CN107998397A - Application of the EMC10 albumen as biomarker in male sterility is diagnosed - Google Patents
Application of the EMC10 albumen as biomarker in male sterility is diagnosed Download PDFInfo
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Abstract
The invention discloses application of the EMC10 albumen as biomarker in male sterility is diagnosed.Experiment proves, male sterility can be prevented by improving the material of EMC10 protein actives and/or expression quantity, treat male sterility, promote sperm and ovum fertilization, promote the ovum fertilization of sperm and zona-free, improve the ability that sperm is merged with after birth, treat sperm function obstacle, promote the maturation of sperm, promote maturation of the sperm in epididymis, improve the locomitivity of sperm, it is horizontal to improve the relevant protein tyrosine phosphatase of capacitation, promote the capacitation of sperm, improve the acrosome reaction ability of sperm, repair cacospermia form, recover the activity of sodium/potassium APT enzymes, maintain ions in spermatozoa balance, maintain Na in sperm+Balance, maintain HCO3 in sperm‑Balance, maintain pH value balance in sperm, improve male fecundity.The present invention has great application value.
Description
Technical field
The present invention relates to biomedicine field, and in particular to EMC10 albumen is as marker in male sterility is diagnosed
Using.
Background technology
The mankind have found that reticulon compound (ER Membrane Protein Complex, EMC) is in ferment first
It is the compound being made of 6 subunits (i.e. Emc1 albumen-Emc6 albumen) related with protein folding in endoplasmic reticulum in mother.Afterwards
Come during the application relevant protein degradation of protein science research endoplasmic reticulum, find in animal EMC be by 10 subunits (i.e.
EMC1 albumen-EMC10 albumen) form compound.EMC is multi-functional, evolution conservative a albumen composition, it is considered
A variety of effects have been played in endoplasmic reticulum degradation, endoplasmic reticulum-mitochondria grappling, membrane-spanning proteins assembling process.For example, EMC6 eggs
Bai Keneng take part in nematode body specific proteins in the degraded of endoplasmic reticulum, it is also possible to participate in adjusting the autophagy of human inner cell.
In addition, EMC1 albumen is found related with cerebellar atrophy with overall development delay, Tendon defection, scoliosis.Although these
Research adds us to the understanding in terms of EMC biology, but the function of EMC also illustrates far away completely.
EMC10 albumen is egg highly conserved in multiple species (including plant, invertebrate, mammal etc.) evolution
It is one of white.The present inventor has cloned the encoding gene of EMC10 albumen from the cDNA storehouses of actrapid monotard's knurl for the first time, and
It is named as INM02 genes.Because EMC10 albumen and the no obvious homology of any of albumen or albumen domain,
EMC10 albumen is considered as a new protein.At present, there are the several a variety of biologies studied and disclose EMC10 albumen
Learn function.The present inventor utilizes ELISA method, and it is one in human serum to have reported EMC10 albumen first in the world
In the secretory protein that can be detected, and it was found that encode the gene of EMC10 albumen (i.e. in mouse islets β cells
Emc10 genes) expression regulated and controled by glucose, prompt it to play an important role in glycometabolism.Hereafter, another
Research group reports the encoding gene that EMC10 albumen is cloned into from the candidate stem cell of people's purifying, which will
It is named as HSS1 genes, and its another Shear criteria is named as HSM1 genes；They study discovery in vitro at the same time,
EMC10 albumen (HSS1 genes) can suppress the propagation of glioma cell line, migration, invasion and attack, while it is thin also to suppress endothelium
The new vessels of born of the same parents is formed, therefore they think that EMC10 albumen is the treatment potential target spot of malignant glioblastoma.Also grind
Study carefully discovery, in a schizophrenia mouse model, elevated Mrita22 genes (the mouse homologous gene of people's EMC10 genes)
It can suppress the development that neuronal cell dendron and ridge are dashed forward, the level by reducing Mrita22 genes can be saved above-mentioned completely
The defects of mouse model hippocampus basivertebral neuron dendron and ridge burst are educated, prompts EMC10 albumen in Mouse Neuron dendron and ridge
Play an important role in prominent forming process.Although result of study is more, the biological action of EMC10 albumen and play a role
Mechanism it is largely still unclear.
Infertile is public health problem more serious in global range, and there is infertile by 15% couple at child-bearing age
The problem of, wherein male sterility accounts for 50%.Now some researches show that many genes have been involved in male sterile pathogenesis.So
And most potential cause is still unclear.
The content of the invention
The technical problems to be solved by the invention are how to prevent and/or treat male sterility.
In order to solve the above technical problems, present invention protection first improves EMC10 protein actives and/or the material of expression quantity exists
Prepare the application in product；At least one of the function of the product can be following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go
The ovum fertilization of oolemma；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7 sperm) is promoted
Maturation；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 it is relevant) to improve capacitation
Protein tyrosine phosphatase is horizontal；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 essence) is repaired
Sub- abnormal morphology；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+
Balance；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
The present invention also protects application of the EMC10 albumen as drug target in product is prepared；The function of the product can
At least one of for following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go
The ovum fertilization of oolemma；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7 sperm) is promoted
Maturation；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 it is relevant) to improve capacitation
Protein tyrosine phosphatase is horizontal；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 essence) is repaired
Sub- abnormal morphology；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+
Balance；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
The present invention also protects application of the EMC10 albumen in product is prepared；The function of the product can be following A1) extremely
At least one of A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go
The ovum fertilization of oolemma；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7 sperm) is promoted
Maturation；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 it is relevant) to improve capacitation
Protein tyrosine phosphatase is horizontal；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 essence) is repaired
Sub- abnormal morphology；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+
Balance；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
In any of the above-described application, the product can be medicine.
At least one of the present invention also protects the application of EMC10 albumen, can be following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go
The ovum fertilization of oolemma；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7 sperm) is promoted
Maturation；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 it is relevant) to improve capacitation
Protein tyrosine phosphatase is horizontal；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 essence) is repaired
Sub- abnormal morphology；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+
Balance；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
In order to solve the above technical problems, the present invention also protects a kind of product, it can contain EMC10 albumen or improve EMC10
The material of protein active and/or expression quantity；At least one of the function of the product can be following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go
The ovum fertilization of oolemma；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7 sperm) is promoted
Maturation；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 it is relevant) to improve capacitation
Protein tyrosine phosphatase is horizontal；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 essence) is repaired
Sub- abnormal morphology；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+
Balance；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
The product concretely medicine.
The present invention also protects following X1) or X2)：
X1 application of the material for) being used to detect EMC10 albumen in the product for preparing diagnosis male sterility；
X2 application of the material for) being used to detect EMC10 albumen in male sterility is diagnosed.
In order to solve the above technical problems, the present invention also protects a kind of kit for being used to diagnose male sterility, it may include uses
In the material of detection EMC10 albumen.
Any of the above-described " material for being used to detect EMC10 albumen " can be the material of detection EMC10 expressing quantities.
It may also include the carrier for recording criterion in mentioned reagent box；The criterion can be：If person under test
The expression quantity of EMC10 albumen is less than the expression quantity of EMC10 albumen in control sperm in sperm, then the person under test is or doubtful is
Male sterility patient；If the expression quantity of EMC10 albumen is not less than the expression of EMC10 albumen in control sperm in person under test's sperm
Amount, then the person under test be not or it is doubtful be not male sterility patient；The control sperm is with normal male fecundity
Sperm.
It is any of the above-described described less than being less than statistically.
The present invention also protects following Y1) or Y2)：
Y1) application of the EMC10 albumen as biomarker in the reagent of exploitation diagnosis male sterility；
Y2) application of the EMC10 albumen as biomarker in male sterility is diagnosed.
Any of the above-described sperm can be b1) or b2)：B1) mouse sperm；B2) human spermatogoa.
Any of the above-described human spermatogoa sperm that concretely people is obtained by masturbation.
Concretely mouse is located at the sperm in cauda epididymis to any of the above-described mouse sperm.
Any of the above-described mouse can be C57BL/6j mouse.
It is demonstrated experimentally that Emc10 gene knockouts cause mouse male sterility, suppress sperm and ovum fertilization, suppress sperm and
The ovum fertilization of zona-free, reduce ability, the maturation of suppression sperm, suppression sperm that sperm merge with after birth in epididymis
Locomitivity that is ripe, reducing sperm, reduce the relevant protein tyrosine phosphatase level of capacitation, suppress obtaining for sperm
Can, reduce sperm acrosome reaction ability, cause cacospermia form, cause ions in spermatozoa it is unbalance, reduce male fertility energy
Power.Male sterility can be prevented and/or treat by improving the material of EMC10 protein actives and/or expression quantity.The present invention has great
Application value.
Brief description of the drawings
Fig. 1 is the experimental result of step 1 in embodiment 2 and embodiment 3.Wherein, Wt Emc10+/+Mouse, Ko are
Emc10-/-Mouse, 8-12w expressions grow to 8-12 week old, and 8-10m represents to grow to the 8-10 monthly ages.
Fig. 2 is the experimental result of step 2 in embodiment 3.Wherein, Wt Emc10+/+Mouse, Ko Emc10-/-Mouse, F
For F patterns, B is B-mode, and AR is AR patterns, and Con represents untreated (control), and A23187 is acrosome reaction derivant A23187,
PG is progesterone.
Fig. 3 is the experimental result of step 3 in embodiment 3.Wherein, Wt Emc10+/+Mouse, Ko Emc10-/-Mouse,
Te represents testis, and Cap represents epididymis head, and Prox.Cor represents the nearly body portion of epididymis, and Distal Cor represent the remote body portion of epididymis,
Cau represents cauda epididymis.
Fig. 4 is the experimental result of step 1 in embodiment 4.Wherein, Wt Emc10+/+Mouse, Ko Emc10-/-Mouse,
Translation is translation, and Translational initiation start for translation, and Cilium morphogenesis are fibre
Hair form is formed, and Spermatogenesis is spermatogenesis, and Cilium assembly are fimbriae assembly, Intraciliary
Transport is transport in cilium, and Protein folding are protein folding, Cell projection organization
Dashing forward assembling for cell, Cell-cell adhesion are cell-cell adhesion, and Protein transport are albumen transport,
TRNAcharging is EIF2 signals for amino acid binding transport RNA, EIF2Signaling, and RAN Signaling believe for RAN
Number, Regulation of eIF4Signaling are regulation and control eIF4 signals, and Protein Ubiquitination are proteins ubiquitin
Change, Unfolded protein response are Non-adhesion inhibition index, and Aldosterone Signaling believe for aldosterone
Number, mTOR Signaling are mTOR signals, and PI3K/AKT Signaling are PI3K/AKT signals, Hypoxia
Signaling is anoxic signal, and Asparagine Biosynthesis I are asparagine biosynthesis I, eNOS
Signaling is eNOS signals, and GDP-L-fucose Biosynthesis I are GDP-L- fucose biosynthesis I,
Endoplasmic Reticulum Stress are er stress, and Fertility is reproduction, and Sperm disorder are sperm
Dysfunction, Cell death of germ cells are reproductive death.
Fig. 5 is the experimental result of step 2 in embodiment 4.Wherein, Wt Emc10+/+Mouse, Ko Emc10-/-Mouse,
Ko+B is 8Br-cAMP, Ko+I IBMX.
Fig. 6 is the experimental result of embodiment 5.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiments, is respectively provided with three repeated experiments, as a result makes even
Average.
In following embodiments all zooperies by Shanghai Inst. of Life Science, CAS biochemistry with it is thin
(credit number is ratified in born of the same parents' biological study institute's tending of animals and committee's agreement of use：SYXK2007-0017).
All mouse in following embodiments are raised and breed at Shanghai model biological study center, and in experimentation
Water and food can be obtained freely.
The step of preparing the antibody of rabbit-anti people ATP1A4 is specific as follows：(1) artificial synthetic polypeptide (amino acid sequence of polypeptide
For (from N-terminal to C-terminal)：CYDEIRKLLIRQHPDGWVERETYY)；A part for this polypeptide behaviour ATP1A4 albumen；(2) with step
Suddenly the large ear rabbit of polypeptide immune health prepared by (1), collects serum, obtains the antibody of rabbit-anti people ATP1A4.
The solute and its concentration of EKRB nutrient solutions are 120mM NaCl, 4.8mM KCl, 1.0mM CaCl2、1.2mM
MgSO4、1.2mM KH2PO4, 5mM glucose, 21mM sodium lactates, 0.25mM Sodium Pyruvates, 25mM NaHCO3And 3mg/mL
BSA；Solvent is distilled water；PH value is natural；115 DEG C of sterilizing 20min.Wherein, NaCl, KCl, CaCl2、MgSO4、KH2PO4, grape
Sugar, sodium lactate, Sodium Pyruvate, NaHCO3It is the product of the highest purity level of Sigma companies production with BSA.
Statistical analysis in following embodiments is to carry out T inspections, P to data with SigmaPlot12.5 softwares<0.05 quilt
Think with statistical significance.
Embodiment 1, the expression of EMC10 albumen, purifying and the preparation of EMC10 polyclonal antibodies
1st, vector construction
EMC10 genes are connected with carrier pET32a, obtain recombinant vector pPROEX HT-EMC10.
Recombinant vector pPROEX HT-EMC10 are sequenced.According to sequencing result, to recombinant vector pPROEX HT-
EMC10 carries out structure and is described as follows：To insertion EMC10 genes between III restriction enzyme site of EcoRI and Hind of carrier pET32a
Nucleotide sequence.
2nd, the expression of EMC10 albumen
(1) recombinant vector pPROEX HT-EMC10 are imported in Escherichia coli ROSSET (DE3), obtains restructuring large intestine bar
Bacterium, is named as ROSSET (DE3)/pPROEX HT-EMC10.
(2) after completing step (1), ROSSET (DE3)/pPROEX HT-EMC10 single bacterium colonies are inoculated in 5mL YTA cultures
Base, 37 DEG C, 250rpm shaken cultivations to OD600nmFor 1.0-2.0, bacterium solution first is obtained.
(3) bacterium solution first is seeded to 500mL YTA culture mediums, 37 DEG C, 250rpm shaken cultivations to OD600nmFor 1.0-2.0,
Obtain bacterium solution second.
(4) bacterium solution second is taken, IPTG is added and makes its concentration in system be 1mM, then 25 DEG C of induced expression 3h, obtain
Bacterium solution third.
(5) bacterium solution third is taken, 4 DEG C, 4000rpm centrifugation 20min, abandon supernatant, collect thalline.
(6) the part thalline for taking step (5) to collect, ultrasonic degradation, centrifugation, obtains bacterial cell disruption precipitation and supernatant.Will
Bacterial cell disruption precipitates and supernatant carries out SDS-PAGE.
The result shows that about have a concentration band at 32kd in molecular weight in bacterial cell disruption precipitation, and expression quantity in supernatant
It is low.Therefore, EMC10 albumen is expressed in inclusion body.
3rd, the purifying of EMC10 albumen
(1) the remaining thalline for taking in step 2 (5) to collect, adds BugBuster (product of Novagen companies) and ultrasound
Bacterium is cracked, centrifugation, collects precipitation.
(2) precipitation for taking step (1) to collect, adds the 1 × binging buffer dissolvings of the urea containing 8M, then will dissolving
Supernatant add through NiSO4The resin columns handled well, with EMC10 albumen in 1 × elute buffer column precipitators after washing, obtain
To the EMC10 albumen of purifying.
1 × binging buffer, 1 × elute buffer be ... the product of company.
4th, prepared by EMC10 polyclonal antibodies
(1) the EMC10 albumen for the purifying for obtaining step 3 carries out SDS-PAGE electrophoresis, then that gel is put into coomassie is bright
Blue dye liquor dyes 4h, takes out gel and is put into 4~8h of decoloration in destainer, purpose band, Ran Houyong are cut off with aseptic operation blade
The PBS buffer of pH7.4,0.1mol/L are washed 2 times, and 4 DEG C save backup.
(2) using new zealand white rabbit as immune animal.White rabbit makees to gather before immunity inoculation blood sample using as negative right
According to.Gel containing 100 μ g EMC10 albumen is pulverized, is emulsified with Freund's complete adjuvant, after the shaving of white rabbit back, disperses 24-
36 site injections are incomplete with the antigen of equivalent and Freund at the 4th week, the 7th week, the 9th week and the 11st week respectively after initial immunity
Booster immunization after adjuvant emulsion.Culling heart blood after final immunization, antibody titer is detected with Western Blot methods.Final immunization
Arteria carotis bloodletting in 10th day afterwards, isolates immune serum, and immune serum adds NaN3To final concentration 0.02%, be sub-packed in 1.5mL from
Heart pipe, -80 DEG C save backup.Antibody titer and quality are detected using Western Blot methods.
(3) Binding/Washing Buffer (volume ratios 1 are added into immune serum：1) serum dilution is obtained.To
The Protein A Resin suspensions of 1mL are added in suction attached column, after resin precipitated, buffer solution is drained, adds 5mL
Binding/Washing Buffer wash a resin, and serum dilution is slowly added into the adsorption column installed and (crosses column flow
For 1mL/min), sample it is complete cross column after, add 30mL Binding/Washing Buffer to wash pillar, added into adsorption column
10mL Elution Buffer antibody elutions, collect eluent, add the Tris-HCl of 1M into eluent immediately, adjust pH
To 7.4~8.0, that is, obtain EMC10 polyclonal antibodies.
Embodiment 2, Emc10 gene knockouts cause mouse male sterility
1st, the acquisition of targeting vector
Using bacterial homologous recombinant technique, two loxp fragments are inserted into Emc10 genes (No. Ensembl：
ENSMUSG00000008140) the both sides of the 2nd exon, then anti-neomycin element is inserted into No. 2 and No. 3 and shows son
Between, obtain targeting vector.The construction method of targeting vector refers to following document：Pentao Liu, Nancy A., Jenkins
and Neal G.Copeland.A highly efficient recombineering-based method for
generating conditional knockout mutations.Genome Res.2003Mar；13(3):476-
84.Chan W, Constantino N, Lee SC, Su Q, Melvin D, Court DL, Liu P.A recombineering
based approach for high-throughput conditional knockout targeting vector
construction.Nucleic Acids Res.2007；35(8)：e64.Epub 2007Apr 10.
2nd, the acquisition of Emc10 gene knockouts homozygote mouse
(1) targeting vector for obtaining step 1 linearizes, and electroporation imports 129Sv/Ev mouse embryo stem cells
(SCR012, Chemicon Ltd.), then separates and expands the embryonic stem cell of anti-neomycin.
(2) after completing step (1), the capsule by the embryonic stem cell microinjection of anti-neomycin to female C57BL/6J mouse
Embryo, then the uterus of false pregnancy female mice is transplanted to, give a birth, obtain gomphosis mouse.
(3) after completing step (2), gomphosis mouse is mated with C57BL/6j mouse, obtains hybrid mice.
(4) after completing step (3), hybrid mice and FLp recombined small-mouses (003800, Jackson lab) is hybridized, obtained
To the mouse for removing anti-neomycin gene.
(5) after completing step (4), will remove the mouse of anti-neomycin gene and EIIa-Cre mouse hybrids (003314,
Jackson lab) (purpose is removal flox genes), obtain Emc10 gene knockout hybrid mices.
(6) after completing step (5), Emc10 gene knockouts hybrid mice is mated mutually, obtains Emc10 gene knockouts
Homozygote mouse (hereinafter referred to as Emc10-/-Mouse) and wild-type mice (hereinafter referred to as Emc10+/+Mouse).
Observe and count Emc10-/-Mouse and Emc10+/+The growth and development situation of mouse.The result shows that Emc10-/-Mouse
With Emc10+/+It is not different in the survival rate of mouse, shape and overall behavior；But male Emc10-/-Mouse infertility (Fig. 1 completely
Middle A), female Emc10-/-The fecundity of mouse declines.
The above results show that Emc10 gene knockouts cause mouse male sterility, and female fertility declines.
Embodiment 3, Emc10 gene knockouts cause the male sterile phenotype research of mouse
First, Emc10 genes may participate in the fertilization process of embryonated egg
1st, 6 male Emc10 are taken-/-Mouse, mates 3 months with some female wild type mouse.Observed during mating female
Property wild-type mice genital tract whether there is smart bolt.
Experimental result is as follows：There is smart bolt in female wild type mouse propagation road, illustrate that mating is normal；But no offspring goes out
It is raw.
2nd, experiment one in vitro fertilization
(1) the pregnant mare serum promoting sexual gland hormone of 5 units is injected intraperitoneally to adult female wild-type mice.
(2) step (1) 48h afterwards is completed, the hCG of 10 units is injected intraperitoneally to the adult female wild-type mice.
(3) step (2) 13h afterwards is completed, the adult female wild-type mice is put to death, then takes out fallopian tubal juxtaposition
In the culture dish (specification 35mm) containing human tubal fluid, cumulus oocyte is finally separating out.
(4) sperm (Emc10 to be measured is taken-/-Mouse sperm or Emc10+/+Mouse sperm), with the PBS of pH7.4,0.1mol/L
Buffer solution washes twice.
(5) sperm to be measured and the separated cumulus oocyte of step (3) that will complete step (4) mix, then 5%CO2、
37 DEG C of incubation 60min.
(6) mixed liquor of step (5) is taken into, fully washs that (purpose is goes with the PBS buffer of pH7.4,0.1mol/L
Except uncombined sperm), then 5%CO2, 37 DEG C of culture 24h.
The solution for completing step (6) is placed in micro- Microscopic observation, and calculates the quantity of two cell stages after insemination.
Experimental result is shown in B and D in Fig. 1.The result shows that Emc10-/-The embryonated egg of mouse sperm insemination cannot develop
The stage of two cell stages, and Emc10+/+The embryonated egg of mouse sperm insemination can then develop the stage to two cell stages.
3rd, experiment two in vitro fertilization
(1) with (1) in step 2.
(2) with (2) in step 2.
(3) with (3) in step 2.
(4) step (3) separated cumulus oocyte is taken, zona-free, obtains the cumulus oocyte of zona-free.
(5) with (4) in step 2.
(6) " cumulus oocyte of zona-free " for obtaining the sperm to be measured for completing step (5) and step (4) is mixed
Close, then 5%CO2, 37 DEG C be incubated 60min.
(7) mixed liquor of step (6) is taken into, fully washs that (purpose is goes with the PBS buffer of pH7.4,0.1mol/L
Except uncombined sperm), then 5%CO2, 37 DEG C of culture 24h.
The solution for completing step (7) is placed in micro- Microscopic observation, and calculates the quantity of two cell stages after insemination.
Experimental result is shown in B in Fig. 1.The result shows that Emc10-/-Mouse sperm can not make the cumulus oocyte of zona-free
Fertilization, and Emc10+/+Mouse sperm can make the cumulus oocyte of zona-free be fertilized.Therefore, Emc10-/-Mouse sperm does not have
The standby ability merged with after birth and activate fertilization.
4th, single-semen injection and fertility experiment in endochylema
(1) with (1) in step 2.
(2) with (2) in step 2.
(3) with (3) in step 2.
(4) sperm (Emc10 to be measured is taken-/-Mouse sperm or Emc10+/+Mouse sperm), the suction pipe driven with Piezo
(product of Piezoelectric actuator, PrimeTech companies) separates head and the afterbody of sperm to be measured, then will treat
The head for surveying sperm is expelled in step (3) separated cumulus oocyte, then the cumulus oocyte of fertilization is trained in KSOM
It is incubated in nutrient solution, treats that embryonic development to two cell stages, is implanted into the fallopian tubal of false pregnancy female mice, observes the fertility feelings of replace-conceive female mice
Condition.
Experimental result is shown in C in Fig. 1.The result shows that Emc10-/-Mouse sperm and Emc10+/+Mouse sperm can make wild
The cumulus oocyte fertilization of type mouse, and develop into normal embryo.
The above results show, Emc10-/-The spermatogenesis function of mouse is normal and carries complete haploid genome.
Emc10 genes may participate in the fertilization process of embryonated egg.
2nd, Emc10 gene knockouts cause sperm function obstacle
Mouse to be measured is male Emc10-/-Mouse or male Emc10+/+Mouse.
1st, Emc10 gene knockouts do not influence spermatogenesis and do not influence the development of testis and epididymis
The testis and epididymis morphosis of mouse to be measured are observed, measures mouse weight and testicular weight to be measured, collects adult
Mouse to be measured is located at the sperm of testis, epididymis head, epididymis near-end, epididymis distal end or cauda epididymis and detects sperm quantity.
As a result it is as follows：Male Emc10-/-Mouse and male Emc10+/+The weight of mouse is without significant difference；Grow to 8-12
During week old, with male Emc10+/+Mouse is compared, male Emc10-/-Sperm quantity is substantially less in mouse testis weight and epididymis；
When growing to the 8-10 monthly ages, with male Emc10+/+Mouse is compared, male Emc10-/-Sperm count in mouse testis weight and epididymis
Amount is then without significant difference；After testicular weight is standardized, the male Emc10 of any age bracket-/-Mouse and male Emc10+/+It is small
Significant difference is not present in the sperm count of allen-Doisy unit testicular weight；With male Emc10+/+Mouse is compared, male Emc10-/-Mouse
Testis and epididymis morphosis no significant difference.The above results show that Emc10 gene knockouts do not influence spermatogenesis and do not influence
The development of testis and epididymis.
2nd, EMC10 has the function that maturation of the sperm in epididymis important
After sperm leaves testis, before interacting with ovum, it is necessary to functional maturation is undergone in epididymis.This
Process includes acquisition, the capacitation of sperm and the acquisition of acrosome reaction ability of Sperm Motility.
(1) Emc10 gene knockouts cause the locomitivity of sperm to be remarkably decreased
The sperm that the mouse to be measured that grows up is located in cauda epididymis is placed in EKRB nutrient solutions, 37 DEG C of incubations；Then in room
Analyzed under the conditions of temperature using HTM-TOX IVOS sperm motilities analyzer (product of Hamilton-Thorn Research companies)
The motion conditions of sperm, i.e. 0min (when the sperm that the mouse to be measured that grows up is located in cauda epididymis is placed in EKRB nutrient solutions), incubate
Educate 30min, the motion conditions for being incubated 60min, being incubated 90min and being incubated 120min analysis sperms.HTM-TOX
The parameter setting of IVOS sperm motility analyzers is as follows：37 DEG C of temperature, two pixels of the smallest cell volume, minimum contrast 50,
Minimum static contrast 25, low VAP point of contacts value 20, low VSL point of contacts value 30, threshold lines degree 50%, static head size 0.3-
1.95, static head intensity 0.5-1.3, magnifying power 0.81,30 frames are obtained with the frame rate of 60Hz.Used in whole analytic process
Playback function is with the accuracy of detection method.
Test result indicates that with Emc10+/+Mouse is compared, Emc10-/-The locomitivity of mouse sperm is remarkably decreased：Including
Master including total locomitivity (A in Fig. 2), path velocity (B in Fig. 2), linear velocity (C in Fig. 2) and orbital velocity (D in Fig. 2)
Kinematic parameter is wanted to be remarkably decreased；But Emc10+/+Mouse and Emc10-/-Similar (94.0 ± the 1.0%vs of sperm motility of mouse
93.7 ± 1.1%).
(2) Emc10 gene knockouts cause the relevant protein tyrosine phosphatase function of capacitation to be damaged
Sperm shows as a series of time dependent protein tyrosine phosphatase levels in the normal capacitation of cauda epididymis
Increase.
The sperm that mouse to be measured is located in cauda epididymis of growing up is taken, total protein is extracted from sperm, in 12% dodecane
It is separated by electrophoresis, is then transferred on polyvinylidene fluoride (PVDF) film, then with tyrosine phosphorus on base sodium sulphate-polyacrylamide gel
Acidifying (Millipore 4G10 are cloned, and 1：10000 dilutions) antibody incubation, the goat that secondary antibody is coupled using horseradish peroxidase
Anti-rabbit antibody (product of Sigma companies), purpose band is shown finally by ECL Plus (Amersham) chemiluminescence.
Experimental result is shown in E in Fig. 2.The result shows that by bull Emc10-/-The sperm that mouse is located in cauda epididymis is put
After being incubated in EKRB nutrient solutions, the relevant protein tyrosine phosphatase of capacitation is horizontal not to be increased with the extension of time
Add；And by bull Emc10+/+Mouse is located at the sperm in cauda epididymis and is placed in EKRB nutrient solutions after incubation, and capacitation is related
Protein tyrosine phosphatase level then increase with the extension of time.Therefore, Emc10 gene knockouts cause capacitation phase
The protein tyrosine phosphatase function of pass is damaged.
(3) Emc10 gene knockouts cause the capacitation of sperm and acrosome reaction ability to be suppressed
A, the sperm that mouse to be measured is located in cauda epididymis of growing up is taken, is dyed with aureomycin (CTC), is then tied according to dyeing
Fruit assesses the capacitation situation and acrosome reaction of sperm.Whole experiment at least have evaluated the different CTC staining methods of 300 sperms：F
Pattern refers to that there is fluorescent staining on whole head, represents the sperm for possessing intact acrosomes but non-capacitation；B-mode refers to except acrosome
Rear portion, other head zones have fluorescent belt, represent the sperm with intact acrosomes and capacitation；AR patterns refer to whole sperm
Head does not have fluorescent staining, represents capacitation and the sperm of acrosome reaction occurs.Three phases can be seen to be had in sperm body portion
Bright fluorescent staining.
Experimental result is shown in F in Fig. 2：With male Emc10+/+Mouse is compared, male Emc10-/-Sperm (the F of the non-capacitation of mouse
Pattern) dramatically increase, the ratio of capacitated sperm (B-mode) substantially reduces, the sperm quantity of spontaneous acrosome reaction (AR patterns)
Also substantially reduce.The result shows that bull Emc10-/-The capacitation that mouse is located at the sperm in cauda epididymis is suppressed, this
One result and the horizontal not increased result with the extension of time of the relevant protein tyrosine phosphatase of capacitation in step (2)
It is completely the same.
B, after completing step a, with acrosome reaction derivant A23187 or the sperm of Progesterone stimulated AR patterns.
Experimental result is shown in G in Fig. 2.The result shows that bull Emc10+/+Reaction of the mouse sperm to A23187 or progesterone
Well, bull Emc10-/-Mouse sperm does not react acrosome reaction derivant A23187 or progesterone.
The above results show that EMC10 has the function that maturation of the sperm in epididymis important.
3rd, Emc10 gene knockouts cause the exception of sperm morphological
The sperm that the mouse to be measured that grows up is located at testis, epididymis head, epididymis near-end, epididymis distal end or cauda epididymis is collected,
Observe under the microscope.Mouse to be measured is male Emc10-/-Mouse or male Emc10+/+Mouse.
Experimental result is as follows：Male Emc10-/-Mouse and male Emc10+/+Mouse is located at sperm in testis in morphology
Upper no difference (A in Fig. 3)；Male Emc10-/-Mouse is located at the sperm in epididymis head or nearly body portion in sperm body portion and afterbody
Junction there is obvious bending (B and C in Fig. 3), male Emc10+/+Mouse does not occur this phenomenon then；Male
Emc10-/-Mouse is located at the sperm of the remote body portion of epididymis or afterbody with the presence of much in the hairpin like folding (Fig. 3 in the engaging portion of body tail
Middle D and E), male Emc10+/+Mouse does not occur this phenomenon then；Male Emc10-/-Mouse is located at the sperm of cauda epididymis
In, 56% there is hairpin like folding (E in Fig. 3) on morphology, and male Emc10+/+Mouse sperm is almost without appearance
Hairpin like folding.The result shows that with male Emc10+/+The sperm of mouse is compared, male Emc10-/-There are hairpin like for mouse
Defective sperm, and in testis to gradually increasing during the dividing a word with a hyphen at the end of a line of epididymis.As it can be seen that Emc10 gene knockouts can cause mouse smart
The modal exception of son, at the same prompt EMC10 to maintain sperm from testis be moved to epididymis during normal morphology have
Important function.
Embodiment 4, Emc10 gene knockouts cause the male sterile Study on Molecular Mechanism of mouse
First, Emc10 gene knockouts cause the proteomic expression profile of sperm to significantly change
Because mature sperm loses the ability of genetic transcription, the present inventor uses the quantitative analysis method of TMT marks
Carried out in protein science level without offset screening analysis.Comprise the following steps that：Take and grow to 8-12 week old mouse to be measured (male
Emc10-/-Mouse or male Emc10+/+Mouse) 17, extract total protein, Trypsin Induced protein, postdigestive peptide fragment
Marked with TMT (Tandem Mass Tag) 6-plex reagents (product of Pierce Biotechnology companies), Ran Houyong
Aquity UPLC systems (product of Waters Corporation companies) separate the polypeptide of mixed mark with high ph-values, then
Analyzed with Q-Exactive mass spectrographs (product of Thermo Fisher Scientific companies).Finally use Proteome
Discoverer software、Mascot、Scaffold Q+、gene ontology(GO)term enrichment
Analysis and Ingenuity Pathway Analysis (IPA) carry out data processing and quantitative analysis.
As a result it is as follows：With male Emc10+/+Mouse is compared, male Emc10-/-Mouse has the expression of 327 protein
More than 1.5 times significantly change.Hotspot graph and volcano figure show in this 327 protein significantly changed, there is 96.6%
(316/327) protein is in male Emc10-/-Expression is up-regulation in mouse sperm, and only 3.4% protein is in male
Emc10-/-Expression is to lower (A in Fig. 4) in mouse sperm.Using DAVID bioinformatics (http://
David.abcc.ncifcrf.gov/) this 327 protein are analyzed, are existed with which clear and definite GO biological process
Male Emc10-/-Significantly changed in mouse sperm.R-Script figures are shown in male Emc10-/-In mouse sperm significantly
First five biological process (C in Fig. 4) changed, is translation, translation startup, cilia morphology formation, spermatogenesis and fibre respectively
Hair assembling.In addition, the enrichment analysis of IPA paths is found, male Emc10-/-EIF2 and PI3K/Akt signal paths quilt in mouse sperm
Significantly activation, and eNOS paths are then suppressed significantly (D in Fig. 4).IPA module analysis shows that Emc10 gene knockouts can cause one
Series is with promoting fertility, suppressing cacospermia and suppressing the up-regulation of apoptosis of germ cells correlative protein expression, the above-mentioned equal energy of reaction
Enough improve male fertility ability (E in Fig. 4).It has also been found that, a series of expression for suppressing cell death related protein are in male at the same time
Emc10-/-Expression in mouse sperm is up-regulation, it means that EMC10 gene knockouts improve the survival rate of cell.It is theoretical
Upper theory, the activation of fecundity and the raising of living spermatozoa percentage are conducive to male Emc10-/-The fertility of mouse, above-mentioned protein groups
Result of study and male Emc10-/-The phenotype of mouse infertility is conflicting, and the present inventor thinks above-mentioned protein groups
It is to cause the compensation response of infertility to EMC10 gene knockouts to learn a series of performances for being conducive to fertility found.In order to probe into hero
Property Emc10-/-The direct molecular mechanism of mouse infertility, it is necessary to study the albumen of those downwards to find male Emc10-/-Mouse essence
The driving factors of subfunction disorder.
With male Emc10+/+Mouse is compared, in male Emc10-/-Only a few (3.4%) egg is only detected in mouse sperm
White matter is lowered, and in these albumen, only form Na/K-ATP enzymes ATP1A4 albumen (the α subunits of Na/K-ATP enzymes) and
The expression quantity of ATP1B3 albumen (the β subunits of Na/K-ATP enzymes) reduces more than 3 times (B in Fig. 4).Prompt EMC10 genes may
Adjusting to Na/K-ATP enzymatic activitys played an important role, and Na/K-ATP enzymes are to maintaining intracellular low sodium state to have weight
Act on.
2nd, Emc10 gene knockouts cause ion imbalance in spermatoblast
Mouse to be measured is male Emc10-/-Mouse or male Emc10+/+Mouse.
1st, EMC10 regulates and controls the expression of Na/K-ATP enzymes in sperm
Take the sperm that mouse to be measured be located in cauda epididymis of growing up, extracted from sperm to total protein, 12% 12
It is separated by electrophoresis on sodium alkyl sulfate-polyacrylamide gel, is then transferred on polyvinylidene fluoride (PVDF) film, uses ATP1A1
(Proteintech, 1：5000 dilution), ATP1A4 (1：1000 dilution), ATP1B3 (Abcam, 1：1000 dilution), tyrosine phosphorus
Acidifying (Millipore 4G10 are cloned, and 1：10000 dilutions) or alpha Actinin (Sigma, 1：20000 dilutions) antibody incubation, two
The anti-goat anti-rabbit antibodies (Sigma) coupled using horseradish peroxidase and goat anti-mouse antibody (Millipore) (dilution
Degree is 1：10000), purpose band is shown finally by ECL Plus (Amersham) chemiluminescence.
The result shows that ATP1A4 albumen and ATP1B3 albumen are in male Emc10-/-Almost without expression (figure in mouse sperm
A and B in 5), and in male Emc10+/+Expression quantity in mouse sperm is higher；ATP1A1 albumen (another α of Na/K-ATP enzymes
Subunit) in male Emc10+/+Mouse and male Emc10-/-Expression quantity in mouse sperm is without significant difference (A in Fig. 5).
2nd, Emc10 gene knockouts cause ion imbalance in spermatoblast
Mouse sperm to be measured is bull Emc10-/-Mouse is located at the sperm or bull Emc10 of cauda epididymis+/+
Mouse is located at the sperm of cauda epididymis.
Measure the Na of mouse sperm to be measured+Concentration.Comprise the following steps that：(1) mouse sperm to be measured is taken, is placed in containing 20 μM
In the PBS buffer of pH7.4,0.1mol/L of SBFI-AM and 0.2% (v/v) Pu Lukang acid, then 5%CO2, 37 DEG C of cultures
60min；(2) system of step (1) is taken into, is washed twice with the PBS buffer of pH7.4,0.1mol/L, then adds and is free of
The EKRB nutrient solutions (the EKRB nutrient solutions without BSA do not contain BSA with differing only in for EKRB nutrient solutions) of BSA, are hanged
Supernatant liquid；(3) produced with fluophotometer (product of BioTek companies) detection suspension when 340nm or 380nm wavelength excites
Fluorescence signal, use OD340nm/OD380nmFluorescence ratio calculate Na+Concentration.
Measure the pH value of mouse sperm to be measured.Comprise the following steps that：(1) mouse sperm to be measured is taken, is placed in containing 1.2 μM
In the PBS buffer of pH7.4,0.1mol/L of BCECF-AM, then 5%CO2, 37 DEG C of culture 15min；(2) step is taken into
(1) system, is washed twice (purpose is the free BCECF-AM of removal) with the PBS buffer of pH7.4,0.1mol/L；Then
Addition is free of HCO3-EKRB nutrient solutions (EKRB nutrient solutions and be free of HCO3-EKRB nutrient solutions differ only in EKRB
25mM NaHCO in nutrient solution3Replace with 25mM NaCl；(3) completed with fluophotometer (product of BioTek companies) detection
The system of step (2) is in fluorescence signal caused by the excitation of 500nm or 439nm wavelength；(4) software (FeliX, version are passed through
1.41) by OD500nm/OD439nmThe fluorescence reading of excitation wavelength is converted into pH value.
As a result it is as follows：With male Emc10+/+Mouse is compared, male Emc10-/-The Na of the sperm of mouse epididymis afterbody+It is horizontal
Significantly rise (C in Fig. 5)；When with without HCO3 -Nutrient solution be incubated Emc10-/-During with wild-type sperm, the two intracellular pH
It is worth no obvious difference, but is containing HCO3 -Nutrient solution be incubated at the beginning of and capacitation after 1h, HCO3 -The pH of induction
Value rise is in Emc10-/-Sperm is significantly inhibited by (D in Fig. 5), and the above results prompting EMC10 take part in the Na in spermatoblast+And HCO3 -Transhipment.
It is many that researches show that cAMP can promote to lack HCO3 -The tyrosine phosphorylation of sperm in nutrient solution, in order to clearly male
Property Emc10-/-Whether the decline of protein tyrosine phosphatase is due to HCO in the sperm of mouse epididymis afterbody3 -The pH value increase of induction
Cause, the present inventor is by male Emc10-/-The sperm of mouse epididymis afterbody is (a kind of to have biology containing 8Br-cAMP
The cAMP analogs of activity) or IBMX (a kind of inhibitor that cAMP can be prevented to be degraded by cyclic nucleotide phosphodiesterase)
It is incubated in capacitation culture medium, E shows male Emc10 in Fig. 5-/-Protein tyrosine phosphatase water in the sperm of mouse epididymis afterbody
Flat decline can be saved completely by 8Br-cAMP or IBMX, further illustrate Emc10-/-The relevant albumen junket ammonia of capacitation
Acid phosphoric acid dysfunction is due to HCO3 -The pH value rise of induction is impaired to be caused.
The studies above the result shows that, Emc10 gene knockouts cause ion imbalance in spermatoblast.EMC10 is for maintaining essence
Na in daughter cell+And HCO3-Balance play an important role.
The locomitivity of EMC10 protein levels and human spermatogoa is proportionate in embodiment 5, sperm
The present embodiment is in accordance with Declaration of Helsinkis in 1964 and the ethical standard in amendment, and it is big to have obtained Fudan University thereafter
Learn the examination ＆ verification approval of attached Huashan Hospital Ethical Committee.Experiment in the present embodiment is on the premise of my informed consent
Carry out, and sign informed consent form.
First, the acquisition of azoospermia patient sperm
27 azoospermia patients of on March 1st, 2017 to the April 30 in the Andriatrics Dept. outpatient service of Huashan hospital are included altogether, and are arranged
Except reproductive system organic disease patient, untreated endocrine disturbance patient, have medicine or alcohol abuse in two years
Patient and the patient for having an impact nitric oxide habits and customs.
Azoospermia patient's masturbation takes essence, collects sperm respectively, then forms harmonic motion sperm group.
2nd, the acquisition of normal male sperm
Normal male masturbation takes essence, collects sperm respectively, then forms proper motion sperm group (as control).
In addition to the locomitivity of sperm, the parameter of harmonic motion sperm group and proper motion sperm group (including age, essence
Liquid product, sperm quantity and sperm morphology etc.) equal no significant differences (table 1).
Proper motion sperm is defined as：A grades+B grades >=50%, harmonic motion sperm is defined as：A+B ＜ 50%.
The characteristic parameter of the clinical sample of sperm of table 1.
| Harmonic motion sperm group | Proper motion sperm group | P values | |
| Quantity | 20 | 7 | - |
| Age | 30.45±3.29 | 29.57±4.47 | 0.601 |
| Sperm (mL) | 4.63±1.73 | 4.00±1.07 | 0.396 |
| Survival rate (%) | 55.50±8.65 | 62.14±9.20 | 0.110 |
| A grades (%) | 12.25±5.80 | 23.57±2.26 | ＜ 0.001 |
| B grades (%) | 17.75±3.70 | 30.00±0.00 | ＜ 0.001 |
| A grades+B grades (%) | 30.00±8.51 | 53.57±2.26 | ＜ 0.001 |
| C grades (%) | 23.00±6.96 | 17.14±2.47 | 0.046 |
| D grades (%) | 47.00±6.40 | 29.29±1.75 | ＜ 0.001 |
| Sperm count (106) | 55.88±26.81 | 70.14±30.10 | 0.270 |
| Form natural rate of interest (%) | 53.00±6.40 | 58.57±6.39 | 0.068 |
| EMC10 protein levels | 0.18±0.30 | 0.65±0.38 | 0.004 |
Note："-" represents to be not present.
3rd, western markings method
Total protein is extracted from the sperm in harmonic motion sperm group and proper motion sperm group, in 12% dodecyl sulphur
It is separated by electrophoresis, is then transferred on polyvinylidene fluoride (PVDF) film on sour sodium-polyacrylamide gel, it is then polyclonal with EMC10
Antibody (1:5000 dilutions) and alpha Actinin antibody (Sigma, 1:20000 dilutions) it is incubated, secondary antibody uses horseradish peroxidase
(dilution factor is 1 to the goat anti-rabbit antibodies (Sigma) and goat anti-mouse antibody (Millipore) coupled:10000), finally
Purpose band is shown by ECL Plus (Amersham) chemiluminescence.
The result shows that the being proportionate property of locomitivity of EMC10 protein expression levels and sperm (A and B in Fig. 6)；Statistics
Learn analysis shows that with proper motion sperm group (A+B>50%) compare, harmonic motion sperm group (A+B<50%) EMC10 in sperm
The level of albumen significantly reduces (C, P in Fig. 6<0.01).The above results show that EMC10 albumen can adjust human spermatogoa movement energy
One of power, and the reason for the reduction of EMC10 protein levels is male sterility in sperm.
Claims (10)
1. application of the material of raising EMC10 protein actives and/or expression quantity in product is prepared；The function of the product is such as
At least one of lower A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go transparent
The ovum fertilization of band；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7) promote sperm into
It is ripe；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 the relevant albumen of capacitation) is improved
Tyrosine phosphorylation level；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 it is different) to repair sperm
Normal form；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+It is flat
Weighing apparatus；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
Application of the 2.EMC10 albumen as drug target in product is prepared；The function of the product is following A1) to A19) in
At least one：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go transparent
The ovum fertilization of band；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7) promote sperm into
It is ripe；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 the relevant albumen of capacitation) is improved
Tyrosine phosphorylation level；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 it is different) to repair sperm
Normal form；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+It is flat
Weighing apparatus；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
Application of the 3.EMC10 albumen in product is prepared；At least one of the function of the product is following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go transparent
The ovum fertilization of band；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7) promote sperm into
It is ripe；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 the relevant albumen of capacitation) is improved
Tyrosine phosphorylation level；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 it is different) to repair sperm
Normal form；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+It is flat
Weighing apparatus；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
At least one of the application of 4.EMC10 albumen, is following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go transparent
The ovum fertilization of band；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7) promote sperm into
It is ripe；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 the relevant albumen of capacitation) is improved
Tyrosine phosphorylation level；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 it is different) to repair sperm
Normal form；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+It is flat
Weighing apparatus；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
5. a kind of product, its material for containing EMC10 albumen or improving EMC10 protein actives and/or expression quantity；The product
At least one of function is following A1) to A19)：
A1 male sterility) is prevented；A2 male sterility) is treated；A3 sperm and ovum fertilization) are promoted；A4) promote sperm and go transparent
The ovum fertilization of band；A5 the ability that sperm is merged with after birth) is improved；A6 sperm function obstacle) is treated；A7) promote sperm into
It is ripe；A8 maturation of the sperm in epididymis) is promoted；A9 the locomitivity of sperm) is improved；A10 the relevant albumen of capacitation) is improved
Tyrosine phosphorylation level；A11 the capacitation of sperm) is promoted；A12 the acrosome reaction ability of sperm) is improved；A13 it is different) to repair sperm
Normal form；A14 the activity of sodium/potassium-APT enzymes) is recovered；A15 ions in spermatozoa balance) is maintained；A16 Na in sperm) is maintained+It is flat
Weighing apparatus；A17 HCO3 in sperm) is maintained-Balance；A18 pH value balance in sperm) is maintained；A19 male fecundity) is improved.
6.X1) or X2)：
X1 application of the material for) being used to detect EMC10 albumen in the product for preparing diagnosis male sterility；
X2 application of the material for) being used to detect EMC10 albumen in male sterility is diagnosed.
7. a kind of kit for being used to diagnose male sterility, including for detecting the material of EMC10 albumen.
8. the kit described in application as claimed in claim 6 or claim 7, it is characterised in that：It is described " to be used to detect
The material of EMC10 albumen " is the material of detection EMC10 expressing quantities.
9. kit as claimed in claim 7 or 8, it is characterised in that：The kit, which further includes, records criterion
Carrier；
The criterion is：If the expression quantity of EMC10 albumen is less than EMC10 albumen in control sperm in person under test's sperm
Expression quantity, then the person under test be or it is doubtful be male sterility patient；If the expression quantity of EMC10 albumen is not in person under test's sperm
Less than the expression quantity of EMC10 albumen in control sperm, then the person under test be not or it is doubtful be not male sterility patient；It is described right
It is the sperm with normal male fecundity according to sperm.
10.Y1) or Y2)：
Y1) application of the EMC10 albumen as biomarker in the reagent of exploitation diagnosis male sterility；
Y2) application of the EMC10 albumen as biomarker in male sterility is diagnosed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711284133.6A CN107998397B (en) | 2017-12-07 | 2017-12-07 | Application of EMC10 protein as biomarker in diagnosis of male infertility |
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| CN108802372A (en) * | 2018-06-19 | 2018-11-13 | 上海伦泽生物科技有限公司 | Detect the kit of reticulon complex subunit 10 in human serum |
| CN108845142A (en) * | 2018-06-19 | 2018-11-20 | 上海伦泽生物科技有限公司 | Application of the EMC10 Protein Detection object in the diagnosis of preparation obesity and scale evaluation and Bariatric effect assessment product |
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| CN107998397B (en) * | 2017-12-07 | 2020-09-04 | 复旦大学附属华山医院 | Application of EMC10 protein as biomarker in diagnosis of male infertility |
| CN108707586A (en) * | 2018-06-19 | 2018-10-26 | 上海伦泽生物科技有限公司 | The monoclonal antibody of anti-reticulon complex subunit 10 and its application |
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| CN108845142A (en) * | 2018-06-19 | 2018-11-20 | 上海伦泽生物科技有限公司 | Application of the EMC10 Protein Detection object in the diagnosis of preparation obesity and scale evaluation and Bariatric effect assessment product |
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