JPH03501203A - Method for in vitro induction of acrosome reaction in sperm, use of this method in sperm testing and treatment of male-related infertility - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method for in vitro induction of photoreactivity in sperm, the use of this method for testing sperm, and Treatment of male-related infertility The present invention relates to a method for in vitro induction of photoreactivity in sperm and an artificial Insemination and test tube insemination! The present invention relates to a method of applying the present method to (engineering vy).

The majority of ejaculated sperm cannot fuse with an egg immediately. Normal case , sperm acquire this fitness ability while ascending the female reproductive tract, especially the uterus and oviduct. . Sperm first undergo a reversible capacitation step and then undergo an irreversible photoreaction.

In in vitro fertilization techniques, loss reactions can be obtained through many routes.

The true order of in vivo photoreaction is not fully understood, and in vitro methods are similar. It is difficult to imagine that this can be achieved by the order of . Biological processes are multivariable and temporary This is suspected to be the reason why the loss of body reaction is reached in vitro through various routes. This is a simple explanation.

Sperm is a highly differentiated cell, and its role is to release and transport eggs to the male sperm. and protection. The structure of mammalian sperm varies widely between species, but The structure of mammals is clear.

Rabbits, pigs, and cows have an oval shape, birds have an elongated cylindrical shape, and mice, hamsters, and cows have an oval shape. and in rats it is curved. There are also changes in size. The head of a human sperm is approximately 5.5 mm long. 0 μM 1 width 3.5 μM and thickness 2.0 μM, whereas the cow one is approximately 3.5 μM wide and 2.0 μM thick. 8.5 μM, width 4.0 PM and thickness C1,3-0.5 μM. These large Changes in size are caused by the cervical membrane around the oocyte and when 1wt sperm selectively invade. is important.

Sperm are taken from the head, which is joined to the tail by a fragile neck. The tail contributes to sperm motility. giving. The sperm head can be divided into two parts: the nucleus and the surrounding membrane structure.

Membrane structures include the plasma membrane that covers the entire structure of the head, the acrosome, and the bag that surrounds the front part of the nucleus. a post-nuclear cap that covers the rear of the nucleus, and a post-nuclear cap that covers the back of the nucleus and a It includes an equatorial region that overlaps with the Kronome.

Through a reversible process of acquiring fertilization, sperm can enter the egg's acellular sugar/white, etc. (Bedfor6゜:roM, 1970, Biol , ReprO+10. (Supplementary light) 2.128-158). The exact mechanism is still It is unclear. Recent models suggest that surface charge, cholesterol deficiency and This suggests dynamic membrane changes.

The photoreaction is a morphological matter, in which the protoplasm and external acrosome membrane are multiple points. Fusion beggar (Green.

D, P, L, 1978, r Mechanism of photoreaction in mammalian development J, Johnson, M, EL, Volume 3, 65-81. Netherlands,A (Akru k, B, R, Mari 1979, GemateRea, 2.1-3) o Basic Hypothesis C: Ca++ ions in the cytoplasm between the protoplasm and the external acrosomal membrane increase. This is an important thing to do immediately before the photoreaction starts. Ca 0 ion for fertility acquisition It includes the preparation of spermatozoa by augmentation of spermatozoa.

Viable l-photoreaction is essential for fusion of egg membrane and sperm during fertilization in stomata It is a requirement.

The first knowledge of the development of sperm function was made in the 1950s, so the species, condition of the specimen, and We have attempted to develop a photoreaction that can be grown under in vitro conditions, taking into account the Many techniques have been developed to imitate it. For example, there are the following technologies.

(1) Preliminary culture of ligated dental implants or rabbit uterus, An8tin, L, R ,,1951,Aunt,,r,SC1,aee,+41581-596 ゜ (2) Albumin or serum? Preculture in supplemented simple or complex media, M iyamoto, L & Chang, M, C,.

1973, T, Reprod, 1rert, 32.193-205 (3) Exposure to high ionic medium, Brackett, B, G, and 011 phant, G. e 1972, Biol, neproa, i2 ゜260-274゜ +41　0&”+ Treatment of spermatozoa with ionophores, Aitken.

R, J. et al., 1984. J, Androl, 5.321-329゜(5 ) Direct microinjection of spermatozoa, La5salle, B., et al., 1987゜Gam ete Res, 1 6 + 69 - 78 + (6) Phosphatidyl Exposure to choline liposomes, Graham, J., K., et al., 1986. Biol, Reprod, 35°413-424° (Synthetic polymers with clear properties and simple media supplemented with high Ca++) Preculture, Tomkinlil, P, T, etc.

1988, RepO (1, 3, 367-376.

(8) Preculture in the presence of glycose aminoglycan, lee, C, N, etc. , 1986. J, Anim, sci, 63°861-867 and (9) TEST - Pre-incubation in egg yolk buffer at 4°C for 48 hours, Bola nos, T, R,, 1983, Fert, Stθri1. e39.536 -540° A laboratory tool increasingly used by clinicians is the so-called sperm penetration test (SPA). ) (Yanagimachi, R, et al.

1976, Biol, Repro 6.15.471-476). this test The experiment was conducted to determine the ability of capacitated human sperm to penetrate fertilizing zone-free hamster eggs. It's about seeing power. Quantitative prediction of penetration rate, polyspermy penetration, and attachment arise. This technique is not standardized, especially if supported by operability data. , showing a good predictive effect of the in vivo fertility of the sample.

Along with other testing methods, this system uses fertilization potential to classify patients complaining of infertility. can be ranked with some accuracy.

Until now, SPA has been limited in its ability to evaluate human insanity, but it has been used in many homes. There is also a current need to expand the tests used to assess infertility in livestock. human sperm and Apart from the fusion of strain-free hamster eggs, fusion of the following species: budgerigars, Koramori, dolphin, mouse, white-footed mouse, rat, guinea pig, rabbit, dog , observed in the sperm of pigs, goats, cows, horses, and cynudels.

Using the SPA system and specific staining, we demonstrated that transmission with albumin or serum 2 It was shown that the systematic sperm capacitation method can only activate cells with cell distribution below 20 cells. Indicated. High ion treatment 3 and TEST-egg yolk 9 in vitro immersion of some samples. Although it can increase repertoire, it is unsuitable for sperm-reduced samples. Ca ++i Exposure to Onophore 4 dramatically accelerates in vitro infiltration of fertility-regulated samples. However, it has no effect on oligozoospermia or many other samples. ionophore It is also difficult to control and its toxicity makes it unacceptable for use in engineering VF or similar programs. It seems that it will not be done. Microinjection of sperm directly into the egg membrane or periyolk space5 is practical as a routine technique, perhaps in large research/clinical centers. Probably different.

The applicant was able to successfully use a medium based on synthetic polymers. death However, although this method can ensure uniformity and rapidity of the reaction, For the obvious subparent substance/donor (5u1) - fertile donor) appears to be of little benefit.

Therefore, at present, it is easy and 100% easy to prepare any kind of sperm for fertilization. There is no surefire way.

Electrotransmission or electroporation (θ18CtrO-p Or ati O n) is a technique that involves the use of high-level electric field pulses that create temporary holes in cell membranes. Ru. This technique specifically introduces biologically active foreign genes into primary rat liver cells. has been used to. Ran Tur-Kaspa et al., 1987, Molecular and Cell Biology ar Biology) (5゜2.716-718 and Cellular Cellu (cell-to Ce’1lfusion) *jime A/? (Zim) mQrmann), σ, and Vienk en,　,r,)　1982　+Journal Membrane Biology (J,　M ezbrane.

Biol,) 67, 165-182゜The applicant has so far reported that the sperm of any species Did you know that electrotransmission has been applied to sperm-egg fusion or sperm-egg fusion? stomach.

One of the objects of the invention is an improved method for preparing any type of spermatozoa for fertilization. It is to offer something.

Another object of the invention is a method for evaluating spermatozoa and thereby assessing male fertility. It is to provide.

Yet another object of the present invention is to provide a method for treating male-induced infertility.

Therefore, the present invention aims to provide a method for causing a photoreaction in sperm. In this method, spermatozoa are exposed to the potential of sperm plasma cells in a test tube. ial) from about -70V to +IVK to allow Ca++ ions to flow in. Regarding normal sperm, it is important to avoid exposing the sperm to electrotransmission to give a sufficient electric field. Penetration of less than 100% is observed in the so-called sperm permeation method (SPA); For o'ligoapermi resamples, less than 75% Penetration was observed.

The present invention also provides a method for evaluating the developmental potential of sperm. The method involves raising the potential of sperm plasma cells to -7 in a test tube. Q: Increase the electric field from mV to +1V to provide enough electric field to cause Ca+ ions to flow in. Exposure to electrotransmission, slot? -) Cinnafree (surrogate 5ona -fre Rehabilitation A , thereby demonstrating the developmental potential of this sperm.

The invention further provides a method for preparing sperm for in vitro fertilization. This method Sperm plasma cell potential (potentia: L) Y approximately -7 [] mV to + 1V to provide an electric field sufficient to allow Ca++ ions to enter. , free from exposure in vitro.

The invention further provides methods of treating male infertility.

This method lowers the potential of sperm plasma cells to approximately -7Q mV. Increase the voltage from 1V to +1V to provide enough electric field to infuse Ca++ ions. Exposure to permeation converts nonviable spermatozoa into viable spermatozoa, thereby It consists of triggering a disembodiment reaction in this sperm.

The present invention further provides a method Z for diagnosing male cause insusceptibility to nyx. this method Is the sperm sample "Y sperm pla, r" in a test tube? cell potential tia'l) Increase Y from -7Dmv to +1v and influx Ca++ ions. exposed to electrical transmission to provide an electric field sufficient to cause rogate zona-rree) /%,! , so that the star penetrates into the egg place! This allows us to assess the developmental potential of this sperm. From showing.

It's settled.

According to the method of the present invention, a large amount of By directly allowing calcium ion influx into cells, capacitance (C Effectively sandstep all stages of apacitization e 5 top). When cells are exposed to a large and temporary DC electric field, the potential Plasma cells with potential are excited from about -73mV to +1V, and the local area of the cell is It will tear and a hole will form. The number, size and persistence of these holes are given Voltage, pulse duration and pulse medium, temperature , and other factors, such as cell size and cholesterol/phospholipids in the cell. It is a function of quality ratio. Main product that directly and quickly infuses Ca ++ io/ into sperm It is the strict capacitance E7 phase (st rict Capa (!1tation phase) There are some cellular changes that can probably be bypassed.

That is, the various stages of the extirpation reaction produce large numbers of and relatively transient spermatozoa, and this Some of them are motile.

The end result of ionophoric exposure is Similar to transmission, but the mechanism is different. An ion permeable carrier is a calcium ion permeable carrier. electrophoresis seems to be more effective. , and its toxicity is much less than that of ionophoric carriers. Usually more than half of the cells It is difficult to control and most of the sub -fertile) sample does not seem to be of benefit. Other conventional pretreatments The system generally uses an electric field that causes only a photoreaction in very few spermatozoa. is 250-1000V in the method of the present invention, which is 625-20V 　D　D　V　cm-”K equivalent Field Strike V7 Gus (fie'ld strength), WK is 400-800V, which is 1000-200 0v cm-” field strength X (field Bstrength) Corresponds to ~ given as a pulse.

This pulse may be caused by either (i) a discharge from a capacitor or (ii) a variable switch in a static power supply. 7. switching or Larr) high voltage power supply. Generator ( pulse generator). The first method is the property The latter two methods provide a clear form of comparison. It gives a clean square pulse and duration. Furthermore, the standard pulse generator Increase the output from the stimulator or pulse stimulator It can also be made wider.

Bioland (registered trademark, Bib-Rad) oene pulsar sar) can be used to output pulses that are exponential in nature. appropriate person The operating conditions for sperm were 25μF, 500V% time constant, 5D OV implementation means 1250V/cm.

In the case of scene pulsar (oene pqlsar), the normal sample is 75 At 0V, 100% penetration is obtained, but polyspermy decreased. On the other hand, at voltages lower than 200V, sup-press hold (aul) -thrθθh01d). For Bioland equipment, the application of a single pulse is It was found to be effective.

Ho fer instrument processor A Progenerator can be used to generate square wave pulses. Ho Proper operating conditions for the Rapro Generator are 450v and 1[1-1-5Q pulses. be. Electrotransmission is usually carried out at 4°C. This is a breakdown (bre akdown) threshold hold (threshhΩld) of the potential be done. Therefore, in the method of the invention, electrotransmission typically occurs at a temperature of 20-25°C. Conducted in the degree range. However, the underlying method is extremely short, only a few minutes. One was done in between, but the other sample was inseminatio ^(inseminatio). Steps up to n) require approximately 1 hour. This eliminates the need for tight temperature control. No longer needed. We assumed that sperm introduction into the SPA was pulsed at sample Y +4°C. 34% reduction when pulsed at 37°C and 49% reduction when pulsed at 37°C. I found it.

Pulse media are ionic, partially ionic or substantially non-ionic media.

The base of a non-ionic medium suitable for use in the method of the invention is a polyol. A monosaccharide or a monosaccharide, especially sucrose. nonionic sucrose The advantage of using it is that local current heating effects are minimized and the Chief Rus is to increase the charges. Mannitol is also suitable for such systems; This also has the advantage of removing hydroxyl radicals. But Manni It was found that toll has a composition that is approximately 20% less efficient than sucrose. . Namely, 137mM Nacx, 2QmM HEPES, 5mM KCI , 0.7mM Na2HPO4, 6mM glucose and 5mM CaCl2 be. A very successful partially ionic medium was developed as follows.

Namely, 21QmM inositol, 30mM KCI, 5mMrncpEs, ID OpM inositol triphosphate, 2mM CaCl2 and 1m51 /s/ BSA.

Both of these media eliminate the need for Bost-Pulse centrifugal washing, and the samples One can simply dilute to the desired concentration as long as the dilution factor is at least x5.

The advantages of ionic Hals medium can be best utilized with the Sufuni γ-wave pulse generator. ing.

The pulsed medium also keeps the sperm from becoming sticky in the reaction chamber that houses them and increases their motility. It should contain drugs to maintain this condition. Suitable such drugs is a polymer such as polyvinyl alcohol. Serum albumin can also be used, but Due to its chelating properties, the concentration of possible ionized C a ++ must be 6 minutes. It will be reduced to 1.

Bost-pulse germ cell interactions are performed using protein-containing media such as albumin-based media. Preferably, it is carried out in a medium.

In the attached drawings: Figure 1 shows the calcium concentration in sucrose-based pulsed medium and SPA score parameters. data, the average intruder, and the decondensation detected in the eggs of each ovarian hamster. 7. : (aekonaensea) measured as the average number of spermatozoa The relationship between sperm and egg ratio is shown. Pron) a represents the average number of intrusions, Pron) is C Regression of sperm/egg ratio on aCl2 (represents regression).

The second factor is the applied pulse voltage (scene-pulsar), the average total detected aquasome Average invader and average sperm/egg ratio as response and SPA score parameters Represents the relationship between The curve ^ represents the average penetration2, and the curves represent the average total penetration and average total penetration of sperm. and, in part, the third factor in the acrosome reaction is the post-pulse incubation time. , the average penetration, and the relationship with the 1n corporation . Plot plots indicate mean % penetration and plot plots indicate mean χ of sperm/egg ratio.

Invasion in SPA is 100% or close to 100% (s/θ ratio is S It is more sensitive than any other parameter measured by PA.

The present invention will be explained in more detail with reference to the following examples.

Example Semen sample request from Gayuway Regional Hospital Infertility Clinic Yoikoshi obtained from a patient who underwent The patients were clearly in the homocynia group, with only 20 % showed obvious secondary infertility. In 15% of couples, the spouse also It was reproducing the problem. The average age of the group was 33.5 ± 0.7 years and the average infertility. The duration was 4.3 years and 0.54 years. Approximately 25% are on the first round (refθrr a1) shows an abnormal spermiogram (Iil) θrmiOgram)'l (shown, The majority of the remaining cases were classified as having unexplained infertility. The control group was slightly smaller, N=5. ), and the average age was 37.2±3.2 years. All have become fathers of one child in the past 10 years. The patient has a normal spermiogram, and there are no medical findings that indicate an unhealthy condition. It was, The semen sample was masturbated into a sterile Sterile IJ 1,1N (registered trademark) 60-container. The sample was collected in a tube and kept warm until it reached the laboratory. Fresh semen samples were received within minutes of collection and liquefied for up to 30 minutes. move Select a group of spermatozoa and place them on a single, discontinuous, two-step psrco11 gradient. It was isolated by spreading sample II of 1-. The lower layer is 954 perco It consists of a 2Q HEPES buffer containing 2.6 M NaC’l, 0.08M KCI, 20mMCaCl2’2 H2Os Contains D-28M glucose and 200mM HEPES Y! and pH 7,3-7 , 4 (permeability 360 mosm.

Density 1.10g/d).

HEPES is a tissue culture grade acid and base solution with a temperature of 7.3-7.4 at 37°C. Prepared as a 1M stock solution by mixing until K-stable. most The final osmolality is 312 mosmo. HEPIJ stock solution can be used as desired. Dilute as necessary to achieve the desired molar concentration. The upper layer is a complete medium (permeable 47.5% pe at 335 mosm, density, 1.5 ji /, t) rc○11.2.5 was made up of HEPES buffered saline. Grady end is Vl ncent, R., and Naaeau, D., (1984) A”1° Blochem, slightly withdrawn for reasons stated in Eng. 41.322-328. It was transparent. Load gradient end at 350μ for 25 minutes at room temperature. It was centrifuged with a drawer. Next, be careful of the pellet of spermatozoa spreading at -36Q mosm. Separate deeply and add 10-liter fresh medium (freshly prepared medium with the same composition and 1-liter fresh medium). Lipi04o1 (registered trademark of May & Baker's electromagnetic impervious oil) 5 turbidity again. and centrifuge 3502 at room temperature with a swing-out rotor. I lost it twice. Sperm are then diluted with WW/DM medium as defined below. and further centrifugal washing.

BWW (hereinafter referred to as BWW/n) is a modified p-aide medium, that is, BWW/n. iggers, Whitten & Whittinghams medium (Bigg er8, etc., 1971) "C-al, non-Koff + 1.94.8 mM N aC1, 4, 77mM KCI, 1.2mM KF! 2PO4,1,2m)l Mg5O, -7H2O, 5,5mM glucose, 25mM NaHCO3,0, 27mM sodium pyruvate, 19.5mM sodium lactate, 0.0D 1 % Penicillin-Streptomycin and 2Q InM dEPBB.

BW'W/DM is a defined medium containing polyvinyl alcohol (PVA) and dextrin. Combination with Try (Tozkins, P., T., et al., 1988!) contains. The complete composition of BWW/DM is shown in Table 1. Sperm pulsed into medium Suspended at a concentration of 0.5 x 108/-. The medium is 0.26M Analar Sucrose, iQ mM analar CaCl2, 2H20, 5mM HE P:S.

At pH 7,3-7,4, 50 μg/- of polyvinyl alcohol CM, W, 12 5.000). 800μm aliquot of suspended sperm Y’Bio - Rad Gene Pu1sar' was applied to high voltage pulses. That Pulse r is 500V, 25μF, time constant 2.5ms (1250V/(17A) Met.

After pulsing, the suspension was diluted with 7 BWW/DM, washed once, and resumed at a concentration of 0. ．． Suspended at 5 x 10'/- and immediately processed through SPA.

A subsequent comparative experiment showed that SPA results for all samples, but if post-pastry germ cell interactions are higher and more consistent when carried out in protein-containing gastric medium. I begged.

Thus, while BWW/DM is proven to be superior to BWW/n, , it appears that sperm were inhibited after being exposed to pulse t. Here, BWW/n is , 0.3-0.5% human serum albumin to induce acrosome reaction. Fusion gene containing fuaiogeniC by pre-incubation Sperm is t@.

A possible explanation is that BWW/DM increases cell leakiness and russ conditions do not adequately preserve cell integrity, and to some extent reduced motility may result. BWW/DM K is better than Bvw/n under Bost-pulse conditions. It's probably because it's big.

The complete composition of BWW/n is shown in Table 1.

Table 1 Culture medium (concentration) Component BWW/n BWW/DM (mM) (mM) NaC19ABC: I 9A, BO KCI A, 77 A, 77 C! ac12・2H201,704,00KH2PO41,2D 1.20 Mg5o4-7H2o 1.20 1.20 Glucose 5.50 5.50 NaHOO325, OD 25.00 Sodium birgate 0.27 0.27 Sodium lactate 19.50 19 ．． 50HEPES 20.00 20.00 pen-strap, (%) 0.001 0.001H8A (qb) 0. 3-0.5-PVA (%) 0.01 Dextran T2O000,3 Taurine (μM) 100.00 Super oxytoic acid mutavi 200.00 (μg ml-1) Catalase (μgm1-1) Egg drop after 100.00 samples arrive The total time until insemination was approximately 1 hour. SPA connectivity, penetration rate ( %) and the pulse of the present invention for normal control and spermic samples. Table 2 shows the ratio s/e of the sulfurized samples.

The 5 hr cap/H9A” group in Table 2 contains 0.5% human serum albumin. (asA)v containing nvw/n followed by normal SPA This is the result of semen from running. Table 2 24 hr Cap/H8A ” group contains 18- These are the results of semen incubated with semen for 24 hours. control semen concentration The degree is 8X IQ7m/-1-7,5XIQ8ml-1, and the ori is spec. The concentration of Luminku sample is IX 10' mZ-1-1゜5 It was mj-1. Details of the experiment can be found in Tomkins, P.T., et al. 198B (supra). In addition to BWW/'DM medium request t・ru , as a modified SPA, low concentrations of hyaluronidase and trypsin, i.e. Yana gimachi, R, at a L, 1976 (supra). A concentration of 0.05% was used for the standard SPA.

Using the electropermeabilization method according to the present invention, four classes of precision Fluid samples, i.e. (1) samples with known fertility; (■) oligospecies; Lumink sample; (iii) Asenospermink sample and OV) Fertility Determine the average penetration rate and average sperm/egg ratio at SPA for samples with unknown force. For this purpose, seven experiments were conducted, and the results are shown in Table 3.

Table 2 Technique Sample Connectivity Penetration rate % s/e ratio 5 hr cap/control le ≦20 50 0.6524 hr cal) / control ≦40 72 0.95 hr cap/Contecour ≦100 90 1.2BW W/DM Pulse control ≦500 100 10.255 hr cap/o Libby ≦5 105 hr cap/oligo-<1 [] <1 [1-BW W/DM Pulse Oligo-5C1-50075j, 2Cap = Capacity Ha A = Human serum albumin DM = Defined medium Oligo = Oligospermincus sample Albumin medium is used because spermink samples do not survive well during culture. The oligospermic samples were not incubated for long periods of time.

FIG. 1 shows the precision according to the invention as measured by the s/e ratio and the penetration rate by SPA. The effect of Oa++ concentration in the medium on the electropermeabilization of the solution show. These results are summarized in Tomkins, P., T., and Houghton.

J, A, Pertil, Stθwi1198 B, 50, 329-3 It has been reported in 36.

The amplified pulse voltage results are shown in the graph of FIG. The standard error is , invasion rate and acrosome response were within ±10%, and the sperm/egg ratio There are 2 units. The acrosome induction level was determined by PSA-F engineering TC fluorescence. Staining (Croas, N, L, stal,.

1 q B 6, Gamete ues, 15 + 213) using quantified directly. The average penetration rate and sperm uptake are similar to a normal bell-shaped curve. there was. The increase in acrosome induction in response to voltage was observed at a fixed concentration of 5 mM Ca”+. The voltage was saturated at 1250 V (m-1 or higher).

The pustule fusion and hydrolase release during the acrosome reaction do not occur instantaneously. There isn't. 3 Acrosome induction occurs in most cells simultaneously with early Ca++ influx. However, the dynamics may differ depending on the semen segment. Ink after pulse This possibility was discussed by amplifying the activation time. Average obtained from two experiments value? This was shown to the 6th prisoner. Post-pulse incubation at 37°C for 3 puffs and continued for over 1 hour. Intrusion responses were significantly reduced when This is the active substance that reacted with the acrosome. This signifies that the sample will grow old over time. Chineep incubation Under these conditions, the motility samples after 3 hours of incubation were 72 ± 8%; After gamete interaction, it decreased to 18±6%.

As is evident from the results shown in Tables 2 and 3, the electrically permeable The induced Acrome reaction was more significant than any known JN vitro method. It is effective, quick, and less harmful. The basis of the method of the present invention is sperm The idea that inducing an acrosomal reaction is based on the results of SPA It can be supported from Induction by the electropernobility method of the method of the present invention It is thought that the membrane of tactile sperm changes to enable fusion. This is a subfu ferti’lθ (oligospermic) sample This is one explanation for the fact that such results are obtained.

Normal IC acrosome-reacted sperm can fuse with the egg cell membrane, so the sperm can It undergoes capacitation and acrome reaction, fuses and is incorporated into the egg.D Determine whether the sample is capable of undergoing NA decondensation. SPA'i can normally be used to From the results in Tables 2 and 3, It can be seen that the method of the present invention increases the invasiveness in SPA. Therefore, the basis of the present invention This method enables grouping and classification based on s/e data. The method of the present invention also allows for the fusion of samples in vitro. SPA is considered to be useful as an aid to the "O test" 2.SPA is currently used in many It is commonly performed at medical centers, but the methodology is not standardized. By applying Zumata false net to existing SPA with a high level of tip response, Each semen sample generates the highest and most optimal response for the test assay. A series of simple and flexible methods are presented to make this possible.

This increases the discriminatory power of the assay and allows for true males exhibiting pathological function as fusion devices. can be isolated.

Conversely, if sperm become unable to pass through the transparent membrane due to induction of mature acrosomes, As long as the tn transfer), engineering VF 11. Couples that benefit from the use of intermediary regeneration techniques It is useful as a pre-screen for isolating.

The basic method of the present invention is to obtain 1nvltro capacitance prior to gamete interaction. It can be used for all Yon methods. The basic method of the present invention is to Publicly known as Sperm Chromoseme Assay (SCA) for establishing otype levels. Makes it possible to use a well-known SPA.

There are still very few animal IMFs, one of the reasons being that important livestock such as cows and horses This is because it is difficult to achieve capacity. of all mammals The basic method of the present invention, which is applicable to spermatozoa, overcomes these difficulties. be able to. The applicability of the method of the invention to mammalian spermatozoa is due to the electrically mediated C It is based on a++-dependent and trigger-like induction.

Different pulse type or amplitude conditions, culture medium used, and gamete handling conditions may vary depending on the species. There are differences depending on

The electropermeabilization method of the present invention includes (!AMP, taurine, and - This effect occurs in subfertile sperm that are deficient in peroxide dismutase, etc. It can also be used to introduce such compounds.

Unlike many mammalian spermatozoa, known fertile human spermatozoa are Incubation iK [no levels of acrosome induction (C, E, 5t ock & L, R, Fraser 1987, Hun, Reprod, 2 , 10q-119). Usually, in vivo, ≦0.001% of sperm Low sperm as well as physical factors because there is no chance of interacting with the egg Synchrony limits the size of the cell pool that can react with acrosomes at an appropriate time. It will be done. Applying electropermeabilization to such samples together with other suitable regeneration techniques to elevate this useful sol. Can be done.

Human Lyzooapermic (po:Lyzooapermic) samples and shapes The sample with a morphologically round head has a higher height than the sample with a round head. Show (W, B, 5chill et aLl 938, Hum, Repr O+1.3.139-145　o　o　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　o　 By combining with the structure, acrosome-induced electropermeation This can be effectively done by IJ session.

Thus, the advantages of the present invention and its potential will be appreciated.

Pure? (No change in content) Pulse field underground CocI2 concentration CmM) continuous pulse voltage (Vcm-') Post-pulse incubation time (min) Procedure amendment (voluntary) April lZ day, 1990

Claims (16)

[Claims] 1. The sperm plasma cell potential is increased from approximately -70mv to +1v to perform so-called sperm immersion. In the transparent method (SPA), penetration of less than 100% is observed for normal sperm. , in oligospermic samples, the method sufficient to allow influx of Ca++ ions such that less than 75% penetration is observed. Characterized by exposing sperm in a test tube to electrotransmission, which involves applying a strong electric field A method to induce a photoreaction in sperm. 2. The potential of the sperm plasma cell was increased from about -70mv to +1v, and the ca++ ion electrotransmission in a test tube, which involves applying an electric field sufficient to allow the influx of exposing sperm, surrogate zona-f ree) Determine the ability of treated sperm to penetrate hamster eggs and The developmental potential of a spermatozoa characterized by exhibiting the developmental potential of said spermatozoa. How to assess competency. 3. Raise the sperm plasma cell potential from about -70mv to +1v to release Ca++ ions. exposing the sperm to electrotransmission, which involves applying an electric field sufficient to allow the influx of inducing a photoreaction in the spermatozoa in a test tube. Method for preparing sperm for in vitro fertilization. 4. Raise the sperm plasma cell potential from about -70mv to +1v to release Ca++ ions. Electrotransmittance the sperm into a test tube, which involves applying an electric field sufficient to allow the influx of The process of converting non-viable sperm into viable sperm by exposing the A method of treating characteristic male infertility. 5. Raise the sperm plasma cell potential from about -70mv to +1v to release Ca++ ions. Electropermeation of the sperm sample involves applying an electric field sufficient to allow the influx of In vitro exposure of the surrogate zonafree na-free) Determining the ability of treated sperm to penetrate hamster eggs , thereby demonstrating the developmental potential of said sperm. How to diagnose sperm. 6. The field strength of the electric field force 625-2000vcm-1 Pulse with a voltage of 250-1000v, corresponding to 6. A method according to any one of claims 1 to 5 applied to. 7. A method according to the preceding clauses, wherein the pulses are exponential in nature. 8. Claim 1- wherein said pulse is a square wave pulse exhibiting a distinct shape and duration. 6. The method according to any one of 6. 9. The applied pulse is time constant. The method of claim 7, wherein the voltage is approximately 2.5 mS and approximately 500 V at 25 μF. . 10. The method of claim 8 in which pulses of 10-50 ms are applied at 450 volts. 11. A method as described in the preceding paragraphs, carried out at a temperature in the range of 20-25°C. 12. The medium of the pulse contains a Ca++ ion source at a concentration in the range of 2.0-20mM. The methods described in the preceding sections, including. 13. The medium is selected from ionic, partially ionic or substantially non-ionic media. The methods described in the preceding sections will be disclosed. 14. The pulsed medium prevents the sperm from sticking to the reaction vessel containing the sperm. and the method described in each of the preceding paragraphs, which includes a drug that maintains sperm motility. 15. Those described in the preceding paragraphs in which the post-pulse gamete interaction is carried out in a protein-containing medium. Law. 16. The method described in each of the preceding items, wherein the spermatozoa are human spermatozoa.