Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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EXAMPLE 1
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
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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
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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.
EXAMPLE 2
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,
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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
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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
EXAMPLE 3
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
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EXAMPLE 4
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
EXAMPLE 5
ALLOGENEIC TRANSPLANTATION OF FRESH OVARIAN TISSUE
FOLLO1JVBD BY IN VITRO FERTILIZATION
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
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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
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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
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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)
EXAMPLE 6
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)
EXAMPLE 7
XENOGENEIC TRANSPLANTATION OF FRESH OR FROZEN OVARIAN
TISSUE FOL1LOWED 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 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.
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Ovarian No. Recipient Mice ReceivingNo. Embryo Collected
Rat
Tissue Ovarian Tissue
Fresh 3 0
Frozen 3 0
