MXPA00009176A - Development of normal offspring from oocytes injected with freeze-dried spermatozoa - Google Patents

Abstract

The invention provides a method for freeze-drying spermatozoa to obtain at least one reconstituted spermatozoon whose head (nucleus) is capable of fertilizing an oocyte to produce a live offspring. The motility of spermatozoa which have been freeze-dried and stored in a vacuum at room temperature is not restored when rehydrated. Their plasma membranes are disrupted and they are all"dead"in the conventional sense. However, when they are injected microsurgically into oocytes, their nuclei transform into male pronuclei and participate in normal embryonic development.

Description

DEVELOPMENT OF NORMAL PROGENIE FROM INOCECTED OOCYTES WITH LIOFILIZED SPERMATOZOIDS BACKGROUND OF THE INVENTION The invention relates to the lyophilization of spermatozoa, the fertilization of oocytes with lyophilized, reconstituted sperm, and the development of live progeny from these. Successful cryopreservation of sperm through the use of cryoprotectants and the ability to store frozen sperm for prolonged periods of time has produced dramatic improvements in animal husbandry as well as in human reproductive medicine. It has been found that frozen-thawed, cryoprotected sperm often return to their motility and fertilize almost as efficiently as fresh sperm. The long-term storage of sperm in liquid nitrogen (-196 ° C) has been routinely used to cryopreserve sperm from cattle and humans. However, the conventional preservation of sperm is very expensive in the long term, due to the need for a REF: 122960 constant supply of liquid nitrogen. In addition, in certain areas of the world it can be inconvenient and / or expensive to store these sperm in liquid nitrogen, such as in developing countries where liquid nitrogen (or even dry ice) may not be easily accessible. The transportation of conventionally frozen sperm is also problematic as it may require the transport of large liquid nitrogen tanks or the use of special shipping containers containing liquid nitrogen or dry ice. In this way, many attempts have been made to preserve the sperm without requiring storage in liquid nitrogen. For example, if sperm competent for fertilization could be stored in a lyophilized state at ambient temperatures or in ordinary refrigerators, the reduction in maintenance and shipping costs could be enormous. It is believed that the first recorded attempt to lyophilize sperm was in 1949, when one milliliter (ml) of poultry semen was mixed with an equal volume of Ringer's solution containing 20-30% glycerol, spread as a thin layer in a distillation flask, and "lyophilized" by eliminating 90% of the water. When the preparations were rehydrated within 2 hours after being put back at room temperature, it was reported that up to 50% of the sperm returned to obtain motility. The fertility of the rehydrated sperm, however, was never determined. Subsequent attempts to produce live progeny using lyophilized sperm have not been successful. It has been reported, for example, that a live calf was born after the artificial insemination of a cow with bull sperm that were reconstituted immediately after lyophilization and showed 50% sperm motility. It was also reported that twelve baits of normal rabbits were obtained by insemination using lyophilized sperm. However, none of these results could be repeated by the original investigators or duplicated and confirmed by others who work in the field. From the studies of the properties and fertility of normal and frozen-thawed sperm, it is known that sperm are not required to be "alive" in the conventional sense (for example, that they have intact plasma membranes) in order that they support normal embryonic development. For example, in a technique of intracytoplasmic sperm injection (ICSI), mobile human sperm are selected. These are - immobilized ("dead") immediately before injection into an oocyte by aggressive abrasion of their tails, resulting in damage to the plasma membrane. It has been reported that immobilization of sperm significantly increases the rate of successful fertilization by ICSI. It has also been reported that when mouse spermatozoa were suspended in medies without any cryoprotectory lue_g-o immediately frozen in liquid nitrogen, 100% of the sperm were "dead" as judged by the staining of live / dead cells, and development still occurred. normal embryo after the microinjection of the thawed sperm heads inside the oocytes. There is also a report of the birth of two normal calves after the miexoinjection, within oocytes, of sperm killed by freezing-thawing • without any cryoprotectant.

Although it is known that hamster and human lyophilized sperm heads, injected into hamster oocytes can form pronuclei that appear normal, it has never been determined whether such sperm heads can support normal embryonic development. In addition, it has been shown that motility loss, acrosome damage and enzyme release occur when sperm are frozen and dehydrated to a moisture content of less than 30%, 7% and 0.5%, respectively. Evidence has also been obtained that cell proteins in sperm are altered by dehydration below 6% moisture. In view of the foregoing, there is a need for a reliable and reproducible method for lyophilizing sperm, whereby freeze-dried sperm retain their competence of fertilization during prolonged periods of storage at room temperature, or at ordinary refrigerator temperatures, or below. There is an additional need for a method for sperm injection, which employs lyophilized sperm, rehydrated to fertilize oocyte vessels, resulting in normal live progeny.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for lyophilizing sperm to obtain at least one reconstituted sperm whose head (nucleus) is capable of fertilizing an oocyte to produce a living progeny. The method of the invention comprises the steps of: (a) collecting live sperm; (b) suspending the sperm in a suspension medium; (c) freeze the sperm suspension; and (d) dehydrating the sperm suspension to a moisture level that is less than 1%, preferably less than 0.01%, more preferably less than 0.001%, and especially less than 0.00001%. The method may further comprise the step of rehydrating the freeze-dried sperm suspension, wherein at least one head of rehydrated sperm retains its genetic integrity and is capable of fertilizing an oocyte to produce living progeny. As discussed below, the method may further comprise the step of storing lyophilized sperm prior to rehydration. To obtain a living progeny, at least the head (nucleus) of a rehydrated sperm is inserted into an isolated oocyte to form a fertilized oocyte. The head of the sperm is inserted into the oocyte by microinjection, preferably by means of piezoelectric-driven microinjection. Preferably, the insertion of the core takes place within one hour after rehydration. The fertilized oocyte is then left to develop into an embryo and implanted in the uterus of a surrogate mother where it develops into a living progeny. In some species (for example, most of the euterian mammals, including humans), the normal embryonic development of fertilized oocytes also requires. the insertion of the progenitor centrosome associated with the sperm nucleus. The centrosome is normally coupled either to the posterior end of the sperm head or to the anterior end of the tail of the sperm, when the head and tail separate. Thus, in embodiments of the invention, a centrosome associated with sperm from another sperm can be inserted simultaneously with the head of the sperm, or it can be inserted by means of simultaneous or consecutive insertion of the sperm. Alternatively, insertion of the sperm nucleus and centrosome can be achieved by inserting a complete rehydrated sperm into the oocyte. It has been found that spermatozoa that are lyophilized by the method of the invention can be stored for at least three months or, more preferably, for at least one year, at temperatures in the range of room temperature (e.g. room) to refrigerator temperatures (for example, approximately 4 ° C) without losing its genetic integrity or fertilization capacity. Thus, lyophilized sperm prepared by the method of the invention, can remain competitive during shipping virtually anywhere in the world at ambient or refrigerator temperatures, and may also suffer short-term storage at ambient or refrigerator temperatures. , in places where liquid nitrogen or dry ice are not readily available. Preferably, long-term (for example, undefined) storage of the lyophilized sperm occurs at temperatures below 4 ° C (e.g., -20 ° C or lower). The method of the invention can be used to lyophilize spermatozoa of invertebrate and vertebrate species including, but not limited to, invertebrates such as sea urchin, lobster, sea ear, shell molluscs and the like, and vertebrates such as fish. , amphibians, reptiles, birds and all mammals.

BRIEF DESCRIPTION OF THE DRAWINGS The presentation of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and with payment of the necessary fees. Figure 1 is a photograph of vacuum sealed ampoules containing lyophilized mouse spermatozoa. The white powder at the bottom of each vial is the dehydrated CZB medium that contains the sperm. Figure 2A is a microphotograph of lyophilized mouse spermatozoa immediately after rehydration. The proportion of sperm heads without tails or with broken tails, varies depending on how smoothly or sharply the dehydrated specimens are handled during storage. Figure 2B is an electron microphotograph showing a longitudinal cross section through the anterior region of a normal sperm head that has not been frozen. Figure 2C is an electron microphotograph showing longitudinal and horizontal cross-sectional views through the anterior region of a lyophilized sperm head after rehydration. The plasma membrane (p) and the acrosome contents (ac) are missing. (ia) = internal acrosomal membrane, (oa) = external acrosomal membrane, (n) = nucleus. Figure 3 is a photograph of three young (black) mice born from a surrogate female CD-1 (albino). These youngsters developed from B6D2F1 oocytes injected with B6D2F1 sperm that had been maintained at room temperature for one month after lyophilization.

DETAILED DESCRIPTION OF THE INVENTION During normal fertilization in mammals, a fertilizing sperm ascends the female genital tract, passes through the coatings of the oocyte, then fuses with an oocyte. The fusion of the sperm with the oocyte triggers the activation of the oocyte. The activated oocyte resumes meiosis and the oocyte chromosomes are transformed into a female pronucleus. Meanwhile, the nucleus of the sperm inside the oocyte is decondensed to become a male pronucleus. The fully developed male and female pronuclei then unite and the chromosomes from these pronuclei intermingle. The resulting zygote develops into a living progeny. The present invention provides a method for lyophilizing spermatozoa which, after rehydration, are capable of fertilizing the isolated oocytes to produce live progeny. The lyophilized spermatozoa produced by the method of the invention retain their genetic and reproductive potential although, when rehydrated, they are motionless and "dead" in the conventional sense. When the complete sperm or the sperm heads are isolated (for example, they contain all the components of the head including the nucleus) or the dismembered sperm or the sperm heads (for example, they retain the nuclei and the perinuclear materials, but that lacking the plasma membranes) of the sperm of the invention, injected directly into the oocytes, normal fertilization and embryonic development occurs, which results in the production of live progeny. Preferably, the sperm head (nucleus) is inserted directly into the cytoplasm of the oocyte. The insertion of the sperm head is by microinjection, preferably by means of piezoelectric driven microinjection. As discussed below, the embryonic development of fertilized oocytes of some species may require the simultaneous or sequential injection of a sperm centrosome. If the centrosome does not survive the lyophilization process, a centrosome of an unfrozen sperm can be harvested for insertion into the oocyte.

The individual steps and sub-steps of the methods of the invention for the preparation of lyophilized sperm competent for fertilization, and their use in an IV fertilization procedure are now presented in greater detail.

Preparation of sperm In order to ensure that as many freeze-dried sperms as possible retain their genetic integrity throughout the lyophilization process of the invention, it is preferred that physiologically mature sperm be used in the method of the invention. In mature sperm, DNA is associated with the basic proteins called protamines. In mammals, protamines are extensively crosslinked by disulfide bonds. This stabilizes the sperm nuclei and makes them very resistant to physical and chemical disorganization. The crosslinking of nuclear prptamines occurs mainly during the transit of the sperm through the epididymis. Thus, mammalian sperm within the epididymis and in ejaculation (semen) are generally physiologically more mature than those within the testes, and are preferred in the methods of the invention, at least in mammals. Mature sperm from invertebrates and vertebrates are collected by methods known to those of skill in the art. For example, mature spermatozoa of rodents, such as mouse, golden hamster (Sirius), guinea pig, rabbit and the like, can be collected from the epididymis; meanwhile, in other species, such as in humans, pigs, horses, bulls, goats, poultry and the like, mature sperm can be isolated from the newly ejaculated semen of fertile males. Fish sperm (for example, sword tail, Xiph oph orus h el l eri) and invertebrates, such as sea urchins (Tripneus t is gra l), can be collected from the testes of mature mammals. Below is an example of a method to obtain sperm from an epididymis cauda. A cauda of the epididymis is removed from a mature male mouse (approximately 8 weeks after birth or older). Blood and adipose tissue are removed from the surface of the epididymis cauda. This is compressed to release a dense mass of sperm. A drop (approximately 2 μl) of the sperm mass is placed in the bottom of 1.5 ml polypropylene centrifuge tubes and spread with 0.5 ml of warm physiological medium (eg, CZB medium, phosphate buffered saline, or isotonic saline solution). After approximately 10 to 20 minutes at 37 ° C, mobile sperm can be collected from the supernatant. An example of a method for obtaining sperm from semen is shown below. The freshly ejaculated human semen is allowed to liquefy for approximately 30 minutes at room temperature (approximately 25 ° C). The semen is then diluted with approximately 10 ml of saline and filtered through approximately two layers of filter paper to remove debris. The filtrate can then be centrifuged at 400 x g for about 10 minutes, and the pelleted sperm are resuspended in a physiological solution at a concentration desired for the lyophilization process. An example of a method for obtaining sperm from the testicles is shown below. An excised testis is placed in an erythrocyte lysis buffer (for example, 155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, 2 mM EDTA, pH 7.2-7.4), shredded using a pair of fine scissors, and filtered through approximately two layers of filter paper to remove debris. The filtrate is then centrifuged (for example 700 x g, 5 minutes) and the button is resuspended in a physiological solution at a desired concentration in the preparation for the lyophilization process. Regardless of the method used to prepare the sperm, more than 50% of the recovered sperm must be mobile. The sperm thus recovered are suspended in a physiological medium, describelow, in preparation for the lyophilization process. Alternatively, the sperm may undergo additional processing to obtain dismembered heads of sperm before lyophilization.

Preparation of heads of dismembered sperm Dismembered sperm heads are detergent-extracted heads that lack all the membranes, including the plasma membrane and the internal and external acrosomal membranes, but retain the nucleus and perinuclear material. For example, sperm heads can be dismembered 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 for the elimination of membrane components under non-denaturing conditions. SDS is an anionic detergent used to solubilize various proteins, including membrane proteins. In the mouse, the heads of the sperm dismembered by the use of Triton X-100 have been shown to be able to activate the oocytes, leading to normal embryonic development. An exemplary method for dismembering the heads of sperm is described below. An aliquot of a sperm suspension, prepared as described above, is sonicated. For example, sperm collected from the epididymis, testis, or semen canals, as described above, can be suspended in 5 ml of BM buffer (75 mM sodium chloride, 24 mM EDTA, and Tris-HCl 50 mM, pH 7.2) and sonicated for 30 seconds at a 70% -80% yield of a Biosonik sonicator (Bronwill Scientific, Rochester, NY). More than 95% of the sperm are decapitated by this treatment. To dismember the sperm heads, the sonicated suspension of sperm is centrifuged at 700 xg for 5 minutes, and the button or concentrate is washed with BM buffer and then treated at room temperature for 5 minutes with a portion of Triton X-100. in the middle NIM. (The NIM medium consists of 123.0 mM potassium chloride, 2.6 M sodium chloride, 7.8 mM sodium diacid phosphate, 1.4 mM potassium diacid phosphate, 3 mM Na2EDTA, which has a pH of 7.2). The heads are then rinsed thoroughly with NIM medium and resuspended in suspension medium of sperm in preparation for the lyophilization process.

Sperm Suspension Medium In the preparation for lyophilization, the sperm (or the dismembered sperm heads) are suspended in a physiological solution that is sufficient to support the integrity of at least the nucleus of the sperm under normal conditions. The solution must be a balanced salt solution with at least one adequate automatic pressure and an adequate pH. There is no simple means that can support the survival of the sperm of all animal species. For example, a suitable solution for marine urchin sperm is seawater, with approximately 1,000 milo-osmoles of osmotic pressure and a pH value of about 8.2. Seawater, however, will kill mammalian sperm instantly. Mammalian sperm require a solution that has an osmotic pressure of approximately 300 milli-osmoles and a pH of 7.0-7.6. Such a solution, however, could kill the sea urchin sperm. In view of the foregoing, the suspension medium for the sperm must be selected according to the species of interest, according to criteria that are well known to those of skill in the art. Such selection can be made without undue experimentation. Because the sperm do not have to have the membrane intact (these can be "dead") in order to successfully fertilize the oocytes by injection, there is no absolute requirement for a cryopreservative such as glycerol or the like, in the suspension medium .

Packing The sperm suspension can be placed in a variety of different containers including, but not limited to glass ampoules or plastic cryotubes (cryofasks) that are capped with a screw cap, or the suspension can be extracted into plastic straws which, after of the lyophilization process, they can be sealed by means of a powder sealant, by heat, or with nylon plugs. The volume of the suspension of sperm in each container is not critical. Typically, a volume of about 50-100 μl is used in a 2 ml ampule.

Lyophilization of sperm Sperm suspension can be frozen slowly or rapidly, by known means. For example, the suspension can be frozen in liquid nitrogen vapor or in the refrigerated air of a mechanical (electric) freezer by methods that are well known in the art. Cooling and freezing can be achieved with a static or gradual manual regime, or in an electronically automated and programmed liquid nitrogen feed system. Various freezing rates can be employed (for example 1 ° C-25 ° C / minute). For example, the freezing step can be carried out at -196 ° C for 10 minutes. Various modifications can be used in the successful freezing of the suspension in straws, ampoules or cryotubes. A metal container (basket) with cigarette-type tubes, or other retainers with straws, or retainers or holders with ampoules or cryotubes, can be placed directly in the vapor of liquid nitrogen through the use of a liquid nitrogen refrigerator. The drying of the frozen sperm suspension in vacuum can be achieved by a variety of different systems known to those skilled in the art. For example, a known apparatus is a VirTis model 10-020 (VirTis Co., Gardiner, NY). The suspension is dried at a moisture level that is less than 15. However, it is preferable that the humidity level is less than 0.01%, more preferably less than 0.001%, and especially less than 0.00001%. The container with lyophilized sperm is preferably sealed in vacuum, or sealed in the presence of an inert gas, such as nitrogen or argon.

Storage The lyophilized sperm vessels are preferably stored in the dark, or wrapped with aluminum foil; or similar. For long-term storage, it may be preferable to store the containers at temperatures of -20 ° C or lower. It is expected that, like comparatively lyophilized bacteria, fungi and the like, lyophilized sperm nuclei will retain their genetic integrity indefinitely under these storage conditions. However, containers can be stored at room temperature (for example, at room temperature) or at ordinary refrigerator temperature (approximately 4 ° C) for periods of time greater than three months without compromising the ability of the sperm nuclei lyophilized to fertilize the oocytes. Therefore, lyophilized sperm can be shipped without the need for special conditions or bulky containers.

Rehydration of lyophilized sperms The lyophilized sperm preparation is preferably rehydrated by the addition of pure water, the volume of which is the same as the original volume of the sperm suspension before lyophilization. Once rehydrated, any physiological saline solution, such as 0.9% saline solution or CZB medium (see below), can be used for dilution. The volume of dilution is not critical. The concentration of the sperm in the final rehydration medium may be sufficient to facilitate the recovery of individual sperm or individual sperm heads for sperm injection purposes within oocytes, as described below. The incidence of oocyte activation and normal fertilization after the injection of the sperm heads seems to decrease as the time after the rehydration of the sperm increases. The allowable period of time between rehydration and injection may vary between species; however, as an example, this time period for mouse sperm is preferably one hour or less.

Microscopic examination of lyophilized, rehydrated sperm The rehydrated, lyophilized sperm are not mobile. The viability of the sperm can be evaluated by using any staining method that is able to distinguish between sperm that are, in the conventional sense, alive or dead. A suitable commercially available viability test kit for use in the invention is Live / dead FertiLight, available from Molecular Probes, Eugen, Oregon, which differentiates between intact cells in the plasma membrane (alive) and damaged cells. in the plasma membrane (dead) according to a fluorescence pattern under a UV microscope after staining with propidium iodide / SYBR 14. The "live" sperm nuclei with intact plasma membranes fluoresce green, while those of the "dead" sperm fluoresce bright orange red. It is expected that all sperm examined will be "dead" in the conventional sense. Figure 2A is a microphotograph of representative freeze-dried mouse sperm immediately after rehydration. The heads and tails of some sperm are separated. The proportion of sperm with broken or missing tails may vary depending on how smoothly or abruptly the dehydrated specimens are handled during storage. Sperm with or without tails are used in the injection procedure described below. The differences between a fresh live (non-lifolized) sperm and the freeze-dried and rehydrated sperm by the method of the invention are illustrated in Figures 2b and 2C, respectively. Each of these Figures represents a longitudinal cross-section through the anterior region of the head of the sperm. Although rehydrated sperm, freeze-dried, has retained the nucleus (n), a portion of the outer acrosomal membrane (a) and a portion of the inner acrosomal membrane (a), the plasma membrane (p) and the acrosome content (ac) ) , They are absent.

The recipient oocytes Recipient oocytes can be obtained, for example, by inducing an animal to ovulate or superovulate by injections of gonadotrophic hormones (eg, the sequential administration of equine and human chorionic gonadotropins), and surgical harvesting of eggs from the oviducts shortly after expected ovulation time (eg, 13-15 hours after the injection of human chorionic gonadotropin into the mouse). Alternatively, ovarian oocytes are harvested and cultured in a medium to allow their maturation, as is known to those skilled in the art. An example of a preferred culture medium is modified Eagle's medium (MEM) supplemented with bovine serum albumin (BSA), as described in Downs, S.M. and A.M. Mastropolo, Devel op. Bi ol. 162: 154-168, 1994 for mouse oocytes.

Sperm components required for successful fertilization in vi tro It is known that, in the mouse, normal fertilization can be carried out by injecting isolated sperm heads into the oocytes, and that the plasma and acrosomal membranes and all the components of the tail are not essential for the development Normal of the embryo. The mouse, and perhaps most common laboratory rodents, are "exceptional" since a sperm centrosome is not required for normal fertilization and, during normal fertilization, the sperm centrosome in the sperm neck region is destined to degenerate within the oocyte after fertilization. In contrast, in most other eutherian mammals, including cattle and humans, the sperm centrosome plays a central role in the formation of the microtubules that are essential for the union of the male and female pronuclei, as well as in the Subsequent splits during embryonic development. Therefore, in these species, the introduction of a sperm nucleus (head) and a centrosome in an oocyte appears to be essential for the production of normal progeny. It is not known to date if the sperm centrosome from all species can survive lyophilization. If not, a centrosome of an unfrozen sperm must be injected into an oocyte together with the lyophilized sperm head in order to ensure normal embryonic development. The introduction of excessive numbers of centrosomes, however, could result in abnormal pronuclear development and abnormal embryonic development. The centrosome is normally coupled either to the posterior end of the sperm head or to the anterior end of the tail of the sperm when the head and tail separate. In this way, the sperm centrosome can be inserted into the oocyte simultaneously with the head of the sperm, or it can be inserted by means of simultaneous or consecutive insertion of the sperm of the tail. Alternatively, insertion of the sperm nucleus and centrosome can be achieved by inserting a complete rehydrated sperm into the oocyte.

Insertion of sperm nuclei in recipient oocytes The complete sperm can be injected into the cytoplasm of the recipient oocyte, but in species in which the sperm are large, an isolated sperm head (nucleus) is preferably injected directly into the cytoplasm of a recipient oocyte by a microinjection technique. In a preferred method of microinjection of a rehydrated sperm head or dismembered, rehydrated sperm heads, in a container oocyte, the piezoelectrically associated micropipette is used. A suitable piezoelectric drive unit is sold under the name Piezo Micromanipulator / Piezo Impact Drive Unit by Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The unit uses the piezoelectric effect to advance, in a highly controlled and fast way, the holder (injection) a very short distance (approximately 0.5 μm). The intensity and duration of each pulse can be varied and regulated by a control unit. For injection into an oocyte, a simple sperm, the tail first, is aspirated into an injection pipette having a flat, short tip, with an internal diameter of approximately 5 μm housed in the piezoelectrically driven unit according to Seller's instructions. The head and tail of the sperm are separated by applying a few piezoelectric pulses to the neck region. The head is then pulled deeply into the pipette. Throughout the injection of the sperm head (nucleus), the oocyte is anchored by a conventional retention pipette. The tip of the injection pipette containing a selected sperm head is placed in intimate contact with the zona pellucida of an oocyte and several piezoelectric pulses are applied (using the adjustment scales of the intensity controller 1-5, speed of 4-6) to advance the pipette while maintaining a slightly negative pressure inside. When the tip of the pipette has passed through the zona pellucida, the plug of the resulting zone is expelled into the perivitelline space and the head of the sperm is pushed forward until it is near the tip of the pipette. The tip of the pipette is then juxtaposed to the plasma membrane and advanced (towards the opposite side of the oocyte) and the retention pipette reaches almost the opposite side of the oocyte cortex. The plasma membrane of the oocyte is now deeply invaginated around the tip of the injection needle. After the application of one to two piezoelectric pulses (intensity 1-2, speed 1), the oolema is punctured at the tip of the pipette, as indicated by a rapid relaxation of the oolema, which can be clearly visible. The head of the sperm is then expelled into the ooplasm with a minimum amount (approximately 6 pL) of the accompanying medium. The pipette is then gently removed, leaving the newly inserted head inside the cytoplasm of the oocyte. This method is performed vigorously, typically in batches of 10 to 15 oocytes which at all times are maintained under culture conditions. Alternative microinjection variants, in which a conventional injection pipette is used, can be used to inject sperm heads. An example of a suitable microinjection method using a conventional pipette to inject a sperm head into the hamster oocyte is described in Yanagida, K., Yanagimachi, R., Perreault, S.D. and R.G.

Kleinfeld, Bi olgy of Reproduc ti on 44, 440-447 (1991), the description of which belongs to such a method, is incorporated herein by reference. The microinjection of the sperm head / dismembered sperm head offers several advantages. Firstly, the administration of the sperm head by microinjection is applicable to a wide variety of sperm types, regardless of size, morphology and the like. Second, microinjection allows carefully controlled coinjection (with the head of the donor sperm) of additional agents into the oocyte at the time of injection of the sperm head. These are exemplified later in the present. Third, in the embodiment of the invention where the insertion of the sperm head is by means of piezoelectrically driven microinjection, rapid and efficient sample processing is provided, thereby reducing trauma to sperm and oocytes undergoing manipulation . The oocytes of some species (eg, mouse) are not suitable for microinjection using conventional needles, while piezoelectrically driven microinjection provides a high success rate.

Activation of fertilized oocytes It is known that the mouse oocyte can be activated by injection of a mouse sperm, intact, simple, or its head isolated. Isolated sperm tails are unable to activate the oocyte. The activating factor (s) of the oocytes carried by the active sperm typically appear during the transformation of the round spermatid into the spermatozoon. The action of these factors is not highly specific to the species because mouse oocytes are activated by the injection of sperm from foreign species, such as hamsters, rabbits, pigs, humans and even fish. It has been reported that an activation factor of this type is a 33 kilodalton protein that resides in the equatorial segment region of the acrosome. This protein, called oscilin, is easily extractable from mature sperm (hamster) by simple freezing and thawing. In addition to oscilin, mature sperm seem to carry another activation factor that is not easily extractable, but can be obtained by sequential treatment of sperm with Triton X-100 and SDS. It is not known if the oscilin is easily extractable and the factors resistant to freeze / thaw extraction are biologically and chemically identical. It is known that sperm heads sonicated in the presence of Triton X-100 lose all components except the nucleus and perinuclear materials. Even when they are microsurgically injected into the oocytes, such sperm heads treated with Triton X-100 (which have the nuclear and perinuclear materials, but not the plasma membranes) can activate the oocytes as efficiently as intact sperm. As described in the following examples, at least in the mouse, the oocyte-activating molecules carried by the sperm must be resistant to lyophilization because most of the oocytes that survive the injection of lyophilized sperm heads they were activated and fertilized in a normal way. If in other species the injection of the sperm head does not serve to activate the oocyte, activation can take place by parthenogenic means, such as by electroactivation, injection of one or more oocyte activating substances, or transfer of the oocytes within the oocyte. medium containing one or more oocyte activating substances. Reagents capable of providing an activating stimulus (or combination of activating stimuli) include, but are not limited to, cytoplasmic sperm activation factor, and certain pharmacological compounds (eg, Ca2 + and other modulators of signal transduction), which can be introduced by microinjection after, or concomitantly with, the injection of the sperm head. Some activating stimuli are provided after the transfer of the fertilized oocytes to the media containing one or the members of a group of activating compounds, including stimulators of Ca2 + release (eg, caffeine, Ca2 + ionophores such as A23187 and ionomycin, and ethanol), modulators of phosphoprotein signaling (eg, 2-aminopurine, staurosporine, and sphingosine), inhibitors of protein synthesis (eg, A23187, cycloheximide), 6-dimethylaminopurine, or combinations of the above ( for example, 6-dimethylaminopurine and ionomycin). In one embodiment of the invention, the activation of mouse oocytes is achieved by culturing for 1 to 6 hours in CaZ + free CZB medium containing 2-10 mM Sr2 +.

Development of embryos to produce viable fetuses and progeny After the formation of the pronucleus, the embryo can be cultured in vitro until it reaches the stage of 2 to 8 cells or the morula / blastocyst stage, at which time the embryo can be transferred to the oviduct or uterus of a substitute mother.

Simultaneous injection of biologically interesting substances with sperm heads In one embodiment of the invention, the microinjection of the sperm head into an oocyte allows the introduction, before, during or after injection of the sperm head into the oocyte, of one or more agents with the potential to alter the effect of embryo development. 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 the recombinant DNA harboring the necessary activation signals ci s may result in the transcription of the sequences present on the recombinant DNA, by resident or co-injected transcription factors, and the subsequent expression of the encoded proteins. , with an antagonistic effect on the inhibitory factors of development, or with a positive effect on the development of the embryo. In addition, the transcript may possess antisense activity against mRNAs that encode developmental inhibitory proteins.

Alternatively, antisense regulation can be achieved by injection of nucleic acids (or their derivatives) and are capable of exerting an inhibitory effect by directly interacting with their nucleic acid target (s) without prior transcription within the oocyte. Recombinant DNA (linear or other) introduced by the method of the invention may comprise a functional replicon containing one or more functional genes, expressed, under the control of a promoter that shows anything from a narrow developmental expression profile up to a large one. For example, the promoter can direct immediate but brief expression where that promoter is active only in the early zygote. The introduced DNA can be either lost at some point during embryonic development, or integrated into one or more genomic loci, to be stably replicated throughout the life of the resulting transgenic individual. In one embodiment, DNA constructs encoding putative "anti-aging" proteins, such as telomerase or superoxide dismutase, can be introduced into the oocyte by microinjection. Alternatively, such proteins can be injected directly.

EXAMPLES The following examples illustrate the method of the invention and the development of live progeny from oocytes injected with lyophilized, reconstituted sperm. In particular, the examples illustrate the development of normal mice from mouse oocytes injected with the heads (nuclei) of mouse spermatozoa, lyophilized, reconstituted, which had been stored either at room temperature (approximately 25 ° C) or in a refrigerator (approximately 4 ° C). The sperm suspension medium before lyophilization was either CZB or DMEM, described below. The examples described herein are intended to be only examples of oocytes and sperm from an animal species, the sperm suspension medium, the freezing protocols, the storage conditions, the rehydration media and the like, which can be used. in the process of the invention, and are not intended to be limiting, since other examples of the embodiments of the invention could easily be recognized by those skilled in the art.

Means and reagents All inorganic and organic compounds were purchased from Sigma Chemical Co. (Saint Louis, MO) unless otherwise stated. Harvested oocytes were maintained in CZB medium (Chatot et al., 1989, J. Reprod Fert. 86, 679-688), prior to sperm injection. The CZB medium comprises 81.6 mM sodium chloride, 4.8 mM potassium chloride, 1.7 mM calcium chloride, 1.2 mM magnesium sulfate, 1.8 M potassium diacid phosphate, 25.1 mM sodium acid carbonate, 0.1 mM Na2EDTA, 31 sodium lactate. mM, 0.3 mM sodium pyruvate, 7 U / ml penicillin G, 5 U / ml streptomycin sulfate, and 4 mg / ml bovine serum albumin. The means for collecting the oocyte from the oviducts, the subsequent treatments and the micromanipulation was a modified CZB containing 20 mM Hepes, a reduced amount of sodium acid carbonate (5 mM) and bovine serum albumin (BSA) at 3 mg / ml. This medium is called Hepes-CZB. For microinjection purposes, it was preferred to replace the BSA in the Hepez-CZB with 0.1 mg / ml of polyvinyl alcohol (PVA, soluble in cold water, average molecular mass 10 X 103) because the PVA maintained the wall of the pipette. less sticky injection over a longer period of time than BSA, and was beneficial during repeated use of a single pipette for multiple transfers of sperm heads / oocytes. Two different means of suspending sperm were used to suspend the spermatozoa before lyophilization: (1) CZB medium without ethylenediaminetetraacetic acid (EDTA) containing 4 mg / ml of BSA; and (2) Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (v / v) (Hyclone, Logan, UT). Preliminary experiments were conducted to investigate what was the "best" means of suspension of sperm for this purpose, and were conducted first by suspending fresh sperm in CZB medium, followed by centrifugation and resuspension in one of the means listed below, and then immediately lyophilized the sperm. The tested sperm suspension medium included (a) distilled water, (b) 34% sucrose in distilled water, (c) 180 mg / ml raffinose plus 5 mg / ml BSA, (d) 0.9% NaCl with 5 mg / ml of BSA, (e) 0.9% sodium chloride with 1 mg / ml of glucose plus 5 mg / ml of BSA, and (f) CZB free of lactate and free of calcium. Only the last means (f) was shown to be as good as the regular CZB in its ability to maintain competent sperm cores for development during lyophilization (data not shown).

Animals The animals used in these examples were maintained in accordance with the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee for the Care and Use of Laboratory Animals of the Institute for Family Research Council for Laboratory Resources ( DHEW publication No. [NIH] 80-23, revised 1985). The animal management and treatment protocol was reviewed and approved by the Committee for Animal Care and Use at the University of Hawaii.

EXAMPLE 1 Preparation of sperm Four different lyophilized sperm preparations were performed in order to illustrate the effects of the suspension medium of sperm, the storage temperature and the period of storage time, on the ability of lyophilized sperm nuclei to participate in the development of progeny live after injection into the oocytes, as described below and illustrated in Table 1. For each preparation, two epididymal strands of a mature B6D2F1 male mouse were used. While pressure was applied with the finger on each epididymis, its distal portion was punctured with acute forceps. A dense mass of epididymal sperm was oozed out and transferred to a 1.5 ml polypropylene tube containing 1 ml of one of the two test media, CZB or DMEM, described above. After incubation for 30 minutes at 37.5 ° C, the top 0.3-0.5 ml of medium was removed from the tube. More than 90% of the sperm in this suspension (approximately 3-10 X 106 per ml) were actively mobile.

EXAMPLE 2 Lyophilization of sperm 100 μl of an aliquot of the sperm suspension was placed in a 2 ml ampule (Heaton Scientific, Millville, NJ, catalog No. 651506) which was directly capped in liquid nitrogen. Ten minutes later, the ampules were placed in a pre-cooled (-50 ° C) freezing flask coupled to a lyophilization system (Model 10-020, VirTis Co., Gardner, NY). The inlet pressure was approximately 1 milliTorr. Approximately 12 hours later, the flask was removed from the system after it had been filled with argon supplied as a gas drying vessel (Fisher Scientific, Pittsburg, PA catalog No. 09-204). Each ampoule was connected to a vacuum pump and structurally sealed after more than 99% of the gas was pumped out of it. The ampules were individually placed with aluminum foil and stored in the dark at room temperature (approximately 25 ° C) or at 4 ° C. The lyophilized mouse sperm ampoules, prepared as described above, are illustrated in Figure 1. The white powder at the bottom of each vial is dehydrated CZB medium containing sperm.

EXAMPLE 3 Rehydration of lyophilized sperm A vial containing 'lyophilized sperm, prepared as described above, was broken and 100 μl of distilled water was added to the ampule to form a reconstituted suspension of sperm. Then 5 μl of the sperm suspension was mixed thoroughly with 50 μl of Hepes-CZB containing 12% (w / v) of polyvinylpyrrolidone (average molecular weight 360,000). The rehydrated sperm were observed microscopically and only those with intact heads and tails were selected for subsequent manipulation.

EXAMPLE 4 Embarking of freeze-dried sperm An experiment was carried out to evaluate whether lyophilized spermatozoa, prepared according to the method of the present invention, could be shipped abroad and still retain their competence to fertilize the oocytes after rehydration. In this experiment, a few ampoules of lyophilized epididymal sperm were taken personally during a three-week trip to Japan, which originated in Honolulu, Hawaii. Before lyophilization, the sperm were suspended in CZB medium. No special precautions were taken to preserve the sperm, except that the vials were wrapped with aluminum foil and kept in a cardboard box during the entire trip. The ambient temperature varied between 5 ° C and 30 ° C. One week after their return to Honolulu, the sperm were rehydrated and used in the method of the invention.

EXAMPLE 5 Preparation of oocytes Mature females B6D2F1 (C57BL / 6 X DBA / 2) were induced to superovulate by injecting to each mouse 7.5 International Units (Ul) of pregnant mare serum gonadotropin, followed by 7.5 IU of human chorionic gonadotropin (hCG) 48 hours later. Fourteen hours after the injection of hCG, complexes of oocyte clusters-oocytes were collected and treated with bovine testicular hyaluronidase (300 USP U / ml, ICN Biochemicals, Costa Mesa, CA) in Hepes-CZB medium for 3 minutes for Disperse the cells of the clusters. Prior to injection with sperm nuclei, the oocytes were rinsed and stored in CZB medium for up to 4 hours at 37 ° C in a 5% C02 atmosphere in air.

EXAMPLE 6 Microinjection of sperm nuclei within oocytes For the injection of the sperm heads into the prepared oocytes, a microinjection chamber was prepared by using the cover (10 mm deep) of a plastic box (100 mm x 15 mm; Falcon Plastics, Oxnard, CA, catalog No. 1001). A row consisting of two round droplets and one elongated droplet were placed along the center line of the box. The first droplet (2 μl, 2 mm diameter) was for pipetting (Hepes-CZB containing 12 [w / v] PVP, average molecular weight 360,000 daltons). The second droplet (2 μl, 2 mm in diameter) was the suspension of lyophilized spermatozoa, rehydrated in CZB or DMEM, as prepared above. The third elongated droplet (6 μl, 2 mm wide and 6 mm long) was Hepes-CZB medium for the oocytes. Each of these droplets was covered with mineral oil (Squibb and sons). The box was placed on the stage of an inverted microscope with optical interference contrast equipment. The microinjection of the sperm nuclei into oocytes was achieved by the piezoelectric microinjection method described previously, using the Piezo Model MB-U Micromanipulator by Prime Tech. Ltd. (Tsukuba, Ibaraki-ken, Japan). This unit uses the piezoelectric effect to advance the portapipeta a very small distance (for example, 0.5 μm) at a time at a very high speed. The intensity and speed of the pulse were regulated by the controller. For injection into an oocyte prepared as described above, a single sperm, the tail first, was sucked into an injection pipette (approximately 5 μM ID at the tip) which had been coupled to the injection unit of the Piezoelectric pipette. The sperm head and tail were separated by applying a few simple piezoelectric pulses to the neck region. The intensity and speed (frequency) of the pulses were regulated by the PMAS-CT01 controller (controller adjustment scales: intensity 2, speed 1). The heads were then pulled tightly towards the pipette and a small volume (approximately 0.5 μl) of mercury was placed on the proximal end of the injection pipette. Meanwhile, an unfertilized, mature oocyte was placed on a microscope slide in the Hepes-CZB medium. The oocyte was maintained by a retention pipette and the tip of the injection pipette was placed in intimate contact with the zona pellucida at the clock position at 3 o'clock. Several piezoelectric pulses were administered. (intensity 1-2, speed 1-2) to advance the pipette, while a slight negative pressure was applied to it. When the tip of the pipette had passed through the zona pellucida, a cylindrical part of the zona pellucida in the pipette was expelled into the perivitelline space. After the head of the sperm was pushed forward until it was near the tip of the injection pipette, the pipette was mechanically advanced until its tip almost reached the opposite side of the oocyte cortex. The oolema was punctured by the application of 1 or 2 piezoelectric pulses (intensity 1-2, speed 1) and the head of the sperm was expelled to the ooplasm with a minimum amount (approximately 6 pL) of the accompanying sperm suspension medium. Subsequently, as much as possible was recovered, the pipette was gently withdrawn, leaving the sperm head inside the ooplasm. All injections were performed in Hepes-CZB at room temperature (23 ° C-27 ° C) within 1 hour of sperm rehydration. Each oocyte was injected with a sperm head. Approximately 5-20 oocytes were microinjected by this method within 10-15 minutes.

EXAMPLE 7 Oocyte examination and embryo transfer Oocytes injected with the sperm head were incubated in CZB at 37 ° C under 5% C02 in air, and examined with an inverted microscope 5 to 6 hours later. Those with two distinct pronuclei and a second polar body were considered normally fertilized and cultured for 4 days in CZB. Those who reached the morula or blastocyst stages were transferred to the uterine horns of female CD-1 recipients (albinos) which had been copulated with vasectomized CD-1 males three days previously to synchronize the stages of embryonic development with those of the interim endometrium. . An average number of eight morulae / blastocysts were transferred into each horn. The females were allowed to develop and breed their surrogate progeny (with black, brown or gray coatings). Some progeny of mature males and females were randomly selected and copulated to examine their fertility.

RESULTS Microscopic examination of lyophilized, rehydrated sperm Microscopic observation of the rehydrated sperm showed that 100% of the sperm were not mobile. The viability of sperm was evaluated by using a commercially available cell viability test kit (Live / dead FertiLight, Molecular Probes, Eugene, Oregon) that differentiates between cells with intact plasma membrane (alive) and cells with damaged plasma membrane - (dead) according to a fluorescence staining pattern under a UV microscope. The nuclei of "live" sperm cells with intact plasma membranes fluoresce green, while those of "dead" sperm fluoresce bright orange red. Over 10,000 sperm from four males were examined after lyophilization and rehydration. All sperm examined were "dead", as assessed by this test.

Development of mouse oocytes injected with lyophilized sperm heads As illustrated in Table 1, 1,236 of 1,353 oocytes (91.4%) survived the microsurgery by injection and, of the survivors, 1,157 (93.6%) were activated by the sperm nuclei and fertilized normally, despite the original suspension medium of sperm (CZB or DMEM), the Storage temperature (25 ° C or 4 ° C), and storage period (1 day, 2 weeks, 1 month or 3 months). The maximum storage period of the ampules in these Examples was 3 months at 4 ° C.

In three experiments, 57 oocytes from three females were injected with sperm stored for 3 months. 95% of the injected oocytes survived the microsurgery, and all of them were fertilized normally. 91% of the fertilized eggs were developed to morula / blastocysts i n vi tro. Fourteen (30%) of 46 embryos transferred to three surrogate mothers were developed into normal adults. The majority (90% -93%) of the fertilized eggs, which had been injected with sperm heads suspended with CZB, which had been lyophilized, were stored for 1 day to 2 weeks at 25 ° C or 4 ° C, and rehydrated, developed in morulae / blastocysts in vi tro and 25-34 (20% -29%) of these developed in normal progeny when transferred to surrogate mothers. Although a lower percentage (76%) of the fertilized eggs that had been injected with lyophilized sperm, suspended in CZB that had been stored for 1 month at 25 ° C before rehydration, they developed in morulae / blastocysts, 16 (18% ) of these were developed to normal progeny when they were transferred to surrogate mothers. Nine (28%) of 32 transferred embryos derived from fertilized eggs that had been injected with sperm heads suspended in CZB, lyophilized and stored at 4 ° C for 3 months, developed into normal progeny. In summary, when freeze-dried sperm, suspended in CZB was employed, a total of 143 live progenies were produced after the transfer of 562 embryos, developed from 664 oocytes that survived microsurgery and fertilized normally, a total success rate of 21.5%. Most of the fertilized eggs (79% -91%), which had been injected with sperm heads suspended in DMEM, which had been lyophilized, stored for 1 day to 1 month at 25 ° C or up to 3 months at 4 ° C , also developed in morula / blastocysts in vi tro. In summary, a total of 92 live progenies were produced after the transfer of 326 embryos, developed from 493 oocytes that survived the microsurgery and were fertilized normally, a total success rate of 18.7%. As illustrated in Figure lb, all progeny developed normally. His sex ratio was approximately 1: 1. Two fully developed females and two males from each of the 12 experimental groups were randomly selected and copulated. All proved to be fertile and normal size baits were produced (8 to 12).

The effect of the shipment of lyophilized sperm on the fertilization competition Of the sperm that were shipped to and from Japan, and reconstituted after their return to Honolulu, 29 randomly selected sperm were individually injected into oocytes. Twenty-three oocytes (79%) survived and were fertilized normally. Nineteen (83%) developed in morulae / blastocysts in vi tro. Three (16%) of those reached the full term after transfer to a surrogate mother. The three (2 females and 1 male) developed in fertile adults. In view of the above Examples, it has been shown that mouse sperm can retain their genetic integrity after lyophilization. There is no reason to believe that sperm from other species, including invertebrates and vertebrates, could behave differently. While the invention has been described herein with reference to the preferred embodiments, it should be understood that it is not intended to limit the invention to the specific forms described. On the contrary, it is intended to cover all modifications and alternative forms that fall within the spirit and scope of the invention.

TABLE 1 Development of Mouse Oocytes Injected with Lyophilized Spermatozoa Storage Medium of Oocytes Injected with Esp-pru. No. of Embryos No. (% total) of Progeny Suspension of Transferred Displays [range] * Viva Espetma Temp. Period of Total No. of Oocytes No. (• /.) Of Oocytes No. (• /.) Of Oocytes No. (%) Reaching Stage (No. of Females (C) Time Injected (Exp. Fertilized Survivors of Protoplasts / Substitute Blastocysts) No.) Normally CZB 25 ° ldia 155 (5) 135 (87) 133 (99) "120 (90) '116 (8) 34 (29) 10-71] 2 weeks 158 ( 4) 144 (91) 141 (98) '131 (93/126 (8) 25 (20) [0-39]' 1 month 130 (4) 20 (92) 117 (98) '89 (76 87 (5 ) 16 (18) [5-33] * 4 ° 2 weeks 137 (4) 126 (92) 123 (98) '114 (93)' 105 (7) 27 (26) [0-45] 1 month 131 (4) 117 (89) 112 (96) ' 98 (88) h 96 (6) 32 (33) [14-65] '3 months 40 (2) 38 (95) 38 (100)' 35 (92/32 (2) 9 (28) [26-31 ] tp 8 DMEM 25 ° ldia 128 (3) 116 (91) 104 (90) '95 (91)' 95 (6) 29 (31) [18-47] '2 weeks 109 (2) 102 (94) 85 (83) '67 (79)' 60 (3) 8 (13) [0-20] "1 month 113 (2) 106 (94) 74 (70) '47 (64)" 47 (3) 8 (17) [0 -24] 4 ° 2 weeks 83 (2) 79 (95) 79 (100) '69 (87 / "56 (3) 20 (36) [21-46]' lmes 152 (3) 137 (90) 133 (99) ' 114 (84/54 (3) 22 (41) [15-60] "3 months 17 (1) 16 (94) 16 (100) 14 (88) 14 (1) 5 (35) [35-35] * Range of progeny% by surrogate mother Significance Statistics: a versus c, d: p <0.005 f versus g, ij: p <0.005 n versus m: p < 0. 01 b versus d: p < 0.005 h versus j: p <0 005 1 versus m: p <0.01 e versus g, j: p <0.005 n versus k: p < 0 .01 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention

Claims (47)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A spermatozoon or lyophilized sperm head, damaged in the membrane, containing a competent nucleus for fertilization, sperm or sperm head has been lyophilized to a moisture level of less than 1% and is capable, after rehydration , of fertilizing an isolated oocyte after microinsertion to produce a living progeny.

2. The spermatozoon or head of lyophilized sperm, damaged in the membrane, according to claim 1, characterized in that the lyophilized sperm or head of spermatozoon is stored for a period of time before rehydration.

3. The lyophilized spermatozoon or sperm head, damaged in the membrane, according to claim 2, characterized in that the freeze-dried spermatozoon or sperm head is stored at room temperature.

4. The spermatozoid or lyophilized sperm head, damaged in the membrane, according to claim 2, characterized in that the lyophilized spermatozoon or sperm head is stored at approximately 4 ° C.

5. The spermatozoon or lyophilized sperm head, damaged in the membrane, according to claim 2, characterized in that the freeze-dried sperm or sperm head is stored at or below -20 ° C.

6. The lyophilized spermatozoid or sperm head, damaged in the membrane, according to claim 2, characterized in that the period of storage time is up to about three months.

7. The spermatozoon or head of lyophilized sperm, damaged in the membrane, according to claim 2, characterized in that the period of storage time is approximately one year or less.

8. The lyophilized sperm or head of spermatozoon damaged in the membrane, according to claim 2, characterized in that the period of storage time is greater than one year.

9. The lyophilized sperm or sperm head, damaged in the membrane, according to claim 1, characterized in that the head is a dismembered head comprising the nuclear and perinuclear materials.

10. The spermatozoid or head of lyophilized sperm, damaged in the membrane, according to claim 1, characterized in that the spermatozoon or sperm head is from an invertebrate.

11. The spermatozoon or lyophilized sperm head, damaged in the membrane, according to claim 10, characterized in that the sperm head is from a sea urchin, a marine ear, or a mollusc with a shell.

12. The spermatozoid or lyophilized sperm head, damaged in the membrane, according to claim 1, characterized in that the spermatozoon or sperm head is of a vertebrate.

13. The spermatozoon or lyophilized sperm head, damaged in the membrane, according to claim 12, characterized in that the spermatozoon or sperm head is of a fish, an amphibian, a reptile, a bird, or a mammal.

14. The spermatozoid or head of lyophilized sperm, damaged in the membrane, according to claim 12, characterized in that the sperm head is from a human.

15. The lyophilized sperm or head of lyophilized sperm, damaged in the membrane, according to claim 12, characterized in that the spermatozoon or sperm head is from a mouse.

16. A vial, characterized in that it contains the lyophilized sperm or head of lyophilized sperm, damaged in the membrane, according to claim 1.

17. A method for obtaining a spermatozoon or head of lyophilized sperm, damaged in the membrane, according to claim 1, characterized in that it comprises the steps of: the collection of live mature sperm; the suspension of the spermatozoa in a medium of physiological suspension; the freezing of the sperm suspension to form frozen sperm; and the dehydration of sperm frozen in vacuum to a moisture level of less than 1% to form lyophilized sperm containing a competent nucleus for fertilization that is capable, after rehydration, of fertilizing an isolated oocyte after the microinsertion to produce a living progeny

18. The method according to claim 17, characterized in that it further comprises the step of dismembering the sperm before the freezing step to form dismembered sperm heads.

19. The method according to claim 18, characterized in that the step of dismemberment comprises the treatment of the spermatozoon with a detergent and, optionally, a surfactant.

20. The method according to claim 17, characterized in that the humidity level is less than 0.01%.

21. The method according to claim 17, characterized in that the humidity level is less than 0.001%.

22. The method according to claim 17, characterized in that the humidity level is less than 0.00001%.

23. The method according to claim 17, characterized in that the sperm are from a vertebrate.

24. The method according to claim 23, characterized in that the sperm come from a fish, an amphibian, a reptile, a bird, or a mammal.

25. The method according to claim 23, characterized in that the sperm are from a human.

26. The method according to claim 23, characterized in that the sperm are derived from a mouse.

27. The method according to claim 17, characterized in that the spermatozoa are derived from an invertebrate.

28. The method according to claim 27, characterized in that the sperm are from a sea urchin, a lobster, a sea ear, or a mollusk with a shell.

29. The method according to claim 17, characterized in that the freezing step is carried out at -196 ° C for 10 minutes.

30. The method according to claim 17, characterized in that it further comprises the step of storing the lyophilized sperm for a period of time before the rehydration step.

31. The method according to claim 30, characterized in that the lyophilized sperm are stored at room temperature.

32. The method according to claim 30, characterized in that the lyophilized sperm are stored at approximately 4 ° C.

33. The method according to claim 30, characterized in that the lyophilized sperm are stored at -20 ° C or at a lower temperature.

34. The method according to claim 30, characterized in that the storage period is up to three months.

35. The method according to claim 30, characterized in that the period of storage time is up to about one year.

36. The method according to claim 30, characterized in that the period of storage time is greater than one year.

37. A method for fertilizing mammalian oocytes with a damaged spermatozoon or sperm head in the membrane, characterized in that it comprises the steps of: collecting live mature sperm from a mammal; the suspension of the spermatozoa in a medium of physiological suspension; the freezing of the sperm suspension to form frozen sperm; dehydration of frozen sperm under vacuum to a moisture level of less than 1% to form lyophilized sperm; the rehydration of lyophilized sperm; the isolation of a spermatozoid or sperm head damaged in the membrane, rehydrated, which contains a competent nucleus for fertilization and which is capable, after rehydration, of fertilizing an isolated oocyte after the microinsertion to produce a living progeny; the isolation of an oocyte from a similar species of the mammal; and the insertion of damaged sperm or sperm head into the membrane, within the isolated oocyte to form a fertilized oocyte.

38. The method according to claim 37, characterized in that the insertion step is achieved by piezoelectrically driven microinjection.

39. The method according to claim 37, characterized in that it also comprises the step of dismembering the spermatozoa before the freezing step, to form dismembered sperm heads.

40. The method according to claim 37, characterized in that the insertion step further comprises the sub-step of inserting a living sperm centrosome.

41. The method according to claim 37, characterized in that the sperm are from a human.

42. The method according to claim 37, characterized in that the sperm are derived from a mouse.

43. The method according to claim 37, characterized in that the humidity level is less than 0.01%.

44. The method according to claim 43, characterized in that the humidity level is less than 0.001%.

45. The method according to claim 44, characterized in that the humidity level is less than 0.00001%.

46. The method according to claim 37, characterized in that the sperm is injected from one to about 60 minutes after rehydration.

47. A method for producing a live non-human mammal progeny, from an isolated oocyte fertilized with a spermatozoid or sperm head damaged in the membrane, rehydrated, according to claim 1, characterized in that it comprises the steps of: mature, mature sperm from a non-human mammal; the suspension of the spermatozoa in a medium of physiological suspension; the freezing of the sperm suspension to form frozen sperm; dehydration of frozen sperm under vacuum to a moisture level of less than 1% to form lyophilized sperm; the rehydration of lyophilized sperm; the isolation of a spermatozoid or sperm head damaged in the membrane, rehydrated, which contains a competent nucleus for fertilization and which is capable, after rehydration, of fertilizing an isolated oocyte after the microinsertion to produce a living progeny; the isolation of an oocyte from a similar species of a non-human mammal; the insertion of the damaged sperm or sperm head into the membrane, within the isolated oocyte to form a fertilized oocyte; and allow the fertilized oocyte to develop into a living progeny.