Note: Descriptions are shown in the official language in which they were submitted.
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PATENT
IN VITRO ~TUR~TION OF BOVINE OOCYTES
Bac]cground of the Invention
In vitro maturation of mammalian oocytes has been -~
studied for many years. The ova have been removed from
the follicles of the mammals and observed to resume
meiosis spontaneously in vitro. Edwards, "Maturation In
Vitro of Mouse, Sheep, Cow, Pig, Rhesus Monkey and Human
Ovarian Oocytes", Nature, Vol. 208, pp.349-351 ~1965);
Leibfried and First, "Characterization of Bovine Folli-
cular Oocytes and Their Ability to Mature In Vitro", J. ~ -
Animal Sci., Vol. 48, pp.76-86 (1979).
With techniques being developed for in vitro
fertilization as well as the more recent nuclear trans-
fer techniques in bovine embryos, a source of readily
available oocytes would compliment the procedures
available for genetic selection and breeding. Immature
ova have been collected from bovine ovaries at abba-
toirs, and the follicles were aspirated to release
cumulus-oocyte complexes. The oocytes have been tested
for maturation in media containing hormones and granu-
losa cells. The interaction of granulosa cells with
cumulus complexes have been determined to contribute to
developmental competence of bovine oocytes. See e. g.
Leibfried-Rutledge et al, "Development Potential of
Bovine Oocytes Matured In Vitro and In Vivo", Biol.
Reprod., Vol. 36, pp.376-384 (1987); Critser et al,
"Acquisition of Developmental Competence During
Maturation In Vitro", Theriogenology, Vol. 25, No. 1,
p.l50 (1986); Critser et al, "Influence of Cumulus Cell
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Association During In vitro Maturation of sOvine Oocytes
on Embryonic Development", siol. Reprod., Vol. 34
(Suppl.1), p.192 (1986).
Investi~ators have reported varying results using
in vitro fertilization to test the developmental
competence of the oocytes. Successful in vitro
maturation systems for bovine oocytes yiQlding fully
developed embryos use granulosa cell co cultures and
estrous cow serum. However, the efficiency of this
procedure is variable. Lu et al, "Pregnancy Established
in Cattle by Transfer of Embryos Derived from In Vitro
Fertilization of Oocytes Matured In Vitro", Vet. Rec.,
Vol. 121, pp.259-260 (1987); Fukui and Ono, "In Vitro
Development to Blastocyst of In Vitro Matured and
Fertilized Bovine Oocytes", Vet. Rec., Vol. 122, p.282
(1988).
The maturation of both the nuclear material and
cytoplasm appears to be important. Activation is the
term used for the initiation of the developmental
program which normally occurs at the time of fertiliza-
tion. Activation can be induced by electropulsation
without fertilization of the oocyte. Control over
activation is important in nuclear transfer to optimize
the time to implant the transferred nuclear material
which incorporates into and "reconditions" in the
presence of recipient maturing ooplasms. The subsequent
development is intended to reflect the genetic make-up
of the transferred nuclear material after insertion into
an enucleated egg. A suitable supply of immature bovine
oocytes exists and is inexpensive. A reliable in vitro
maturation procedure would give synchronous and
measurable oocyte development. Therefore, a mature
supply of oocytes would be available for use for genetic
selective breeding or cloning.
In vitro matured oocytes for nuclear transfer in
cattle has been reported, although embryonic development
was limited. Prather et al, "Nuclear Transplantation in
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the Bovine Embryo: Assessment of Donor Nuclei and
Recipient Oocytes", Biol. Reprod., Vol. 37, pp.859-866
(1987). The oocytes were aspirated from 1-5 mm folli-
cles. The culture medium contained ovine luteinizing
hormone (NIADDK oLH-24, 0.012 units/ml), ovine follicle-
stimulating hormone (NIADDK oFSH-15, 0.01 units/ml), and
estradiol-17B (1 ug/ml). The maturation time was 21 to
27 hours. The hormones used are naturally occurring
purified product which contains some comtaminants.
However, maintenance of pregnancy from an in vitro
matured ooctye was not reported by Prather et al.
Summary of the Invention
The present invention is a new method for in vitro
maturation of hovine oocytes. The process can be used
to produce oocytes for nuclear transfer, and pregnancies
have been confirmed.
Bovine ovaries are collected at abattoirs. Compact
cumulus oocyte complexes (COC) were selected preferably
from the aspirate of 3-8 mm antral follicles. The COC
were placed in a maturation medium containing the
gonadotropins luteinizing hormone (LH) and follicle-
stimulating hormone (FSH), both purified and recombinant
LH and FSH were used. The medium also contained estra-
diol.
In vitro fertilization was performed with oocytes
matured with (1) recombinant FSH and LH, and (2) only
the recombinant FSH to determine the activity of the
recombinant hormones. These oocytes were not used in
nuclear transfer. For the oocytes used in nuclear
transfer and other procedures, the cumulus cells were
stripped after 20 to 24 hours. The oocytes were incubat-
ed in a co-culture of medium plus bovine oviductal
cells. The oocytes used for nuclear transfer were aged
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from 26 to 43 hours after initial placement in the
maturation medium.
The procedure developed for bovine oocyte in vitro
maturation with the co-culture feature is successful for
use in the nuclear transfer process to produce cloned
animals. The use of unique recombinant gonadotropins is
another feature of the new process to mature the bovine
oocytes.
Detailed Description of the Invention
The following is a description of the process and
methods of the invention including the best mode. There
may be modifications made to some of the steps used to
practice the invention that are apparent to those
skilled in the art.
The bovine ovaries were collected at abattoirs and
maintained and transported in Dulbeccos PBS at 32 to
39C. The collection and transport time ranged from 1.5
to 3.75 hours past death. The compact cumulus oocyte
complexes ~COC) were selected from the aspirate of
antral follicles. Some follicles were dissected as
discussed below.
The follicle size was investigated as to the best
source of oocytes. The most useful range is 3-8 mm.
The preferred follicle size determined to give the best
oocytes for further developmental competence were 6-8mm.
Although the 1-2mm follicles yielded some oocyctes that -
would mature to develop to morula and blastocyst stage
after in vitro fertilization, the development was at a -
- . . ~
lower frequency. The follicle size was examined in
dissected ovaries because 2mm and 3-5mm follicles are ~-
difficult to distinguish when measured at the ovarian
surface.
The COC were washed three times in Tyrodes-Hepes -~-
medium (Bavister, et al, "Development of Preimplantation
Embryos of the Golden Hamster in a Defined Culture
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Medium", Biol. Reprod., Vol. 2~, pp.235-247 (1983))
which is incorporated hy reference herein, containing
3mg/ml bovine serum albumin and antibiotic 1~ penicillin
streptomycin solution (Gibco). After washing, the COC
are placed in the preferred maturation media of Tissue
Culture Medium (TCM) 199 supplemented with 10% heat
inactivated fetal calf serum (HTFCS) and 0.01 units/ml
bFSH (recombinant FSH) resuspended in BSA and mannitol
solution which is equivalent to 0.01 units of
NIADDK-oFSH standard. The recombinant FSH was obtained
from Integrated Genetics, Inc., Chappel et al, "Bovine
FSH Produced by Recombinant DNA Technology", Theriog.,
Vol. 29, No. 1 (1988). Esch et al, "Cloning And DNA
Sequence Analysis of The DNA for The Precursor of The
Chain of Bovine Follicle Stimulating Hormone", Proc.
Nat'l Academy Sci. USA, Vol. 83, pp.6618-6621 (1986).
The NIADDK-oFSH is a purified natural FSH from the
National Institute of Arthritis, Diabetes and Digestive
and Kidney Diseases (NIADDK), Baltimore, Maryland. The
maturation media also contained lug/ml estradiol and 1%
penicillin streptomycin solution. The COC are placed in
the maturation media for at least 20 hours.
A comparison of other alternative maturation media
was performed by subjecting the oocytes from the dif-
ferent media to in vitro fertilization and monitoring
subsequent development. The maturation media described
above was supplemented with 0.01 units/ml bLH (recombi-
nant LH from Integrated Genetics, Inc.) resuspended in ;~
BSA and mannitol solution which is equivalent to 0.01 --~
units of NIADDK-oLH standard. The NIADDK-oLH is a
purified natural LH from the National Institute of
Arthritis, Diabetes and Digestive and Kidney Diseases,
Baltimore, Maryland. Alternative commercially available
FSH and LH such as pituitary FSH (pFSH) and pituitary LH
(pLH) can be used. ;~
The oocytes were matured in TCM 199, lug/ml
estradiol and either 0.01 units/ml of bFSH or 0.01 and
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0.012 units/ml of bFSH and bLH, respectively, supple-
mented with 10% HTFCS. The oocytes were cultured at
39C 5~ CO2 and air. The oocytes were then assayed for
developmental competence by in vitro fertilization
followed by culture in ovine oviducts for six days. The
addition of bLH adversely affected developmental compe-
tence of in vitro matured oocytes although there was
some development to the blastocysts and morula stage.
TABLE 1 is a summary of the ln vitro fertilization
results with use of the recombinant gonadotropins.
TABLE 1
RECOMBINANT GONADOTROPINS
IN VITRO FERTILIZATION
Development to
Morula or Blastocyst
Media Fertilization (%) and (%)
bFSH 42/50 (84%) 24/75 (32~)
bFSH + bLH 38/51 (74.5%) 21/128 (16.4%J
In addition, the use of recombinant gonadotropins
bFSH and bLH in maturation media was compared to the
media with the natural gonadotropins oFSH and oLH
supplied by NIADDK. Maturation was carried out in a
maturation culture media TCM 199 with 10% HTFCS, 1%
penicillin and streptomycin, 1 ug/ml estradiol, 0.01
units/ml of NIADDK-oFSH, 0.012 units/ml of NIADDK-oLH in
one media preparation and the equivalent amounts of bFSH
and bLH in another media preparation. Bovine oocytes
were matured as described above and subjected to in
vitro fertilization. The fertilized oocytes were
. .
cultured in ovine oviducts and a development to morula
or blastocysts was comparable.
The following TAB~E 2 shows the results of the
NIADDK gonadotropins and the recombinant gonadotropins.
The recombinant gonadotropins compare favorably to the
NIADDK standards.
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TABLÆ 2
COMPARISON OF GONADOTROPINS
Development to
Morula or Blastocyst
MediaFertilization (~) and (%)
NIADDK
Gonadotropins 26/33 (78.7~) 46/103 (44.6%)
Recombinant
Gonadotropins 22/30 (73.3%) 26/77 (33.7%)
In vitro oocytes were placed in maturation culture
containing both oFSH and oLH to study the ability to
activate. Activation was defined as the completion of
meiosis and the progression of a metaphase II oocyte to
a pronuclear egg. The ln vitro matured oocytes were
removed from the maturation culture media over intervals
of 26 to 32 hours as shown in TABLE 3. The oocytes were
electropulsed under the same conditions as those used
for fusion according to "Bovine Nuclear Transplanta-
tion", cited below and incorporated by reference. The ~ -
following data shows that oocytes 30 hours and older
have increased activation potential. The results of the
activation procedure are shown below in TABLE 3
TABLE 3
OOCYTE ACTIVATION
Oocyte Age Metaphase II Pronuclear Lysed
2615/38 (39%) 18/38 (47%) ~/38 (13%)
2815/39 (3~%) 24/39 (61%) 1/39 ( 2%)
302/32 ( 6%) 26/32 (81%) 4/32 (12%)
321/34 ( 3%) 27/34 (79%) 6/34 (17
Additional aging beyond the period required for
acquisition of activation competence (30 hours post
introduction into maturation medium) was found to
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enhance the developmental potential of the in vitro
matured oocytes used in nuclear transfer.
The ln vitro oocyte maturation process which
sustains nuclear transfer and embryonic development
includes a further co-culture procedure. The COC were
removed from the maturation media after 20 to 24 hours,
preferably 22 hours, and stripped of cumulus cells by
vortexing (Vortex Genie 2; shake setting #8) for 2
minutes, 15 seconds in 2ml of Tyrodes-Hepes medium in a
15ml conical tube. About 15 to 30 denuded oocytes of
medium color with a polar body were selected and placed
into 23ul microdrops and co-cultured with bovine oviduc-
tal cells. The polar bodies were visualized with a
dissecting microscope 60-120X. The oviductal cell
co-culture consisted of 3ul of packed oviductal cells in
20ul of co-culture media TCM 199 with 10% HTFCS and 1%
penicillin and streptomycin.
The bovine oviductal epithelial cells were collect~
ed from oviductal flushings 36 to 48 hours, preferably
36 hours, after injection of HCG. The preferred oviduc-
tal cells for co-culture are predominantly elongated, -
multicellular, ciliated clumps. The oviductal cells
were preconditioned in the co-culture media for 22 to 24
hours prior to the addition of the oocytes. The hand-
ling time is 1 to 2 hours prior to introducing the
selected oocytes with the co-culture. The oocyte-ovi-
ductal cell co-cultures were then incubated at 39C 5%
C2 for about 3 to about 5 hours. ;~
The co-culture is supplemented with an equal volume
of fresh TCM 199 with HTFCS between 3 to 5 hours after ; -
initiating of the co-culture. The co-culture with
oocytes was incubated up to 19 hours. Some oocytes were
withdrawn at earlier intervals.
Nuclear transfer was performed on oocytes that had ~
been incubated in maturation culture with recombinant -
bFSH and bLH and co-culture. The oocytes were matured
in maturation media for 20 to 24 hours, preferably 22
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hours, co-cultured for 3 hours prior to micro-manipula-
tion and then cultured an additional 2 hours in TCM 199
with 10% HTFCS prior to fusion.
Other groups of cells remained in the co-culture
for 17 to 19 hours before micro-manipulation for nuclear
transfer, fusion and embyronic culture. A comparison
was made on the cells in co-culture for about 3 hours
to about 5 hours.
The micro-manipulation procedure to enucleate the
matured oocytes and transfer nuclear material takes
about 4 hours. The nuclear transfer procedure was
followed according to "Bovine Nuclear Transplantation",
International Application No. PCT/US 88/01906, Interna-
tional Publication No. W088/091816, 15 December, 1988,
which published patent application is incorporated by ; -
reference herein. -
The preferred method of micro-manipulation in
addition to the general technique disclosed in "Bovine
Nuclear Transplantation" includes a staining procedure
to visualize the female chromatin. The preferred
technique provides for removing about 1/2 of the ooplasm
from an oocyte and placing the removed ooplasm into a
foreign zona pellucida creating two egg halves each with
a surrounding zonae. One half should include the female
chromatin and one-half should not. With light micro-
scopy, it is impossible to discern the enucleated half
without chromatin which is the preferred recipient egg
half for the donor nuclear material.
In the preferred procedure, the in vitro matured
oocyte halves are placed in phosphate saline supplement-
ed with 0.4% BSA, 1~ antibiotic penicillin/streptomycin
(MPBS) and 5ug/ml Hoechst stain (33342, bisbenzimide
trihydrochloride) at 37C for about 30 minutes. The
oocyte halves are then placed in fresh MPsS without
stain several minutes and viewed under a fluorescent
microscope with the appropriate excitation and barrier
filters. Each oocyte half is viewed at 200-400X
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magnification using white light. The white light is
shut off and the oocyte half is exposed to UV light.
The chromatin fluoresces a bright blue and should be
located as yuickly as possible to reduce the exposure
time to UV light. The oocyte halves with chromatin are
discarded. The oocyte halves without chromatin are
recipients of donor nuclear material.
In some cases, only one of each of the egg halves
was stained. In the event the half stained contained
the chromatin, the other enucleated half was not exposed ~
to the Hoechst stain. E~owever, the enucleated halves ~-
exposed to the Hoechst stain were viable and developed
normally.
The fused cells were placed in TCM 199 with 10%
HTFCS 0 to 10 hours. The embryos were then cultured
according to the procedure described in "Bovine Nuclear
Transplan~ation." The results are shown below in TABLE
4.
TABLE 4
NUCLEAR TRANSFER
IN VITRO MATURED OOCYTES
In Vitro Oocyte
Age atMorula & Blastocysts/Embryos
ManipulationRecovered (%)
Appro~imate ~ours
25 - 26 17/253 ( 6.7%)
40 - 43 18/153 (11.7%)
": '
Part of the nuclear transfer study compared in
vitro oocytes to _ vivo recovered oocytes and their
capability to support development of embryos after
nuclear transfer. The nuclear material came from the
same blastomeres of one donor embryo. The following
TABLE 5 shows the results of the use of in vivo matured
oocytes and in vitro matured oocytes.
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TABLE 5
COMPARISON IN VIVO AND IN VITRO OOCYTES
Morula ~ Blastocysts/
Treatment Embryos Recovered (~
In vivo oocyte 3/12 (25%)
In vitro oocyte 6/33 (18%~ -
There were 36 embryos developing to morula or
blastocysts stage. All viable embryos were transferred
to synchronous recipient cows. Seven pregnancies have
been confirmed.
The method of this invention is a successful in ~-~
vitro oocyte maturation process which can be used for
nuclear transfer or other genetic manipulation or ~-
fertilization techniques. The method offer an alterna- `
tive to use of ln vivo oocytes.
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