CA2350592A1 - Method for the production of mammalian organisms - Google Patents

Abstract

A method is provided for enhancing the production of mammalian organisms and tissues thereof by preserving female and male mammalian reproductive tissues, and combining gametes derived from the preserved reproductive tissues to produce mammalian organisms or tissues, cells, or cellular constituents of the mammalian organisms. The method comprises preserving female ovarian tissue and testicular tissue by cryopreservation, or by transplantation into live female and male host mammals, and producing a mammalian organism or mammalian tissues by combining oocytes and spermatozoa derived from the cryopreserved or nonfrozen reproductive tissues. The method also provides for the use of cryopreserved or unfrozen spermatozoa for the artificial insemination or in vitro fertilization of oocytes derived from transplanted ovarian tissue. The method is used for the effective production of mammalian organisms or tissues, and may result in a "single-step rederivation" of the germ line of desired organisms or in rapid rederivation of desired tissues.

Description

METHOD FOR THE PRODUCTION OF MAMMALIAN ORGANISMS
Field of the Invention The invention relates to a method of producing mammalian organisms S and tissues by combining fennale and male gametes derived from reproductive tissues preserved by cryopreservation or transplantation into host mammals. More particularly, mammalian organisms or tissues are produced by combining (i.e., by allowing the animals to mate;, by artificial insemination, or by in vitro fertilization) oocytes derived from ovarian tissue that has been freshly transplanted into a female host mammal or cryopreserved and subsequently transplanted, with spermatozoa derived from testicular tissue that has been freshly transplanted into a male host mammal or cryopreserved and subsequently transplanted. Alternatively, oocytes from the ovarian tissue are combined, by artificial insemination or in vitro fertilization, with spermatozoa that have been cryopreserved or freshly isolated from male mammals.
Background and Summary of the Invention The preservation of mammalian reproductive tissues for the production of mammalian organisms and tissues is important for a number of uses, including the maintenance of human reproductive tissues for later use in production of offspring and the continuance of the germs line of important species of laboratory, agricultural, or wild animals. In the context of continuance of the germ line of important animal species, the capacity to preserve both female and male reproductive tissues and to combine gametes derived from preserved reproductive tissues to produce offspring avoids the time and expense; of maintaining the germ line by continuous breeding, and results in a one-step rederivation of the germ line avoiding the necessity of rederiving the germ line by extensive backcrossing. The need for effective methods of preserving both female and male reproductive tissues for the production of mammalian organisms and tissues has become more critical because of the rapid development of technologies for genetically engineering animals (e.g., transgenic animal technologies and techniques for directed DNA mutation) resulting in increased numbers of laboratory and agricultural animal lines. T'he maintenance of these genetic lines through standard breeding techniques is producing increasing and often intolerable strains on facilities and budgets. The preservation of mammalian reproductive tissues for the later production of organisms and tissues also has application in the fields of conservation biology (e.g., preservation of endangered species), and human clinical medicine (e.g., maintenance of human reproductive tissues for later use). For example, there is a need for preserving reproductive tissues of valuable species, such as endangered species, to maintain these species at times of risk of loss to protect biodiversity. An example, in human clinical medicine, of tlhe importance of developing effective methods for the preservation of mammalian reproductive tissues for the later production of offspring is the preservation of ovarian tissue of female oncology patients or male reproductive tissues of male oncology patients for later use by patients who face loss of ovarian or male reproductive function due to chemotherapy and/or radiation therapy.
Another important area related to preservation of female and male reproductive tissues is for use of these reproductive tissues in the production of organisms such as embryos, embryonic cells, fetuses, newborn animals, and adult animals, or their tissues. The; production of transgenic animals utilizes early one-cell embryos into which gene constructs are inserted. In addition, recent procedures to isolate totipotent or pluripotc:nt embryonic stem cells for specific cell lineages, as well as differentiated cells and tissues, such as pancreatic islets and dopaminergic neurons from later fetal stages, provide important cell and tissue transplantable biomaterials for the treatment of a multitude .of diseases (Thompson, J.A., Science 282, pp.
1145-1147, 1998). For example, isolation of hematopoeitic stem cells from mammalian embryos, __ fetuses, newborn animals, or adult animals, and their development into a cultured cell line, could provide a renewable alternative to bone marrow for the transplant treatment of oncology patients. Identilication and isolation of pancreatic islet stem cells of more developed fetal islet tissue could provide a source of materials for the treatment of insulin-dependent (Type 1 ) dliabetes. Similarly, identification and isolation of neuronal stem cells or more developed fetal neuronal tissue could provide a source of materials with which to treat Parkinson's or Alzheimer's patients (Thompson, J.A., Science 282, pp. 1145-1147, 1998). Finallly, analogous stem cells for cardiac tissue could be developed for the treatment of victims of myocardial infarction (Klug, M.G., J. Clin.
Inves. 98, pp. 216-224, 199Ei). Thus, there is a need for a reliable and cost-effective method of preserving mammalian reproductive tissues for the efficient production of mammalian offspring and tissues.
Various methods of cryopreservation of ovarian tissue, mature sperm cells and spermatogonial genm cell tissues, and embryos have been developed.
These methods employ cryoprotectants to protect reproductive cells or embryos from the high solute concentrations which develop as water precipitates as ice (Gwnasena, K.T., Human Repro. 12(1), pp. 101-106, 1997). These methods also employ controlled cooling of the reproductive cells or embryos with the method of cooling depending on the species {Critser, J.K., Fertility and Sterility 47(4), pp. 656-663, April 1987).
Current procedures for freeziing embryos from many mammalian species work with high efficiency, but are relatively costly. Recently, cryopreservation of ovarian tissue has been demonstrated to allow long-term preservation of reproductive tissues from mice (Gwnasena, K. T., Human Repro. 12( 1 ), pp. 101-106, 1997), but an effective method of preserving oocyte~s has not been developed. In an analogous manner, methods of cryopreservation of spermatogonial stem cells (sperm precursor cells) have recently been developed for the mouse (Brinster, R.L., U. S. Patent No.
5,817,453), but methods of cryopreservation of spermatozoa (mature sperm cells) currently are ineffective far many important laboratory species (e.g., mouse and rat).
Furthermore, in vitro fertilization and artificial insemination of oocytes utilizing cryopreserved sperm results in a fecundability rate (the monthly probability of conception) as low as 10 (Critser, J.K., Repro. Medicine Rev. 4, pp. 9-17, 1995).
A method of alogeneic transplantation of cryopreserved or fresh ovarian tissue from mice into the ovarian bursa of an immune-incompetent mouse (e.g.
an Athymic Nude nu/nu mouse) has been previously reported (Gwnasena, K.T., Human Repro. 12(1), pp. 101-106, 1997; Gunesena, K.T., HumanRepro. 57, pp. 226-231, 1997; Gunesena, K.T., Anim~xl Repro. Sci. 53, pp. 265-275, 1998). In these experiments, female mice with ovarian tissue transplants bore live offspring after mating with a male mouse that had not undergone transplantation, demonstrating that, after transplantation, cryopreserved or fresh ovarian tissue is capable of regaining both steroidogenic endocrine (production of steroid hormones) and gametogenic (production of oocytes) fiznction. However, litter size was reduced in transplanted animals compared to control animals and the percentage of matings resulting in offspring was low (about 10~%). In the same studies, xenogeneic transplantation of cryopreserved pieces of ovarian tissue (e.g., sheep to mice and elephants to mice) resulted in the development of antral follicles, but oocyte fertilization and live births were not attained. Similarly, allogeneic or xenogeneic transplantation of donor spermatogonial stem cells from a fertile testis to an infertile recipient mouse has been reported (Brinster, R.L., Pra~c. Natd. Acad. Sci. USA, 91, pp. 11303-11307, 1994;
Brinster, R.I,., Proc. Natl. A~cad. Sci. USA, 91, pp. 11298-11302, 1994). In the case of the allogeneic transplants, the donor cells established spermatogenesis in the seminiferous tubules of the host, and the recipient mouse, when mated with a female mouse that had not undergone transplantation, sired offspring. In the case of the xenogeneic transplants (rats to mice), rat spermatogenesis occurred, but oocyte fertilization and live births were not attained.
The invention. relates to a method of producing mammalian organisms or tissues by combining ovarian tissue transplantation and spermatogonia transplantation (with cryopre;served or freshly transplanted ovarian or testicular tissue) and one of three approaches to fertilization: 1 ) artificial insemination (with unfrozen or cryopreserved spermatozoa), 2) in vitro fertilization (with unfrozen or cryopreserved spermatozoa), ar 3) mating. Alternatively, ovarian tissue transplantation (with cryopre;served or freshly transplanted ovarian tissue) may be combined with one of two approaches to fertilization (i. e., in vitro fertilization or artificial insemination) using fresh or cryopreserved sperm.
The method of the present invention results in the production of mammalian organisms and tissues by utilizing procedures for combining gametes derived from preserved ar unfrozen female and male reproductive tissues. The female reproductive tissue comprises ovarian tissue and the male reproductive tissue comprises testicular tissue (e:.g., spermatogonial stem cells and spermatogonia) and spermatozoa. The female and male reproductive tissues are cryopreserved, preserved by transplantation into live animals, or are used unfrozen. In the cases where ovarian or testicular tissue is preserved, mammalian organisms and tissues are produced by combining gametes derived iirom the cryopreserved reproductive tissues after transplantation into host mammals, or from the reproductive tissues stored in live host mammals, by such processes as allowing animals to mate, in vitro fertilization, and -S-artificial insemination. In cases where spermatozoa are used, cryopreserved or freshly isolated sperm are combined, by artificial insemination or in vitro fertilization, with oocytes derived from ovarian tissue. Performance of the method involves harvesting ovarian tissue and testicular tissue from donor mammals, and preserving the tissues by S cryopreservation followed by transplantation into host mammals, or transplantation of unfrozen tissues into host mammals for storage. At a time when mammalian organisms or tissues are required, the oocytes produced in the female host mammal from the transplanted ovarian tissue are fertilized with spermatozoa produced in the male host mammal from the transplanted testicular tissue by such processes as allowing the female and male host mammals to mate, in vitro fertilization, or artificial insemination.
In an alternate embodiment of the invention, spermatozoa are collected from a donor mammal and may be cryopreserved or may be used unfrozen to fertilize, by such methods as artificial insemination or in vitro fertilization, oocytes derived from transplanted ovarian tissue. After implantation of the fertilized oocyte into a female host mammal, sufficient time is allowed for the development of the mammalian organism or its tissues, and the mammalian organism or its tissues are collected.
The method fizrther provides for the transplantation of ovarian tissue and testicular tissue into animals of the same species as the donor mammal (allogenenic transplantation) or into mammals of a different species (xenogeneic transplantation).
In cases of xenogeneic transplantation where the mammalian embryo cannot fully develop in the host mammal because of such differences as size, length of gestation, _. and type of placentation, embryos are harvested from the host mammal and placed in a recipient mammal of the same species as the mammalian organism or a gestationally similar species for further development. In cases of xenogeneic transplantation where the mammalian organism can fully develop in the host mammal, or in cases of allogeneic transplantation, the mammalian organism or its tissues are allowed to develop sufficiently in the host mammal species before collection.
In one embodiment of this invention, ovarian tissue is harvested from a female donor mammal and testicular tissue is harvested from a male donor mammal and the reproductive tissues .are cryopreserved for storage prior to thawing and transplantation into female and male host mammals of the same species as the donor mammals (allogeneic transpl;~ntation), or into female and male host mammals of a different species than the donor mammals (xenogenic transplantation). The oocytes produced in the female host mammal from the transplanted ovarian tissue are then fertilized by combination with spermatozoa produced in the male host mammal from the transplanted testicular tissue. Fertilization is accomplished by methods such as S allowing the female and male host mammals to mate, in vitro fertilization, and artificial insemination. After implantation of the fertilized oocyte in the female host mammal and allowing sui~cient time for the mammalian organism to develop, the mammalian organism or its tissues are collected. In cases where the donor species is too dissimilar to the host species to allow the embryo to fully develop in the host mammal, embryos are harvested from the host reproductive tract and may be utilized or allowed to develop after implantation into a recipient mammal of the same species as the embryo or a gestationally similar species. In cases where there is sui~cient similarity between the donor and host species, :live oi~spring of the tissue donor species can be derived by allowing gestation and parturition in the host mammal.
In another embodiment of this invention, ovarian tissue is harvested from a female donor marnm,al and testicular tissue is harvested from a male donor mammal, and, instead of storing the tissues by cryopreservation, the reproductive tissues are transplanted into female and male host mammals, respectively, for storage in the live animals. Allogeneic or xenogeneic transplantation can be performed. The oocytes and the spermatozoa produced from the transplanted tissues are combined by one of the methods described above. After implantation of the fertilized oocyte in a female host mammal, the mammalian organism is allowed to develop and the organism or its tissues are collected. In cases where the embryo cannot fully develop in the host mammal, embryos may be collected or embryo transfer may be performed as described above.
In an alternate embodiment of this invention, combinations of preservation methods are used. Ovarian tissue is harvested from a female donor mammal and is cryopreserved prior to transplantation into a female host mammal, and testicular tissue is harvested from a male donor mammal and is transplanted into a male host mammal for storage in the live animal. Alternatively, harvested female ovarian tissue is transplanted into a female host mammal for storage in the live animal, and harvested male germ cell ti;>sue is cryopreserved prior to transplantation into a male _'7_ host mammal. Allogeneic or xenogeneic transplantation can be performed. The oocytes produced in the female host mammal are fertilized with the spermatozoa produced in the male host rrca.mmal as described above, and the fertilized oocyte is implanted in a female host mammal, allowed to develop into a mammalian organism, and the organism or its tissues are collected. Embryo transfer may or may not be performed as required.
In another embodiment of this invention, ovarian tissue is harvested from a female donor mamunal and the ovarian tissue is either cryopreserved and subsequently transplanted into a female host mammal, or is directly transplanted into a live animal for storage. Simularly, testicular tissue is harvested from a male donor mammal and the male germ cell tissue is either cryopreserved and subsequently transplanted into a male host mammal, or is transplanted directly into a live animal for storage. Allogeneic or xenogeneic transplantation can be performed. In the case of the ovarian tissue transplants, the ovarian tissue can be transplanted to an orthotopic site (a site in the body where the tissue is normally located) in the female host mammal, or a heterotopic site (a site iin the body where the tissue is not normally located). In the case of heterotopic transplantation, the ovarian tissue transplant will be used only to store ovarian tissue, with or without the production of oocytes, and will not be used for development of the manunalian organism until subsequent transplantation or oocyte harvesting for in vitro fertilization. The oocytes produced by orthotopic or heterotopic transplants are then fertilized by combination with the spermatozoa produced from the - transplanted testicular tissue, and development and collection of the mammalian organism or tissues, or embryo transfer, is as described above.
In accordance with one other embodiment of this invention, there is provided a method of producing mammalian organisms or tissues thereof. The method comprises the steps of harvesting ovarian tissue from a female donor mammal, harvesting testicular tissue from a male donor mammal, preserving the ovarian tissue from the female donor mammal by either transplanting the ovarian tissue directly into a female host mammal or initially cryopreserving the ovarian tissue and subsequently transplanting the cryopreserved ovarian tissue into a female host mammal, and preserving the testicular tissue from the male donor mammal by transplanting the testicular tissue directly into a male host mammal or initially cryopreserving the _g_ testicular tissue and subsequently transplanting the cyropreserved testicular tissue into a male host mammal. The method further comprises the steps of fertilizing an oocyte produced in the female host mammal from the transplanted ovarian tissue with a spermatozoa produced in the male host mammal from the transplanted testicular tissue, allowing for the development of the fertilized oocyte to produce a mammalian organism, and collecting the mammalian organism or tissues thereof.
In accordance with still one other embodiment of the invention, there is provided a method of producing mammalian organisms or tissues thereof. The method comprises the steps of harvesting ovarian tissue from a female donor mammal, harvesting spermatozoa from a male mammal, preserving the ovarian tissue from the female mamanal, fertilizing an oocyte obtained from the preserved ovarian tissue with a spermatozoa from the male mammal by artificial insemination or in vitro fertilization, transferring the fertilized oocyte to a female host mammal for the development of a mammalian embryo or a mammalian offspring, and collecting the mammalian embryo I S or mammalian offspring, or tissues thereof.
Detailed Description of the Invention Specific language is used to describe several embodiments of this invention to promote an understanding of the invention and its principles. It must be understood that no specific limitation of the scope of this invention is intended by using this specific language. Any alteration and further modification of the methods or devices and any application of the principles of this invention that would normally occur to one skilled in this art are also intended.
The present invention relates to a method of producing mammalian organisms and tissues by using a combination of 1) a method of preserving ovarian tissue and testicular tissue, including cryopreservation followed by transplantation or direct transplantation into live animals, and 2) a method of producing mammalian organisms or tissues by combining gametes derived from the transplanted tissues by such processes as mating, in vitro fertilization, and artificial insemination.
An alternate embodiment of the invention utilizes cryopreserved or fresh sperm collected from a male mammal to fertilize, by artificial insemination or in vitro fertilization, oocytes derived from preserved ovaJrian tissue. The preservation of mammalian reproductive tissues is useful for such purposes as maintenance of human reproductive tissues for the later production of offspring, and reclaiming the germ line of important species of laboratory, agricultural, or wild animals without the necessity of breeding.
The mammalian organisms arid tissues produced are particularly useful for the isolation of important biomaterials.
The method of the present invention is utilized to produce at least one mammalian organism or tissues thereof including, but not limited to, a blastocyst, cells of a blastocyst, blastocyst cell constituents, an embryo, embryonic tissues, embryonic cells including embryonic stem cells, embryonic cell or tissue constituents, a fetus, fetal tissues, fetal cells, fetal cell or tissue constituents, a newborn organism, tissues of a newborn organism, cells of a~ newborn organism, constituents of the cells or tissues of a newborn organism, an adult organism, tissues of an adult organism, cells of an adult organism, and constituents o~f cells or tissues of an adult organism. The mammalian organism can also be a genetic variant produced by art-recognized genetic engineering techniques. The present mel:hod can be used to produce cells at early developmental stages, such embryonic stem cells, or cells at later developmental stages, such as fetal cells. The cells produced can be from any tissue, including, for example, neuronal cells, epithelial cells, muscle cells, cells of the liver, kidney, pancreas, heart, reproductive system, hematopoietic system, endocrine system, urinary system, alimentary canal, such as the oral and pharyngeal mucosa, the cells lining the stomach, small intestine, or large intestine, and cells of the respiratory system. The cellular constituents produced can ir.~clude, for example, DNA, RNA, nucleic acids, amino acids, peptides, proteins, glycoproteins, apoproteins, enzymes, enzyme inhibitors, coenzymes, hormones, neurotransmitters, antibodies, cytokines, lipids, phospholipids, vitamins, minerals, carbohydrates, prostaglandins, chromosomes, small organic molecules, and any other biological molecule associated with cells or tissues.
The donor mammals of the present invention are female mammals from which ovarian tissue is harvc;sted, or male mammals from which testicular tissue or spermatozoa (mature sperm cells) are harvested. The host mammals of the invention are female mammals into wtuch ovarian tissue harvested from female donor mammals is transplanted, female macmmals in which oocytes fertilized with spermatozoa, by processes such as in vitro fertilization or artificial insemination, are implanted, or male mammals into which testicul~~r tissue harvested from male donor mammals is transplanted. The present invention is applicable to any species of donor mammal with female mammals having ovarian tissue capable of producing oocytes following transplantation or with male mammals having spermatozoa or cells capable of differentiating into spermatozoa following transplantation. Thus, the present invention can be applied to all mammalian species, with the exception of the two egg laying mammalian species of the Order Monotremata. The donor mammals that can be utilized include, but are not liimited to humans, laboratory animals such as rodents {e.g., mice, rats, hamsters, etc.), rabbits, transgenic animals, monkeys, chimpanzees, dogs, cats, agricultural animas such as cows, horses, pigs, sheep, goats, wild animals or animals in captivity such a.s bison, deer, antelope, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, whales, marmosets, or any endangered mammal. The female donor mammal can be at any stage of development where ovarian tissue capable of producing oocytes following transplantation is present, and the male donor mammal can be at any stage of development where spermatozoa or cells capable of differentiating into spermatozoa following transplantation are present.
In the case of allogeneic transplantation, the donor mammal can be of the same species as the host mammal, or the dlonor and host mammal can be the same animal.
Alternatively, the donor and host mammals can be of different species (xenogeneic transplantation).
In accordance with the method of the present invention, ovarian tissue is harvested from a female donor mammal and testicular tissue is harvested from a male donor mammal for preservation by freezing followed by transplantation into host mammals, or for preservation by transplantation of unfrozen tissues into host mammals. Alternatively, in the case of spermatozoa, the gametes may be cryopreserved after harvesting or used unfrozen. Suitable ovarian tissues from the female donor mammal for transplantation into a host mammal include, but are not limited to, whole ovaries and pieces of ovarian tissue, or any ovarian tissue capable of producing oocytes following; transplantation into a female host mammal. The reproductive tissues from the male donor mammal for transplantation into a male host mammal can include, for ex~unple, totipotent stem cells, pluripotent stem cells, embryonic stem cells, gonocytes, spermatogonial stem cells, proliferative WO 00/32042 PCT/US99/t8059 spermatogoriia, and other sperm precursor cells or any other testis-derived cells capable of producing spermatozoa following transplantation into a male host mammal.
It is contemplated that the ovarian tissue from a female donor mammal and the testicular tissue or spermatoaoa from a male donor mammal can be harvested from the donor mammal by any art-recognized survival surgical procedure with or without anesthesia, surgical procedure with euthanasia, or non-surgical procedure.
Euthanasia techniques are species-specific and include such techniques as cervical dislocation in mice and COz asphyxiation in rats. Art-recognized techniques for harvesting testicular tissue (e.g., spermatogonial stem cells or spermatozoa) include such methods as surgical methods wherein the spermatogonia are isolated from a surgically removed testis.
The host mammals of the present invention harbor the transplanted tissue for storage and/or for the production of mammalian organisms or tissues. The host mammals can be either female or male mammals transplanted with reproductive tissues from donor mammals or female mammals in which oocytes fertilized with spermatozoa, by processes ,uch as in vitro fertilization or artificial insemination, are implanted. The present invention can be applied to host mammals including, but not limited to, humans, laboratory animals such rodents (e.g., mice, rats, hamsters, etc.), rabbits, transgenic animals, monkeys, chimpanzees, dogs, cats, agricultural animals such as cows, horses, pigs, sheep, goats, wild animals or animals in captivity such as bison, deer, antelope, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, whales, marmosets or an endangered mammal. In the case of allogeneic transplantation, the host mammal will be of the same species as the donor mammal, or the host and donor mammal can be the same animal. Alternatively, the host and donor mammals can be of different species (xenogeneic transplantation). Allogeneic host mammals may be selected based on various factors, including ease of handling, ease of breeding, expense, size, availability, and a variety of other factors.
Xenogeneic host mammals may be chosen for the same factors with size and cost of maintenance being particularly important considerations.
In a preferred embodiment of the present invention, the female host mammal is ovariectomized (the ovaries of the female mammal have been surgically removed). Similarly, it is contemplated, but not required, that the male host mammal utilized in the present invention is infertile. The male host mammal may be genetically infertile or infertility may result from destruction of the native germ cell population by physical processes, such as radiation, or by chemical treatment, or any other art-recognized method for destroying the endogenous male germ cell population. It is preferred, but not required, for application of the present invention that the host mammals be immuno-incompetent (e.g., SCID mice or nude mice) or at least immunodeficient to avoid rejc;ction of the transplanted tissues. In the case of xenogeneic transplantation, tranplantation of ovarian tissue or testicular tissue into an immuno-incompetent or immunodeficient host may be utilized to avoid rejection of the transplanted tissues. In the case of allogeneic transplantation, transplantation of ovarian tissue or testicular tissue into an immuno-incompetent or immunodeficient host is preferred, but not required., where the donor mammal is genetically altered (e.g., the donor mammal is a transgenic animal), and may be utilized where the donor mammal is not genetically altered.
In accordance with the method of the present invention, ovarian tissue and testicular tissue are preserved by cryopreservation followed by transplantation into host mammals for the production of mammalian organisms or tissues, or are preserved by transplantation of unfrozen tissues into live host mammals for storage until mammalian organisms or tissues are required. Spermatozoa may be cryopreserved or used unfrozen. Cryopreservation of ovarian tissue, testicular tissue, and spermatozoa can be performed by any art-recognized techniques, and may utilize cryoprotectants and controlled cooling.
An illustrative; procedure for cryopreservation of ovarian tissue, not intended to limit the scope ofthe application in any manner, utilizes dimethylsulfoxide as the cryoprotective agent, but any other cryoprotectant can be used including, but not limited to, ethylene glycol, propylene glycol, and glycerol. The tissue is held at room temperature in the cryoprotectant-containing solution for 5 minutes.
Vials containing the ovarian tissue in the cryoprotectant-containing solution are then placed in a programmable rate freezer and the temperature is cooled from 25°C
to 10°C at 1°C/minute, then to -7°C at a rate of 0.5°C/minute, and the temperature is held at -7°C
for S minutes. Ice nucleation is induced manually and the temperature is held at -7°C
for another 5 minutes. The temperature is then reduced to -55°C at a rate of WO 00/32042 PCT/US99/2$059 0.5°C/minute, and the vial is placed into liquid nitrogen and stored at -196°C.
Cryopreservation of male germ cell tissue (e.g., spermatogonial stem cells) or spermatozoa can be perfarmed by any art-recognized techniques utilizing cryoprotectants and controlled cooling. An illustrative procedure for controlled cooling of testicular tissue or' spermatozoa that is not intended to limit the scope of the application in any manner involves slow cooling from room temperature to 0°C, and then rapid cooling (e.g., 10 to 100°C/minute) to an intermediate temperature (e.g., between -40 and -80°C) before storing at -196°C.
Transplantation of ovarian tissue into a female host mammal and of testicular tissue into a male b.ost mammal is achieved, in the method of the present invention, by using any art-recognized survival surgical procedure with or without anesthesia. In a preferred embodiment of the invention, ovarian tissue from the female donor mammal is placed into the ovarian bursa of the female host mammal to achieve orthotopic transplantation for the production of mammalian organisms or tissues. In an alternate but equally preferred embodiment of the invention, it is contemplated that the site of transplantation in the host mammal will be used only to store ovarian tissue and will not be used for development of the mammalian organism. In this alternate embodiment, orthotopic or heterotopic transplantation can be performed with alternative heterotropic transplantation locations including, but not limited to, under the kidney capsule and subcutaneous transplantation. Subcutaneous transplantation is particularly practical in combination with ultrasound-guided oocyte aspiration. In another preferred embodiment of the invention, testicular tissue is introduced into the seminiferous tubules of the male host mammal by procedures including, but not limited to, injection into the vas deferens and epididymis or severing the seminiferous tubules inside the testicular covering and injecting testicular tissue into the seminiferous tubules. The host mammals receiving transplanted ovarian tissue or testicular tissue harbor the transplanted tissue for storage and/or for the production of mammalian organisms or tissues, and the host mammals can be any female or male host mammal of any developmental stage or species capable of harboring the transplanted tissue for storage and/or for the production of mammalian organisms or tissues.
The method of the present invention provides that the oocytes produced in the female host mammal from the transplanted ovarian tissue are fertilized with spermatozoa produced in the male host mammal from the transplanted testicular tissue by processes such as allowing the female and male host mammals to mate, artificial in vitro fertilization with implantation of a fertilized oocyte into a female host mammal, or artificial insemination in vivo. Oocytes may also be fertilized with spermatozoa, by processes such as in vitro fertilization and artificial insemination, collected from a male mammal of the appropriate germ line that ha.s not received a transplant of testicular tissue.. In vitro fertilization is performed by any art-recognized technique, and the technique is species-specific. Oocyte retrieval for in vitro fertilization rnay be accompliished by any technique known to those skilled in the art including, for example, ultrasound-guided oocyte aspiration and surgical exposure of the ovarian tissue, with or without removal of ovarian tissue from the female host mammal, followed by manu~d oocyte aspiration. In the case where ovarian tissue is removed from the female host mammal, the transplanted ovarian tissue can be replaced with the same or new ovarian tissue. The fertilized oocyte may be implanted in the female host mammal from which the oocyte was removed or into another female host mammal. Spermatozoa far use in in vitro fertilization or artificial insemination may be collected from male host mammals or male mammals that have not received a transplant of testicular tissue; by any art-recognized techniques including ejaculation and electro-ejaculation. Artificial insemination can be performed by any art-recognized survival surgical procedures with or without anesthesia, including, for example, intrauterine insemination and intra-oviductal insemination, or, alternatively, by any non-surgical techniques known to those skilled in the art such as vaginal insemination and cervical insemination. ~~ccording to the method of the present invention, following fertilization the fertilized oocyte is implanted in a female host mammal and sufficient time is allowed for the development of the mammalian organism or its tissues before collecting the mammalian organism or its tissues. The time sufficient for the development of the mammalian organism or its tissues can be from about 0 to about 3600 days.
The mammalian organism can be collected from the host mammal by birth or by using any art-recognized survival surgical procedure with or without anesthesia or a surgical procedure with euthanasia. In the case of the collection of cells, tissues, and constituents of cells or tissues, the organism may be removed from the host mammal or present within the host mammal when the cells, tissues, or constituents of cells or tissues are collected and the cells, tissues, or constituents of cells or tissues can be removed by any art-recognized techniques. The cells, tissues, or constituents of cells or tissues can be collected from a recently deceased organism provided that, in the case of cells or tissues, the cells or tissues are collected priorto cell death. Cellular constituents can be collected prior to or after cell death.
In some cases of xenogeneic transplantation, the method of the present invention provides for removal of the mammalian embryo from the female host mammal, and implantation of the embryo into a female recipient mammal of the same species as the embryo or a ge;stationally similar species for further development. A
recipient mammal is a female mammal into which an embryo from a host mammal is implanted for further development, and the recipient mammal may be of the same species as the embryo or a geatationally similar species. A gestationally similar species is a recipient mammal of a dilTerent species than the embryo, but with placentation and 1 S a gestation time compatible with required development of the embryo. It is contemplated that this step will be necessary in cases of xenogeneic transplantation where the mammalian organism cannot develop to the extent required in the host mammal because of such differences as size, length of gestation, and type of placentation. Embryo transfer according to the method of the present invention can be performed by any art-recognized survival surgical procedure with or without anesthesia. Alternatively, embryo transfer can be performed by any non-surgical _ techniques known to those skilled in the art, or by a combination of a survival surgical procedure and a non-surgical technique.
As shown by '.the following examples, the method of the present invention results in the production of mammalian organisms and tissues at a useful fecundability rate by utilizing oocytes derived from preserved ovarian tissue transplanted into allogeneic or xenogenic animals, and, thus, among other uses, allows for the preservation of the germ line of important species of laboratory animals.

COLLECTION AND CR;YOPRESERVATION OF DONOR SPERMATOZOA
AND ARTIFICIAL INSEMINATION USING SPERM
Collection of Donor spermatozoa. Donor spermatozoa are collected by a variety of methods dependiing upon the species. The examples given below are intended to serve as illustration only and are not intended to limit the scope of the overall application in any manner. Rodents (e.g., mice, rats): Spermatozoa from rodents are routinely collectf:d from the epididymides and vas differentia after euthanasia. The excised epididymides and vas diferentia are punctured and spermatozoa are released into a tissue culture medium. Cattle: Domestic bull sperm is routinely collected by using ~m artificial vagina (AV) or electroejaculation.
In the first approach, a bull is trained to mount a dummy device (simulating a cow) and the penis manually diverted into an AV into which the bull ejaculates. The sperm is then collected from a test tube placed at the end of the AV. Collection via electroejaculation is performed by placing the bull in a restraining chute and inserting a probe into the rectum. An electric current is administered through the probe, stimulating the reproductive tract directly below the rectum and the bull ejaculates.
The ejaculate is collected in .a container held over the protruding penis. S, been:
Domestic ram sperm is routinely collected using electroejaculation as described above.
Horses: Domestic horse sperm is routinely collected using an AV as described above.
Primates: Primate sperm a.re; often collected using a manual self masturbation _. approach (e.g., humans) or am electroejaculation approach. Swine: Domestic boar sperm is routinely collected using a "gloved hand" method. Similar to the AV
procedure described above for bulls, boars are trained to mount a dummy.
However, instead of diverting the penis into an AV, a technician grasps the boar penis and applies pressure, causing the boar to ejaculate into a collection container.
Spermatozoa Cryopreservation. Sperm may be cryopreserved using a wide variety of procedures dlepending upon the species. The examples given below are intended to serve as illustratiions and are not intended to limit the scope of the overall application in any manner. yodents (e.g., mice): Mouse sperm is typically placed into a balanced, buffered salt solution to which skim milk, chicken egg yolk (egg yolk) and glycerol are added (extender medium). Sperm are generally cooled slowly from room _ 17-temperature to 0 ° C and then cooled relatively rapidly (e. g., 10 to 100 ° C/min) to an intermediate temperature (e.;~., between -40 and -80°C) before being stored at a low temperature (usually in liquid nitrogen (LNz) at -196°C). Cattle: Bull sperm is typically placed in an extender medium, consisting of, for example, a balanced, buffered salt solution to which egg yolk and glycerol are added. The exact composition of the extender medium may vary and many different extenders are routinely used with good results. The bull sperm samples are cooled slowly from room temperature to about 0 ° C and then relatively rapidly (between 10 and 100 ° C/min) to an intermediate temperature (usually between -30 and -80°C) before being stored at a low temperature (usually in L N2). S, heen: Ram spermatozoa are typically cryopreserved by procedure.. similar to those described above for cattle.
Horses:
Horse sperm can be cryopreserved by procedures similar to those described above for cattle. Primates: Human spermatozoa is typically cryopreserved by placing the cells in a balanced, buffered solution. to which glycerol (3-10% V/V) and egg yolk is added.
The samples can be rapidly cooled (e.g., abruptly cooled) from room temperature to about to 0 ° C, then cooled at a relatively rapid rate (e. g., 10 to 100 ° C/min) to an intermediate low temperature (e.g., between -20 and -80°C) before storage at a low temperature (usually in LNZ). Swine: Boar sperm can be cryopreserved by procedures in which 1) the sperm is placed in an extender medium (as described above), or 2) the sperm is cooled slowly to 15 ° C and allowed to remain at 15 ° C
for several hours, then cooled slowly to about 0°C. Subsequently, the sperm are cooled rapidly (e.g., 100°C/min) to an intermediate temperature (usually between -30 and -80°C) before storage at a low temperature: (usually in LNZ).
Artificial Insemination in Mice. Approaches to artificial insemination (AI) in mice include insemination by vaginal, cervical, intrauterine and intra-oviductal routes: V~ final AI is performed using a speculum through which an inseminating pipette or cannula is passed into the vagina where the sperm is deposited.
Cervical insemination is performed u;>ing a method generally similar to that used for vaginal inseminations. A speculum its inserted into the vagina and the cervical os visualized. A
small diameter pipette or cmnula is passed through the speculum into the cervical os and the spermatozoa are deposited into the cervix. Intrauterine insemination is performed surgically with the mouse anesthetized as described below under ovarian tissue transplantation. Briefly, an incision is made thorough the skin and peritoneum, exposing the uterus. The sperm (2-5 x 104 sperm in 5-10 microliters) are inserted into the uterine lumen (one or both uterine horns) using a hypodermic needle and syringe containing the sperm or puncturing the uterine horns) using a hypodermic needle and then passing a pipette or canmula containing the sperm through the puncture.
Intra-oviductal insemination is another surgical approach to AI in the mouse. As with intrauterine insemination, with the female under general anesthesia, an incision is made thorough the skin and peritoneum, and exposing the female reproductive tract.
Sperm (2-S x 104 sperm in 2-S microliters) are inserted into the oviduct (one or both oviducts) using a pre-warmed hypodermic needle and syringe (a glass pipette or plastic cannula could also be used) containing the sperm.

TRANSPLANTATION OlF OVARIAN TISSUE FROM 101-Rl OR 129-CT MICE
INTO NL!/NU MICE FOLLOWED BY MATING
Collection of the donor ovarian tissue. Ovarian tissue is collected using either a survival surgical procedure or after euthanasia. Surgical and euthanasia (e.g., cervical dislocation in mice, COz asphyxiation in rats, etc.) procedures are species-specific. Using the mouse as an example, and 101-Rl or 129-CT mice in particular, in the case of survival surgery, anesthesia is induced with an intraperitoneal (IP) injection of ace promazine and Ketas~et (Fort Dodge Laboratories, Fort Dodge, Iowa) diluted with phosphate buffered saline (Gibco, Grand Island, NY) or other appropriate _. anesthesia methods (e.g., S '% 2-bromo-2-chloro-1, 1, 1-trifluoroethane and maintenance on 12% Halothane in oxygen).
Each ovary and surrounding fat pad is exposed through a small dorsolateral incision and kept moist with sterile HEPES-buffered Tyrode's lactate (TL-HEPES). A small slit is made in the ovarian bursa, and the ovary excised, taking care to remove all ovarian tissue. Excessive bleeding can be reduced by applying pressure with a sterile gauze until a clot forms. Ovaries are then removed from the bursa, and, whether for immediate (non-cryopreserved) or later (cryopreserved) transplantation are placed in a sterile tissue culture medium (e.g., TL-HEPES) at 37°C.
Immediate transplants are performed within 5-10 min. of removal (see transplantation section below). Whole ovaries may be processed in mice while in the case of larger species, pieces (approximately 1 mm x 1-1.5 mm) of the cortical region of the whole ovary is dissected and processed.
Ovarian tissue; cryopreservation. Ovaries are placed in a suitable cryogenic storage vessel (e.g. a 1.2 ml cryovial (Corning, Corning, N~ or a 0.25 or 0.5 ml plastic freezing straw) with 1 ml of 1-1.5 M dimethyl sulphoxide (DMSO}
or other cryoprotective agent (e:.g., ethylene glycol, propylene glycol, glycerol or the like (Sigma, St. Louis, MO)) in T°L-HEPES medium and held at room temperature for S
min. The vials are sealed, placed in a programmable rate freezer (e.g., Planer, Sunbury-on-Thames, UK), and cooled from 25 ° C to 10 ° C at 1 ° C/min, then at a rate of 0. 5 ° C/min. to -7 ° C, and held at -7 ° C for 5 min.
Ice nucleation is induced manually using precooled forceps and the temperature is held at -7°C for a further 5 min., for dispersion of the latent heat of fusion. The ovarian tissue is then cooled to -S 5 ° C at a rate of 0. 5 ° C/min., plunged into liquid nitrogen (LNz) at -19b ° C, and stored under LN2.
Ovarian tissue thawing. The ovarian tissue is thawed by removing the cryovials from LNz and placing the vials at room temperature (e.g., 20-22°C) until all visible ice has melted (15-20 min.). The tissue is then transferred to 5 ml of fresh culture media (e.g., TL-HEPES) at room temperature for 10 min. with gentle shaking to promote ei~lux of the cryoprotectant from the tissue. The ovarian tissue is placed in tissue culture medium at 37 ° C: until used for transplantation. Using this approach, the actual thaw rate is approximately 5 0 ° C/min for the first 2 min., 9 ° C/min. for the next 8 min, and 1 ° C/min for the last 10 min. at which point all ice has melted.
Ovarian tissue transplantation. With the recipient mouse under anesthesia, fresh or frozen and thawed ovarian tissue is placed into the ovarian bursa after removal of the native ovarian tissue as described above. The bursa is secured with a suture (e.g., 8-0 vicryl suture from Ethicon, Somersville, NJ), the fat pat is replaced, and the incision closed. In the case of mouse-to-mouse transplants, a whole or half ovary may be placed into the recipient bursa. In the case of larger species, pieces of ovarian tissue are used (e.g., 1 mm x 1-1.5 mm pieces).
After ovarian. tissue collection from 101-Rl or 129-CT mice, the tissues were transplanted into immumo-incompetent nude mice (Nu/Nu), with or without cryopreservation, and oocytes derived from the transplanted ovarian tissues were fertilized by allowing the female mammals to mate with nude (Nu/Nu) male mice.
-Sham-operated (Sham) and ovariectomized (Ovex) mice were used as control animals.
Ovarian No. RecipientStrain No. Preg No. DeliveredNo. Pups Tissue Frozen 3 101-R1 2 1 2 Frozen 3 129-CT 1 I 2 Fresh 4 ICR 2 2 7 Sham 4 nude 3 3 15 Ovex 4 nude 0 0 0 ALLOGENEIC TRANSPLANTATION OF FRESH OVARIAN TISSUE
FOLLOWED BY ARTIFICIAL INSEMINATION
The same general procedures for ovarian tissue transplantation and artificial insemination were followed as set forth in examples 1 and 2. After ovarian tissue collection from immunocompetent mice, the tissues were transplanted into nude mice (Nu/Nu), without cryopreservation, and oocytes derived from the transplanted ovarian tissues were fertilized by artificial insemination with fresh or frozen sperm.
Either an infra-oviductal or iintrauterine route was used for artificial insemination.
-. No. Pregnant Sperm Site of InseminationNo. RecipientMice (%) No. Pups Fresh Intra-oviductal4 1 (25) 4 insemination Frozen Intra-oviductal3 0 (0) 0 insemination Fresh Intrauterine 4 1 (25) 5 insemination Frozen Intrauterine 5 1 (20) 4 insemination ALLOGENEIC TRAl'JSPLANTATION OF FROZEN OVARIAN TISSUE
FOLLOWED BY ARTIFICIAL INSEMINATION
The same general procedures for ovarian tissue transplantation and artificial insemination were f=ollowed as set forth in examples 1 and 2. After ovarian tissue collection from 101-R.l or 129-CT mice, the tissues were transplanted into nude mice (Nu/Nu) after cryopreservation, and oocytes derived from the transplanted ovarian tissues were fertilized by artificial insemination with frozen sperm.
Mouse No. Pregnant Strain No. Recipient Mice No. Pups (%) 101-R1 3 0 (0) 0 l29-CT 3 0 (0) 0 ALLOGENEIC TRANSPLANTATION OF FRESH OVARIAN TISSUE

Oo rte Retrieval Procedures. Oocyte retrieval (OR) can be accomplished using several approaches. The examples given below are intended to serve as illustrations are not intended to limit the scope of the overall application in any manner. In many cases, the ovarian tissue host is an immune-incompetent mouse (e.g., an Athymic Nude or a SCII) mouse). The ovarian tissue is placed within the host's ovarian bursa (as described above) and Grafian (fluid filled) ovarian follicles develop.
One approach to OR is to place the host under anesthesia and use ultrasound methods to identify the size and location of the ovarian follicles. Once identified, these follicles a.re aspirated using ultrasound-guided insertion of a retrieval needle through the peritoneum and into the follicle lumen. Once inside the follicle lumen, negative pressure is applied and the follicular fluid and cells from the follicle wall lining (including the oocyte surrounded by the corona and cumulus cells) are recovered into a collection vessel (usually a conical test tube). Tissue culture medium (as described above) is then introduced and removed several times, creating a "washing"
action to increase the likelihood that retrieval of the oocyte is accomplished.
Alternative -z2-approaches include surgical exposure of the ovarian tissue in situ {e.g., within the host's ovarian bursa) and manual aspiration of the tertiary, fluid-filled follicles on the surface of the ovarian tissue transplant, or unilateral or bilateral surgical removal of the ovarian tissue from the host followed by manual follicle aspiration as described above.
In the latter case, new ovarian tissue could be replaced into the bursa following removal of older, tertiary follicle-containing tissue.
In Vitro Fertilization Procedures. In vitro fertilization (IVF) conditions are species-specific and may vary within a given species. The examples given below are intended to serve as illustrations only and are not intended to limit the scope of the overall application in any manner. Oocytes and spermatozoa are obtained as described above. Mice: Oocytes can lbe placed into 25-50 microliter drops of tissue culture medium (e.g., Whitten's medium, Ham's F10 medium, or M199 medium) at 10-20 oocytes per drop under washed oil. A small aliquot (5-15 microliters) of sperm is added so that the final sperni concentration is between 1 x 104 to 2 x 105/ml.
The oocyte-sperm mixtures are incubated for 3-6 hours at 35-37°C. After 3-6 hours, normally fertilized oocytes will contain two pronuclei which are clearly visible through the transparent cytoplasm oiFthe ooplasm of this species. Oocytes containing fewer or more than two pronuclei are: not normally fertilized. Cattle: IVF in cattle is performed in a manner similar to mouse IVF. A sperm concentration of 0.5-1 x 106 sperm/ml of tissue culture medium (e.g., TALP ) containing the oocytes will, within 12-18 hours of co-incubation at 3 9 ° C, result in 60-70% normal fertilization rates as assessed by the _. presence of two polar bodies (pronuclei cannot be seen in the cow oocyte due to the dark, lipid dense nature of tlhe ooplasm). Sheen: Current IVF procedures in sheep are very similar to those used fcrr cattle. Oocytes are co-incubated with approximately 1 x 106 sperm/ml in 25-50 microliter drops of tissue culture medium (e:g., MEM, SOF, HSOF) for 12-20 hours at 3 9 ° C. The fertilization rates achieved by using this approach are 40-60% of normal as assessed by the presence of two polar bodies (pronuclei cannot be seen in the ewe oocyte due to the dark, lipid dense nature of the ooplasm). Mare: In vitro fertilization of horse oocytes is more difficult and less well developed than in the species described above. To date, while IVF can be achieved using methods similar to those described for cattle and sheep above, capacitation of the spermatozoa with calcium ionophore has also been used. Primates: In vitro fertilization has been achieved in several species of primates including the lowland gorilla, the Rhesus monkey, and is routinely performed in the human. Using the human as an example of this group, IVF procedures for the human utilize sperm-egg co-culture systems similar to those described above. Oocytes are placed in small (20-50 mucroliter) drops and 5-10 nucroliters of sperm are added in a final concentration of between 1 x 104 to I x 105/n~l. The sperm and oocytes are co-incubated for approximately 14-16 hours at 37°C and normal fertilization is determined by visualization of two pronuclei (as in the mouse, the pronuclei can be seen in human zygotes). Swine: Methods for IVF in the pig utilize sperm concentrations between 1 x 105 and 1 x 10'/ml in the sperm-egg co-incubation system. The gametes are co-incubated in tissue culture medium (e.g., NCSU, TALP) for 5-14 hours at 37°C and normal fertilization can be estimated by the incidence of two polar bodies (as with cattle, pronuclei cannot be visualized in the pig oocyte).
Embryo Transfer Procedures. In vitro fertilization conditions are species-specific and may vary within a given species. The examples given below are intended to serve as illustrations only and are not intended to limit the scope of the overall application in any manner. Mice: Embryo transfer is performed with the recipient under anesthesia and the embryos (e.g., 10-IS) are placed surgically either into the oviducts (if the embryos are at an early stage of development) or into the uterus (later stage embryos). Cattle: Embryo transfer is performed non-surgically with the recipient standing in a restraining chute. An insemination device loaded with an embryo is passed through the cervix of the cow and into one uterine horn where the embryo is deposited. Sheep: Embryo transfer is often performed surgically as described for the mouse. Horses: Embryo transfer is performed non-surgically as described above for the cow. Primates: A non-surgical method is often used in which the embryos) is loaded into a flexible cannula and passed through the cervix into the uterus using ultrasound-guided direction.
The same general procedures for ovarian tissue transplantation were followed as set forth in example 2. After ovarian tissue collection from immunocompetent mice, th<; tissues were transplanted into nude mice (Nu/Nu), without cryopreservation, and oocytes derived from the transplanted ovarian tissues were fertilized by in vitro fertilization with unfrozen sperm. Nine zygotes resulted from the oocytes with two pronuclei, and the zygotes were transferred to one pseudopregnant female mouse and five pups were born.

No. RecipientRecip. No. Oocyte No. Oocyte Sperm Mice; Ovulated Collected with 2PN
(%) Fresh 4 3 13 9 (70) XENOGENEIC TRANSPLANTATION OF FRESH OR FROZEN OVARIAN
TISSUE FOLLOWED BY IN VITRO FERTILIZATION
The same general procedures for ovarian tissue transplantation and in vitro fertilization were followed as set forth in examples 2 and 5. After ovarian tissue collection from immunocom.petent rats, xenogeneic transplantation was performed by transplanting the ovarian tissues into nude mice (Nu/Nu). The rat ovarian tissues were 1 S cryopreserved or were transplanted without freezing, and oocytes derived from the transplanted ovarian tissues were fertilized by in vitro fertilization with fresh sperm.
Eleven embryos resulted from the fourteen oocytes with two pronuclei.
Ovaria No. Recipient Recip. No. Oocyte No. Oocyte lVfice n Receiving Rat Ovulated Collected with 2PN
(%) Tissue Ovarian Tissue Fresh 5 2 I 1 8 (72) _. Frozen 6 2 11 6 (55) XENOGENEIC TRANSPLANTATION OF FRESH OR FROZEN OVARIAN

The same general procedures for ovarian tissue transplantation and artificial insemination were followed as set forth in examples 1 and 2. After ovarian tissue collection from immunocompetent rats, xenogeneic transplantation was performed by transplanting the ovarian tissues into nude mice (Nu/Nu). The rat ovarian tissues were cryopreserved or were transplanted without freezing, and oocytes derived from the transplanted ovarian tissues were fertilized by artificial insemination with fresh sperm.

Ovarian No. Recipient Mice ReceivingNo. Embryo Collected Rat Tissue Ovarian Tissue Fresh 3 0 Frozen 3 0

Claims (17)

CLAIMS:

1. A method of producing mammalian organisms or tissues thereof comprising the steps of:
a. harvesting ovarian tissue from a female donor mammal;
b. harvesting testicular tissue from a male donor mammal;
c. preserving the ovarian tissue from the female donor mammal by either transplanting the ovarian tissue directly into a female host mammal or initially cryopreserving the ovarian tissue and subsequently transplanting the cryopreserved ovarian tissue into a female host mammal;
d. preserving the testicular tissue from the male donor mammal by transplanting the testicular tissue directly into a male host mammal or initially cryopreserving the testicular tissue and subsequently transplanting the cryopreserved testicular tissue into a male host mammal;
e. fertilizing an oocyte produced in the female host mammal from the transplanted ovarian tissue with a spermatozoa produced in the male host mammal from the transplanted testicular tissue;
f. allowing for the development of the fertilized oocyte to produce a mammalian organism; and g. collecting the mammalian organism or tissues thereof.

2. A mammalian organism or tissues thereof produced by the method of claim 1.

3. The method of claim 1 wherein the ovarian tissue and the testicular tissue are both initially cryopreserved and thereafter transplanted.

4. The method of claim 1 wherein the ovarian tissue is preserved by direct transplantation into a female host mammal, and the testicular tissue is preserved by direct transplantation into a male host mammal.

5. The method of claim 1 wherein the ovarian tissue is initially cryopreserved and thereafter transplanted and the testicular tissue is preserved by direct transplantation into a male host mammal.

6. The method of claim 1 wherein the ovarian tissue is preserved by direct transplantation into a female host mammal and the testicular tissue is initially cryopreserved and thereafter transplanted into a male host mammal.

7. The method of claim 1 wherein the ovarian tissue from the female donor mammal is transplanted into a female host mammal of the same species and the testicular tissue from the male donor mammal is transplanted into a male host mammal of the same species.

8. The method of claim 1 wherein the oocyte is fertilized with a spermatozoa produced in the male host mammal by a process selected from the group consisting of mating of the female host mammal and the male host mammal, artificial in vitro fertilization, and artificial insemination in vivo.

9. The method of claim 1 wherein the ovarian tissue from the female donor mammal is transplanted into a female host mammal of a different species than the female donor mammal and the testicular tissue from the male donor mammal is transplanted into a male host mammal of the different species.

10. The method of claim 9 wherein an oocyte produced in the female host mammal is fertilized with a spermatozoa produced in the male host mammal by a process selected from the group consisting of mating of the female host mammal and the male host mammal, artificial in vitro fertilization, and artificial insemination in vivo.

11. The method of claim 10 wherein the mammalian organism is removed from the female host mammal and implanted in a female recipient mammal of the same species as the mammalian organism or a gestationally similar species for further development.

12. A method of producing mammalian organisms or tissues thereof comprising the steps of:
a. harvesting ovarian tissue from a female donor mammal;
b. harvesting spermatozoa from a male mammal;
c. preserving the ovarian tissue from the female mammal;
d. transplanting the ovarian tissue from the female mammal;

e. fertilizing an oocyte obtained from the transplanted ovarian tissue with a spermatozoa from the male mammal by artificial insemination or in vitro fertilization;
f. transferring the fertilized oocyte to a female host mammal for the development of a mammalian embryo or a mammalian offspring; and g. collecting the mammalian embryo or the mammalian offspring, or tissues thereof.

13. The method of claim 12 wherein the ovarian tissue is preserved by either directly transplanting the ovarian tissue into a female host mammal or initially cryopreserving the ovarian tissue and subsequently transplanting the cryopreserved ovarian tissue into a female host mammal.

14. The method of claim 12 wherein the spermatozoa are initially cryopreserved.

15. The method of claim 12 wherein the ovarian tissue is preserved by transplantation into a female mammal of the same species as the female donor mammal.

16. The method of claim 12 wherein the ovarian tissue is preserved by transplantation into a female mammal of a different species than the female donor mammal.

17. The method of claim 12 wherein the mammalian organism is removed from the females host mammal and implanted in a female recipient mammal of the same species as the mammalian organism or a gestationally similar species for further development.