2. Materials and methods
2.1. In Õitro maturation IVM Ovaries were collected from Holstein cows and heifers at a local slaughterhouse and
were transported to the laboratory in 0.9 saline at 22–258C. Immature oocytes were collected from 2 to 5 mm diameter follicles using an 18-gauge needle and a 5-ml
syringe. The oocytes were washed three times with 3 ml of 10 mM hepes-buffered Ž
. TL-HEPES medium Gibco BRL, Life Technologies, Grand Island, NY , based on
tyrode-lactate medium and supplemented with 0.3 BSA and 2 mM NaHCO , and once
3
with maturation medium, TCM-199 with Eagle’s salts and
L
-glutamine supplemented Ž
. Ž
. with 10 vrv heat-inactivated fetal bovine serum FBS; Gibco BRL and 25 mM
NaHCO . After washing, 20 to 30 oocytes with evenly granulated cytoplasm surrounded
3
by compact cumulus cells were transferred into 0.5 ml of maturation medium containing Ž
. Ž
1 ugrml oestradiol Sigma, St Louis, MO and 1 mgrml FSH-Pe Schering-Plough .
Ž .
Animal Health, Kenilworth, NJ in a 4-well multidish Nunclon, Roskilde, Denmark and cultured for 22 to 24 h at 38.58C, 5 CO in air.
2
2.2. In Õitro fertilization Ž
At the end of IVM, oocytes were rinsed twice with TALP Bavister and Yanagi- .
machi, 1977 and placed in 50 ml of fertilization medium, which consisted of modified Ž
. Tyrode-Lactate medium Fert-TALP; Bavister and Yanagimachi, 1977 . Frozen semen
from two Holstein bulls was thawed at 378C in water for 30 s and layered on a 45–90 Ž
. Percoll Sigma gradient in a 15-ml centrifuge tube. The Percoll gradient was composed
of 2 ml of 90 Percoll overlaid with 2 ml of 45 Percoll, both of which were prepared Ž
. with Sperm TALP SP-TALP; Parrish et al., 1988 . After 20 min of centrifugation at
2000 = g, the top layers were removed and the sperm pellet was suspended in SP-TALP. After 10 min of centrifugation at 700 = g, the sperm pellet was resuspended
by the same method. The sperm suspension was introduced into a 50-ml drop of fertilization medium containing 10 oocytes to make the final sperm concentration
Ž
6
. 2 = 10 rml . When sperm were added to the fertilization drops, 2 mgrml heparin, 20
Ž .
mM penicillamine, 10 mM hypotaurine and 1 mM epinephrine PHE were also added. After 18 h of insemination, the cumulus investment was removed by repeated pipetting
in 2 ml TL-hepes medium in a 3-mm petri dish and the oocytes were then placed in CR1aa medium before DNA injection. CR1aa medium was formulated according to
Ž .
Rosenkrans et al. 1993 , and supplemented with 1 mM glutamine and 1 = Eagle’s Ž
. essential amino acids solution Gibco BRL .
2.3. Microinjection of DNA The DNA used for microinjection was a 5.5-kb fragment of pBL1, which consisted of
the bovine b-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation Ž
. signal Kim et al., 1994 . The DNA concentration was adjusted to 4 mgrml in a buffer
of 10 mM Tris–HCl, pH 8.0, and 0.1 mM EDTA. Microinjection was conducted
Ž similarly to the methods previously described for pig and sheep embryos Hammer et
. al., 1985 . In order to visualize the pronuclei, cumulus-free oocytes were centrifuged in
Ž TL-hepes medium at 12,000 = g for 7 min in a microcentrifuge Vision Scientific,
. Korea . The embryos were then microinjected into one pronucleus with DNA solution
21 to 25 h after insemination. Swelling of the pronucleus indicated a successful injection. After DNA injection, surviving zygotes were cultured for 2 days in 50 ml
drops of CR1aa medium supplemented with 3 mgrml BSA under light mineral oil Ž
. Sigma .
2.4. MEF monolayers Ž
. MEF monolayers were prepared as described by Joyner 1993 . Briefly, fetuses were
collected aseptically at necropsy from Day 14 to 16 pregnant mice and then finely Ž
. minced using micro-scissors. The minced tissues were washed three times with PBS y
free of Ca
2q
and Mg
2q
ions, and trypsinized in 0.1 trypsinr0.05 EDTA solution Ž
while being stirred. Suspended cells were sieved through a stainless steel mesh Ikemoto, .
Tokyo, Japan and centrifuged at 700 = g for 5 min. The cell pellet was re-suspended in Ž
. DMEM Gibco BRL containing 10 FBS and the cell suspension was placed in a
100-mm petri dish. The primary fibroblasts were cultured for 2 days until confluent at 378C, 5 CO in air, proliferated through two subsequent passages as described above
2
and then frozen at y708C. Freezing medium used for fibroblasts consisted of 10 Ž
. Ž
. vrv glycerol and 50 vrv FBS in PBS. Frozen MEF were thawed in 378C water
and then grown in 100 mm petri dishes for 2 days until confluent. MEF were inactivated Ž
. by treatment with 10 mgrml Mitomycin C Sigma for 2.5 h and then harvested. To
co-culture bovine embryos, Mitomycin C-treated cells were rinsed three times with PBS, re-suspended in DMEM containing 10 FBS and then plated at a concentration of
6 = 10
5
cellsrml placing 0.5 ml of the suspension in each well of a 4-well dish to form monolayers. Approximately 4 h later, all the DMEM was removed and replaced with
0.75 ml of CR1aa medium containing 10 FBS at least 2 h before use. 2.5. In Õitro culture
DNA-injected or IVF-derived bovine zygotes were cultured in 50 ml drops of CR1aa medium supplemented with 3 mgrml BSA under light mineral oil. Embryos were
selected at approximately 72 h after insemination and used for the culture experiments. At this time, 4- to 6-cell stage embryos and embryos with 6-cells were placed in
culture groups denoted as 4- and 8-cell stage embryos, respectively. Approximately half of the cleaved embryos were further co-cultured with MEF and the remainder were
cultured without MEF for another 5 days in 0.75 ml of CR1aa medium supplemented with 10 FBS. All in vitro cultures were under oil at 38.58C in a humidified atmosphere
of 5 CO . A total of 10 to 15 embryos were subjected to each culture experiment. At
2
Day 5 of culture, glucose was added to bring the final concentration to 5.56 mM and Ž
. 1 = GMS-X supplement Gibco BRL was added to each culture medium. GMS-X
supplement consisted of 1.0 mgrml insulin, 0.67 mgrml sodium selenite, 0.55 mgrml transferrin and 0.2 mgrml ethanolamine. In both culture systems, 0.5 ml medium per
well was replaced with fresh CR1aa medium at 48 h intervals. Embryo development was evaluated by morphological appearance under an inverted Nikon light microscope. The
day of IVF was designated as Day 0.
2.6. Embryo transfer Blastocysts that developed from DNA-injected embryos were transferred to recipi-
ents. The embryos were transported for about 4 h prior to transfer to recipients in PBS medium supplemented with 15 FBS at 35–378C in a thermos bottle. All recipients
were virgin, 14- to 18-month-old Holstein heifers. They were observed twice daily for the onset of standing oestrus and heifers with spontaneous oestrus were used as
recipients. Prior to embryo transfer, the recipients were palpated rectally for the presence
Ž .
Ž of a functional corpus lutea CL . A sterile embryo transfer gun 0.25 ml straw; IMV,
. Ž
L’aigle Cedex, France ensheathed in a sanitary sleeve
IMV, Chemise Sanitaire, .
France was used to perform non-surgical embryo transfer. One or two blastocysts, depending on the availability of embryos each day, were transferred to the ipsilateral
uterine horn of a recipient heifer on Day 7 or 8 after spontaneous oestrus. Following transfer, the heifers were observed for signs of oestrus, and pregnancy was confirmed by
rectal palpation at approximately Day 60 of gestation. The pregnant recipients were normally managed to deliver their calves at term.
2.7. Experimental designs 2.7.1. Experiment 1
To examine the effect of MEF on in vitro development of IVF-derived bovine embryos, immature oocytes were matured, fertilized and cultured in vitro. After 2 days
of culture, approximately half of the cleaved embryos were further co-cultured with MEF and the remainder was cultured without MEF for 5 days. These experiments were
contemporaneously repeated seven times in each group.
2.7.2. Experiment 2 The development ability of DNA-injected bovine embryos was also compared
between two different culture systems. The data for in vitro development of DNA-in- jected embryos were pooled from results of experiments which were performed in our
Ž .
laboratory over approximately one and half years March, 1995 to October, 1996 . 2.7.3. Experiment 3
To investigate whether MEF are effective on in vivo development of DNA-injected bovine embryos, DNA-injected blastocysts that developed from the two different culture
systems were transferred to recipients. 2.8. Statistical analysis
The developmental rates to the blastocyst stage of IVF-derived embryos were compared by the Duncan’s group test using the Generalized Linear Models procedure in
Table 1 Comparison of in vitro development of Day 3 IVF-derived bovine embryos co-cultured with or without MEF
for 5 days Group
Cell stage No. of embryos
No. of embryos Developmental
a
Ž . cultured
developed to rate
b b
b b
EB MB
EX HB
d,e
CR1aa 4-cell
91 1
1 2
4.4
d
8-cell 103
1 2
15 6
23.3
f
Sub-total 194
1 3
16 8
14.4
e
MEF 4-cell
96 1
1 2.1
c
8-cell 105
1 25
21 44.8
g
Sub-total 201
1 26
22 24.4
a
No. of total blastocystsrno. of embryos cultured=100.
b
EB: early, MB: mid, EX: expanded, HB: hatched blastocysts.
c,d,e
P - 0.05,
f,g
P - 0.05.
SAS. Differences in preimplantation development of DNA-injected embryos between the two culture systems were analyzed by Student’s t-test in SAS.
3. Results
Kategori