RU2244525C2 - Method for growing normal descendants from oocytes injected with spermatozoa treated by drying with freezing - Google Patents

Abstract

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

Description

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

The US government has a paid license for this invention and the right under limited conditions to require the patent holder to grant the license under reasonable conditions to other persons, as stipulated by the terms of contract No. R01-HD-03402 with the National Institutes of Health, Public Health Service.

The present invention relates to freeze-drying of sperm, the fertilization of oocytes with sperm, recovered after freeze-drying, and the development of living offspring from them

Successful cryopreservation of sperm using cryoprotectants and the ability to store frozen sperm for a long time made a dramatic revolution in both animal breeding and human reproductive medicine. It was found that cryopreserved fatigue thawed after freezing often regains its mobility and fertilizes almost as efficiently as fresh sperm.

Long-term storage of sperm in liquid nitrogen (-196 ° C) is usually used for cryopreservation of livestock and human sperm. However, the usual conservation of sperm over time is very expensive because it requires a constant supply of liquid nitrogen. Moreover, in some parts of the world, it is difficult and / or expensive to store sperm in liquid nitrogen; for example, in developing countries where it is not easy to get liquid nitrogen (or even dry ice). Transportation of sperm cells frozen in the usual way is also problematic, as it requires the transport of large containers for liquid nitrogen or the use of special ship containers containing liquid nitrogen or dry ice. Therefore, many attempts have been made to preserve sperm without the need for their storage in liquid nitrogen.

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

It is believed that the first recorded attempt to sublimate (i.e., freeze and dehydrate) spermatozoa was made in 1949, when 1 milliliter (ml) of rooster seed was mixed with an equal volume of Ringer's solution containing 20-30% glycerol, applied thinly to the distillation the flask and “sublimated” by removing 90% of the water. When the drug was rehydrated 2 hours after warming to room temperature, it was noted that up to 50% of sperm cells remained motile. However, the ability of rehydrated sperm to fertilize has not yet been determined.

Subsequent attempts to obtain live offspring using sublimated sperm did not bring success. It was reported, for example, that after artificial insemination of a cow with bull sperm, which were restored immediately after sublimation and showed 50 percent sperm motility, a live calf was born. It was also reported that twelve broods of normal rabbits were obtained after insemination using sublimated sperm. However, none of these results could be repeated by the authors, or duplicated and confirmed by other specialists working in this field.

From a study of the properties and fertility of normal and restored spermatozoa, it is known that spermatozoa do not need to be “alive” in the usual sense of the word (that is, have intact plasma membranes) in order to maintain normal embryonic development. For example, when using the sperm injection technique inside the cytoplasm (ISVC), human motile spermatozoa are selected. They are immobilized (“killed") immediately before injection into the oocyte by vigorously removing the tail parts, which leads to a violation of the plasma membrane. It has been noted that sperm immobilization significantly increases the success rate of IVEC fertilization. There is also a report that when mouse sperm were suspended in a medium without any cryoprotectant and then immediately frozen in liquid nitrogen, 100% of the sperm were “dead”, as determined using live / dead cell labeling, however, after microinjection of the heads thawed sperm into oocytes occurred normal embryonic development. There is also a message about the birth of two normal calves after microinjection into the oocytes of spermatozoa that died during thawing after drying by sublimation in the absence of any cryoprotectants.

Although it is known that freeze-dried hamster and human sperm heads injected into hamster oocytes can form externally normal pronuclei, it has never been previously determined whether such sperm heads can support normal embryonic development. Moreover, it was shown that sublimation of spermatozoa to a moisture content of less than 30%, 7% and 0.5% leads, respectively, to loss of mobility, disruption by acrosome and leakage of enzymes. Evidence has been obtained that cell proteins in sperm change when dehydrated below a moisture content of 6%.

In view of the above, there is a need for a reliable and reproducible method of sublimation (freeze-drying) of spermatozoa, in which sublimated sperm retain their ability to fertilize during long-term storage at room temperature or in ordinary refrigerators, or at lower temperatures. In addition, a sperm injection method is required, in which sperm rehydrated after sublimation are used to fertilize recipient oocytes resulting in normal live offspring.

According to the invention, there is provided a method of freeze-drying sperm to obtain at least one recovered sperm, whose head (core) has the ability to fertilize an oocyte to produce live offspring. The method of the invention includes the steps of: (a) collecting live sperm; (b) suspension of sperm in a suspension medium; (c) freezing the sperm suspension; and (d) dewatering the sperm suspension to a moisture level of less than 1%, more preferably less than 0.01%, even better less than 0.001%, and in particular less than 0.00001%. The method further may include the step of rehydrating the freeze-dried sperm suspension, where at least one rehydrated sperm head retains its genetic integrity and the ability to fertilize an oocyte to produce live offspring. As will be discussed below, the method may further include the step of storing the freeze-dried sperm until rehydration.

To produce live offspring, at least the head (core) of the rehydrated sperm is introduced into an isolated oocyte to form a fertilized oocyte. The sperm head is introduced into the oocyte by microinjection, preferably by piezoelectric microinjection. Preferably, the introduction of the nucleus occurs within one hour after rehydration. The fertilized oocyte is then given the opportunity to develop into an embryo and implanted in the uterus of a surrogate mother, where it develops into a living offspring.

In some species (for example, in most terrestrial mammals, including humans), the normal embryonic development of a fertilized oocyte also requires the introduction of paternal centrosomes associated with the sperm nucleus. When the head and the tail are separated, the centrosomes usually remain attached either to the posterior end of the sperm head or to the front end of the tail sperm. So, in one embodiment of the invention, the sperm centrosome of another sperm can be introduced simultaneously with the sperm head, or it can be introduced by the simultaneous or subsequent introduction of the tail of the sperm. Alternatively, the introduction of the sperm nucleus and centrosome can be accompanied by the introduction of the whole rehydrated sperm into the oocyte.

It was found that sperm freeze-dried according to the method of the invention can be stored for at least three months or, more preferably, at least for a year at temperatures ranging from ordinary (i.e. room temperature) to temperatures existing in refrigerators (i.e., about 4 ° C) without loss of their genetic integrity or ability to fertilize. So, freeze-dried spermatozoa can retain their “abilities” during transporting them to almost anywhere in the world at ordinary or existing temperatures in refrigerators and can also endure short-term storage at ordinary temperatures or temperatures existing in refrigerators, in places where liquid nitrogen or dry ice are not readily available. Preferably, the long-term (i.e., indefinitely long) storage of freeze-dried sperm occurs at temperatures below 4 ° C (e.g., 20 ° C or lower).

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

The materials of this patent contain at least one pattern made in color. Copies of this color-coded patent may be provided by the Patent and Trademark Office in response to a request and after payment of the required fees.

Figure 1 is a photograph of evacuated ampoules containing freeze-dried sperm. The white powder at the bottom of each ampoule is CZB medium containing sperm.

Fig. 2A is a micrograph of a freeze-dried mouse sperm immediately after rehydration. The proportion of sperm heads without tailings or with broken tailings varies depending on how carefully or roughly the dried samples were handled during storage.

2B is an electron micrograph showing a longitudinal section through the front end of a normal sperm head that has not been frozen.

2C is an electron micrograph showing longitudinal and horizontal sections through the front of the head of a freeze-dried sperm after rehydration. The plasma membrane (p) and the contents of the acrosome (ak) are lost, (to you) - the inner acrosomal membrane, (to us) - the outer acrosomal membrane, (n) - the nucleus.

Figure 3 is a photograph of three young (black) mice born to a CD-1 (albino) surrogate mother. These cubs developed from B6D2F1 oocytes injected with B6D2F1 sperm that were stored at room temperature for one month after freeze-drying.

During routine fertilization in mammals, the fertilizing sperm rises through the female genital tract, penetrates the oocyte membrane and then merges with the oocyte. The fusion of sperm with the oocyte causes activation of the oocyte. The activated oocyte continues delayed meiosis, and the oocyte chromosomes are transformed into the female pronucleus. Meanwhile, the sperm nucleus inside the oocyte is decondensed to transform into the male pronucleus. The fully developed male and female pronuclei fuse, and the chromosomes from these pronuclei combine. The resulting zygote develops into a living descendant.

The present invention provides a method of freeze-drying (i.e., freeze-drying) spermatozoa that, after rehydration, are able to fertilize isolated oocytes to produce live offspring. Freeze-dried sperm obtained by the method of the invention retain their genetic and reproductive potential even when, after rehydration, they are immobile and in the usual sense are “dead”. When whole sperm or isolated sperm heads (i.e. heads containing all components, including the nucleus) or lacking sperm membranes or sperm heads (i.e. retaining the core and perinuclear material but not having a plasma membrane) according to the invention are injected directly into oocytes, normal fertilization and embryonic development occurs, which can lead to live offspring. Preferably, the sperm head (nucleus) is inserted directly into the oocyte cytoplasm. The introduction of the sperm head is carried out by microinjection, preferably by piezoelectric microinjection. As will be discussed below, the embryonic development of a fertilized oocyte of some species may require simultaneous or subsequent injection of a sperm centrosome immediately after this.

If the centrosome is not subjected to freeze-drying, centrosomes from unfrozen spermatozoa can be selected for insertion into the oocyte.

The following are presented in more detail the individual stages and substages of the method of the invention for the preparation of fertilizable freeze-dried sperm and their use in the in vitro fertilization procedure.

Sperm preparation

In order to ensure as many freeze-dried sperm as possible maintain their genetic integrity during freeze-drying, it is preferable to use physiologically mature spermatozoa in the method of the invention. In mature spermatozoa, DNA is bound to major proteins called protamines. In mammals, protamines are repeatedly stitched with disulfide bonds. This stabilizes sperm nuclei and makes them very resistant to physical and chemical destruction. Nuclear protamine flashing occurs mainly during sperm movement through the epididymis. Thus, the mammalian sperm in the epididymis and ejaculate (seed) are generally physiologically more mature than inside the testes, and is preferred for the method of the invention, at least in the case of mammals.

Mature spermatozoa of invertebrates and vertebrates are harvested by methods well known in the art. For example, mature sperm of rodents, such as a mouse, a golden (Syrian) hamster, guinea pig, rabbit and the like, can be collected from caudal epididymes; while in other species, such as humans, pigs, horses, bulls, goats, roosters, and the like, mature sperm can be isolated from fresh ejaculated seed from prolific males. Sperm from fish (such as the horseshoe crab, Xiphophorus helleri) and invertebrates such as sea urchins (Tripneustes gratilla) can be selected from the testicles of mature males.

As an example, a method for producing spermatozoa from caudal epididymis is described below. Caudal epididymis is taken from a sexually mature male mouse (approximately 8 weeks after birth or older). Blood and adipose tissue are removed from the surface of the caudal epididymis. Then it is squeezed to release a dense mass of sperm. A drop (about 2 μl) of sperm mass is placed on the bottom of a centrifuged polypropylene tube with a volume of 1.5 ml and 0.5 ml of warm physiological medium (for example, CZB medium, saline with phosphate buffer, or isotonic saline) is placed on top. After 10-20 minutes at 37 ° C, motile spermatozoa can be taken from the supernatant.

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

The following is an example of a method for producing sperm from the male sex glands. The removed glands are placed in an erythrocyte-lysing buffer (i.e. 155 mM NH ₄ Cl, 10 mM KHCO ₄ , 2 mM EDTA, pH 7.2-7.4), ground with sharp scissors and filtered through two layers of filter paper to remove impurities. Then, the filtrate is centrifuged (for example, 700 × g, 5 minutes) and the precipitate is re-suspended in physiological saline to the desired concentration in preparation for freeze-drying.

Regardless of the sperm preparation method used, more than 50% of recovered sperm must be motile.

Thus restored sperm are suspended in a physiological environment, described below, to prepare for the freeze-drying process. Alternatively, sperm can be further processed before freeze drying begins to produce membrane-free sperm heads

Preparation of membrane-free sperm heads

Membrane-free sperm heads are detergent-extracted heads that are devoid of all membranes, including the plasma membrane and the inner and outer acrosomal membranes, but retaining nuclear and perinuclear material. For example, sperm heads may be deprived of membranes by treatment with Triton X-100 with or without SDS (sodium dodecyl sulfate). Triton X-100 is a well-known non-ionic surfactant that is widely used to remove membrane components under conditions without denaturation. SDS is an anionic detergent used to dissolve various proteins, including membrane proteins. It was shown that in mice, sperm heads lacking membranes with Triton X-100 are able to activate oocytes, which leads to normal embryonic development.

The following is a typical method for removing sperm head membranes. Multiple sperm suspensions prepared as described above are exposed to sound. For example, spermatozoa taken from caudal epididymis, gonads, or sperm, as described above, can be suspended in 5 ml of BM buffer (75 mM NaCl, 24 mM EDTA and 50 mM Tris-HCl, pH 7.2) and exposed to sound for 30 seconds at 70% -80% of the power of the device Biosonic sonicator company (Bronwill Scientific, Rochester, NY). Over 95% of sperm are beheaded during this treatment. To remove sperm head membranes, the sperm suspension after sound exposure is centrifuged for 5 min at 700 g, and the precipitate is washed with BM buffer, and then treated with 1% Triton X-100 in BAT medium at room temperature (BAT medium consists of 123 mM KCl, 2.6 mM NaCl, 7.8 mM NaH ₂ PO ₄ , 1.4 mM KH ₂ PO ₄ , 3 mM Na ₂ EDTK, pH 7.2) Then the heads are thoroughly washed with BAT and re-suspended in preparation for freeze-drying.

Sperm Suspension Medium

In preparation for freeze-drying, sperm (or membrane-free sperm heads) are suspended in physiological saline, which is sufficient to maintain at least sperm nuclei integrity under normal conditions. The solution should be a balanced saline solution having at least a suitable osmotic pressure and pH. There is no single environment that could support the survival of sperm of all animal species. For example, a suitable solution for sperm of a sea urchin is seawater, having an osmotic pressure of about 1,000 milli-osmol and a pH of about 8.2. Sea water, however, will instantly kill mammalian sperm. Mammal sperm require a solution with an osmotic pressure of about 300 milli-osmol and a pH of 7.0-7.6. Such a solution, however, will kill the sperm of the sea urchin.

In view of the foregoing, a sperm suspension medium should be selected according to the species of interest, taking into account criteria well known to those skilled in the art. Such a choice can be made without experimentation. Since sperm cells do not have to be whole membranes (they can be “dead”) to successfully fertilize oocytes by injection, there are no strict requirements for cryoprotectants, such as glycerol or the like, in suspension medium.

Packaging

Sperm suspension can be placed in many different containers, including, but not limited to glass ampoules, plastic cryotubules (cryobubbles) with screw caps, or the suspension can be placed in plastic straws that can be sealed with powder sealant after freeze-drying, sealed or closed with nylon caps . The volume of the sperm suspension in each reservoir is not important. Typically, a volume of about 50-100 μl is placed in a 2 ml ampoule.

Freeze-dried sperm.

The sperm suspension can be frozen quickly or slowly by well-known methods. For example, the suspension can be frozen in liquid nitrogen vapor or in the air of a mechanical (electrical) refrigerator by methods well known in the art. Cooling and freezing can be carried out in manual static or staged mode, or in an electronic automated and programmable system that supplies liquid nitrogen. Various freezing rates can be used (e.g. 1 ° C-25 ° C / min). For example, the freezing step can be carried out at a rate of -196 ° C for 10 minutes.

To successfully freeze the suspension in straws, ampoules or cryotubes for a couple, you can use various options. A metal container (canister) with cigar-like tubes or other holders with straws, or holders, or racks with ampoules, or cryotubes can be placed directly in pairs of liquid nitrogen using a liquid nitrogen refrigerator.

Drying a frozen sperm suspension in vacuo can be accomplished using a variety of different systems known to those skilled in the art. For example, the apparatus of the model VirTis 10-020 of the company (VirTis Co., Gardiner, NY) is known. The suspension is dried to a moisture level of less than 1%. However, it is preferable that the moisture level is less than 0.01%, even better less than 0.001%, and especially good less than 0.00001%. The freeze-dried sperm container is preferably packaged under vacuum or in the presence of an inert gas such as nitrogen or argon.

Storage

Containers with freeze-dried sperm are preferably stored in the dark or wrapped in aluminum foil or the like. For long-term storage, it is preferable to store containers at -20 ° C or lower. It is assumed that, similarly to freeze-dried bacteria, fungi, and the like, freeze-dried sperm nuclei under these storage conditions will fully retain their genetic integrity. However, for some time longer than three months, containers can be stored at ordinary temperatures (i.e. room temperature) or at temperatures of ordinary refrigerators (about 4 ° C) without impairing the ability of freeze-dried sperm nuclei to fertilize oocytes. Therefore, freeze-dried sperm can be transported without the need for special conditions or the use of bulky containers.

Sublimated Sperm Rehydration

It is preferable to rehydrate preparations of freeze-dried sperm with pure water, the volume of which is equal to the initial volume of the suspension of sperm before drying by freezing. After rehydration, any physiological saline solution, such as 0.9% saline or CZB medium (see below), can be used for dilution. The volume of diluent is not standardized.

The concentration of sperm in the final rehydrated medium should be sufficient to allow recovery of individual sperm or individual sperm heads for injection of sperm into oocytes, as described below.

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

Microscopic examination of spermatozoa rehydrated after freeze-drying

Freeze-dried sperm are immobile. Sperm viability can be determined using any staining method that distinguishes sperm that are normally dead and alive. A suitable viability test device that can easily be purchased for use in the invention is Live / dead FertiLight, Molecular Probes, Eugene, Oregon, which allows to distinguish cells with undisturbed plasma membranes (living) from cells with damaged plasma membranes (dead) by the nature of the fluorescence under a UV microscope after staining with propidium iodide / SYBR 14. The nuclei of “living” spermatozoa with undisturbed plasma membranes fluoresce green, while those of “dead” sperm fluoresce brightly orange nym. It is expected that all studied sperm cells will be “dead” in the usual sense.

Fig. 2A is a micrograph of a typical freeze-dried mouse sperm immediately after rehydration. The heads and tails of some sperm cells are separated. The proportion of spermatozoa with broken tail parts or without tail parts may vary depending on how carefully or roughly treated the samples during storage. Spermatozoa with or without tail sections were used in the injection procedure described below.

The differences between the fresh (not freeze-dried) sperm and the freeze-dried and sperm rehydrated by the method of the invention are shown in FIGS. 2B and 2C, respectively. Each of these figures represents a latitudinal cross section through the front of the sperm head. Although freeze-dried rehydrated sperm retained the core (s), part of the outer acrosomal membrane (us) and part of the inner acrosomal membrane (you), both the plasma membrane (pm) and the contents of the acrosome (a) are missing.

Recipient Oocytes

Recipient oocytes can be obtained, for example, by inducing ovulation or ovulation in an animal by injection of gonadotropin hormones (for example, by sequential administration of equine and human chorionic gonadotropins) and surgically removing eggs from the oviduct shortly after the expected ovulation time (for example, 13-15 hours after injection of human chorionic gonadotropin mouse). Alternatively, oocytes can be taken from the ovaries and cultured in a medium until they mature, as is known to those skilled in the art. An example of a preferred culture medium is a modified Eagle's medium (BCI) enhanced with bovine serum albumin (ASB), described by Downs, S.M. and A.M. Mastropolo, Develop. Biol. 162: 154-168, 1994 for mouse oocytes.

Sperm components necessary for successful in vitro fertilization

It is known that normal fertilization can be achieved in mice by injecting isolated sperm heads into oocytes and that plasma and acrosomal membranes and all tail components are not essential for normal embryonic development. The mouse and perhaps the most common laboratory rodents are “exceptions” in that they do not require a centrosome of sperm for normal fertilization, and during normal fertilization of the centrosome, the sperm in the sperm neck after fertilization is doomed to degenerate inside the oocyte.

In contrast, in most terrestrial mammals, including livestock and humans, the sperm centrosome plays a central role in the formation of microtubules, which are necessary for the union of male and female pronuclei, as well as for subsequent fragmentation during embryonic development. Thus, in these species, the introduction of both the sperm nucleus (head) and the centrosome into the oocyte was necessary to obtain normal offspring. It is currently unknown whether sperm centrosomes in all species can survive freeze-drying. If not, then to guarantee the normal embryonic development of the centrosome, unfrozen sperm should be injected into the oocyte together with a freeze-dried sperm head. The introduction of a larger number of centrosomes, however, will lead to abnormal development of pronuclei and abnormal embryonic development.

When the head and tail are separated, the centrosome usually remains attached either to the posterior end of the sperm head or to the front end of the sperm tail. Therefore, the centrosome sperm can be introduced into the oocyte simultaneously with the sperm head, or it can be introduced by simultaneous or subsequent administration of the tail of the sperm. Alternatively, the introduction of the sperm nucleus and centrosome may be accompanied by the introduction of a whole rehydrated sperm into the oocyte.

Introduction of the sperm nucleus into the recipient oocyte

A whole sperm can be introduced into the recipient’s oocyte cytoplasm, but for those species in which the sperm are large, it is preferable to directly insert the sperm into the cytoplasm using the microinjection technique. In a preferred method for microinjection of a rehydrated sperm head or rehydrated membrane-free sperm head into a recipient oocyte, a piezoelectric micropipette is used.

A suitable piezoelectric controlled instrument is sold under the name Piezo Micromanipulator / Piezo Impact Drive Unit by Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The device uses a piezoelectric effect for very fast, controlled advancement of the holder (injection) pipette over a very short distance (approximately 0.5 micrometers). The intensity and duration of each pulse can be varied and adjusted using the remote control.

For injection into an oocyte, a separate sperm is aspirated with the tail part forward into an injection pipette with a short flat tip, the inner diameter of which is about 5 micrometers, placed in a piezoelectric controlled device in accordance with the manufacturer's instructions. The sperm head is separated from the tail by the application of one or more piezoelectric pulses in the neck region. Then the head is sucked into the pipette.

During injection of the sperm head (nucleus), the oocyte is held with a suitable retention pipette. The tip of the injection pipette containing the selected sperm head is brought into close contact with the oocyte pellucide zone and several piezo pulses are applied to advance the pipette (using the control panel, set the intensity on a scale of 1-5, speed 4-6), while creating a small negative pressure inside. When the tip of the pipette passes the zone of the pellucide, the resulting cork of the zone is pushed into the perivitelline space, and the sperm head is pushed forward until it is near the tip of the pipette. The tip of the pipette is then applied to the plasma membrane and pushed forward (towards the opposite side of the oocyte), and the retention pipette almost reaches the opposite side of the oocyte cortex. Now the plasma membrane is deeply concave around the tip of the injection needle. When one or two piezoelectric pulses are applied (intensity 1–2, speed 1), the olemma at the tip of the pipette breaks, as indicated by the clearly visible rapid relaxation of the olemma. The sperm head is then pushed into the ooplasm with a minimal amount (about 6 picoliters) of the accompanying medium. Then, the pipette is carefully removed, leaving the newly introduced head inside the oocyte cytoplasm. This operation is carried out quickly, usually on a group of 10-15 oocytes that have been kept under culture conditions all this time.

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

Microinjection of a sperm head / membraneless sperm head has several advantages. First, sperm head transfer by microinjection is applicable to a wide range of sperm types, regardless of size, morphology, or the like. Secondly, microinjection allows, together with the injection of the sperm head (with the head of donor sperm), to carry out a controlled injection of additional agents into the oocyte. This is shown below with examples. Thirdly, in the implementation of the invention, where the introduction of the sperm head is carried out by piezoelectric microinjection, a quick and efficient processing of samples is possible, thus, trauma to sperm and oocytes subjected to manipulation is reduced. Oocytes of certain species (for example, mice) are not susceptible to microinjection using conventional needles, while piezoelectric microinjection gives a high level of success.

Fertilized Oocyte Activation

It is known that mouse oocyte can be activated by injection of a single intact mouse sperm or its isolated head. The isolated tail of the sperm cannot activate the oocyte. An active, sperm-derived oocyte-activating factor (s) usually appears during the transformation of a spherical spermatid into a sperm. The action of these factors is not strictly species-specific, since mouse oocytes are activated by injection of spermatozoa of other species, such as a hamster, rabbit, pig, human and even fish. There is a report that one such activating factor is a protein, 33 kilo-daltons in size and located in the region of the equatorial segment of the acrosome. This protein, called oscillin, is easily extracted from mature sperm (hamster) by simple freezing and thawing. In addition to oscillillin, a mature sperm cell obviously carries another activating factor, which is not easy to extract, but can be obtained by sequential treatment of the sperm cell with Triton X-100 and SDS. However, it is not known whether the easily extractable oscillins and frozen / thawed, resistant to extraction factors are biologically and chemically identical.

It is known that sperm heads treated with sound in the presence of Triton X-100 lose all components except the nucleus and perinuclear materials. However, after microsurgical introduction into oocytes, such Triton X-100-treated sperm heads (having a nucleus and perinuclear material, but without plasma membranes) can activate oocytes as efficiently as undamaged spermatozoa.

As described in the examples below, at least the sperm-derived mouse oocyte-activating molecules must be freeze-dried, since most oocytes that survived the injection of freeze-dried sperm heads were normally activated and fertilized.

If sperm injection does not cause oocyte activation in other species, activation can be carried out by parthenogenetic agents, such as electroactivation, injection of one or more oocyte-activating substances, or transfer of oocytes to a medium containing one or more oocyte-activating substances. Reagents capable of providing an activating stimulus (or a combination of activating stimuli) include, but are not limited to: sperm cytoplasmic activating factor and certain pharmacological compounds (e.g., Ca ²⁺ and other signal transduction modulators) that can be administered by microinjection after or simultaneously with the injection sperm heads. Some activating stimuli are used following the transfer of fertilized oocytes to an environment containing one of the members of a subset of activating compounds, including Ca ²⁺ release stimulants (e.g. caffeine, Ca ²⁺ , ionophores such as A 23187 and ionomycin, and ethanol), phosphoprotein signal modulators (e.g. 2-aminopurine, staurospurin and sphingosine), protein synthesis inhibitors (e.g. A 23187, cyclohexamide), 6-dimethylaminopurine, or a combination of the above (e.g. 6-dimethylaminopurine and ionomycin). In one embodiment of the invention, mouse oocyte activation was achieved by culturing for 1-6 hours in a Ca ²⁺ CZB-free medium containing 2-10 mM Sr2 +.

Embryo development with a viable fetus and offspring

Following the formation of the pronucleus, the embryo can be cultured in vitro until it reaches the stage of 2-8 cells or the morula / blastocyst stage, at which the embryo can be transferred to the oviduct or uterus of the surrogate mother.

Simultaneous administration of biologically significant substances with sperm heads

In one embodiment of the invention, microinjection of the sperm head into the oocyte allows one or more agents to affect the yield of developed embryos to be injected before, during, or after the sperm head is injected into the oocyte. For example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) can be introduced into the oocyte by microinjection before or after injection of the sperm head. For example, injection of recombinant DNA carrying the necessary cis-active signals can lead to transcription of sequences present on the recombinant DNA by transcription factors resident or injected with it and subsequent expression of encoded proteins with antagonistic effects on developmental inhibitors or with a positive effect on embryonic development. Moreover, the transcript may have antisensory activity against mRNAs encoding development inhibitory proteins. Alternatively, antisensory regulation can be obtained by injection of nucleic acids (or their derivatives), which are able to influence the inhibitory effect by direct interaction with their targets, nucleic acids, without prior transcription inside the oocyte.

Recombinant DNA (linear or other) introduced by the method of the invention can carry a functional replicon containing one or more expressed functional genes under the control of a promoter that exhibits everything from narrow to wide, developmental expression profiles. For example, a promoter may cause instant 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 at one or more genomic loci in order to stably replicate during the life of the resulting transgenic individual. In one embodiment, DNA can be introduced simultaneously with microinjection into the oocyte, building encoded, presumably “anti-aging” proteins, such as telomerase or superoxide dismutase. Alternatively, such proteins can be injected directly.

EXAMPLES

The following examples illustrate the method of the invention and the development of a live offspring from an oocyte injected with sperm restored after freeze-drying. In particular, the example illustrates the development of normal mice from mouse oocytes injected with heads (cores) recovered after freeze-drying of mouse sperm, which were stored either at room temperature (about 25 ° C) or in a refrigerator (about 4 ° C). The medium for suspending sperm before freeze-drying was CZB or DMEM media described below.

The examples given here should be considered only as examples of oocytes and spermatozoa of certain animal species, sperm suspension media, freezing schemes, storage conditions, rehydration media and the like that can be used in the process of the invention, but should not be limited to them, since other examples of methods for implementing the invention can be easily determined by specialists.

Medium and Reagents

All inorganic and organic compounds were purchased from Sigma Chemicals Co. (St. Louis), unless otherwise specified.

The collected oocytes were stored in CZB medium before sperm injection (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 CaCl ₂ , 1.2 mm MgSO ₄ , 1.8 mm KH ₂ PO ₄ , 25.1 mm NaHCO ₃ , 0.1 mm sodium lactate, 0.3 mm sodium pyruvate, 7 units / ml penicillin G , 5 units / ml streptomycin sulfate, and 4 mg / ml bovine serum albumin. The medium for collecting oocytes from the oviducts, subsequent processing and micromanipulations was a modified CZB medium containing 20 mM Hepes, a reduced amount of NaHCO ₃ (5 mM), and bovine serum albumin (ASB) - 3 mg / ml. This medium is here referred to as Hepes-CZB. For microinjection purposes, it is preferable to replace the ASB in Hepes-CZB with 1 mg / ml polyvinyl alcohol (PVA dissolved in cold water, average molecular weight 10 × 10 ³ ), because the PVA keeps the walls of the injection pipette less sticky for longer than the ASB , which is better when repeatedly using the same pipette for multiple transfers of sperm heads to oocytes.

Two different media were used to suspend the sperm cells before freeze-drying: (1) CZB medium without ethylenediaminetetraacetic acid (EDHC) containing 4 mg / ml ABS; and (2) Dulbecco-modified Eagle medium (DMIS) enhanced with 10% (v / v) bovine fetal serum (Hyclone, Logan, UT).

In order to determine which medium would be “the best” for sperm suspension, preliminary experiments were carried out; for this purpose, initial suspension of fresh sperm in CZB medium was carried out, followed by centrifugation and re-suspension in one of the media described below, and then immediately followed by freezing of sperm. Test media for sperm suspension included (a) distilled water, (6) 34% sucrose in distilled water, (c) 180 mg / ml raffinose plus 5 mg / ml ASB, (g) 0.9% NaCl with 5 mg / ml ASB, (e) 0.9% NaCl with 1 mg / ml glucose plus 5 mg / ml ASB; and (e) lactate and calcium free CZB. Only the latter medium (e) turned out to be as good as the usual CZB in its ability to preserve the sperm core, capable of developing during freeze-drying (data not shown).

Animals

The animals that were used in these examples were kept in accordance with the instructions of the Laboratory Animal Service of the University of Hawaii and the guidelines prepared by the Laboratory Animal Containment and Use Committee of the Institute for Laboratory Resources of the National Research Council (DHEW, publication No. {NIH} 80-23, revised in 1985). The treatment and handling of animals has been reviewed and approved by the Animal Care and Use Committee of the University of Hawaii.

EXAMPLE 1

Sperm preparation

Four different preparations were prepared in order to illustrate the effect of the sperm suspension medium, storage temperature and storage time on the ability of nuclei dried by freezing sperm to participate in the development of live offspring after injection into the oocyte, as described below and illustrated in table 1. For each two caudal epididymes of the mature B6D2F1 male mouse were used. Simultaneously with squeezing each epididymis with fingers, its distal part was pierced with sharp tweezers. The dense mass of sperm released from the epididymis was transferred into a 1.5 ml polypropylene tube containing 1 ml of one or two of the test media described above, 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 sperm in this suspension (approximately 3-10 × 10 ⁶ per ml) were actively motile.

EXAMPLE 2

Sperm Freeze Drying

An aliquot (100 μl) of the sperm suspension was placed in a 2 ml ampoule (Wheaton Scientific, Millville, NJ, catalog No. 651506), which was lowered directly into liquid nitrogen. Ten minutes later, the ampoules were placed in pre-cooled (-50 ° C) refrigeration flasks connected to a freeze-drying unit (Model 10-020, VirTis Co., Gardner, NY). The inlet pressure was approximately 1 milliTorr. The flasks were disconnected from the system after about 12 hours when it was filled with argon supplied through a gas dehydration chamber (Fisher Scientific, Pittsburg, PA catalog number 09-204). Each ampoule was connected to a vacuum pump and, after more than 99% of the gas was evacuated, sealed. The ampoules were individually wrapped in aluminum foil and stored in the dark at room temperature (about 25 ° C) or at 4 ° C.

Ampoules with freeze-dried sperm prepared as described above are shown in FIG. The white powder at the bottom of each ampoule is a dry CZB medium containing sperm.

EXAMPLE 3

Rehydration of Freeze-dried Sperm

Ampoules containing freeze-dried sperm prepared as described above were opened and 100 μl of distilled water were added to the ampoule to obtain a reconstituted suspension of sperm. Then 5 μl of the suspension was thoroughly mixed with 50 μl of Hepes-CZB containing 12% (w / v) polyvinylpyrrolidone (average molecular weight 360,000). Rehydrated sperm were examined under a microscope and only those with intact heads and tails were selected for further manipulations.

EXAMPLE 4

Freeze-dried sperm transport

An experiment was conducted to verify that freeze-dried sperm prepared in accordance with the present invention could be transported abroad, while maintaining the ability to fertilize oocytes after rehydration. In this experiment, several freeze-dried vials of epididymic spermatozoa were transported by hand during a three-week trip to Japan, which began in Honolulu, Hawaii. Prior to freeze-drying, the sperm cells were suspended in CZB medium. No special precautions were taken to preserve sperm besides the fact that the ampoules were wrapped in aluminum foil throughout the journey and kept in cardboard boxes. The ambient temperature varied from 5 ° C to 30 ° C. One week after returning to Honolulu, sperm were rehydrated and used according to the method of the invention.

EXAMPLE 5

Oocyte preparation

In sexually mature females, B6D2F1 (C57BL / 6 × DBA / 2) was over-ovulated by injecting 7.5 international units (ME) of pregnant mare gonadotropin into each mouse, followed by an injection of 7.5 ME human chorionic gonadotropin (hGT) after 48 hours. Fourteen hours after hGT injection, cumulus-oocyte complexes were taken from the oviducts and treated with testicular bovine hyalorunidase (300 USP units / ml; ICN Biochemicals, Costa Mesa, CA) in Hepes-CZB medium for 3 min to separate cumulus cells.

Prior to sperm injection, the oocytes were washed and stored in CZB medium for 4 hours in an atmosphere containing 5% CO ₂ in air.

EXAMPLE 6

Microinjection of sperm nuclei into oocytes

For injection of sperm heads into prepared oocytes, a microinjection chamber was prepared using a lid (10 mm deep) for a plastic cup (100 mm × 15 mm; Falcon Plastics, Oxnard, CA, catalog number 1001). A row consisting of two round drops and one oblong drop was placed along the center line of the cup. The first drop (2 μl; 2 mm in diameter) is for pipet flushing (Hepes-CZB containing 12% (w / v) PVA, average molecular weight 360,000 daltons). The second drop (2 μl; 2 mm in diameter) was a suspension of rehydrated, freeze-dried sperm in CZB or DMEM prepared as described above. The third, oblong drop (6 μl; 2 mm wide and 6 mm long) was Hepes-CZB medium for oocytes. Each of these drops was coated with mineral oil (Squibb and sons). The cup was placed on the stage of an inverse microscope with interference contrast optics.

Microinjection of sperm nuclei into oocytes was carried out as previously described by the piezoelectric microinjection method using the MB-U model piezo micromanipulator from Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The device uses the piezoelectric effect for a single advancement of the pipette holder at a very small distance (for example, 0.5 μm) at a very high speed. The intensity and speed of the pulses were controlled using the control panel.

For injection into an oocyte prepared as described above, into an injection pipette (inner tip diameter of about 5 μm), which was attached to a device controlling a piezoelectric pipette, a separate sperm was selected with the tail part forward. The sperm head and tail were separated by the application of one or more piezoelectric pulses in the neck region. The intensity and speed (frequency) of the pulses was regulated by the PMAS-CT01 remote control (scales are set on the remote control: intensity 2, speed 1). Then the heads were sucked deep into the pipette and a small volume (about 0.5 μl) of mercury was placed at the proximal end of the injection pipette.

Meanwhile, a mature, unfertilized oocyte was placed on a microscope stage in Hepes-CZB medium. The oocyte was fixed with a holding pipette, and the tip of the injection pipette was brought into close contact with the zone of the pellucid at the 3 o'clock position. Several piezo pulses (intensity 1-2, speed 1-2) were applied to advance the pipette while creating a slight negative pressure inside When the tip of the injection pipette passed the pellicidal zone, a cylindrical piece of the pellicidal zone in the pipette was pushed into the perivitelline space. After the sperm head was pushed forward until it was near the tip of the pipette, the pipette was mechanically advanced forward until its tip almost reached the opposite side of the oocyte cortex. The olemma was pierced by the application of 1 or 2 piezoelectric pulses (intensity 1-2, speed 1) and the sperm head was pushed into the ooplasm with a minimum amount (about 6 pl) of the accompanying medium for the suspension of sperm. After removing the maximum possible amount of medium, the pipette was carefully removed, leaving the sperm head inside the ooplasm. All injections were performed in Hepes-CZB medium at room temperature (23-27 ° C) for one hour after rehydration. One sperm head was injected into each oocyte. About 5-20 oocytes were microinjected for 10-15 minutes.

EXAMPLE 7

Inspection of oocytes and embryo transfer

Oocytes injected with sperm heads were incubated in Hepes-CZB medium at 37 ° C at 5% CO ₂ in air and examined under an inverse microscope after 5-6 hours. Sperm cells that had two distinct pronuclei and a second polar body were considered normally fertilized and cultured in Hepes-CZB medium for 4 days. Spermatozoa that have reached the morula or blastocyst stage were transferred to the horns (tubes) of the uterus of the recipient female CD-1 (albinos), which were paired with the vasectomized CD-1 males three days earlier to synchronize the stages of embryonic development with the development of uterine endometrium. On average, eight morul / blastocysts were moved to each horn (tube). Females were given the opportunity to bear and grow their surrogate offspring (with black, brown or gray skins). Several sexually mature males and females from the offspring were randomly selected and crossed in order to check their fecundity.

RESULTS

Microscopic examination of rehydrated freeze-dried sperm

Microscopic examination of rehydrated sperm showed that 100% of the sperm were immobile. Sperm viability was determined using a cell viability kit, which can be easily purchased (Leave / dead FertiLight, Molecular Probes, Eugene, Oregon) and which detects cells with a whole plasma membrane (live) and with a damaged plasma membrane (dead) in accordance with the nature of fluorescence under a UV microscope. The nuclei of “living” sperm cells with whole plasma membranes fluoresce green, while the nuclei of “dead” fluoresce bright red-orange. After freeze-drying and dehydration, more than 10,000 spermatozoa from four males were examined. All sperm tested were “dead,” as determined using this test.

As illustrated in Table 1, 1236 of 1353 oocytes (91.4%) survived injection microsurgery and 1157 (93.6%) of the survivors were activated by the sperm nucleus and normally fertilized, regardless of the initial suspension medium (CZB or DMEM), storage temperature (25 ° С or 4 °), and storage time (1 day, 2 weeks, 1 month or 3 months).

The maximum storage time of the ampoules in these examples was 3 months at 4 ° C. In three experiments, 57 oocytes from three females were injected with spermatozoa that had been stored for 3 months, 95% of the injected oocytes survived microsurgery, and all of them were normally fertilized. 91% of fertilized eggs developed to the morula / blastocyst stage in vitro. Fourteen (30%) of the 46 embryos transferred to three surrogate mothers developed into normal adult organisms.

Most (90% -93%) of fertilized eggs that were injected with sperm heads suspended in CZB medium, freeze dried, stored from 1 day to 2 weeks at 25 ° C or 4 ° C and rehydrated, developed to the morula / blastocyst stage in vitro and 25-34 (20% -29%) of them developed to normal offspring after transfer to surrogate mothers. Although a smaller percentage (76%) of fertilized eggs that were injected with CZB suspended in freeze-dried sperm, stored until rehydration for 1 month at 25 ° C, developed to the morula / blastocyst stage, 16 (18%) of them developed to normal offspring after transfer to surrogate mothers. Nine (28%) of 32 transferred embryos originating from fertilized eggs, which were injected with sperm suspended in CZB medium, freeze-dried and stored at 4 ° C for 3 months, developed into normal offspring. In total, using freeze-dried sperm suspended in CZB medium, a total of 143 live offspring were obtained after transferring 562 embryos that developed from 664 oocytes that survived microsurgery and fertilized normally with an average success rate of 18.7%.

As illustrated in FIG. 1b, all offspring grew normally. The sex ratio was 1: 1. Two fully grown females and two males from each of the 12 experimental groups were randomly selected and mated. All proved their fertility and produced broods of usual sizes (from 8 to 12).

Of the sperm that were sent to and back from Japan and recovered after they returned to Honolulu, 29 randomly selected sperm were individually injected into oocytes. Nineteen (83%) developed to the in vitro morula / blastocyst stage. Three (16%) of them reached full development after transfer to a surrogate mother. All three (2 females and 1 male) grew up in prolific adults.

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

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

Table 1
The development of mouse oocytes injected with freeze-dried sperm Sperm Suspension Medium Sample storage Sperm Injected Oocytes Number of transplanted embryos (number of surrogate females) Number (total%) of live descendants (range) * Temperature (C) Period of time The total number of injected oocytes (expert No.) The number (%) of surviving oocytes Number (%) of normally fertilized oocytes The number (%) reached the stage of morula / blastocyst Czb 25 ° 1 day 155 (5) 135 (87) 133 (99) ^a 120 (90) ^e 116 (8) 34 (29) {0-71} 2 weeks 158 (4) 144 (91) 141 (98) ^a 131 (93) ^f 126 (8) 25 (20) {0-30} ^k 1 month 130 (4) 120 (92) 117 (98) ^a 89 (76) ^g 87 (5) 16 (18) {5-33} ^k 4 ° 2 weeks 137 (4) 126 (92) 123 (98) ^a 114 (93) ^e 105 (7) 27 (26) {0-45} 1 month 131 (4) 117 (89) 112 (96) ^a 98 (88) ^h 96 (6) 32 (33) {14-65} ^l 3 months 40 (2) 38 (95) 38 (100) ^b 35 (92) ^h 32 (2) 9 (28) {26-31} DMEM 25 ° 1 day 128 (3) 116 (91) 104 (90) ^b 95 (91) ^e 95 (6) 91 (31) {18-47} ^l 2 weeks 109 (2) 102 (94) 85 (83) ^c 67 (79) ⁱ 60 (3) 8 (13) {0-20} ^m 1 month 13 (2) 106 (94) 74 (70) ^d 47 (64) ^j 47 (3) 8 (17) {0-24} 4 ° 2 weeks 83 (2) 79 (95) 79 (100) ^a 69 (87) ^h 56 (3) 20 (36) {21-46) ^l 1 month 152 (3) 137 (90) 135 (99) ^a 114 (84) ^h 54 (3) 22 (41) {15-60} ⁿ 3 months 17 (1) 16 (94) 16 (100) 14 (88) 14 (1) 5 (35) {35-35} * - scatter of data (%) of offspring from a surrogate mother Statistical significance: but against c, d: p <0/005 f against g, i, j; p <0.005 n vs m: p <0.01 b vs d: p <0.005 h vs j: p <0.005 l vs m: p <0/01 e vs g, j: p <0.005 n vs k: p <0.01

Claims (39)

1. Product for artificial insemination, containing spermatozoa with a broken membrane or a sperm head treated with freeze-dried to a moisture level of less than 1%, the core of which retained genetic integrity sufficient for fertilization, and after rehydration and microinjection into an isolated oocyte, these sperm heads inseminate an isolated oocyte while the preserved genetic integrity is sufficient for fertilization with the receipt of live offspring. 2. The product for artificial insemination according to claim 1, characterized in that the sperm or sperm head after freeze-drying is stored for some time until rehydration. 3. The product for artificial insemination according to claim 2, characterized in that the sperm or sperm head is stored after freezing drying at normal temperature. 4. The product for artificial insemination according to claim 2, characterized in that the sperm or sperm head is stored at a temperature of about 4 ° C. 5. The product for artificial insemination according to claim 2, characterized in that the sperm or sperm head is stored at a temperature of minus 20 ° C or lower. 6. The 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 a year, or more than one year. 7. The product for artificial insemination according to claim 1, characterized in that the membraneless head contains a core and perinuclear materials. 8. The product for artificial insemination according to claim 1, characterized in that the sperm or sperm head is obtained from an invertebrate animal. 9. The artificial insemination product of claim 8, wherein the sperm or sperm head is obtained from a sea urchin, lobster, shell or mollusk. 10. The product for artificial insemination according to claim 1, characterized in that the sperm or sperm head is obtained from a vertebrate animal. 11. The artificial insemination product of claim 10, wherein the sperm or sperm head is obtained from fish, amphibian, reptile, bird or mammal. 12. The product for artificial insemination according to claim 10, characterized in that the sperm or sperm head is obtained from a person. 13. The artificial insemination product of claim 10, wherein the sperm or sperm head is obtained from a mouse. 14. An ampoule containing a product for artificial insemination, characterized in that the product for artificial insemination is a product according to claim 1. 15. The method of obtaining a product for artificial insemination according to claim 1, comprising collecting live mature sperm, suspending sperm in a physiological suspension medium, freezing a suspension of sperm to obtain frozen sperm, drying the frozen sperm or sperm heads under vacuum to a moisture level of less than 1% s obtaining freeze-dried sperm or sperm heads with a broken membrane, rehydration of sperm or sperm heads membrane-damaged atozoids, the selection of sperm or sperm heads that have a nucleus that has preserved their genetic integrity, and after microinjection into an isolated oocyte, the oocyte is fertilized with live offspring. 16. The method according to p. 15, characterized in that it further includes the step of removing the sperm membranes to the stage of freezing with obtaining deprived membranes of sperm heads. 17. The method according to clause 16, wherein the step of removing the membranes comprises treating the sperm with a detergent and possibly a surfactant. 18. The method according to p. 15, characterized in that the drying of the frozen sperm or head is carried out to a moisture level of less than 0.01%, preferably less than 0.001%, more preferably less than 0.00001%. 19. The method according to clause 15, wherein the sperm is obtained from a vertebrate animal. 20. The method according to claim 19, characterized in that the sperm are obtained from fish, amphibians, reptiles, birds or mammals. 21. The method according to claim 19, characterized in that the sperm are obtained from a person. 22. The method according to claim 19, characterized in that the sperm are obtained from the mouse. 23. The method according to clause 15, wherein the sperm is obtained from an invertebrate animal. 24. The method according to item 23, wherein the sperm is obtained from a sea urchin, lobster, shell or mollusk. 25. The method according to p. 15, characterized in that the freezing of a suspension of sperm is carried out at minus 196 ° C for 10 minutes. 26. The method according to clause 15. characterized in that it further includes the step of storing freeze-dried sperm for some time until the rehydration stage. 27. The method according to p. 26, characterized in that the freeze-dried sperm stored at ordinary temperature. 28. The method according to p. 26, characterized in that the freeze-dried sperm stored at a temperature of about 4 ° C. 29. The method according to p. 26, characterized in that the freeze-dried sperm stored at a temperature of minus 20 ° C or lower. 30. The method according to p, characterized in that the storage time of the sperm is not more than three months, or up to about one year, or more than one year. 31. The method of fertilizing mammalian oocytes with a product for artificial insemination according to claim 1, containing a sperm with a broken membrane or a sperm head, including collecting live mature mammalian spermatozoa, suspending sperm in physiological medium for suspension, freezing a suspension of spermatozoa, to obtain frozen spermatozoa, for frozen under vacuum to a moisture level of less than 1% to obtain freeze-dried sperm, reagent sperm cells after freeze-drying, isolation of such a rehydrated sperm with a damaged membrane or sperm head, which contains a nucleus that has retained genetic integrity and is capable of fertilization, and is capable of re-hydration by fertilization as a result of microinjection of an isolated oocyte with the formation of living offspring and isolation of living offspring, introduction of a sperm with a damaged membrane or sperm head into an isolated oocyte with the formation of fertilized oocyte. 32. The method according to p, characterized in that the sperm with a damaged membrane or the sperm head is injected into the oocyte by piezoelectric microinjection. 33. The method according to p, characterized in that it further includes the removal of sperm membranes to the stage of freezing with the formation of sperm heads devoid of membranes. 34. The method according to p, characterized in that when the sperm head is introduced into the oocyte, the centrosome of the sperm is additionally introduced. 35. The method according to p, characterized in that the sperm are obtained from a person. 36. The method according to p, characterized in that the sperm are obtained from the mouse. 37. The method according to p, characterized in that the humidity level of the sperm frozen under vacuum is less than 0.01%, preferably less than 0.001%, more preferably less than 0.00001%. 38. The method according to p, characterized in that the isolated sperm is injected into the oocyte for 1-60 minutes after rehydration. 39. A method for producing live offspring of a non-humanoid mammal from an isolated oocyte fertilized with a rehydrated artificial insemination product according to claim 1, comprising a sperm with a damaged membrane or a sperm head, including collecting live mature sperm from non-human mammals, suspension of spermatozoa in physiological cells, suspension of sperm to obtain frozen sperm, drying frozen sperm p vacuum to a moisture level of less than 1% to obtain freeze-dried sperm, rehydration of freeze-dried sperm, isolation of such a rehydrated sperm with a damaged membrane or sperm head, which contains a preserved genetic integrity and is capable of fertilization and can be insulated after re-hydration with live offspring, isolation of an oocyte of similar non-humanoid mammals Introduction of sperm with damaged membrane or sperm head into an isolated oocyte to form a fertilized oocyte; cultivation of a fertilized oocyte to obtain live offspring.

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