Method for growing normal descendants from oocytes injected with spermatozoa treated by drying with freezing

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

 

This application is based on priority application for U.S. patent No. 09/177,391, filed October 23, 1998, which in turn has priority on provisional application U.S. No. 60/078,925 dated March 20, 1998, and No. 60/089,938 dated June 19, 1998

The U.S. government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to grant a license on reasonable terms to other persons, as specified in the terms of contract number R01-HD-03402 with National Institutes of Health, Public Health Service.

The present invention relates to drying by freezing sperm, fertilization of oocytes with spermatozoa recovered after freeze-drying, and the development of them live offspring

Successful cryopreservation of semen with the use of cryoprotectants and the opportunity for a long time to store frozen sperm produced dramatic revolution in animal breeding and reproductive medicine man. It was found that kriokhraneniya spermatozoide, thawed after freezing, often restore their mobility and fertilized nearly as effective as fresh sperm.

For cryopreservation of spermatozoa of cattle and humans typically use long-term storage of spermatozoa in liquid nitrogen (-196°C). However, the usual conservation of semen during long remeniscent very expensive because it requires a continual supply of liquid nitrogen. Moreover, in some areas of the globe is difficult and/or expensive to store sperm in liquid nitrogen; for example, in developing countries, where it is difficult to get liquid nitrogen (or dry ice). Transport of spermatozoa frozen in the usual way, is also problematic, as it requires transporting large containers for liquid nitrogen or the use of special marine containers containing liquid nitrogen or dry ice. So were many attempts to preserve sperm without the need for storage in liquid nitrogen.

For example, if capable of fertilization, the sperm could be stored in a freeze dried (frozen-dehydrated state at ordinary temperatures or in simple refrigerators, reducing operating and transportation costs could be huge.

It is believed that the first recorded attempt sublimirovanny (i.e. freezing and dehydration) of spermatozoa was done in 1949, when 1 milliliter (ml) of the seed rooster mixed with an equal volume of ringer's solution containing 20-30% glycerol, caused by thin layer distillation flask and “sublimated”by removing 90% of water. When the drug rehydratable 2 h after warming to room temperature, was about the observed, up to 50% of spermatozoa preserved mobility. However, until now, has not been determined ability rehydration sperm to fertilize.

Subsequent attempts to obtain live offspring using freeze-dried sperm was not successful. It was reported, for example, that after artificial insemination of cows with bull sperm were recovered immediately after sublimation and showed a 50 percent motility of sperm, was born of a live calf. It was also reported that it had obtained the twelve litters of normal rabbits after insemination with the use of freeze-dried spermatozoa. However, none of these results could not be repeated by the authors, or duplicated and confirmed by others working in this field.

From studying the properties and fertility normal and reduced sperm it is known that the sperm does not need to be “alive” in the usual sense of the word (i.e. to have intact plasma membrane) in order to support normal embryonic development. For example, using the methods of injection of sperm into the cytoplasm (ISWC) select motile sperm of the man. Their immobilizer (“kill”) immediately before injection into the oocyte by forceful removal of the tail parts, which then leads to disruption of the plasma membrane. It was noted that the immobilization of sperm increases the level of success ISWC fertilization. There is also a message that when the mouse sperm was suspensively in the environment without any cryoprotectant, and then immediately frozen in liquid nitrogen, 100% of the sperm were dead, as was determined using the marking of live/dead cells, but after microinjection heads thawed spermatozoa into oocytes was normal embryonic development. There is also a message about the birth of two normal calves after microinjection into oocytes sperm killed when thawed after drying by sublimation in the absence of cryoprotectants.

Although it is known that dried by freezing spermatozoa heads hamster and man, injected into hamster oocytes, can form an outwardly normal pronuclei, has never been determined whether such spermatozoa heads to support normal embryonic development. Moreover, it was shown that sublimemovies sperm to a moisture content of less than 30%, 7% and 0.5% lead, respectively, to the loss of mobility, disruption acrosome and leakage of enzymes. Have provided evidence that cellular proteins in the sperm cells are altered by dehydration of the lower moisture content of 6%.

Due perechislennogo there is a need for reliable and reproducible method of sublimation (freeze-drying) of sperm, in which the sublimated added, retain their ability to fertilize during long storage at room temperature or in conventional refrigerators, or at lower temperatures. Furthermore, the method of injection of the sperm, which use rehydration after the sublimation added for fertilization recipient oocytes with results in normal living offspring.

According to the invention, a method of freeze-drying spermatozoa to obtain at least one recovered sperm, whose head (core) has the ability to fertilize an oocyte to obtain a living offspring. The method of the invention involves the following stages: (a) collection of live spermatozoa; (b) suspending the spermatozoa in suspesiouse environment; (C) freezing the suspension of spermatozoa and (d) dewatering the suspension of spermatozoa to the moisture level of less than 1%, preferably less than 0.01%, better still less than 0.001% and in particular less than 0,00001%. The method further may include the stage of rehydration of the dried suspension freezing of sperm, when at least one head registrationpage sperm retains its genetic integrity and the ability to fertilize the oocyte to obtain a living offspring. As will be discussed below, the method can give is further include storage phase dried by freezing sperm before rehydration.

To obtain a living offspring, at least the head (nucleus) registrationpage sperm is injected into an isolated oocyte for the formation of a fertilized oocyte. The head of the sperm is injected into an oocyte microinjection, preferably, the piezoelectric microinjection. Preferably, the introduction of the core occurs within one hour after rehydration. Fertilized, the oocyte then provide the opportunity to develop in the embryo and implanted into the uterus of a surrogate mother, where he develops in the living descendant.

In some species (such as most land mammals, including humans) normal embryonic development of the fertilized oocyte requires the introduction of paternal centrosomes associated with the nucleus of the sperm. When the head and the tail separated, the centrosome usually remain attached either to the rear end of the head of the sperm, or to the front end of the tail of the sperm. Thus, in one implementation of the invention associated with the sperm centrosome other sperm can be injected simultaneously with the head of the sperm or can be entered by simultaneous or subsequent administration tail of the sperm alternatively, the introduction of the nucleus of the sperm and the centrosome may be accompanied by the introduction of a whole registrationpage sperm into the oocyte.

Was obnarujeno, what spermatozoa subjected to drying by freezing according to the method of the invention can be stored for at least three months or, more preferably, at least one year at temperatures varying from normal (i.e. room temperature) to the temperature existing in the refrigerator (i.e. about 4° (C) without losing their genetic integrity, or the ability to fertilize. So, dried by freezing spermatozoa can retain their "abilities" during their transportation to almost anywhere in the globe at the usual or existing in refrigerators temperatures and also may make short-term storage at ordinary temperatures or the temperatures existing in the refrigerators, in places where liquid nitrogen or dry ice are not readily available. Preferably, the long-term (i.e. indefinitely) storing dried by freezing sperm occurs at temperatures less than 4°With (for example, 20°or lower).

The method of the invention can be used for freeze-drying spermatozoa as invertebrates and vertebrates, including, but not limited to invertebrates, such as sea urchin, lobster, lander, mollusks and the like, and vertebrates, such as fish, amphibians, reptiles, birds and all mammals.

In the mate who ialah of this patent contains at least one drawing, made in color. Copies of this patent with color drawing can be provided by the Patent and Trademark office in response to the request and after payment of the required fees.

Figure 1 is a photograph vacuumed vials containing dried by freezing sperm. White powder at the bottom of each ampoule is CZB medium containing spermatozoa.

Figa is a micrograph dried by freezing sperm mouse immediately after rehydration. The proportion of sperm heads without the tail parts or with a broken tail parts varies depending on how carefully or mistreated dried samples during storage.

Figv is an electronic micrograph showing a longitudinal section through the front end of the head normal sperm, which was not frozen.

Figs is an electronic micrograph showing a longitudinal and horizontal section through the front portion of the head dried by freezing sperm after rehydration. Plasma membrane (p) and the contents of the acrosome (AK) lost, (you) - internal acrosomal membrane (us) - outer acrosomal membrane (n) is the nucleus.

Figure 3 is a photograph of three young (black) mice born to CD-1 (albino) surrogate mother. These cubs times who were from B6D2F1 oocytes, injected sperm B6D2F1, which was kept at room temperature for one month after freeze-drying.

During normal fertilization in mammals fertilizing sperm rises on the female sexual tract, penetrate the membrane of the oocyte and then fuses with the oocyte. The fusion of the sperm with the oocyte causes activation of the oocyte. The activated oocyte continues delayed meiosis and oocyte chromosomes are transformed into the female pronucleus. Meanwhile, the core of sperm inside the oocyte decondensed for transformation into a male pronucleus. Fully developed male and female pronuclei blend in, and chromosomes of these pronuclei unite. The resulting zygote develops into a living descendant.

The present invention provides a method of sublimation (i.e. freeze-drying) of sperm that after rehydration able to fertilize the isolated oocyte to obtain a living offspring. Dried by freezing sperm cells obtained by the method of the invention, retain their genetic and reproductive potential even when after rehydration they are fixed in the usual sense “dead”. When a sperm or isolated sperm head (i.e. head, containing all components, including the kernel) or devoid of membranes stole the AI or the head of the sperm (i.e. preserving the core and perinuclear material, but not with plasma membrane) according to the invention inherits directly into the oocyte is normal fertilization and embryonic development, which may lead to the production of live offspring. Preferably the head of the sperm (the kernel) is injected directly into the cytoplasm of the oocyte. Introduction of the head of the sperm is carried out by microinjection, preferably piezoelectric microinjection. As will be discussed below, embryonic development of fertilized oocytes of some species may require simultaneous or immediately after injection of the sperm centrosome.

If the centrosome is not subjected to the process of freeze-drying, for introduction into the oocyte is possible to select the centrosome of thawed sperm.

Next, a more detailed individual phases and sub-phases of the method of the invention for the preparation of fertile dried by freezing sperm and their use in the process of fertilization carried out in vitro.

Preparation of sperm

In order to provide more dried by freezing sperm preserve their genetic integrity in the process of freeze-drying, in the method of the invention, it is preferable to use physiologically Mature spermatozoa. In La the entire sperm DNA is associated with basic proteins, called Protamine. In mammals, Protamine repeatedly bound by disulfide bonds. This stabilizes the nucleus of the sperm and makes them very resistant to physical and chemical destruction. Firmware nuclear protamines occurs mainly during the movement of sperm through epididymis. Thus, mammalian spermatozoa in the epididymis and semen (seed) mainly physiologically more Mature than the inside of the testes, and it is preferable for the method of the invention, at least in the case of mammals.

Mature sperm cells of invertebrates and vertebrates collected by methods well known in the art. For example, the Mature sperm rodents, such as mouse, Golden (Syrian) hamster, Guinea pig, rabbit and the like, may be collected from the tail epididymis; whereas in other species, such as humans, pigs, horses, bulls, goats, cocks and the like, Mature sperm can be isolated from fresh traveled by ejaculated seed fertile males. Sperm fish (e.g., horseshoe crabs, Xiphophorus helleri) and invertebrates such as sea urchins (Tripneustes gratilla) can be extracted from the testes of Mature males.

In the example below describes how to obtain sperm from the caudal epididymis. The caudal epididymis collected from sexually Mature male mice (approximately weeks after birth or older). Blood and fatty tissue is removed from the surface of the tail of the epididymis. Then squeeze to release the dense mass of sperm. A drop (about 2 µl) of the mass of semen is placed on the bottom polypropylene centrifuge tubes with a volume of 1.5 ml and placed on top of 0.5 ml of warm physiological environment (e.g., CZB medium, saline phosphate buffer, or isotonic saline). After 10-20 min at 37°With motile sperm can be selected from the supernatant liquid.

The following is an example of obtaining sperm from a seed. Freshly traveled by ejaculated human semen allow us to go into a liquid state for 30 min at room temperature (about 25°). Then the seed is diluted with about 10 ml of saline and filtered through two layers of filter paper to remove impurities. The filtrate can then be ottsentrirovat at 400×g for 10 minutes, and the precipitated sperm re suspensionthe in saline to the desired concentration for the process of freeze-drying.

The following is an example of a method of obtaining a sperm from the male sexual glands. Remote gland placed in the erythrocyte-lyse buffer (i.e. 155 mm NH4Cl, 10 mm KHCO4, 2 mm add, pH 7.2-7.4), shredded sharp scissors and filtered through two layers of filter b is mages to remove impurities. Then the filtrate is centrifuged (for example, 700×g, 5 min) and the precipitate re-suspension in saline to the desired concentration in preparation for the process of freeze-drying.

Regardless of the used method for preparation of sperm for more than 50% of recovered sperm should be moving.

Restored thus sperm suspension in the physiological environment, described below, to prepare for the process of freeze-drying. Alternative prior to the freeze-drying spermatozoa can be subjected to further processing to obtain deprived of membranes of sperm heads

Preparation deprived of membranes of sperm heads

Deprived of the membranes of spermatozoa heads are extracted by detergent heads, deprived of all membranes, including the plasma membrane and the inner and outer acrosomal membrane, but kept nuclear and perinuclear material. For example, the head of the sperm may be deprived of the membranes by treatment with Triton X-100 with or without LTOs (dodecyl sodium sulfate). Triton X-100 is a well-known non-ionic surface-active agent, which is widely used for removal of membrane components without denaturation. LTO is an anionic detergent, the use of which has been created for the dissolution of various proteins, including membrane proteins. It has been shown that mouse sperm head, devoid of membranes by Triton X-100, is able to activate the oocyte, resulting in normal embryonic development.

The following is a typical method of removing the membranes of the sperm heads, Multiple suspensions of sperm, prepared as described above, is subjected to the action of sound. For example, sperm cells selected from the caudal epididymis, gonads or sperm, as described above, can be suspensionthe in 5 ml of BM buffer (75 mm NaCl, 24 mm add and 50 mm Tris-HCl, pH 7.2) and to expose sound for 30 seconds at 70%-80% of the capacity of the device Biosonic sonicator company (Bronwill Scientific, Rochester, NY). More than 95% of the sperm obuslavlivaya under this treatment. To remove membranes heads of spermatozoa suspension of sperm after sound exposure centrifuged for 5 min at 700 g and the precipitate washed with BM buffer, and then at room temperature is treated with 1% Triton X-100 in the environment (environment IT consists of 123 mm KCl, 2.6 mm NaCl, 7.8 mm NaH2PO4, 1.4 mm KH2PO4, 3 mm Na2Add, pH 7.2) and Then head thoroughly washed by environment THEM and re-suspension in preparation for the process of freeze-drying.

The suspension medium for sperm

In preparation for drying by freezing sperm (or devoid of membranes golokopytenko) suspension in physiological solution, which is sufficient to maintain in normal conditions of integrity, at least, of the nuclei of the sperm. The solution should be balanced salt solution with at least an appropriate osmotic pressure and pH. There is no single medium that could support the survival of spermatozoa of all species of animals. For example, a suitable solution for sea urchin sperm is sea water has an osmotic pressure of about 1,000 Milli-osmole and pH value of about 8.2. Sea water, however, will instantly kill the sperm mammals. Sperm mammals require a solution with an osmotic pressure of about 300 Milli-osmole and pH 7.0-7.6. This solution, however, will kill the sperm of the sea urchin.

With this in mind, the medium for suspending the spermatozoa should be selected according to interest the mind, taking into consideration criteria well known in the art. Such a choice can be made without conducting experiments. As for the successful fertilization of oocytes by injection of the sperm does not have to be with intact membranes (they may be “dead”), there are no strict requirements to the cryoprotectants, such as glycerol or the like, in the medium for suspension.

Packaging

Suspension of sperm can be placed in many different what's containers, including, but not limited to, glass vials, plastic Citrobacter (krivozerkale) with screw-caps, or the suspension can be placed in a plastic straws, which after a process of freeze-drying can be sealed powder sealant, sealed or closed nylon tubes. The volume of the suspension of spermatozoa in each tank is not important. Usually the amount of approximately 50-100 μl placed in a 2 ml ampoule.

Drying by freezing sperm.

Suspension of spermatozoon you can freeze quickly or slowly well-known ways. For example, the suspension can be frozen in the vapor of liquid nitrogen or in air mechanical (electric) refrigerator ways, well known to specialists. Cooling and freezing can be performed in static or manual stage mode, or in an electronic automated and programmable system that supplies liquid nitrogen. You can use different freezing rate (for example, 1°25°C/min). For example, the stage of freezing can be performed with a speed of up to -196°C for 10 minutes.

For successful freezing the suspension at straws, ampoules or Citrobacter a couple can use the various options. Metal container (canister) with cigarettebuy tubes or other holders with straws, and the holders, or rack of vials, or Citrobacter can be placed directly into a pair of liquid nitrogen using gelatinosa refrigerator.

Drying the frozen suspension of sperm in vacuum can be implemented using many different systems known in the art. For example, the known apparatus VirTis model 10-020 company (VirTis Co., Gardiner, NY). The suspension is dried to a moisture level of less than 1%. However, it is preferable that the humidity level was less than 0.01%, better still less than 0.001% and particularly well - less than 0,00001%. The container with the dried freezing sperm preferably packaged under vacuum or in the presence of inert gas, such as nitrogen or argon.

Storage

The containers are dried by freezing sperm is preferable to store in the dark or wrapped in aluminum foil or in kind. In the case of long term storage it is preferable to store the containers at -20° or below. It is assumed that similarly dried by freezing bacteria, fungi and the like, and dried by freezing nuclei of sperm under these storage conditions will fully retain their genetic integrity. However, some time in excess of three months, the containers can be stored at normal temperatures (i.e. room temperature) or tempera is Urach conventional refrigerator (about 4° (C) without compromising the ability of dried freezing nuclei of sperm to fertilize oocytes. Therefore, dried by freezing the added, can be transported without special conditions or bulky containers.

Rehydration of lyophilized sperm

Drugs dried by freezing sperm is preferable to registrationall clean water, the volume of which is equal to the initial volume of the sperm suspension before drying by freezing. After rehydration for dilution, you can use any physiological salt solution, such as 0.9% saline or medium CZB (see below). The amount of diluent is not standardized.

The sperm concentration in the final rehydration environment should be sufficient to ensure recovery of individual sperm or separate heads of sperm for the purpose of injection of the sperm into the oocyte, as described below.

It turned out that the number of cases of activation of oocytes and normal fertilization following the injection head of sperm decreases with time after rehydration of sperm. The valid time interval between rehydration and injection of different species can vary; however, as an example, for mouse sperm this interval is preferably equal to one hour or even less.

p> Microscopic examination of spermatozoides, rehydration after drying, sublimation

Dried by freezing sperm immobile. The viability of sperm can be determined using any method of staining, which allows to distinguish the sperm, which in the usual sense are the living and the dead. Suitable test device for vitality, which can be readily purchased for use in the invention is Live/dead FertiLight, the company Molecular Probes, Eugene, Oregon, which allows us to distinguish cells with intact plasma membranes (live) from cells with damaged plasma membranes (dead) character fluorescence under a UV microscope after staining iodide propidium/SYBR 14. The kernel of live spermatozoa with intact plasma membranes fluoresce green, while those of dead sperm cells fluoresce bright orange-red. It is expected that all investigated sperm will be “dead” in the usual sense.

Figa is a micrograph of a typical dried by freezing sperm mouse immediately after rehydration. Head and tail of some spermatozoa separated. The proportion of spermatozoa with a broken tail parts or without tail parts can vary depending on how carefully or GRU is on the treated samples during storage. Spermatozoa with tail parts or without them was used in the injection procedure, described below.

Differences between fresh (not dried by freezing) sperm and dried by freezing and rehydration according to the method of the invention sperm shown in figv and 2C, respectively. Each of these figures represents the latitudinal cross-section through the front portion of the head of the sperm. Although dried by freezing registrationentry sperm retained the core (I), part of the outer acrosomal membrane (us) and part of the inner acrosomal membrane (you), as a plasma membrane (PM)and the contents of the acrosome (a) no.

The oocyte recipient

The oocyte recipient can be obtained, for example, causing the animal ovulation or sirovasthi effect of injections of gonadotropin-releasing hormone (for example, sequential assignment horse and human hormonally gonadotropin) and selecting surgically egg from the oviduct shortly after the expected time of ovulation (for example, after 13-15 hours after injection of human hormonales gonadotropin mouse). Alternatively, oocytes can be extracted from the ovaries and cultured in the environment prior to their maturity, as is known in the art. An example of a preferred environment for cultivation is modified environment IG is a (ALL), enhanced serum albumin bull (ASB), described by Downs, S.M. and A.M. Mastropolo, Develop. Biol. 162: 154-168, 1994 for mouse oocytes.

Components of semen required for a successful in vitro fertilization

It is known that mouse normal fertilization can be achieved inhazinue isolated head of the sperm into the oocyte, and that the plasma and acrosomal membrane and all of the components of the tail is not essential for normal embryonic development. Mouse and possibly the most common laboratory rodents are “exceptions” that they are for normal fertilization is not required, the sperm centrosome, and during normal fertilization, the sperm centrosome in the cervix sperm after fertilization doomed to degeneration within the oocyte.

In contrast, in most terrestrial mammals, including cattle and humans, the sperm centrosome plays a Central role in the formation of microtubules, which are necessary for the unification of male and female pronuclei, as well as for subsequent crushing during embryonic development. Thus, these species introduction as the nucleus of the sperm (head), and microtubules in the oocyte was found to be necessary for normal offspring. It is currently unknown whether all types of centrosome sperm can survive drying by freezing. If not, then to guarantee n is Smolnogo embryonic development centrosome thawed sperm must be injected into the oocyte together with dried by freezing the head of the sperm. The introduction of larger numbers of centrosomes, however, will lead to abnormal development of pronuclei and abnormal embryonic development.

When the head and the tail separated, the centrosome usually remains attached either to the rear end of the head of the sperm, or to the front end of the tail of the sperm. Therefore, the sperm centrosome may be introduced into the oocyte at the same time with the head of the sperm, or can be entered by simultaneous or subsequent administration tail of the sperm. Alternatively, the introduction of the nucleus of the sperm and the centrosome may be accompanied by the introduction of a whole registrationpage sperm into the oocyte.

The introduction of the nucleus of the spermatozoon into the oocyte recipient

In the cytoplasm of the oocyte recipient, you can enter a sperm, but for those species in which sperm are large, preferably directly into the cytoplasm by microinjection technique introduces an isolated cylinder (core) of the sperm. In a preferred method, microinjection rehydration head of the sperm or rehydration deprived membranes heads of sperm into the oocyte recipient uses piezoelectric introduction.

Suitable piezoelectricity managed device sold under the name of Piezo Micromanipulator/Piezo Impact Drive Unit firm Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The device uses a piezoelectric effect for PTS is ery fast controlled advancement of the holder (injection) pipette at a very small distance (approximately 0.5 micrometers). The intensity and duration of each pulse can be varied and controlled via remote control.

For injection into an oocyte, a single sperm is sucked tail forward in the injection pipette with a short flat tip, an inner diameter of about 5 micrometers, placed in piezoelectricity the managed device in accordance with the manufacturer's instructions. The head of the sperm is separated from the tail by the application of one or more pietinalho in the cervical area. Then the cylinder is sucked into the pipette.

During the injection head (nucleus) of sperm oocyte hold a suitable holding pipette. The tip of the injection pipette containing the selected cylinder, sperm, put in close contact with the pellucid zone of the oocyte and to promote the eyedropper to put a few pietinalho (using the remote control, set the intensity on a scale of 1-5, rate 4-6), while creating a slight negative pressure inside. When the pipette tip is pellucid zone, the resulting cork area push in perivitelline space, and the head of the sperm is pushed forward until, until it is near the tip of the pipette. The pipette tip is then applied to the plasma membrane and moves forward (toward protivopul the Noah side of the oocyte), and holding pipette almost reaches the opposite side of the cortex of the oocyte. Now plasma membrane deeply concave around the tip of the injection needle. When the application of one or two pietinalho (intensity 1-2, speed 1), oolemma at the tip of the pipette bursts, as indicated by clearly visible fast relaxation of the oolemma is broken. The head of the sperm is then forced into ooplasm with a minimal amount (about 6 picolitres) accompanying environment. Then the pipette carefully removed, leaving the newly introduced head inside the cytoplasm of the oocyte. This operation is carried out quickly, usually in a group of 10-15 oocytes that all this time are in terms of culture.

Alternative microinjection options for injection of sperm heads you can use a regular injection pipette. An example of a suitable microinjection method using a conventional pipette for injection head of the sperm into the oocyte hamster described in Yanagida, K., Yanagimachi, R., Perreault, S.D. and R.G. Kleinfeld, Biology of reproduction 44, 440-447 (1991), the disclosure of which relating to such method, included in the references.

Microinjection of sperm head/devoid of the membrane of the sperm head has several advantages. First, the transfer head of the sperm microinjection is applicable to a wide range of types of sperm, regardless of size, morphology and the fact is such. Secondly, microinjection allows together with the injection of the sperm head (head of donor sperm) to carry out a controlled injection of additional agents in the oocyte. Below this is shown in the examples. Thirdly, in the implementation of the invention, where the entry of the sperm head is a piezoelectric microinjection, fast and efficient processing of samples, thus reducing the injury of sperm and oocytes manipulated. The oocytes of some species (e.g., mouse) not amenable microinjection using conventional needles, whereas the piezoelectric microinjection gives a high level of success.

Activation of fertilized oocytes

It is known that the mouse oocyte can be activated by injection of a single intact mouse sperm or isolated head. Isolated the tail of the sperm is not able to activate the oocyte. Active originating from sperm oocyte - activating factor(s) usually occurs during the transformation of spherical Spermatid in sperm. The effect of these factors is not strictly species-specific, because the mouse oocytes are activated by injection of spermatozoa of other species, such as the hamster, rabbit, pig, man and even fish. There is a message that one such activating factor is a protein, the size of 33 kilo-Dalt is on and staying in the area of the Equatorial segment of the acrosome. This protein, called oscillon, easily extracted from the Mature sperm cells (hamster) simple freezing and thawing. In addition ozilline Mature sperm, obviously, is another triggering factor, which is not easy to extract, but you can get the serial processing of sperm Triton X-100 and SDS. However, it is unknown whether easily extracted oscillon and frozen/thawed, resistant to extraction factors of biologically and chemically identical.

It is known that the head of the sperm, the processed sound in the presence of Triton X-100, lose all parts except engine and perinuclear materials. However, after microsurgical the introduction of such oocytes treated with Triton X-100 heads of sperm (having a core and perinuclear material, but without the plasma membrane) can activate oocytes as effectively as intact spermatozoa.

As described in the examples below, at least going from sperm oocyte-activating molecules mouse must be resistant to drying, freezing, since the majority of oocytes that survived after injection dried by freezing sperm heads were normally activated and fertilized.

If other types of injection head of the sperm does not cause activation of the oocyte, the activation signal is to be parthenogenetic means, such as electroactivity, the injection of one or more oocyte-activating substances or transfer of oocytes in an environment containing one or more oocyte-activating substances. Reagents capable of giving an activating stimulus (or a combination of activating stimuli), include, but are not limited to: activating factor in the cytoplasm of the sperm and certain pharmacological compounds (e.g., CA2+and other modulators of signal transduction), which can be introduced by microinjection after or simultaneously with the injection of the sperm head. Some of the activating stimulus is applied after the transfer of the fertilized oocyte in a medium containing one of the members of the subgroups activating compounds, including stimulants release of CA2+(for example caffeine, CA2+the ionophores, such as a 23187 and ionomycin, and ethanol), modulators phosphoprotein signals (for example, 2-aminopurine, staurosporin and sphingosine), inhibitors of protein synthesis (for example, 23187, cyclohexamide), 6-dimethylaminophenyl or combinations listed (for example, 6-dimethylaminoborane and ionomycin). In one method of carrying out the invention the activation of mouse oocytes was achieved by culturing for 1 to 6 hours in free CA2+CZB medium containing 2-10 mm Sr2+.

The development of the embryo with obtaining a viable fetus and flux the STV

Following the formation of pronucleuses the embryo can be grown in vitro until they reach the stage 2-8 cell stage or the morula/blastocysts in which the embryo can be transferred into the oviduct or uterus of a surrogate mother.

Simultaneous introduction with the heads of sperm biologically significant substances

In one example implementation of the invention microinjection head of the sperm into the oocyte allows you to enter before, during, or after the injection head of the sperm into the oocyte one or more agents that affect the output developed embryos for Example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)can be introduced into the oocyte by microinjection before or after the injection of the sperm head. For example, injection of recombinant DNA carrying the necessary cis-active signals, can lead to transcription of sequences present in the recombinant DNA of the resident or injected together with the transcription factors and the subsequent expression of the encoded proteins with antagonistic action on the factors inhibiting the development of, or with a positive effect on embryonic development. Moreover, the transcript may have antisense activity against mRNA encoding proteins that inhibit development. Alternatively, antisense regulation can be obtained by injection nuclein what's acids (or their derivatives), who are able to influence the inhibitory effect through direct interaction with their target nucleic acids without prior transcription within the oocyte.

Recombinant DNA (linear or other), introduced by the method of the invention may carry a functional replicon containing one or more expressed functional genes under the control of the promoter, showing everything from narrow to wide, the expression profiles of development. For example, the promoter can cause a sudden, but brief expression when the promoter is active only in the early zygote. The introduced DNA can either be lost at some point during embryonic development, or integrated into one or more genomic loci to stably replicated during the lifetime of the resulting transgenic individuals. In one example implementation simultaneously with microinjection into oocytes can be injected DNA building coded supposedly “anti-aging” proteins, such as telomerase or supercena dismutase. Alternatively, such proteins can be injected directly.

EXAMPLES

The following examples illustrate the method of the invention and development of the living descendant of an oocyte injected recovered after freeze-drying sperm. In particular, PR the measures illustrates the development of normal mice from oocytes of the mouse, injected heads (nuclei) recovered after freeze-drying spermatozoa mice that were kept either at room temperature (about 25°C)or in the refrigerator (about 4°). Medium for suspending the sperm to freeze-drying were medium CZB or DMEM described below.

The examples given here should only be considered as examples of oocytes and spermatozoa of certain species of animals, environments for suspending the sperm, schemes freezing, storage conditions, environments for rehydration, and the like, which may be used in the process of the invention, but should not be limited, as other examples of the methods of the invention can be easily defined.

Environment and reagents

All inorganic and organic compounds were purchased from Sigma Chemicals Co. (St. Louis), if it is not specifically mentioned.

The collected oocytes prior to injection of sperm stored in CZB medium (Chatot et al., 1989. J. Reprod. Pert. 86, 679-688). The CZB medium contains 81.6 mm NaCl, 4.8 mm KCl, 1.7 mm Cl2, 1.2 mm MgSO4, 1.8 mm KH2PO4, 25.1 mm NaHCO3, 0.1 mm sodium lactate, 0.3 mm sodium pyruvate, 7 units/ml penicillin G, 5 units/ml streptomycin sulfate, and 4 mg/ml albumin serum bull. Environment to collect an egg from the fallopian tubes, the subsequent processing and micromanipulation was modified with the food CZB, containing 20 mm Hepes, reduced the number Panso3(5 mm), and serum albumin bull (ASB) 3 mg/ml of This medium is here referred to as Hepes-CZB. For the purposes of microinjection it is preferable to replace the SSA in Hepes-CZB 1 mg/ml polyvinyl alcohol (PVA dissolved in cold water, the average molecular weight of 10×103because PVA saves wall injection pipettes less sticky over a longer time than the SSA that it is better if you repeatedly use the same pipette for multiple transfers of the heads of the sperm into the oocyte.

Were used two different environments for suspending the spermatozoa before freeze-drying: (1) medium CZB without ethylenediaminetetraacetic acid (ADGC)containing 4 mg/ml anti-lock brakes; and (2) modified Dulbecco medium Eagle (DMIS), reinforced with 10% (vol./percent). serum bovine fetus (Hyclone, Logan, UT).

In order to determine which environment is best for suspending the spermatozoa were conducted preliminary experiments; to this end we conducted a primary suspending fresh sperm in CZB medium, followed by centrifugation and re-suspending in one of the environments described below, and then immediately followed by drying by freezing sperm. Test environment for suspending the sperm included (a) distilled water, (6) 34%sucrose in distilled water, (in) 180 mg/ml raffinose plus 5 mg/ml ASB, (g) 0.9% NaCl with 5 mg/ml ASB, (d) 0.9% NaCl with 1 mg/ml glucose plus 5 mg/ml ASB and (e) free from lactate and calcium CZB. Only last Wednesday (e) was as good as usual CZB on the ability to keep the nucleus of the sperm, capable of development during freeze-drying (data not shown).

Animals

Animals that were used in these examples contained in accordance with the instructions of the laboratory service animal from the University of Hawaii and guidelines prepared by the Committee on the content and use of laboratory animals Institute of laboratory resources, National Research Council (DHEW publication No.{NIH} 80-23, revised 1985). The scheme of treatment of animals and were reviewed and approved by the Committee on the content and use of animals at the University of Hawaii.

EXAMPLE 1

Preparation of sperm

It was prepared by four different drug in order to illustrate the influence of the environment for suspending sperm, storage temperature and storage time on the ability of kernels, dried by freezing sperm, to participate in the development of live offspring after injection into oocytes as described below and illustrated in table 1. For each drug were used in two caudal, epidode the ISA Mature B6D2F1 male mice. Simultaneously with the compression of each epididymis fingers, its distal part pierced with sharp tweezers. A dense mass of spermatozoa released from the epididymis was transferred into a 1.5 ml polypropylene tube containing 1 ml of one or two above the studied environments, CZB or DMIS. After incubation for 30 minutes at 37,5°C, the upper 0.3-0.5 ml of medium was removed from the tube. More than 90% of the spermatozoa in the suspension (approximately 3-10×106per ml) were actively moving.

EXAMPLE 2

Drying by freezing sperm

Aliquot amount (100 μl) of the sperm suspension was placed in 2 ml vials (Wheaton Scientific, Millville, NJ, catalog No. 651506), which was put directly into liquid nitrogen. Ten minutes later, the ampoules were placed in a pre-cooled (-50° (C) cooling the flask is connected to the drying by sublimation (freezing) (Model 10-020, VirTis Co., Gardner, NY). The inlet pressure was approximately equal to 1 millitorr. The flask was disconnected from the system after about 12 hours, when it is filled with argon, is fed through the chamber for gas dehydration (Fisher Scientific, Pittsburg, PA catalog number 09-204). Each ampoule was connected to the vacuum pump and, after more than 99% of the gas was pumped out, was sealed. Capsules individually wrapped in aluminium foil and kept in the dark at room temperature (about 25° (C) or PR is 4° C.

Capsules with dried by freezing sperm, prepared as described above, is shown in figure 1. White powder at the bottom of each ampoule is a dry environment CZB containing spermatozoa.

EXAMPLE 3

Rehydration of dried by freezing sperm

Capsules containing dried by freezing sperm, prepared as described above was exposed and added to the vial 100 ál of distilled water to obtain the recovered suspension of sperm. Then 5 μl of the suspension was thoroughly mixed with 50 μl of Hepes-CZB containing 12% (wt./about.) polyvinylpyrrolidone (average molecular weight of 360,000). Rehydration the added, viewed under a microscope for further manipulations were selected only those who had intact head and tail parts.

EXAMPLE 4

Transportation dried by freezing sperm

An experiment was conducted to verify whether prepared in accordance with the present invention is dried by freezing the sperm to be transferred abroad, while retaining the ability to fertilize oocytes after rehydration. In this experiment, several vials dried by freezing epididymis sperm transported manually during a three-week trip to Japan that began in the fees the Ulu, Hawaii. Before drying by freezing sperm were suspended in the medium CZB. No special precautions to preserve spermatozoa were not observed except that the capsules during the entire trip were wrapped in aluminum foil and kept in cardboard boxes. The ambient temperature varied from 5°With 30°C. one week after returning to Honolulu sperm rehydratable and used according to the method of the invention.

EXAMPLE 5

Preparation of oocyte

The Mature female B6D2F1 (C57BL/6×DBA/2) were generated sirovasthi by injection to each mouse 7,5 international units (ME) gonadotropin serum of pregnant mares, then after 48 h followed by injection of 7.5 ME human hormonales gonadotropin (CGT). Fourteen hours after injection CGT of the oviduct were selected complexes Cumulus-oocyte and for the Department of Cumulus cells were treated with testicular hyaluronidases bull (300 USP units/ml; ICN Biochemicals, Costa Mesa, CA) in Hepes-CZB for 3 minutes

Before injection of the nucleus of the sperm, oocytes were washed and kept in CZB medium for up to 4 h in an atmosphere containing 5% CO2in the air.

EXAMPLE 6

Microinjection of sperm nuclei into oocytes

For injection of sperm heads into the prepared oocytes prepared the camera for microinjection using caps (10 mm deep) for plastic is howling Cup (100 mm× 15 mm; Falcon Plastics, Oxnard, CA, catalog number 1001). A number consisting of two round drops and one oblong drops were placed along the center line of the Cup. The first drop (2 μl; 2 mm diameter) for rinsing pipettes (Hepes-CZB containing 12% (wt./about.) PVA, average molecular weight 360,000 daltons). Second drop (2 μl; 2 mm in diameter) was presented a suspension rehydration dried by freezing sperm in CZB or DMEM, prepared as described above. Third, oblong drop (6 μl; 2 mm wide and 6 mm long) was a Wednesday Hepes-CZB for oocytes. Each of these drops was covered with mineral oil (Squibb and sons). The Cup was placed on the table inverted microscope with interference contrast optics.

Microinjection of sperm nuclei into oocytes was performed in the way described earlier piezoelectric microinjection using piezotransducer model MB-U from Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The device uses a piezoelectric effect for a single promotion holder pipette at a very small distance (for example, 0.5 μm) at a very high speed, Intensity and speed of the pulses was controlled by means of remote control.

For injection into an oocyte, prepared as described above, in the injection pipette (internal tip diameter of about 5 μm), which was attached to the device, Manager piezoelectric pipette, selected individual sperm tail forward. The head and the tail of the sperm were separated by application of one or more pietinalho in the cervical area. The intensity and speed (frequency) pulses regulated by remote PMAS-CT01 (on the remote control set scale: intensity 2, speed 1). Then the head was sucked deep into the pipette and a small volume (about 0.5 ml) of mercury was placed at the proximal end of the injection pipette.

Meanwhile Mature unfertilized oocyte was placed on the table of the microscope on Wednesday Hepes-CZB. The oocytes were fixed holding pipette and the tip of the injection pipette was brought into close contact with the pellucid zone at the position of 3 o'clock Lodged several pietinalho (intensity 1-2, speed 1-2) to promote the pipette while also creating a light negative pressure inside When the tip of the injection pipette was held the pellucid zone, a cylindrical piece of the pellucid zone in the pipette was pushed in perivitelline space. After the sperm head pushed forward, until it is near the tip of the pipette, the pipette mechanically pushed forward until its tip almost did not reach the opposite side of the cortex of the oocyte. Oolemma pierced Appendix 1 or 2 pietinalho (and what tensionally 1-2, speed 1) and the head of the sperm is pushed in ooplasm with a minimal amount (about 6 PCL) accompanying environment for the sperm suspension. After you remove the maximum possible amount of protection, the pipette was carefully removed, leaving the head of the sperm inside ooplasm. All injections were performed in the environment Hepes-CZB at room temperature (23-27° (C) within one hour after rehydration. Each oocyte was injected with one head of the sperm. Approximately 5-20 oocytes were micronational for 10-15 minutes.

EXAMPLE 7

Examination of oocyte and embryo transfer

Injected heads of spermatozoa, oocytes were incubated in medium Hepes-CZB at 37°at 5% CO2in the air and examined under inverted microscope after 5-6 hours. The sperm that had two clear pronucleuses and the second polar body, considered normally fertilized and cultured in medium Hepes-CZB for 4 days. The sperm that reached the morula stage or blastocyst, transferred in the trumpet females receptive female CD-1 (albino)that were paired with vasectomypenis CD-1 males three days before this synchronization stages of embryonic development with the development of endometrial cancer. On average, eight moral/blastocysts were transferred into each horn (trumpet). Females were given the opportunity to stand and to grow their surrogate children (black, brown or grey skin). Several adult males and females from the offspring were randomly selected and crossed in order to test their fertility.

RESULTS

Microscopic examination rehydration dried by freezing sperm

Microscopic examination rehydration sperm showed that 100% of the sperm were fixed. The viability of sperm was determined using a kit for determining the viability of cells that can be readily purchased (Leave/dead FertiLight, Molecular Probes, Eugene, Oregon) and which identifies cells with a plasma membrane (live) and with a damaged plasma membrane (dead) in accordance with the nature of the fluorescence under a UV microscope. The kernel of live spermatozoa with intact plasma membranes flyuorestsiruyut green, whereas kernel “dead” flyuorestsiruyut bright red-orange. After freeze-drying and dehydration, was investigated over 10,000 sperm from four males. All investigated sperm were “dead”, as it was determined with this test.

As illustrated in table 1, 1236 from 1353 oocytes (91.4%) experienced injection microsurgery and surviving 1157 (93,6%) were activated by the nucleus of the sperm and normally fertilized, regardless of the original with the food for suspending (CZB or DMEM), temperature storage (25°or 4°), and storage time (1 day, 2 weeks, 1 month or 3 months).

The maximum time of storage vials in these examples was 3 months at 4°C. In three experiments, 57 oocytes from three females were injected with spermatozoa stored for 3 months, 95% of injected oocytes survived the surgery, and they were normally fertilized. 91% of fertilized eggs developed to the stage of morula/blastocysts in vitro. Fourteen (30%) of the 46 embryos transferred three surrogate mothers, has developed into normal adults organisms.

The majority (90%-93%) fertilized eggs that were injected with sperm heads, suspended in the medium CZB, freeze dried, preserved from 1 day to 2 weeks at 25°or 4°and rehydration, has evolved to the stage of morula/blastocysts in vitro and 25-34 (20%-29%) of them developed to a normal offspring after transfer into surrogate mothers. Although a smaller percentage (76%) of fertilized eggs that were injected suspended in the medium CZB dried by freezing sperm stored before rehydration therapy for 1 month at 25°With developed to the stage of morula/blastocysts, 16 (18%) of them developed to a normal offspring after transfer into surrogate mothers. Nine (28%) of 32 of transferred embryos originating from fertilized eggs, is the quiet were injected with sperm, suspended in the medium CZB, dried by freezing and stored at 4°within 3 months, has developed into normal offspring. In General, when using suspended in the medium CZB dried by freezing sperm, they received a total of 143 of live offspring after transfer 562 embryos developed from 664 oocytes that survived the surgery and were fertilized normally with an average success rate of 18.7%.

As illustrated in fig.1b, all offspring grew normally. The sex ratio among them was 1:1. Two fully grown females and two males from each of the 12 experimental groups were randomly selected and paired. All have proven their fertility and produced broods normal size (8 to 12).

Sperm that were sent to Japan and back and restored after their return to Honolulu, 29 randomly selected spermatozoa were individually injected into the oocytes. Nineteen (83%) developed to the stage of morula/blastocysts in vitro. Three (16%) of them reached full development after transfer into a surrogate mother. All three (2 females and 1 male) were grown in fertile adults.

From the point of view of the following examples it was demonstrated that mouse sperm can maintain their genetic integrity after freeze-drying. There is no reason to believe that the special is malotidi other species including invertebrates and vertebrates, will behave differently.

Although the above described invention relates to the preferred forms of implementation, it should be understood that the invention is not limited to the above forms. On the contrary, it is assumed that the present invention covers all modifications and alternatives falling within the spirit and scope of this invention.

Table 1

The development of mouse oocytes, injected dried by freezing sperm
Wednesday suspending spermStoring samplesThe oocytes injected with spermThe number of embryos transferred (number of surrogate females)Qty (total %) living descendants (range)*
Temperature (C)Period of timeThe total number of injected oocytes (no expert.)Number (%) of surviving oocytesNumber (%) of normally fertilized oocytesNumber (%) reached the stage of morula/blastocysts
CZB25°1 day155(5) 135(87)133(99)a120(90)e116(8)34(29) {0-71}
  2 weeks158(4)144(91)141(98)a131(93)f126(8)25(20) {0-30}k
  1 month130(4)120(92)117(98)a89(76)g87(5)16(18) {5-33}k
 2 weeks137(4)126(92)123(98)a114(93)e105(7)27(26) {0-45}
  1 month131(4)117(89)112(96)a98(88)h96(6)32(33) {14-65}l
  3 months40(2)38(95)38(10) b35(92)h32(2)9(28) {26-31}
DMEM25°1 day128(3)116(91)104(90)b95(91)e95(6)91(31) {18-47}l
  2 weeks109(2)102(94)85(83)c67(79)i60(3)8(13) {0-20}m
  1 month13(2)106(94)74(70)d47(64)j47(3)8(17) {0-24}
 2 weeks83(2)79(95)79(100)a69(87)h56(3)20(36) {21-46)l
  1 month152(3)137(90)135(99)a114(84)h 54(3)22(41) {15-60}n
  3 months17(1)16(94)16(100)14(88)14(1)5(35) {35-35}
* - data variation (%) of offspring from a surrogate mother
Statistical significance:and against, d:p<0/005f against g,i,j;p<0.005n against m: p<0.01
 b against d:p<0.005h against j:p<0.005l vs. m: p<0/01
 e against g,j:p<0.005n against k:p<0.01 

1. The product of artificial insemination, containing processed freeze-drying to a moisture level of less than 1% of sperm cells with impaired membrane or the head of the sperm, the core of which retained the genetic integrity is th sufficient for fertilization, and after rehydration and microinjection in an isolated oocyte these heads of sperm to fertilize an isolated oocyte, retain the genetic integrity sufficient for fertilization with getting a live offspring.

2. Product for artificial insemination according to claim 1, characterized in that the sperm or the sperm head after freeze-drying store some time before rehydration.

3. Product for artificial insemination according to claim 2, characterized in that the sperm or the sperm head store after freeze-drying at ordinary temperature.

4. Product for artificial insemination according to claim 2, characterized in that the sperm or the sperm head is kept at a temperature of about 4°C.

5. Product for artificial insemination according to claim 2, characterized in that the sperm or the sperm head is kept at a temperature of minus 20°With or below.

6. Product for artificial insemination according to claim 2, characterized in that the storage time is not more than three months, or about one year, or less than one year, or more than one year.

7. Product for artificial insemination according to claim 1, characterized in that it is devoid of membrane head contains the nucleus and perinuclear materials.

8. The product is La artificial insemination according to claim 1, characterized in that the sperm or the sperm head get from bespozvonochnykh animal.

9. The product of artificial insemination of claim 8, characterized in that the sperm or the sperm head get from sea urchin, lobster, lander or mollusk.

10. Product for artificial insemination according to claim 1, characterized in that the sperm or the sperm head get from a vertebrate animal.

11. The product of artificial insemination of claim 10, wherein the sperm or the sperm head get from fish, amphibian, reptile, bird or mammal.

12. The product of artificial insemination of claim 10, wherein the sperm or the head of the sperm get from the person.

13. The product of artificial insemination of claim 10, wherein the sperm or the head of the sperm get from the mouse.

14. The ampoule containing the product of artificial insemination, characterized in that the product of artificial insemination is a product according to claim 1.

15. A method of obtaining a product for artificial insemination according to claim 1, including the collection of live Mature spermatozoa, suspending the spermatozoa in the physiological environment for suspending, freezing the suspension of spermatozoa for the teachings of frozen sperm, drying of frozen spermatozoa or sperm heads under vacuum until the moisture level of less than 1% with obtaining dried by freezing of spermatozoa or sperm heads with broken membrane, rehydration of spermatozoa or sperm heads with broken membrane, the selection of spermatozoa or sperm heads, which have preserved the genetic integrity of the kernel, and after microinjection in isolated oocytes fertilized oocyte with getting a live offspring.

16. The method according to item 15, characterized in that it further includes a step of removal of the membranes of the sperm to the stage of freezing with getting deprived of membranes of sperm heads.

17. The method according to item 16, characterized in that the stage of removal of the membranes includes the handling of sperm detergent and possibly surface-active substance.

18. The method according to item 15, wherein the drying of the frozen sperm or head is carried out until the moisture level of less than 0.01%, preferably less than 0.001%, more preferably less than 0,00001%.

19. The method according to item 15, wherein the sperm get from a vertebrate animal.

20. The method according to claim 19, characterized in that the sperm get from fish, amphibian, reptile, bird or mammal.

21. The method according to claim 19, characterized in that the SPE is malotidi get from a person.

22. The method according to claim 19, characterized in that the sperm get from the mouse.

23. The method according to item 15, wherein the sperm get from bespozvonochnykh animal.

24. The method according to item 23, wherein the sperm get from the sea urchin, lobster, lander or mollusk.

25. The method according to item 15, wherein the freezing of the sperm suspension is carried out at minus 196°C for 10 minutes.

26. The method according to clause 15. characterized in that it further includes a step of storing dried by freezing sperm for some time before being rehydrated.

27. The method according to p, characterized in that the dried freezing spermatozoa stored at ordinary temperature.

28. The method according to p, characterized in that the dried freezing spermatozoa stored at a temperature of about 4°C.

29. The method according to p, characterized in that the dried freezing spermatozoa stored at a temperature of minus 20°With or below.

30. The method according to p, characterized in that the retention time of the sperm is not more than three months, or up to one year, or more than one year.

31. Method of fertilization of mammalian oocytes product for artificial insemination according to claim 1, containing sperm with compromised membrane or the head of the sperm, including the surrounding collection of live Mature spermatozoa of mammals, the suspending of sperm in the physiological environment for suspending, freezing the suspension of spermatozoa to obtain frozen sperm, drying the frozen sperm under vacuum until the moisture level of less than 1% with obtaining dried by freezing sperm, rehydration of spermatozoa after freeze-drying, isolation of such registrationpage sperm with damaged membrane or head of a spermatozoon that contains preserving genetic integrity and able to fertilize the core, and capable after rehydration of fertilization in the microinjection isolated oocyte with getting a live offspring, isolating oocytes similar to mammalian species and the introduction of sperm with damaged membrane or the sperm head in an isolated oocyte with the formation of a fertilized oocyte.

32. The method according to p, characterized in that the sperm with damaged membrane or the head of the sperm is injected into an oocyte piezoelectric microinjection.

33. The method according to p, characterized in that it further includes removing the membranes of spermatozoa to the stage of freezing with the formation deprived of membranes of sperm heads.

34. The method according to p, characterized in that when injected into the oocyte, the sperm head on the additional injected the sperm centrosome.

35. The method according to p, characterized in that the sperm get from the person.

36. The method according to p, characterized in that the sperm get from the mouse.

37. The method according to p, characterized in that the moisture level of the frozen under vacuum sperm is less than 0.01%, preferably less than 0.001%, more preferably less than 0,00001%.

38. The method according to p, wherein the isolated sperm cell is injected into an oocyte for 1-60 min after rehydration.

39. The way to obtain living offspring neumannova mammal from an isolated oocyte, fertilized rehydration product for artificial insemination according to claim 1, containing sperm with damaged membrane or the head of the sperm, including the collection of live Mature spermatozoa from negumanoidnyh mammals, suspending the spermatozoa in the physiological environment for suspending, freezing the suspension of spermatozoa with getting frozen sperm, drying the frozen sperm under vacuum until the moisture level of less than 1% with obtaining dried by freezing sperm, rehydration dried by freezing sperm, isolating such registrationpage sperm with damaged membrane or head of a spermatozoon that contains preserved gene is political integrity and able to fertilize the core and capable of after rehydration to fertilize an isolated oocyte in the microinjection with getting a live offspring, isolation of oocytes similar species negumanoidnyh mammals, the introduction of sperm with damaged membrane or the sperm head in an isolated oocyte to education fertilized oocyte; culturing the fertilized oocyte to obtain a living offspring.



 

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