized in that, comprises step:

Test substance is applied to the non-human mammal of test group;

Detect the expression of Prss37 gene or the activity of albumen; With

Compare with control group, if wherein described test substance causes the rise of Prss37 genetic expression or protein-active, represent that then test substance is the candidate substances that promotes that sperm is combined with ovum; If described test substance causes the downward modulation of Prss37 genetic expression or protein-active, represent that then test substance is the candidate substances that Inhibit sperm is combined with ovum.

8. method as claimed in claim 7 is characterized in that, also comprises step:

To the candidate substances that the promotion sperm that obtains is combined with ovum, further test it and can treat or improve the male fertility obstacle; And/or

To the candidate substances that the Inhibit sperm that obtains is combined with ovum, can further test it as contraceptive.

9. a Prss37 gene or the promotor of albumen or the purposes of inhibitor is characterized in that, for the preparation of the conditioning agent that affects sperm and be combined with ovum.

10. a method of screening the ripe promotor of ADAM3 is characterized in that, comprises step:

Test substance is applied to the animal model of the described method preparation of claim 1; With

Detect the content of ripe ADAM3 in the mature sperm albumen extract of described animal model;

If compared with the control, the content of described ripe ADAM3 raises, and then described test substance is the ripe promotor of ADAM3.

11. one kind with the described method of claim 1 male fertility obstacle animal model preparation, non-human mammal, it is characterized in that, described animal model has following characteristic:

(a) produce ripe sperm, but described sperm can not be combined with the ovum with zona pellucida effectively;

(b) in testis tissue, produce the ADAM3 precursor, but do not produce ripe ADAM3.

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