METHOD FOR OBTAINING MATURED OVA FROM IMMATURE OVA USING Rad51 PROTEIN

The present invention refers to (a) inter-injecting spermatozoa separated then it is fine to Rad51 protein; and (b) obtained in step (a) including the mature spermatozoa spermatozoa in vitro maturation then it is then it is a method for obtaining spermatozoa from number [...] substrate.

The present invention the victims of the spermatozoa in vitro maturation mitochondrial activity wherein the new function Rad51 gene in a cancer cell. The number of spermatozoa GV Rad51 gene expression by injecting fine Rad51 dsRNA when meiosis (MI arrest) was causing stop MI billion. Rad51 - depleted ATP generating reduced the spermatozoa, showed increased DNA degradation of a A2 and reduction potential. The present invention the number of victims of the Map1lc 3b - positive point (puncta) derived from an increase at Rad51 - depleted when the spermatozoa, this number of mitochondria (autophagy) Rad51 billion by magnetic damage associated with the activity that allowed for clinical use other. Mitochondrial damage inducing aggregation of failures when the chromosome alignment errors (chromosomal misalignment) and spindle (spindle aggregation), cytoplasmic mitosis was cause incomplete. The, e toe of mitochondrial number and quality maintained by the spermatozoa in phage Rad51 protein functions, the maturation of spermatozoa (meiotic maturation) that simultaneously control has been found by the present invention.

The present invention refers to inter-injecting spermatozoa separated [i.e., step (a)] then it is fine to Rad51 protein without using a tool.

DNA repair protein (DNA repair protein) Rad51 protein is known as a double cutting (double strand breaks, DSBs) mitotic and meiosis is formed in DNA homologous recombination (homologous recombination) recovery of conserved protein mediated (conserved eukaryotic protein) are disclosed. In addition, the garment number increases mitochondrial DNA Rad51 stress promote synthesis substrate. Rad51 G2/M (G2/M transition) in embryonic stem cells is in addition preserving cell cycle (cell cycle progression) conversion by adjusting substrate. Human Rad51 protein ISOform 1 (isoform 1), ISOform 2 (isoform 2), ISOform 3 (isoform 3), and typed number 4 (isoform 4) in the form of a known, each sequence number 1 (NP_002866. 2), Sequence number 2 (NP_597994. 3), Sequence number 3 (NP_001157742. 1), And sequencing of number 4 (NP_001157741. 1) Amino acid sequences. In addition, each encoding said ISOform as gDNA sequences, each sequence number 5 (1 ISOform encoding bio-sequence listing, NM_002875. 4), Sequence number 6 (ISOform 2 encoding bio-sequence listing, NM_133487. 3), Sequence number 7 (3 ISOform encoding bio-sequence listing, NM_001164270. 1), And sequencing of number 8 (4 ISOform encoding bio-sequence listing, NM_001164269. 1) Is known. Thus, in one embodiment said Rad51 protein sequence number 1 to 4 amino acid sequence can be selected from the group consisting protein.

Said Rad51 protein is commercially available protein or proteins can be prepared by the number by conventional genetically engineered method. E.g., said Rad51 Rad51 protein encoding gene (e.g., the sequence numbers 5 to 8 comprising a nucleotide sequence selected from the group consisting of human Rad51 protein cDNA polynucleotide or polynucleotide) according to the method of double-plasmid DNA vector (e.g., various commercially available expression vectors) to recombinant plasmid DNA vector includes the following performing cloning, E. Coli transformed host cells such as can be obtained by cultivation of protein. In addition, said Rad51 protein is commercially available recombinant Rad51 protein [Recombinant Rad51 protein (Abcam, Sigma non-Aldrich, Thermo Fisher Scientific, Novus Biologicals] using disapproval.

Said "inter-spermatozoa separated then it" is infertility treatment program for women to in vitro maturation (In a-vitro maturation, IVM) pager spermatozoa separated from inter-then it is said substrate. Then it is publicly known method of separation of the egg (e.g. Cha, KY, et al. , Fertil. Steril. Vol. 55, No. 1, 1991) Can be performed according to a. Said female inter-GV "spermatozoa (immatured oocytes) then it is" multi-step separated spermatozoa (Germinal Vesicle stage) of said substrate.

Said conventional syringe for microinjection of microinjection (microinjection) using spermatozoa separated into Rad51 protein inter-then it is can be performed by (or scanning). The amount of said injected Rad51 protein per 20 ∼ 60 pg then it can be a fine range of spermatozoa, the one number are not disclosed.

The present invention refers to obtained in step (a) transgenic then it is [i.e., step (b)] the spermatozoa in vitro maturation without using a tool. Said pager and the like be assayed in maturation in vitro maturation investigation relates procedure (In a-vitro maturation, IVM) method is provided can be performed according to a method. The in vitro maturation of said publicly known method (e.g. Cha, KY, et al. , Fertil. Steril. Vol. 55, No. 1, 1991) Can be performed according to a.

The present invention refers to in addition to a separate inter-Rad51 protein obtained by microinjection into spermatozoa then it is, then it is for spermatozoa in vitro maturation number [...] substrate.

The present invention according to in vitro maturation then it is for spermatozoa, the spermatozoa as described in Rad51 protein and extracorporeal split into and then it is said, also described in the can be performed according to a microinjection in addition said.

Hereinafter, the present invention in the embodiment through a more detailed as follows. The present invention is exemplified in the embodiment these is thus, in the embodiment of the present invention range and not the limited to these.

1. Material and test method

(1) Test animals

ICR mouse (male and magnetic) tag includes a core yarn (Koatech, invest, a compensation) from it receives, ball number, research medical science cruelty school difference (CHA Research Institute) as the chamber Pig embryos are obtained. All animal experiments to medical science cruelty school process disclosed herein has been addressed by the ethical Committee (Institutional Animal Care and Use Committee) and authorizing, effective use management guidelines (Guiding Principles for the Care and Use of Laboratory Animals) has been performed in accordance.

(2) Collection spermatozoa

Before ovulation (germinal vesicle stage) for separating spermatozoa from follicular fluid of step (preovulatory follicles) then it is of GV, ICR mouse serum 3 week zero magnetic (female) 5 IU 46 the e being-invertebrate hormone (pregnant mare's serum gonadotropin, PMSG; sigma - the [tu reach which it will know) has been completed with respect to the fiber is lethal. 27 - Gauge syringe with difficulty nine cells before ovulation using follicular puncture to obtain the spermatozoa from - composite (cumulus-a enclosed oocyte complexes, COCs) that were unsafe. 0. 2 Mm 3 - isobutyl - 1 - methyl - [...] (3 a-isobutyl-a 1 a-methyl-a xanthine, IBMX; sigma - the [tu reach which it will know) containing M2 medium (sigma - the [tu reach which it will know) inhibiting (germinal vesicle breakdown, GVBD) using his difficulty karyomere collapse. Through repetitive extraction by pipette (fine a-bore pipette), difficulty nine cells (cumulus cells) was mechanically from a recovering spermatozoa.

MII step to obtain spermatozoa, the FEP-invertebrate hormone (equine chorionic gonadotropin, eCG) 5 IU ([...]) intercooler connection 46 5 IU human chorionic-invertebrate hormone (human gonadotropin, hCG) after time that scans a scanning magnetic mouse was. After hCG MII with ovulation it was controlled from step 16 after the injection time contain spermatozoa are obtained. With it is a [ni, oh oh base number (hyaluronidase) (300 U/ml) COCs to processing, step number been difficulty nine cells surrounding the MII spermatozoa alone. Quick refrigeration (snap-a frozen) separated spermatozoa to -70 °C until RNA separation was installed application.

(3) MRNA Separation

Dynabeads mRNA DIRECT kit (with [thu [ceyn enhancing) number using mRNA from different development step was formed in the triangular beam separating spermatozoa and embryos. I.e., 300 micro l spermatozoa (lysis/binding buffer) dissolved/binding buffer (100 mm Tris a-HCl [pH 7. 5], 500 Mm LiCl, 10 mm EDTA, 1% LiDS, five [ssu [thol (dithiothreitol, DTT) dithiocarbamates and 5 mm) have been suspended during 5 minutes at room temperature. After dicing (vortexing) [...], 20 micro l [...] oligo dT previously cleaned die₂₅ (Dynabeads oligo dT₂₅ ) Is mixed with and dissolving, 5 minutes agitating-gate annealing at room temperature. Magnetic particle concentrator (Dynal MPC-a S magnetic particle concentrator) Dynal MPC-a S separating using beads, 14 micro l Tris a-HCl(10 mm Tris a-HCl, pH 7. 5) 73 °C in poly (A) by incubation in 2 minutes⁺ Eluting the RNAs have been.

(4) Inverse - transfer Polymerase chain reaction(Reverse -Transcriptase Polymerase chain reaction, RT a-PCR)

The positive number mRNA and 0. 5 Micro g oligo (dT) primer mixing, 70 °C cDNA by incubation in 10 minutes and copiers. PCR analysis was used as a single egg - equivalent cDNAs (single oocyte provided equivalent cDNAs) templates. Gene encoding for table 1 PCR primer sequence and conditions such as disclosed.

<표 1>

Then, PCR product 1. 5% Direct recovery system and separated by electrophoresis, Gel Doc^TM EZ Imager (Bio-a Rad) was analyzed. The relative gene expression levels H1fooGene expression levels of compensating him. Test 3 was all periodical repetition.

(5) Quantitative RT - embodiment liverPCR(Quantitative real time RT -PCR)

MtDNA transfer Mtnd6Quantitative RT-a PCR analysis of liver mRNA embodiment (Bio-a Rad Laboratories Inc, Hercules, CA, USA) was performed on the iCycler iQTM detection system. IQ SYBR Green Supermix PCR amplification using reagent (Bio-a Rad) monitor [...], iCycler iQ result^TM Software was analyzed. The reaction mixture consists of cDNA, forward and reverse primer 20 pmol, and SYBR Green Supermix 2 (100 mm KCl, 40 mm Tris a-HCl [pH 8. 4], 0. Each dNTP 4 mm, 50 U/ml iTaq DNA polymerase, 6 mm MgCl₂ , SYBR Green I, 20 nm phosphor (fluorescein), and stabilizing number) black. Table 1 primer sequences used for gene analysis such as disclosed. Modified (40 seconds, 95 °C), annealing (40 seconds, 60 °C), and extension (40 seconds, 72 °C) template was 40 cycles of amplification. After completion of the PCR, samples to the 60 °C 95 °C in 0. 5 °C intervals fluorescent material-gate queue monitor continuously. Melted by curve (melting curves) for identifying non-specific PCR product was used. Rad51 - silence in spermatozoa (Rad51 non-silenced oocytes) Mtnd6 The mRNA expression level of the H1fooGene expression levels of compensating him. C level of target gene expression_T Method [Yuan, J. S. Et al. , BMC bioinformatics 7, 85, Doi: 10. 1186/1471 - 2105 - 7 - 85 (2006); And Livak, K. J. Et al. , Methods 25, 402 - 408, Doi: 10. 1006/Meth. 2001. 1262 (2001)] Have evaluated using, all analysis process was 3 times.

(6) Rad51 and GFPOf dsRNA Number bath

For RNAi experiments, a high pressure liquid coolant Rad51 dsRNA for his number. Two different primer set i.e., endogenous transfer of each dsRNA number four knocks-down Rad51 provided A and Rad51 provided B was used for confirmation (of Figure 1 A and table 1) bath. GFP GFP cDNA amplification using primer Rad51 Rad51 provided A and each portion and then, SpeI linearized pGEM-a T Easy (Promega) to benzylic (linearized) and to him. T7 RNA polymerase MEGAscript RNAi kit (Ambion) and single - stranded RNA (ssRNA) is determined by orientation and copiers. Complementary RNAs 5 minutes after mixing in the queue hemp cloth which is [syen grudge 75 °C was cooled down to room temperature. The formation of the dsRNA (mobility) dsRNA by electrophoresis mobility of 1% direct recovery system has been confirmed that the mobility of a ssRNA compared (of Figure 1 B). Fine implantation, a 2 RNAs. 9 Micro g/final concentration was micro l dilution. Injecting GFP RNAi was used as regulated.

(7) And microinjection In vitro Culture

0. 2 Mm IBMX M2 GV Rad51 dsRNA in a medium containing spermatozoa to fine of his. Inserting a dsRNA scanning pipette solution formula 1 egg, a constant flow system (constant flow system) using a 10 pl of dsRNA (Transjector; Eppendorf) fine of his. For evaluating the implant damage (injection damage), as well as regulated as the injection GFP dsRNA injected spermatozoa. For measuring in vitro maturation rate, 0 spermatozoa. Culturing in a medium containing 2 mm IBMX M16 then 8 hours, 16 hours in a medium 5% CO M16₂ 37 °C and about him as. After RNAi experiments, morphological changes in vitro maturation rate and Kim, K. H. Et al. PloS One 6, E23304, doi: 10. 1371/Journal. Pone. 0023304 (2011) According to the method under the condition of a recording. I.e., the maturation of spermatozoa (GV) or presence (MII) difficulty karyomere (germinal vesicle) a pole body (polar body) stored in the score. Both the pole body and difficulty karyomere MI spermatozoa as not stored in the score.

(8) Time video microscopy (Time lapse video microscopy)

The change speed of the phenotype in mature spermatozoa in vitro culture process according to keep track of the time video observation Lee, S. Y. Et al. PloS One 8, E70708, doi: 10. 1371/Journal. Pone. 0070708 (2013) According to the method described by dielectrophoresis. Time elapsed microscope (time-a lapse microscope) - (JuLI^TM ; Digital Bio) 5% CO a₂ And 37 °C incubator is placed, spermatozoa in culture which plate is rested on the microscope. 36 Image automatically photographing time 5 minutes interval then, sequential Image points JuLI operating software was converted into flat file (movie files).

(9) The [wey the [su it shook offBlot(Western blot)

Protein extract (lane two 150 per egg) using a 12% SDS-a PAGE separating, nitride (polyvinylidene difluoride) membrane (Amersham Biosciences) was transferred polyvinylidene transducer base five. 5% Nonfat dried milk (non non-fat dry milk) - containing tris buffer saline - twin (Tris-a buffered saline-a Tween) [TBST; 0. 2 M NaCl, 0. 1% Tween-a 20, and 10 mm Tris (pH 7. 4)] 1 Blocking time-gate in said film. Blocked film [lay comb will be and it will grow in TBST monoclonal anti - Rad51 antibody (1:1000; sc provided 8349, Santa Cruz Biotechnology) or mouse monoclonal anti - α - [thyu phosphorus antibody (1:1000; sc provided 8035, Santa Cruz Biotechnology) incubation with on-gate. After incubation, it ladles and the jade it is sour, oh - conjugated anti - [lay comb [...] hose film number - IgG (1:5000; #7074, Cell Signaling Technology) or anti - mouse IgG (1:2000; A provided 2554) was incubation at room temperature in a TBST 1 time. After each stage, film to have a codeword TBST completely cleaning, chemiluminescent detection system (Amersham Biosciences) coupled antibodies formed in the triangular number detected using irradiated (enhanced chemiluminescence detection system).

(10) Nose [meys Analysis (Comet assay)

DSBs nose [meys analysis (neutral version) (Trevigen) number by detecting irradiation balance described according to DNA damage was assessed. Mote road solution (Tyrode's solution) (pH2. 5) By dissolving in transparent layer from egg (zona pellucida. ZP) number was a stand-alone. Rad51 - for reparing over 1% low-melting-point agar with five [cu 37 °C to 1:10 (low melting point agarose, LMA) in silence spermatozoa ZP - number ratio of suspending, micro l 70 pre-coated slide (Pre-a coated Slide (Trevigen) on pipetting-gate. (Adherence) 40 minutes in place of said slide 4 °C incubation is then attached, Trevigen 4 °C (lysis solution) dissolved solution of 4 hours to cool water. A stand-alone and slide number from solution dissolved, then 30 minutes to 4 °C 1X TAE Buffer immersing, 30 V 40 minutes (1 V/cm, 11 - 12 mA) to electrophoretic-gate. After electrophoresis, DNA slide precipitation solution (1M ammonium acetate) at room temperature 30 minutes then immersing, 75% ethanol at room temperature 30 minutes immersing was stationary. After fixing, then completely dried in about 20 minutes until the queue hemp cloth which is [syen grudge slide 42 °C LMA agar with five [cu, 1X SYBR^® Dyeing solution dyeing Green I was 5 minutes. Dyed slide is observed under fluorescence microscopy. (Comets) quantify a nose [meys each egg, a user Konca CASP PC Image analysis, K. Et al. Mutation research 534, 15 - 20 (2003) Scoring method described according to conducting analysis. Tail moment (tail moment, TM), tail length and percent tail DNA, i.e. the nose [meys TM=TL × (% DNAT) stored in the score.

(11) Immune fluorescent Dyeing (Immunofluorescence Staining)

A number spermatozoa a difficulty nine cells alone (denuded oocytes) 0. 1% Polyvinyl alcohol, formaldehyde content 0 and 4%. Dulbecco's PBS (PBS-a PVA) containing 2% Triton X-a 100 placed in next, 40 minutes was fixed at room temperature. 10 Minutes 3 times each fixed spermatozoa PBS-a PVA washed and, supplemented with 1% BSA - in PBS-a PVA (BSA-a PBS non-PVA) was stored at storage. 3% BSA-a PBS-a PVA 1 time block and then to spermatozoa, mouse monoclonal anti - α - [thyu phosphorus antibody (1:100 dilution, sc provided 8035; Santa Cruz Biotechnology) or [lay comb will be and it will grow monoclonal anti - Map1lc 3b antibody (1:100 dilution, ab48394; Abcam) stored at 4 °C with incubation in-gate. After washing, secondary antibodies (Molecular Probes) incubation at room temperature with time and spermatozoa Alexa Fluor 488 2 1, DNA (Promega) to a propy galactooligosaccharides iodide (propidium iodide, PI; sigma - the [tu reach which it will know) or DAPI was counter dyeing.

(12) Mitochondrial for dyeing

Rad51 RNAi after, step (Molecular Probes; enhancing with [thu [ceyn) was dyed MitoTracker Orange CMTMRos Rad51 - silence MI spermatozoa. MitoTracker dyeing mitochondria in living cells and is, this important tool for evaluating the distribution of mitochondria which, accumulation of mitochondrial membrane potential (membrane potential) in dependence on the other. The MitoTracker 0. At a concentration of 300 nm 37 °C during 30 minutes in place as well as M16 containing 3% BSA. After washing 3 times with PBS-a PVA medium, is used to fix an egg, then immunized with anti - α - [thyu phosphorus antibodies in various colors, DAPI (sigma - the [tu reach which it will know) was counter to dyeing. Confocal microscopy analysis of until, the egg was dyed 4 °C hereinafter of the installed application. Laser - scanning confocal around elder brother america (laser scanning confocal microscope) (LSCM; Leica) spermatozoa using observed. MitoTracker helium/neon laser 543 nm and filter G-a 2 A is (576 nm excitation and 551 nm light emitting) observed using orange - fluorescent dye are disclosed. The group consisting of minimum 30 each RNAi spermatozoa to LSCM observed.

(13) JC-1 A2 by (Δ Ψ) of potential Measuring

For measuring mitochondrial activity of spermatozoa, potential sensitive fluorescence probes JC-a 1 A2 (5, 5 ', 6, 6' - process for the - 1, 1, 3, 3 '- (5, 5', 6, 6 '- tetrachloro-a 1, 1, 3, 3' - tetraethylbenzimidazolycarbocyanine iodide) 2 the ethyl it cuts the [cu already is and a car [lu time signal which will boil down it is not iodide (Thermo Fisher Scientific) mitochondrial potential analysis using conducting. 10 Micro g/mL dissolution with a stock solution (DMSO) dimethyl dye JC-a 1 and to a process for crude number, [...] M16 micro g/mL was good storage at 120 to 1 dilution. In a light place, 5% CO spermatozoa in step 37 °C MI Rad51 - silence₂ Incubator in 20 minutes in a medium to expose a M16 JC provided 1, PBS-a PVA was washed. Fabrics JC-a 1 relative fluorescence intensity [phul base five [ley new use of an isothiocyanate (fluorescein isothiocyanate, FITC, green) and spermatozoa with was and it pushed use of an isothiocyanate (rhodamine isothiocyanate, RITC, red) stored in the LSCM images on and methods. Two fluorescent Image (green and enemy lifestyle fluorescent) is greatest diameter plane (plane) obtained in spermatozoa. Quantitative analysis of green and red fluorescent signal intensity was confocal software enabling these two analyzing the images. Computing a ratio of a transparent conductive layer of each egg RITC to FITC, as well as the endpoint for Δ Ψm (endpoint).

(14) Measurement of ATP content

Combelles, C. M. , Et al. , Biology of reproduction 68, 812 - 821 (2003) And Van Blerkom, J. , Et al. , Hum Reprod 10, 415 - 424 (1995) Described a method of the deformed slightly, EDMOND lung phosphorus [...] EDMOND - number reaction based on spermatozoa using assay kit (Bioluminescent Somatic Cell Assay Kit, FL non-ASC; sigma - the [tu reach which it will know) in ATP content were measured. I.e., two water (ultrapure water) 40 spermatozoa 160 micro l to quickly and refrigeration (snap-a frozen), the installed -70 °C was at the time of analysis. ATP for analysis, the thawed sample solution 50 - 100 micro l ice cools each micro l Cell ATP-a Releasing Reagent followed by addition, then the queue hemp cloth which is [syen grudge 5 minutes on ice, ice cools 100 micro l - ATP Assay Mix (1:25) was added. 10 Minutes incubation at room temperature the reaction mixture in place for chemiluminescent light-gate. Each analysis each (0 to 5. Including a 0 pmol ATP) 11 - point standard curve made the bill. The amount of ATP in spermatozoa roh meter luminary (luminometer) (Centro XS3 LB 960; Berthold Technologies) of each sample using bio light emitting (bioluminescence) were measured. In spermatozoa spermatozoa pmol/ATP content have been designated.

(15) Statistical analysis

A transparent conductive layer at least 3 times each experiments, the Stowe trident t - test (Student's t-a test) conducting using statistical analysis. 3 Obtained from at least two separate independent experiments was average ± SEM data display. P value is calculated based on an average of GFP RNAi Rad51 RNAi group and the group when the paired t - test, p<0. 05 Value was deemed statistically significant.

2. Results of this test

(1) GV in spermatozoa Rad51 of The silence (silencing) MI stop His cause

In the present invention it has been determined that the expression level of the mature spermatozoa Rad51 initially by users. As shown in of Figure 2 A, Rad51 transfer comprises meiosis are expressed throughout. Specifically, expression of GV MI Rad51 mRNA has been continue at step to step, in reduced MII (of Figure 2 A). Based on form, 76. 5% Of spermatozoa in the silence Rad51 - MI halted and, 23. 5% Egg number 1 (first polar body extrusion) of the pole body shown (of Figure 2 B and C) release. While, in the meiosis process terminates when the microinjection GFP dsRNA - regulated spermatozoa, conjunction with his develop normally to step number 1 [...] MII spermatozoa (91. 5%; Of Figure 2 B and C). Rad51 RNAi Rad51 mRNA and protein levels of has been stopped after MI - in spermatozoa were identified (of Figure 2 D).

(2) Rad51 of The silence was meiosis in DNA damage Neck

Rad51 homologous DNA strand exchange reaction plays an important role in DNA repair in recombinant and used for. Rad51 expression in mature spermatozoa DNA (DNA integrity) affecting whether the deletion of the original state were measured. In nose [meys analysis for detection of single or dual - means and strand damage, such analysis in analyzing DNA damage was Rad51 - ZP - number for reparing over nose [meys silence spermatozoa. In spermatozoa Rad51 expression is compared to DNA damage and thus can be spermatozoa billion number showed EDM electrode, this silence Rad51 - spermatozoa are of an DNA damage by a goniophotometer (3 also). The, Rad51 - silencing induced by DNA damage, DNA and/or mitochondria DNA (mtDNA) is a major cause of the stop MI spermatozoa can be.

(3) Rad51 Depletion of a spindle and chromosome orientation and A MTOCWith respect to the collapse

Rad51 - difficulty karyomere collapse in silence spermatozoa (germinal vesicle break down, GVBD) is generated but, 76. 5% Egg of the meiosis MI does not been stopped at the terminating step. Rad51 - mature spermatozoa spermatozoa in nuclear cytoplasmic division terminating because not silence, stationary spindle structure and chromosome was assessed by fluorescence of meiosis immune egg orientation (of Figure 4 A). Microinjection GFP dsRNA - an explicit form of meiosis (clear normal barrel shape) normal spermatozoa regulated MI barrel spindle and concentrator plate (metaphase plate) (of Figure 4 A a and b) and their aligned - well in chromosome. The in contrast, which are washed with chromosome after Rad51 RNAi MI spindle is in addition been (of Figure 4 A c and d) and are mixed.

Mouse spermatozoa which lack a central lip (centrioles), has a paperboard tube - engine centers (microtubule-a organizing centers, MTOCs). Said center [lip proteins (pericentriolar material (PCM) proteins) that of the ambient centers paperboard tube - engine is made up of a, spindle pole located (poles), microtubule nucleation centers (microtubule nucleating center) functions as a substrate (Szollosi, D. , Et al. , Journal of cell science 11, 521 - 541 (1972); Calarco provided Gillam, P. D. , Et al. , Cell 35, 621 - 629 (1983); Maro, B. , Et al. The Journal of cell biology 101, 1665 - 1672 (1985)). Rad51 expression reduction leading into a spindle assembly of since, when the PCM gene expression of several essential, Tacc3 [...] 6 a number of PCM gene (Aurka, Aurkc, Bora, Cep192, Pcnt and Plk1) is stopped has been found being, reduced predominantly in silence MI Rad51 - spermatozoa (of Figure 4 B).

(4) Rad51 of Reduction mitochondrial activity and ATP generation Disfigured

Since the abnormal DNA damage and spindle observed, of the number and distribution of mitochondrial mammalian spermatozoa were measured. The amount of and distribution mitochondrial mammalian spermatozoa, silence MI compared to GFP - spermatozoa, reduced in silence MI Rad51 - spermatozoa (of Figure 5 A). After GFP RNAi, egg has been concentrated mainly around the meiosis in the development of mitochondrial regulated spindle, the remaining part of the egg cytoplasm was generally uniformly distributed (of Figure 5 A a and g). Conversely, abnormal spindle sprocket and a silence MI Rad51 - formula 1 in spermatozoa as well as reduced without accumulation of mitochondrial around that amount of inhibin (of Figure 5 A b and h).

Next, in order to identify the abnormal mitochondria (abnormalities), A2 (Δ Ψm) influence of potential for a long time (of Figure 5 B) in spermatozoa Rad51 down - control. As a result, silence Rad51 - red and green fluorescence in range of the spermatozoa can be effectively L-band, this potential reduction of A2 exhibits (of Figure 5 C; 1. 09 Vs. 0. 86, P<0. 05). In addition, measuring the change in generation of mitochondrial ATP Rad51 - silence when the spermatozoa, and spermatozoa average ATP concentration in each result GFP-a RNAi Rad51 provided RNAi 0. 64 ± 0. 013 Pmol and 0. 38 ± 0. 014 Pmol fluorescence, this statistically significance has been the reduction (of Figure 5 D). In addition, mtDNA levels closely associated Mtnd6 transfer of expression is known as having a mitochondrial - and thus can be coded in an improved reduction (of Figure 5 E) Rad51 - silence spermatozoa. The, generally same when considered, the mtDNA levels reduced egg Rad51 - silencing, and low levels of mitochondrial ATP to induce the production and reduction of potential A2, this mitochondrial dysfunction in an amount and exhibits reduced Rad51 provided RNAi in spermatozoa.

(5) Rad51 of With the programmable for screening The predatory magnetic- Associated E toe phageHis cause

E toe gripping (mitophagy) (autophagy) leading through selective isolation of mitochondria is damaged and magnetic predatory decomposition are disclosed (Ashrafi, G. Et al. , Cell death and differentiation 20, 31 - 42, Doi: 10. 1038/Cdd. 2012. 81 (2013)). Rad51 - silencing observed in mitochondria was assessed whether e toe gripping - dependent reduction in the spermatozoa. Green onion petty Map1lc 3b mitochondria-e toe and a recombinant film is resistant, e toe [...] are used widely as a marker (Mizushima, N. , Et al. , Annual review of cell and developmental biology 27, 107 - 132, Doi: 10. 1146/Annurev provided cellbio provided 092910 provided 154005 (2011)). Rad51 RNAi after, the entire number of spermatozoa MI (puncta) has been stopped by the formula 1 was an increase or decrease of Map1lc 3b point counted Map1lc 3 point analysis (of Figure 6 A). Advantageously interesting, the depletion of the EDM electrode formation Rad51 Map1lc 3b point, this mitochondrial number (of Figure 6 B) exhibits substantially induction resistance plate in silence Rad51 - e toe spermatozoa.