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Parthenogenetic activation of mouse and pig oocytes matured in vitro
THERIOGENOLOGY
PARTHENOGENETICACTIVATION OF MOUSE AND PIG OOCYTES MATURED IN VITRO
B. A. Didion, M. J. Martin and C. L. Markert Department of Animal Science North Carolina State University Raleigh, NC 27695-7621 Received for publication: Accepted:
September February
22, 9,
1989
1990
ABSTRACT The objective of this study was to determine if mouse and pig oocytes matured in vitro undergo parthenogenetic activation following exposure to various activation stimuli. Cumulus-intact, germinal vesicle-stage mouse oocytes (n = 151) were collected from pregnant mare serum gonadotropin primed mice and incubated overnight in Brinster’s medium. This culture system allowed an 85% maturation to Metaphase II. Pig oocytes (n = 242) were gathered from ovaries collected at an abbattoir and incubated in vitro for 48 h to allow maturation to occur (51% maturation to Metaphase II). Following maturation, mouse and pig oocytes were exposed to various activation stimuli. Mouse oocytes were treated with medium containing ethanol (7%), eiectricity (85 V, 30 us, one time), or medium; then they were incubated for 6 to 8 h to allow for activation. Pig oocytes were treated with medium containing ethanol (lo%), electricity (85 V, 30 us once or twice), ethanol followed by electricity, or medium then incubated for 18 h to allow for activation. A portion of the mouse and pig oocytes were fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342. Chi-square procedures were used to analyze the data. The proportion of mouse oocytes which underwent activation was higher (PcO.01) for ethanol and electricity than for the medium (22, 30 and O%, respectively). The proportion of pig oocytes which underwent activation was higher (P
activation,
mouse, pig
INTRODUCTION The cloning of superior animals would be economically worthwhile to the livestock industry (1, 2). Recently, progress has been made in the cloning of sheep, cattle and rabbit embryos (3-6). However, the efficiency of cloning embryos is low for at least two reasons. First, several animals must be maintained to serve as a
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source of in vivo matured oocytes. Second, success of the cloning procedure is dependent upon the activation of recipient oocytes. In vitro matured (IVM) oocytes are fertilizable in vitro and can lead to the birth of live young following transfer to suitable recipients, as shown in mice (7), cattle (8,9), sheep (10) and pigs (11). Therefore IVM oocytes might also be suitable as nuclear transfer recipients. This approach could significantly increase the practicality of cloning, since a large supply of oocytes is available from abattoir ovaries. Ovulated mouse oocytes can undergo parthenogenetic activation following exposure to various stimuli (12). In vitro matured cattle oocytes can be activated following exposure to ethanol (13), ionophore, and electric current (14). Little information exists about the activation potential of ovulated and IVM oocytes of other farm animals. Our objective was to determine if IVM mouse and pig oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli.
MATERIALS AND METHODS Collection of Immature Mouse Oocytes Cumulus enclosed, germinal vesicle-stage mouse oocytes were obtained from the ovaries of 8 to 14-wk-old FVB female mice. The mice received 10 IU of pregnant mare serum gonadotropin 48 to 52 h prior to removal of the ovaries. The ovaries were placed in a culture dish containing 1 ml of warm (37°C) Brinster’s medium (BM, 15). Oocytes were collected by puncturing the ovary repeatedly with a 30gauge needle. The oocytes were rinsed twice in IOO-ul droplets of BM and transferred to a 50-1~1droplet of BM under paraffin oil in a 5% CO2 in air atmosphere at 37°C until used (within 15 min of collection). Collection of Immature Pig Oocytes Pig ovaries were collected at a local abattoir, placed in PBI medium (16) at 30°C and transported to the laboratory within 2 h. Ovaries were perforated repeatedly with an 1&gauge needle to recover cumulus-enclosed oocytes. Oocytes were collected, rinsed three times in PBl at 25”C, placed in a 200-ul droplet of PBl in a dish (Falcon ##3002) and maintained on a warming plate (37°C) until used. Oocytes were allocated to treatment groups within 15 min of collection. Experiment
1
The objective of this experiment was to determine the optimal incubation time for the production of IVM mouse and pig oocytes. Onehundred and forty cumulusintact, germinal vesicle-stage mouse oocytes were allocated to one of three groups: 1) O-h incubation, 2) 9-h incubation, and 3) 18-h incubation. Oocytes were transferred to a 50-ul droplet of BM (15 to 20 oocytes per droplet) under oil and incubated at 37°C in a 5% CO2 in air atmosphere. Following incubation, oocytes were placed in fixative (3% gluteraldehyde in 0.1 M cacodylate buffer, pH 7.4)
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THERIOGENOLOGY under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-ul dH20, strippped of their cumulus with a mouth-controlled pipette, transferred to a glass slide, and stained with Hoechst 33342. Onehundred and fifty-six cumulus-intact, germinal vesicle-stage pig oocytes were allocated to one of three groups:l) O-h incubation, 2) 24-h incubation, and 3) 48-h incubation. Oocytes were transferred to a 50-ul droplet of BM (15 to 20 oocytes per droplet) under oil and incubated at 37°C in a 5% CO2 atmosphere. Following incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-1~1dH20, stripped of their cumulus with a mouth-controlled pipette, transferred to a glass slide, and stained with Hoechst 33342. Experiment
2
The objective of this experiment was to determine if IVM mouse oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli. Onehundred and fifty-one oocytes exhibiting a polar body (approximately 85% of the total) were transferred to one of four groups:l) 50-ul BM containing 7% ethanol for 5 min, 2) 200-ul droplet of BM and exposed to electric current (once using a DC pulse of 85 V for 30 us, using a high voltage cell processor, Bioelectronics Corporation, Troy Mich.), and 3) 50-ul BM media control: a fourth group of oocytes was placed in fixative (fixed) immediately after culture to serve as a control. Oocytes in Groups 1-3 were washed twice in 50-ul droplets of BM and incubated in vitro as above for 6 to 8 h. After incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-ul dH20, transferred to a glass slide and stained with Hoechst 33342. Experiment
3
The objective of this experiment was to determine if IVM (cultured for 48 h) pig oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli. Use of a dissecting microscope (40x magnification) made visualization of the first polar body difficult. Therefore, all oocytes (n = 242) were randomly allocated to one of six groups:l) 50-1~1BM, 2) 50-ul BM containing 10% ethanol for 10 min, 3) 200-ul droplet of BM and treated with electric current (once at 85 V for 30 us), 4) 200-ul droplet of BM and exposed to electric current (twice at 85 V for 30 us, 15 min apart), and 5) ethanol treatment followed by electric current 15 min later. A sixth group of oocytes, which served as a control, was placed in fixative immediately after IVM. After treatment, the oocytes in Groups l-5 were washed twice in 50-1~1droplets of BM and incubated in vitro as above for 18 h. Following incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. Oocytes were rinsed in 200-ul dH20, transferred to a glass slide and stained with Hoechst 33342. Oocyte Staining A modification of the Hoechst 33342 staining method for embryos (17) was used to examine the nuclear status of oocytes. After the oocytes were fixed in gluteraldehyde and rinsed in dHp0, they were transferred to a glass slide in 10 to 20 ul of dH20. A coverslip with spots of Vasoline on its corners was placed over the oocytes to anchor them. Oocytes were stained with 0.01% trypan blue for 2 min by drawing a solution of trypan blue under the coverslip. Oocytes were then stained for
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THERIOGENOLOGY 5 to10 min in a similar way using Hoechst 33342. The Hoechst working solution was made by adding 20-1~1of stock Hoechst (lmg HoechstIml water) to 980~ul of a 3:l PBS:ethanol solution. Statistical analysis was performed using Chi-square.
RESULTS The in vitro maturation rate of germinal vesicle-stage mouse oocytes to Metaphase II was 85% when incubated for 18 h (Table 1). Nuclear staining of these oocytes revealed a normal appearance of the Metaphase II plate located paratangentially to the oocyte periphery (Figure 1, top). The first polar body was usually located adjacent to the Metaphase II plate (Figure 1, top). In approximately 30% of these oocytes the first polar body was located some distance away (20 to 30 u) from the Metaphase II plate. Oocytes not scored as Metaphase II were categorized as germinal vesicle-stage or Metaphase I oocytes. There was no evidence of spontaneous activation during the maturation period. The activation rates of IVM mouse oocytes are shown in Table 2. The proportion of IVM oocytes which underwent activation was greater (PcO.01) for ethanol and electric current treated oocytes than for the media control oocytes (22, 30, and O%, respectively). Approximately 80% of the activated IVM oocytes contained a single haploid pronucleus, as evidenced by two polar bodies and one pronucleus (Figure 1, bottom). The in vitro maturation rate of germinal vesicle-stage pig oocytes to Metaphase II was 51% when incubated for 48 h (Table 1, bottom). Nuclear staining of these Metaphase II oocytes revealed a normal appearing Metaphase II spindle located adjacent to the oocyte membrane (Figure 2, top). The first polar body was usually located next to the Metaphase II plate. In approximately 25% of the Metaphase II oocytes the first polar body was located some distance away (30 to 50 u) from the Metaphase II plate. Oocytes not scored as Metaphase II were categorized as germinal vesicle-stage or Metaphase I oocytes. There was no evidence of spontaneous activation during the maturation period. Activation rates of IVM pig oocytes are shown in Table 3. Activation rates were higher (PcO.05) for electric current (twice, 15’ apart) and alcohol followed by electric current oocytes than for media control oocytes (14, 16, and O%, respectively). Most of the activated oocytes were diploid parthenotes, as evidenced by one polar body and one pronucleus (Figure 2, bottom). There was no evidence of spontaneous activation of pig oocytes during the maturation period.
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Table
1. Effect of incubation time on the proportion mouse and pig oocytes reaching Metaphase for various time periods
Incubation time
of m
(hours)
No. of Oocytes
0 9 18
GVS(%)a
75 90 91
70(93) 12(13) 7(7)
. .
MI (%)b
MI1 ((%)c
5(6) 76(U) 7(7)
0 3(3) 77( 85)
p&
. .
Incubation time (hours)
No. of oocytes
0
GVS(%)
77 90 89
24 48
75(97) 20(22) 22(25)
Mouse and pig data was gathered a GVS = germinal vesicle-stage. b MI = Metaphase I of meiosis. c MI1 = Metaphase II of meiosis.
Table 2. Activation stimuli
ID
of germinal vesicle-stage II when cultured in vitro
response
from
four
of rrlf;lps1s MI (%)
MI1 (%a)
2(3) 65(72) 21(23)
46(51)
separate
of in vitro matured
experiments.
mouse
oocytes
to various
vitro matured Nuclear Statusa
Treatment
No. of Oocytes
Fixed
No. Activated(%)
No. of Haploid
No. of Diploid
No. of IC
25
Ob
27
Ob
7% Ethanol
50
1 l(22)C
9
2
0
Electric shock (once 85 V, 30 us)
49
15(3O)C
10
3
2
Media
control
--Data was gathered from three separate experiments. IC = immediate cleavage. aBased on activated oocytes. bcValues not sharing a common superscript within columns (P>O.Ol).
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are different
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THERIOGENOLOGY
Figure 1. In vitro matured mouse oocyte (top photo) showing the first polar body (1 PB) and Metaphase II plate (MP). Parthenogenetic mouse oocyte (bottom photo) containing haploid pronucleus (PN), second polar body (2PB) and IPB.
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THERIOGENOLOGY
Figure
2.
In vitro matured pig oocyte (top photo) showing the first polar body (lPB), Metaphase II plate (MP) and cumulus cells (CC). Parthenogenetic pig oocyte (bottom photo) containing diploid pronucleus (PN) and a polar body (PB).
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THERIOGENOLOGY Table
3. Activation
IJI vitro
response
of in vitro matured
pig oocytes
to various
stimuli
matured Nuclear Statusa
Treatment
No. Oocytes
No. of Activated(%)
No. of Haploid
No. of Diploid
32
O(0)b
40
O(O)b
7% Ethanol
42
3(7)bJ
0
3
85 V 30~s (once)
43
3(7)bJ
2
1
85 V 30~s (twice)
4 1
6(14)c
3
3
Ethanol electric
44
7(16)c
2
5
Fixed Media
Control
plus current
Data was gathered from three separate experiments. aBased on activated oocytes. bcValues not sharing a common superscript within (P
columns
are different
DISCUSSION In vitro maturation of germinal vesicle-stage mouse oocytes to Metaphase II of meiosis was highest (85%) when oocytes were incubated for 18 h. Donahue (18) found that maturation in vitro of mouse oocytes to Metaphase II begins at 11 h (14%) and continues to increase until 17 h (88%) of incubation. In the present study, the chromosomes of IVM oocytes appeared morphologically normal, with a low incidence of chromosome scatter (~7%) as evidenced by nuclear staining with Hoechst 33342. The first polar body was usually located adjacent to the Metaphase II plate. However, in approximately 30% of the oocytes the polar body was located some distance away from the Metaphase II plate. The efficiency of enucleating the Metaphase II plate is probably influenced by this polar body migration (19). Spontaneous activation did not appear to occur from the maturation period or activation period (media control group). In contrast, mouse oocytes of the LT/Sv strain undergo spontaneous activation within the oviduct and following in vitro maturation (20). The in vitro maturation rate of germinal vesicle-stage pig oocytes to Metaphase II was highest (51%) when cultured for 48 h. Motlik et al., (21) reported that oocytes liberated from follicles ~0.8 mm in diameter do not resume meiosis since the oocyte is not fully developed. However, 76% of the oocytes recovered from 3 to 5 mm-follicles reach Metaphase II following 48 h of incubation (21). The inclusion of small follicles (co.8 mm), and therefore small oocytes, may have lead to the reduced IVM rate in our study. The chromosomes of the IVM pig oocytes appeared morphologically normal, with only a low incidence of chromosome scatter or
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clumping (~10%). Spontaneous activation of oocytes did not appear to occur during the maturation or activation periods (media control group). In vitro matured bovine oocytes, however, undergo spontaneous activation when cultured beyond the time required for maturation to Metaphase II (13, 22-24). The response of IVM mouse and pig oocytes to activation stimuli was low (25% for the mouse, 14 to16% for the pig) following both ethanol and electric current exposure. In vitro matured pig oocytes did not activate following exposure to calcium ionophore (B.A. Didion et al., unpublished observations) Ovulated mouse oocytes (15 to 17 h post-hCG treatment) exposed to similar stimuli have high activation rates: 76% for electric current (25), and 85% for ethanol (26). Similarly, ovulated pig oocytes exposed to an electric stimulus have high activation rates: 93% (27) and 82% (B.A. Didion et al., unpublished observations). Yang and Yanigamachi (28) found preovulatory (Metaphase II) hamster oocytes less responsive to calcium ionophore (37% activation) than ovulated oocytes (93% activation). Therefore, the in vivo matured oocyte appears to activate more readily than the IVM oocyte with respect to response to stimuli. Reasons for the disparity in activation rates between ovulated and IVM oocytes are not known. It is possible that the release of intracellular calcium in IVM mouse and pig oocytes is inadequate or abnormal at the time of exposure to activation stimuli (29). Since high in vitro fertilization rates (and therefore activation rates) are obtainable in IVM mouse (73%; 7) and pig oocytes (78%; 11) it appears that the methodology used to activate IVM oocytes influences their response. Therefore, activating IVM mouse and pig oocytes by means other than sperm-induced activation may compromise the oocyte’s ability to respond. The incidence of parthenogenetic activation increases with post-ovulatory age (30). However, when pig oocytes were matured for 56 versus 48 h and exposed to activation stimuli there was no increase in parthenogenetic activation (B.A. Didion et al., unpublished observations). In contrast, an age dependency for activation of IVM bovine oocytes has been reported previously (13, 14). In summary we have shown that IVM mouse and pig oocytes respond to activation stimuii but at a low rate. Why these oocytes have a lower activation rate in comparison to ovulated oocytes is not known. Determining the requirements needed for optimizing the activation of IVM oocytes would make embryo cloning more practical for use in biotechnological research and industry.
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Marker-t, C. L. and Seidel, G. E. Parthenogenesis, identical twins, and cloning in mammals. In: Brackett, B. G., Seidel, G. E. and Seidel, S. M (eds.), New Technologies in Animal Breeding. Academic Press, New York, (1981) pp.1 81-200 .
2.
Markert, C. L. Cloning mammals: Current reality and future prospects. Theriogenology a:60-67 (1984).
3
Willadsen, S. M. Nuclear transplantation 65(1986).
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in sheep embryos. Nature m:63-
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4.
Smith, L. C. and Wilmut, I. Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. Biol. Reprod. =:1027-1035 (1989).
5.
Prather, R. S., Barnes, F. L., Sims, M. M., Robl, J. R. Eyestone, W. H. and First, N.L. Nuclear transplantation in the bovine embryo; assessment of donor nuclei and recipient oocyte. Biol. Reprod. x:859-866 (1987).
6.
Stice, S. L. and Robl, J. M. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol. Reprod. =:657-664 (1988).
7.
Schroeder, A. C. and Eppig, J. J. The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev. Biol. j&$493497(1 984).
8.
Xu, K. P., Greve, T., Callesen, H. and Hyttel, P. Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fertil. &f&501-504 (1987).
9.
Lu, K. H., Gordon, I., Gallaher, M. and McGovern, H. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet. Rec. jZl: 259-260 (1987).
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Crozet, N., Hunecew, S., Desmedt, D., Theron, Y., Szollosi, M. C., Torres, D. and Sevellec, C. In vitro fertilization with normal development in sheep. Gam. Res. I&:159-170 (1987).
11.
Mattioli, M., Bacci, M. L., Galeati, G. and Seren, E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology =:1201-1207 (1989).
12.
Kaufman, M. H. Early Mammalian Development: Parthenogenetic Studies. Cambridge University Press, Cambridge, England (1983).
13.
Nagai, T. Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gam. Res. j&243-249 (1987).
14.
Ware, C. B., Barnes , F. L., Maiki-Laurila, M. and First N. L. Age dependence of bovine oocyte activation. Gam. Res. 2:265-275 (1989).
15.
Brinster, Ft. L. Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J. Reprod. Fert. 1p:227-240 (1965).
16.
Whittingham, D. G. and Wales, R. G. Storage of two-cell mouse embryos in vitro. Aust. J. Biol. Sci. 2:1065-1068 (1969).
17.
Pursel, V. G., Wall, R. J., Rexroad, Jr., C. E., Hammer, R. E. and Brinster, R. L. A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenology u:687-689 (1985).
18.
Donahue, D. P. Maturation of the mouse oocyte in vitro. Sequence and timing of (1969). nuclear progression. J. Exp. 2001. =:237-250
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THERIOGENOLOGY 19.
Robl, J. M. and Stice, S. L. Prospects for the commercial cloning of animals by nuclear transplantation. Theriogenology a:7584 (1989).
20.
Eppig, J. J. Developmental potential of LT/Sv parthenotes derived from oocytes matured in vivo and in vitro. Dev. Biol. f&244-249 (1978).
21.
Motlik, J., Crozet, N. and Fulka, J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J. Reprod. Fertil. E:323-328 (1984).
22.
Xu, K. P., Greve, T., Smith, S., Liehman, P., Callesen, H., Hyttle, P. Parthenogenetic activation of cattle oocytes matured in vitro and cultured in rabbit. Theriogenology a:21 8 abstr. (1986).
23.
Sato. E., Kawamura, N. and Ishibashi, N. Chemicals influencing maturation, activation and degeneration of bovine oocytes in culture. J. Dairy Sci. =:3482-3488 (1988).
24.
King, W. A., Sirard, M. A., Greve, T., Leclerc, P., Lambert, Ft. D. and Jacques, P. Cytogenetic study of parthenogenetically activated bovine oocytes matured in vitro and in vivo. Gam. Res. a:265274 (1988).
25.
Onodera, M. and Tsunoda, Y. Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. Gam. Res. =:277-283 (1989).
26.
Cuthbertson, K. S. R. Parthenogenetic activation of mouse oocytes with ethanol and benzyl alcohol. J. Exp. Zool. =:31 I-314 (1983).
27.
Prather, R. S., Sims, M. L. and First, N. L. Nuclear transplantation early porcine embryo. Theriogenology a:290 abstr. (1988).
28.
Yang, C. H. and Yanagimachi, R. Differences between mature ovarian and oviductal oocytes: a study using the golden hamster. Human Reprod. 4:63-71 (1989).
29.
Cuthbertson, K. S. R., and Cobbold, P. H. Phorbl ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca+2. Nature ;LL6:541-542 (1985).
30.
Graham, C. F. The production of parthenogenetic mammalian embryos and their use in biological research. Siol. Rev. &&399-422 (1974).
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in the
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PARTHENOGENETICACTIVATION OF MOUSE AND PIG OOCYTES MATURED IN VITRO
B. A. Didion, M. J. Martin and C. L. Markert Department of Animal Science North Carolina State University Raleigh, NC 27695-7621 Received for publication: Accepted:
September February
22, 9,
1989
1990
ABSTRACT The objective of this study was to determine if mouse and pig oocytes matured in vitro undergo parthenogenetic activation following exposure to various activation stimuli. Cumulus-intact, germinal vesicle-stage mouse oocytes (n = 151) were collected from pregnant mare serum gonadotropin primed mice and incubated overnight in Brinster’s medium. This culture system allowed an 85% maturation to Metaphase II. Pig oocytes (n = 242) were gathered from ovaries collected at an abbattoir and incubated in vitro for 48 h to allow maturation to occur (51% maturation to Metaphase II). Following maturation, mouse and pig oocytes were exposed to various activation stimuli. Mouse oocytes were treated with medium containing ethanol (7%), eiectricity (85 V, 30 us, one time), or medium; then they were incubated for 6 to 8 h to allow for activation. Pig oocytes were treated with medium containing ethanol (lo%), electricity (85 V, 30 us once or twice), ethanol followed by electricity, or medium then incubated for 18 h to allow for activation. A portion of the mouse and pig oocytes were fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342. Chi-square procedures were used to analyze the data. The proportion of mouse oocytes which underwent activation was higher (PcO.01) for ethanol and electricity than for the medium (22, 30 and O%, respectively). The proportion of pig oocytes which underwent activation was higher (P
activation,
mouse, pig
INTRODUCTION The cloning of superior animals would be economically worthwhile to the livestock industry (1, 2). Recently, progress has been made in the cloning of sheep, cattle and rabbit embryos (3-6). However, the efficiency of cloning embryos is low for at least two reasons. First, several animals must be maintained to serve as a
JUNE 1990 VOL. 33 NO. 6
1165
THERIOGENOLOGY
source of in vivo matured oocytes. Second, success of the cloning procedure is dependent upon the activation of recipient oocytes. In vitro matured (IVM) oocytes are fertilizable in vitro and can lead to the birth of live young following transfer to suitable recipients, as shown in mice (7), cattle (8,9), sheep (10) and pigs (11). Therefore IVM oocytes might also be suitable as nuclear transfer recipients. This approach could significantly increase the practicality of cloning, since a large supply of oocytes is available from abattoir ovaries. Ovulated mouse oocytes can undergo parthenogenetic activation following exposure to various stimuli (12). In vitro matured cattle oocytes can be activated following exposure to ethanol (13), ionophore, and electric current (14). Little information exists about the activation potential of ovulated and IVM oocytes of other farm animals. Our objective was to determine if IVM mouse and pig oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli.
MATERIALS AND METHODS Collection of Immature Mouse Oocytes Cumulus enclosed, germinal vesicle-stage mouse oocytes were obtained from the ovaries of 8 to 14-wk-old FVB female mice. The mice received 10 IU of pregnant mare serum gonadotropin 48 to 52 h prior to removal of the ovaries. The ovaries were placed in a culture dish containing 1 ml of warm (37°C) Brinster’s medium (BM, 15). Oocytes were collected by puncturing the ovary repeatedly with a 30gauge needle. The oocytes were rinsed twice in IOO-ul droplets of BM and transferred to a 50-1~1droplet of BM under paraffin oil in a 5% CO2 in air atmosphere at 37°C until used (within 15 min of collection). Collection of Immature Pig Oocytes Pig ovaries were collected at a local abattoir, placed in PBI medium (16) at 30°C and transported to the laboratory within 2 h. Ovaries were perforated repeatedly with an 1&gauge needle to recover cumulus-enclosed oocytes. Oocytes were collected, rinsed three times in PBl at 25”C, placed in a 200-ul droplet of PBl in a dish (Falcon ##3002) and maintained on a warming plate (37°C) until used. Oocytes were allocated to treatment groups within 15 min of collection. Experiment
1
The objective of this experiment was to determine the optimal incubation time for the production of IVM mouse and pig oocytes. Onehundred and forty cumulusintact, germinal vesicle-stage mouse oocytes were allocated to one of three groups: 1) O-h incubation, 2) 9-h incubation, and 3) 18-h incubation. Oocytes were transferred to a 50-ul droplet of BM (15 to 20 oocytes per droplet) under oil and incubated at 37°C in a 5% CO2 in air atmosphere. Following incubation, oocytes were placed in fixative (3% gluteraldehyde in 0.1 M cacodylate buffer, pH 7.4)
1166
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THERIOGENOLOGY under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-ul dH20, strippped of their cumulus with a mouth-controlled pipette, transferred to a glass slide, and stained with Hoechst 33342. Onehundred and fifty-six cumulus-intact, germinal vesicle-stage pig oocytes were allocated to one of three groups:l) O-h incubation, 2) 24-h incubation, and 3) 48-h incubation. Oocytes were transferred to a 50-ul droplet of BM (15 to 20 oocytes per droplet) under oil and incubated at 37°C in a 5% CO2 atmosphere. Following incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-1~1dH20, stripped of their cumulus with a mouth-controlled pipette, transferred to a glass slide, and stained with Hoechst 33342. Experiment
2
The objective of this experiment was to determine if IVM mouse oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli. Onehundred and fifty-one oocytes exhibiting a polar body (approximately 85% of the total) were transferred to one of four groups:l) 50-ul BM containing 7% ethanol for 5 min, 2) 200-ul droplet of BM and exposed to electric current (once using a DC pulse of 85 V for 30 us, using a high voltage cell processor, Bioelectronics Corporation, Troy Mich.), and 3) 50-ul BM media control: a fourth group of oocytes was placed in fixative (fixed) immediately after culture to serve as a control. Oocytes in Groups 1-3 were washed twice in 50-ul droplets of BM and incubated in vitro as above for 6 to 8 h. After incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. The oocytes were rinsed in 200-ul dH20, transferred to a glass slide and stained with Hoechst 33342. Experiment
3
The objective of this experiment was to determine if IVM (cultured for 48 h) pig oocytes undergo parthenogenetic activation following in vitro culture or exposure to activation stimuli. Use of a dissecting microscope (40x magnification) made visualization of the first polar body difficult. Therefore, all oocytes (n = 242) were randomly allocated to one of six groups:l) 50-1~1BM, 2) 50-ul BM containing 10% ethanol for 10 min, 3) 200-ul droplet of BM and treated with electric current (once at 85 V for 30 us), 4) 200-ul droplet of BM and exposed to electric current (twice at 85 V for 30 us, 15 min apart), and 5) ethanol treatment followed by electric current 15 min later. A sixth group of oocytes, which served as a control, was placed in fixative immediately after IVM. After treatment, the oocytes in Groups l-5 were washed twice in 50-1~1droplets of BM and incubated in vitro as above for 18 h. Following incubation the oocytes were placed in fixative under oil for 1 to 72 h at room temperature. Oocytes were rinsed in 200-ul dH20, transferred to a glass slide and stained with Hoechst 33342. Oocyte Staining A modification of the Hoechst 33342 staining method for embryos (17) was used to examine the nuclear status of oocytes. After the oocytes were fixed in gluteraldehyde and rinsed in dHp0, they were transferred to a glass slide in 10 to 20 ul of dH20. A coverslip with spots of Vasoline on its corners was placed over the oocytes to anchor them. Oocytes were stained with 0.01% trypan blue for 2 min by drawing a solution of trypan blue under the coverslip. Oocytes were then stained for
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THERIOGENOLOGY 5 to10 min in a similar way using Hoechst 33342. The Hoechst working solution was made by adding 20-1~1of stock Hoechst (lmg HoechstIml water) to 980~ul of a 3:l PBS:ethanol solution. Statistical analysis was performed using Chi-square.
RESULTS The in vitro maturation rate of germinal vesicle-stage mouse oocytes to Metaphase II was 85% when incubated for 18 h (Table 1). Nuclear staining of these oocytes revealed a normal appearance of the Metaphase II plate located paratangentially to the oocyte periphery (Figure 1, top). The first polar body was usually located adjacent to the Metaphase II plate (Figure 1, top). In approximately 30% of these oocytes the first polar body was located some distance away (20 to 30 u) from the Metaphase II plate. Oocytes not scored as Metaphase II were categorized as germinal vesicle-stage or Metaphase I oocytes. There was no evidence of spontaneous activation during the maturation period. The activation rates of IVM mouse oocytes are shown in Table 2. The proportion of IVM oocytes which underwent activation was greater (PcO.01) for ethanol and electric current treated oocytes than for the media control oocytes (22, 30, and O%, respectively). Approximately 80% of the activated IVM oocytes contained a single haploid pronucleus, as evidenced by two polar bodies and one pronucleus (Figure 1, bottom). The in vitro maturation rate of germinal vesicle-stage pig oocytes to Metaphase II was 51% when incubated for 48 h (Table 1, bottom). Nuclear staining of these Metaphase II oocytes revealed a normal appearing Metaphase II spindle located adjacent to the oocyte membrane (Figure 2, top). The first polar body was usually located next to the Metaphase II plate. In approximately 25% of the Metaphase II oocytes the first polar body was located some distance away (30 to 50 u) from the Metaphase II plate. Oocytes not scored as Metaphase II were categorized as germinal vesicle-stage or Metaphase I oocytes. There was no evidence of spontaneous activation during the maturation period. Activation rates of IVM pig oocytes are shown in Table 3. Activation rates were higher (PcO.05) for electric current (twice, 15’ apart) and alcohol followed by electric current oocytes than for media control oocytes (14, 16, and O%, respectively). Most of the activated oocytes were diploid parthenotes, as evidenced by one polar body and one pronucleus (Figure 2, bottom). There was no evidence of spontaneous activation of pig oocytes during the maturation period.
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Table
1. Effect of incubation time on the proportion mouse and pig oocytes reaching Metaphase for various time periods
Incubation time
of m
(hours)
No. of Oocytes
0 9 18
GVS(%)a
75 90 91
70(93) 12(13) 7(7)
. .
MI (%)b
MI1 ((%)c
5(6) 76(U) 7(7)
0 3(3) 77( 85)
p&
. .
Incubation time (hours)
No. of oocytes
0
GVS(%)
77 90 89
24 48
75(97) 20(22) 22(25)
Mouse and pig data was gathered a GVS = germinal vesicle-stage. b MI = Metaphase I of meiosis. c MI1 = Metaphase II of meiosis.
Table 2. Activation stimuli
ID
of germinal vesicle-stage II when cultured in vitro
response
from
four
of rrlf;lps1s MI (%)
MI1 (%a)
2(3) 65(72) 21(23)
46(51)
separate
of in vitro matured
experiments.
mouse
oocytes
to various
vitro matured Nuclear Statusa
Treatment
No. of Oocytes
Fixed
No. Activated(%)
No. of Haploid
No. of Diploid
No. of IC
25
Ob
27
Ob
7% Ethanol
50
1 l(22)C
9
2
0
Electric shock (once 85 V, 30 us)
49
15(3O)C
10
3
2
Media
control
--Data was gathered from three separate experiments. IC = immediate cleavage. aBased on activated oocytes. bcValues not sharing a common superscript within columns (P>O.Ol).
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are different
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THERIOGENOLOGY
Figure 1. In vitro matured mouse oocyte (top photo) showing the first polar body (1 PB) and Metaphase II plate (MP). Parthenogenetic mouse oocyte (bottom photo) containing haploid pronucleus (PN), second polar body (2PB) and IPB.
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Figure
2.
In vitro matured pig oocyte (top photo) showing the first polar body (lPB), Metaphase II plate (MP) and cumulus cells (CC). Parthenogenetic pig oocyte (bottom photo) containing diploid pronucleus (PN) and a polar body (PB).
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THERIOGENOLOGY Table
3. Activation
IJI vitro
response
of in vitro matured
pig oocytes
to various
stimuli
matured Nuclear Statusa
Treatment
No. Oocytes
No. of Activated(%)
No. of Haploid
No. of Diploid
32
O(0)b
40
O(O)b
7% Ethanol
42
3(7)bJ
0
3
85 V 30~s (once)
43
3(7)bJ
2
1
85 V 30~s (twice)
4 1
6(14)c
3
3
Ethanol electric
44
7(16)c
2
5
Fixed Media
Control
plus current
Data was gathered from three separate experiments. aBased on activated oocytes. bcValues not sharing a common superscript within (P
columns
are different
DISCUSSION In vitro maturation of germinal vesicle-stage mouse oocytes to Metaphase II of meiosis was highest (85%) when oocytes were incubated for 18 h. Donahue (18) found that maturation in vitro of mouse oocytes to Metaphase II begins at 11 h (14%) and continues to increase until 17 h (88%) of incubation. In the present study, the chromosomes of IVM oocytes appeared morphologically normal, with a low incidence of chromosome scatter (~7%) as evidenced by nuclear staining with Hoechst 33342. The first polar body was usually located adjacent to the Metaphase II plate. However, in approximately 30% of the oocytes the polar body was located some distance away from the Metaphase II plate. The efficiency of enucleating the Metaphase II plate is probably influenced by this polar body migration (19). Spontaneous activation did not appear to occur from the maturation period or activation period (media control group). In contrast, mouse oocytes of the LT/Sv strain undergo spontaneous activation within the oviduct and following in vitro maturation (20). The in vitro maturation rate of germinal vesicle-stage pig oocytes to Metaphase II was highest (51%) when cultured for 48 h. Motlik et al., (21) reported that oocytes liberated from follicles ~0.8 mm in diameter do not resume meiosis since the oocyte is not fully developed. However, 76% of the oocytes recovered from 3 to 5 mm-follicles reach Metaphase II following 48 h of incubation (21). The inclusion of small follicles (co.8 mm), and therefore small oocytes, may have lead to the reduced IVM rate in our study. The chromosomes of the IVM pig oocytes appeared morphologically normal, with only a low incidence of chromosome scatter or
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clumping (~10%). Spontaneous activation of oocytes did not appear to occur during the maturation or activation periods (media control group). In vitro matured bovine oocytes, however, undergo spontaneous activation when cultured beyond the time required for maturation to Metaphase II (13, 22-24). The response of IVM mouse and pig oocytes to activation stimuli was low (25% for the mouse, 14 to16% for the pig) following both ethanol and electric current exposure. In vitro matured pig oocytes did not activate following exposure to calcium ionophore (B.A. Didion et al., unpublished observations) Ovulated mouse oocytes (15 to 17 h post-hCG treatment) exposed to similar stimuli have high activation rates: 76% for electric current (25), and 85% for ethanol (26). Similarly, ovulated pig oocytes exposed to an electric stimulus have high activation rates: 93% (27) and 82% (B.A. Didion et al., unpublished observations). Yang and Yanigamachi (28) found preovulatory (Metaphase II) hamster oocytes less responsive to calcium ionophore (37% activation) than ovulated oocytes (93% activation). Therefore, the in vivo matured oocyte appears to activate more readily than the IVM oocyte with respect to response to stimuli. Reasons for the disparity in activation rates between ovulated and IVM oocytes are not known. It is possible that the release of intracellular calcium in IVM mouse and pig oocytes is inadequate or abnormal at the time of exposure to activation stimuli (29). Since high in vitro fertilization rates (and therefore activation rates) are obtainable in IVM mouse (73%; 7) and pig oocytes (78%; 11) it appears that the methodology used to activate IVM oocytes influences their response. Therefore, activating IVM mouse and pig oocytes by means other than sperm-induced activation may compromise the oocyte’s ability to respond. The incidence of parthenogenetic activation increases with post-ovulatory age (30). However, when pig oocytes were matured for 56 versus 48 h and exposed to activation stimuli there was no increase in parthenogenetic activation (B.A. Didion et al., unpublished observations). In contrast, an age dependency for activation of IVM bovine oocytes has been reported previously (13, 14). In summary we have shown that IVM mouse and pig oocytes respond to activation stimuii but at a low rate. Why these oocytes have a lower activation rate in comparison to ovulated oocytes is not known. Determining the requirements needed for optimizing the activation of IVM oocytes would make embryo cloning more practical for use in biotechnological research and industry.
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