Effect of co-culture with somatic cells on the in vitro developmental potential of pig parthenogenetic and nuclear transferred oocytes
receiving blastomeres from in vivo fertilized embryos
Yoko Katol), Masaki Hata1.2" Isao YonemuraJl , Katsuhiro Ookoshil.4>, Yasuhiro Morita5" Toshiyuki Kojima6J , Yukio Tsunodall
1) Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, 631-8505, 2) present address; KAN Research Institute Inc., S10-1-8-3, Minamikaneden, Suita, 564-0044,
3) Tottori Swine and Poultry Experiment Station, Saihaku, Tottori, 683-0364,
4) present address; National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 5) Nara Prefectural Livestock Experimental Station, Ouda, Nara, 633-2113 and
6) National Livestock Breeding Center, Ministry of Agriculture, Forestry and Fisheries, Odakurahara-1, Nishigoh, Nishishirakawa, Fukushima, 961-8511, Japan
Short title; Parthenogenesis and nuclear transfer in the pig Key words; parthenogenesis, nuclear transfer, pig, activation
(4%) and even without co-culture (10%). When SUMMARY
nuclear transferred embryos cultured overnight were transferred to 8 recipient gilts, estrus of gilts Effect of co-culture with somatic cells on the in
vitro developmental potential of parthenogenetic retwned on day 20 to 58 days and no pregnancy and nuclear transferred embryos in pig was was observed.
examined. Single blastomere from 4- to 32- cell-
stage embryos, which were collected from INTRODUCTION
artificially inseminated female gilts at days three
to four, was fused with enucleated oocytes Recently, it was reported that somatic cells of fetuses in sheep (1), bovine (2) and goat (3), and matured in vitro. Fused oocytes were cultured
with somatic cells for 8 days in vitro, or some adult somatic cells in sheep (1), mouse (4) and nuclear transferred embryos were transferred to bovine (5) developed to normal young after nuclear synchronized recipient gilts after overnight culture transfer. Now, many attempts are done to produce in vitro. Results demonstrated that nuclear cloned or copied animals from somatic cells in transferred embryos cleaved at a significantly farm animals for not only animal reproduction but higher rate under co-culture system with pig also transgenic research. Especially, cloning of pig cumulus (41 vs 63%) and bovine oviductal cells (41 is one of the hottest topics because the size of pig vs 59%). Later development beyond 4-cell stage organs is considered most likely to be compatible was, however, limited and similar (5 vs 7 and with humans for xenotransplantation. In pig, 10%), and no blastocyst was obtained. While, however, successful nuclear transfer is very parthenogenetic oocytes developed to blastocysts limited until recently even though using with pig cumulus cells (7%), bovine oviductal cells preimplanted embryos as donors; only one piglet
was produced by nuclear transfer using 4-cell stage embryos (6). Very recently, successful production of piglets from somatic nuclear transfer has been reported from different three laboratories (7, 8, 9), the success rates are still low. Although the reasons for the poor development in the pig nuclear transfer are unclear, obvious problem for the pig embryos is a high sensitivity against in vitro environment. The in vitro maturation culture sy tern of immature oocytes and in vitro culture of zygotes to blastocysts with developmental ability were nol yet fully established (10, 11). Thus, in vivo matured oocytes had been used for recipient cytoplasm for pig nuclear transfer (7, 8, 12, 13) t'xcept the report of Betthauser et al., (9). Nuclear transferred embryos were often cultured in vivo (14) or transferred to recipient with minimal culture period. It is necessary to establish reliable in vitro culture system in pig embryos for the convenience of experiments. It has been demonstrated that co-culture with somatic cells (15, 16, 17) and culture in oviducts of other speci (18) improved the developmental potential of pig embryos in vitro. However, it is unclear whether co-culture with somatic cells is effective for the development of nuclear transferred embryos. In this experiment, it was examined whether developmental potential of pig parthenogenetic and nuclear transferred embryos was improved under co-culture with somatic cells.
MATERIALS AND METHODS
Recipient oocytes
Ovaries were obtained from a local abattoir and brought to the laboratory in physiological saline at 27 to 33 . Cumulus-oocytes complexes were aspirated through a 20G needle from 2 to 5 mm follicles with Dulbecco's modified phosphate buffered saline (mPBS) containing 3 mglml bovine erum albumin (BSA). Oocytes with cumulus cells were collected and washed 3 times with TCM-199 supplemented with 3.05 mM glucose, 2.92 mM alcium lactate, 0.91 mM sodium pyruvate and 10% heat inactivated calf serum. The cumulus-
oocytes complexes were transferred to 100 p I of mTCM-199 supplemented with 10 IU/ml pregnant mare serum gonadotrophin (PMSG) and 10 I Iml human chorionic gonadotrophin (hCG) covered with mineral oil and cultured for 42-47 hrs at 39 in 5% C02 and 95% air. At the end of the maturational culture, oocytes were denuded by treatment with hyarulonidase (3001 Iml). Oocytes with a polarbody were judged as matured.
Matured oocytes were selected and used for activation or nuclear transfer. The chromosomes at the second metaphase stage were mechanically removed from oocytes in PB 1 medium (19) supplemented with 7.5 fl glml cytochalasin Band 20% calf serum (CS) and then the enucleated oocytes were used as recipient oocytes for nuclear transfer.
Donor embryos
In vivo fertilized embryos were collected from thirteen 150-215 days old crossbred gilts (LWD:
Landrace x Large White x Duroc or LW:
Landrace x Large White), one 170 days old Landrace, one 215 days Duroc and one 723 days Landrace gilts. They were superovulat d and inseminated following the previous report (20).
Briefly, they were administered intramuscularly with 1,000 to 2,000 LV. of PMSG followed 48-72 later by 500 to 750 LV. of hCG. The donor gilts were inseminated at 24, 36 and 48 h after thl' injection of hCG. Gilts were killed on days 3 of the estrous cycle (Day 0 = onset of estrous). Collected embryos were stored at 37 in mKRB medium (21) and transported from Yonago to Nara at distance of 380km. In some experiments, embryos were cultured in vitro for one day due to the experimental convenience.
Parthenogenetic activation and nuclear transfer Enucleated oocytes and matured oocytes were treated with 5% ethanol for 1 min following electrically two sets of direct current (DC) pulse of 0.5 kv Icm for 25 f) sec 3 times at 20 min interval (22). In the preliminary experiments effective concentrations and exposing duration of ethanol
were examined (0-10 % for 0-10 min), and it was revealed that the treatment with 5% ethanol for 1
7 min had similar effect on the incidence of parthenogenetic activation and In vitro development (activation rate: 58-670/0, blastocyst rate: 3-5%). Activated oocytes were cultured in mKRB medium supplemented with 100/0 CS and 10 ,II g/ml cychrohexmide for 5 h. For nuclear transfer embryos, blastomere of donor embryos was introduced into the perivittelline space of activated oocytes and fused each other by three times of DC pulf'e~ of 0.75 kv/cm for 50 f1 sec at 20 min interval. Activated or nuclear transferred oocytes were cultured with cytochala in B supplemented medium for 5 h.
In vitro culture
After cytochalasin B treatment, oocyt s were co
cultured with pig cumulus cell , pig and bovine oviductal cells in mKRB at 39- in 50/0 CO~, 5%
Nt and 900/0 air for 8 days. We had previously found that mKRB medium wa the most uitable medium for the in vitro culture of pig parthenogenetic oocytes among M16, ClB, CRl
aa, TCM-199 and modified Whitten npdium;
developmental rate into blastocyst stag was 13 0/0 in mKRB, 30/0 in M16, 20/0 in ClB, 10/0 in CR1-aa, 0% in TCM-199 and modified Whitten m dium, respectively (unpublished ob"'ervation). I olation of oviductal cells and co-culture w.re perform d according to the report by Eyeston and First (23) with a little modification. Briefly, pig Or bovine oviducts obtained from a local slaughterhouse were cleaned, washed and mucosae were extracted by mechanical pressure with a glass slides along the oviducts or flushing of ovidu .ts by PBS. Mucosal tis ues containing epithelial cell clump were washed and epithelial cell clumps were collected. Mobil epith lial ell clump before attachment to the bottom of the culture dish were used for co-culture. Pig cumulus cells were the same cells to be used for th maturational culture. In the preliminary experiments where effe ts of the concentrations of epithelial cell clumps and cumulus c II on the
dev lopment of pig activat d oocyte w r exam in d, ffective concentrations were 1.7 x 105/ml for cumulus cells, and 2-3 x 102/ml for bovine epithelial cell clumps (unpubli h d observation).
In vivo culture of nuclear transferred oocytes Nuclear transf rred oocyt cultured overnight were surgically transf rred into Iigat d rabbit oviduct following hCG injection four days before.
After 4 days in vi 0 culture, rabbit were killed and oviducts were flushed with PBI medium for embryo recovery. Recovered embryos were evaluated for the developmental tage and cultured in vitro for another 3 to 4 days.
Embryo transfer
The nuclear transferred oocyt s cultured in vitro for 1 or 2 days were transf rred again from ara to Yonago and then transf rred urgically to ovidu ts of 8 cro bred gilts. Th ir strous wa induced to be the day of on 1 day after nu lear transfer by th injection of 1,000 1 PMSG and 001 hCG according to the previous report (20).
Each gilt r ceived 14 to 119 embryos at 1- to 8
cell tage. Recipient pig were monitor d daily for return to estrus. Itrasonic Doppler, ultra onic cho methods and echography were used as the pregnant t t.
Statistical analysis
The developmental rat s w re analyzed by X~ analysis.
RESULTS
When a ti at d oocytes wer fixed 5 h after lh treatm nt, 77·100 % of ooc tes fomled pronuclei in all groups (data not shown). The d velopm ntal tages of donor embryos us d for nucl ar tran f r were 3- to 6-cell stag s on day 3 and 7 to 33-cell tages on day 4. Data f nu lear transfer with day 3 and 4 embry were mix d because the dev lopmental ability of nuclear transferred oocyt s was similar.
As shown in Table 1, parthenogenetic oocytes developed to 4-cell stage at a high rate with or without co-cultures. Activated oocytes in all groups developed to blastocysts (4 to 12%), but the developmental ability in co-culture group with bovine oviductal cells was significantly low compared with that in control group (4 vs 10%).
Combination of co-culture cells, such as pig cumulus with oviductal cells (6%) or pig cumulus with bovine oviductal cells (7%) did not increase the developmental potential of parthenogenetic oocytes. Table 2 showed the in vitro developmental potential of nuclear transferred oocytes. Nuclear transferred oocytes cleaved at a significantly or higher rate under co-culture system with pig cumulus (P
<
0.01, 63%) and bovine oviductal cells (59%) compared with control (41 %). Later development beyond 4-cell stage, however, was low and similar (19 vs 28 and 30%), and no blastocyst was obtained in all groups. For in vivo culture, eighty reconstituted oocytes developed to the 1- to 3-cell stages were transferred to 2 rabbits, 45 embryos were recovered (45/80, 56%). However, development was quite poor and only one degenerating 8-cell stage and two 4-cell stage embryos were observed.After in vitro culture of nuclear transplants with pig cumulus cells for 1- or 2 days, 54% of embryos were still at I-cell stage and others cleaved to 2- cell (26%), 3-4-cell (14%) and 6-8-cell (6%) stages.
Totally 449 nuclear transferred embryos were transferred to 8 gilts, but no pregnancy was observed and their estrus returned on day 20 to day 58.
DISCUSSION
In the present study, the development of nuclear tra nsferred oocytes to blastocysts was not observed in all groups. It suggested that the reprogramming of donor nuclei after fusion with oocytes matured in vitro was insufficient. Co- culture of pig early stage embryos with somatic
cells such as endometrial cells, oviductal epithelial cells and granulosa cells is one of the methods to enhance the in vitro development of bovine and pig embryos (24-26). In this experiment, however, co-culture of pig parthenogenetic and nuclear transferred oocytes with pig oviductal, cumulus and bovine oviductal cells was not effective for the in vitro and in vivo development.
It has been demonstrated that nuclear transplants with a blastomere from preimplanted embryos fertilized in vivo have a high developmental ability in vitro than those with fertilized in vitro. In this experiment, in vivo fertilized embryos were used as donor nuclei. Although, the reasons for the poor development of nuclear transplants were unclear, candidates were as follows.
First, insufficient of cytoplasmic maturation of recipient cytoplasm was strongly suggested. All the reports on the successful development of nuclear transferred pig oocytes to blastocysts (14, 27) or to live young (6, 7, 8) used ovulated oocytes as recipient cytoplasm except the report of Betthauser et aI., (9) who used in vitro matured oocytes. Nagashima et al. (13) improved the system of in vitro maturation of pig oocytes with addition of growth factors and gonadotrophins into the medium and successfully obtained blastocysts from in vitro matured oocytes following subzonal sperm injection. When the developmental ability was compared with in vivo matured oocytes, it was still significantly lower. The incomplete maturation of oocytes would extend to the incomplete reprogramming of donor nuclei.
Second, condition of artificial activation of oocytes and of in vitro culture seemed to be insufficient for not only nuclear transplants but also parthenones. In the current experiment, the highest proportion of blastocysts developed from parthenogenetic oocytes was 15%. Many attempts were done to activate pig oocytes effectively by combined treatments (28). Recently, Machaty et al (29) clarified that oocytes treated with thimerosal,
which induced repetitive Ca transients similar to those by the sperm at fertilization, and dithiothreitol (DDT) treatment, which blocks the Ca oscillations, developed to morulae to blastocysts at a high rate (42%). Dinnyes et al. (30) reported that combined electrical and butyrolactone I, specific cyclin-dependent kinase inhibitor, treatment enhanced the activation of pig oocytes. They also observed that increased when oocytes were cultured in mNCSU 37 (50%) than in Whitten's medium (5%). Although appropriate conditions of artificial activation and in vitro culture of oocytes might be different between parthenones and nuclear transplants because of the difference of genomic compositions, combined treatments and different culture media should be examined further.
Third, donor and nuclear transferred embryos were transported for a long way in this experiment. But the trip did not affect the embryo quality. Because transported 4- to 16-cell stage embryos developed to the blastocyst stage at a high rate, when they were cultured in vitro 3 to 5 days (data not shown). In mice, we found that serial nuclear transfer was effective for the development of nuclear transferred embryos in vitro and in vivo (31-33). In such experiments, karyoplasts of nuclear transferred embryos were again transferred into the enucleated cytoplasms of the same stage embryos that developed from in vitro fertilization. The developmental potential of the nuclear transfer embryos was improved by the serial nuclear transfer in mice, suggesting that being worth trying also in the pig nuclear transfer.
Very recently, Polejaeva et al. (7) obtained piglets by serial nuclear transfer using cultured adult granulose cells, but several reports (7-9) succeeded to produce piglets by single nuclear transfer of cultured fetal fibroblast cells.
In conclusion, it was clear in this study that co- culture of pig nuclear transplants with somatic cells was not effective for in vitro development.
Further studies concerning in vitro culture
systems for oocyte maturation and embryo development, and parthenogenetic activation methods should be required for the success of pig nuclear transfer.
ACKNOWLEDGMENT
The authors thank to members of Tottori Swine and Poultry Experiment Station for embryo transfer. This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the Ministry of Education, Science, and Culture (13308054, 14034259, 15039233), Ito Kinen Foundation and Nakajima International Interchange Foundation.
References
Wilmut I, Schnieke AE, McWhir J, Kind N, Campbell KHS. Nature 1997; 380: 64·66.
2 Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, F Abel Ponce de Leon, Robl JM. Science 1998; 280: 1256-1258.
3 Baguisi A, Benboodi E, Melican OT, Pollock JS, Oestrempes MM, Canunuso C, Williams JL, Nims SO, Porter CA, Midura P, Palacios M, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstom EW, Echelard Y. Nature Biotech 1999; 17: 456-46l.
4 Wakayama T, Perry ACF, Zuccotti M, Johnson KR, Yanagimachi R. Nature 1998; 394: 369·374.
5 Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Ooguchi H, Yasue H, Tsunoda Y. Science 1998; 282:
2095-2098.
6 Prather RS, Sims MM, First NL. BioI Repord 1989; 41:
414-418.
7 Polejaeva lA, Chen SH, Vaught TO, Page RL, Mullins J, Ball S, Oai Y, Boone J, Walker S. Ayares OL, Colman A, Campbell KH.Nature. 2000: 407(6800):86-90.
8 Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC.Science 2000;
289(5482): 1188-90.
9 Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, JurgeUa G.
Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Thompson S, Bishop M. Nat Biotechnol. 2000 ; 18: 1055-9.
10 Reed ML, iJIera MJ, Petters RM. Theriogenology 1992;
37: 95-109.
11 Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil199~;~ll: 61-73.
12 Nagashima H, Yamakawa H, Saito S. J Reprod Dev 1992; 38:73-78.
13 agashima H, Grupen CG, Ashman R], Nottle MB. Mol Reprod Dev 1996; 45:359-363.
14 aito S, Yamakawa H, Nagashima H. Assis Reprod Tech/Andro 1992; 259:257-266.
15 Krisher RL, Petters RM, Johnson BH, Bavister BD, Archibong AE. J Exp Zool 1989; 249: 235-239.
16 Whit KL, Hehnke K, Rickards LF, outhern LL, Thompson DL, Wood TC. Bioi Reprod 1989; 41:425- 430.
17 Allen RL, Wright RW Jr. J Anim Sci 1984; 59:1657- 1661.
18 Prather RS, Sims :vI:vI, First NL. Theriogenology 1991;
35:1147-1151.
19 Whittingham DG. Nature 1971; 233: 125-126.
20 Yonemura I, Fujino Y, Irie S, Miura Y. Transcervical transfer of porcine embryos under practical conditions. J Reprod Dev 1996; 42: 89-94.
21 Davi DL, Day BN. J Anim Sci 1978; 16: 1043-1053.
22 Hata M, Ohkoshi K, Kato Y, Tsunoda Y. Jpn J Fertil teril 1996; 41: 305-310.
23 Eyestone WH, First NL. J Reprod F rtil 1989; 85: 715- 720.
24 Ellington JE, Farrell PB, Simkin ME, Foote RH, Goldman EE, McGrath AB. J Reprod F rtil 1990a; 89:
293-299.
25 Ellington JE, Carney EW, Farrell PB, Simkin ME, Foote RH. BioI Repro<! 1990b; 43: 97-104.
26 Reed WA, Suh TK, Bunch TD, White KL.
Theriogenol gy 1996; 45: 439-449.
27 Nagashima H, Ashman RJ, Bottle MB. \101 Repro<! Dev 1997; 48: 339-343.
2 Petr J, Grocholova R, Rozinek J, ] ilek F.
Theriogenology 1996; 45: 1473-1478.
29 Machaty Z, Wang WH, Day B?\, Prather RS. Bioi R pord 1.997; 57:1123-1127.
30 Dinnyes A, Hirao Y, Nagai T. Cloning 1999/2000; 1:209- 216.
31 Tsunoda Y, Kato Y. J Exp Zool 1997; 278: 250-254.
32 Tsunoda Y, Kato Y. J Reprod Fertil 1998; 113:181-184.
33 Kato Y, Yabuuchi A, Motosugi N, Kato ], Tsunoda Y.
Bioi Reprod 1999; 61: 1110-1114.
Talbe1 Effc o c uet f o‑c urltewihst oma cc so hell nted ooc syte
d re i t o .f
lt cu u
l
eveopmentalabilityof l a hepgPrt nogenetl Noofo tesdevelopedto(%)
la b s Co‑C山t d
t re u h wi
No ocy
2‑cell 4‑cell
ts te Cy
ts COO S
to s cys tocys 662n(con oり｢t 506(76) 372(56)
no
pl U 山sc s 350
5
gC mU ell 270 2 6(73) 182(52) lgOV u
ov eov p唱 CUmU
id lu ld in p b
cells 225 3) (8 188(70) ct
uc c st ell 331
255(77) 209 3) (6 90s十〇v uid cells 79 (68) 60(8 7)ct 72
63(88) p唱 CUmUlus
+
bov eOln V ug up ld ro
‖S t
c ce 46(64)
p<0.01wihc n lt o ot｢
Ta lbe2 D o al ar d
wl u d
★ 195 124
re lt 亡l ta
re h t l e
ev of lpgnucle a ferr o . No ns
tr ed‑oocy亡esinvitro
】pott‑Ln pmcn
Co‑C山tu foocy ste
deveopedtJ o(%) cu
2‑ceH 3‑4‑cell 5‑8‑cell
(1 0 la
) l) (7 (l )
) (1 (2 (3 )a
)b )b (6 (4 (6 (5
b
4) 29
1
3 8 4
9 0 6 b
5) 205 84 39 9) 10 )(5a
lgCUmU山sce 0 ov eOV uc
Hs p
b
54 34 15
henogene id ln
｢t
lce cacttav te
Hs i t
ta 54 32 16 0
a s
a‑ ;b p<001 .
ブタ単為発生卵と核移植卵の体外発生能に及ぼす共培養の影響
★p doocyte
3 )
･2
1･米村功:
加藤容子 H･秦雅樹
一･大越勝広
1,l)"
‑
森 田恭弘･･小島敏之 ‑ i 一 ･角 田幸雄
1) I:. l T F･
近故 大草 凸II都市鹿?･叔聾 BLl野地 :K∧N研究fr･ 5取舵,
卜. l
t
所
本研究では,ブ タ単為発生卵 と初期艦割球 を ドナー核 とした核移植卵の体外発生能 に及ぼす共培哉紳胞 の影矧 こついて検討 した｡火培超細胞には,ブ タ卵管細胞,ブ タ卵丘細胞,ウシ卵管細胞を用いた｡ その 胞にブ タ卵 丘細胞あ るいは ウシ卵管細胞 を用いる と,核移植卵の分割率を有意 に向 上させ るこ とがで きた｡ しか しなが ら,旺盤胸への発/率を向 卜させ るには至 らず,今後,更に検討を要するこ
4EE lr小'#諮.t^玖機 ･現所岨 独 立行政故 人曲 菜JE物や源 研'
U +
奈良県招座 談輸切 ･衷 TS改 良t'ン '/‑ 1近
故k
竿 段':部t5席fT=数寄結 巣,共培うSA
とが明 らか とな った｡
