MOLECULAR REPRODUCTION AND DEVELOPMENT 30:214-219 (1991)
Developmental Potential and Chromosome Constitution of Strontium-InducedMouse Parthenogenones G.T. O’NEILL,l L.R. ROLFE? AND M.H. KAUFMAN2 ‘Institute of Animal Physiology and Genetics Research, Roslin, Scotland; ‘Department of Anatomy, University Medical School, Edinburgh, Scotland The brief exposure of recently ovuABSTRACT lated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr”) for 2-10 min was observed t o induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure t o M16 Sr2+ was increased from 2 t o 30 min. Under these conditions a greater proportion of the activated oocytes developed as twopronuclear diploid parthenogenones, due t o failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes t o calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr2+-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr2+induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities t o improve the efficiency of embryo cloning techniques with the ruminant species.
Key Words: Sr2+-induced parthenogenesis, Oocyte, Preimplantation embryo
INTRODUCTION Ovulated mouse oocytes can be induced to initiate parthenogenetic development following their in vitro exposure to a variety of physical or chemical agents (Kaufman, 1983). Two agents t h a t are recognised to be efficient parthenogenetic stimuli are hyaluronidase (Kaufman, 1973; O’Neill and Kaufman, 1988) and dilute solutions of ethanol (Kaufman, 1982; O’Neill and
0 1991 WILEY-LISS, INC.
Kaufman, 1989). A high incidence of hyaluronidaseinduced parthenogenesis is observed only after ovulated oocytes have aged in vivo for at least 8-10 h r prior to their exposure to this agent. Although postovulatory aging prior to either fertilisation or parthenogenetic activation is recognised to interfere with polar body extrusion (Austin, 1970; Kaufman, 1975), hyaluronidase-induced activation has not been found to induce chromosome segregation errors, i.e., aneuploidy, during the completion of the second meiotic division (O’Neill and Kaufman, 1988), Postovulatory aging has also been reported to reduce the potential of mouse embryos to progress through the cleavage stages of early development (Smith and Lodge, 1987). Exposure to dilute solutions of ethanol can induce the activation of more recently ovulated oocytes but its use as a parthenogenetic stimulus may be disadvantageous in some studies, in that a significant proportion of the resultant parthenogenones are recognised to carry numerical chromosomal anomalies (Kaufman, 1982; ONeill and Kaufman, 1989; O’Neill et al., 1989). The majority of activated oocytes that initiate development following exposure to this stimulus develop a single haploid pronucleus following the extrusion of the second polar body. Several studies have also reported that postovulatory aged oocytes incubated in media of low osmolarity (Kaufman and Surani, 1974) or deficient in calcium ions (Surani and Kaufman, 1977; Whittingham and Siracusa, 1978; Whittingham et al., 1978) may also initiate parthenogenetic development. These studies showed that a significant proportion of the activated oocytes developed into two-pronuclear diploid parthenogenones a s their incubation in culture media with a reduced concentration of calcium ions for 1-2 h r interfered with the modifications to the oocyte cytoskeleton required for second polar body extrusion. The exposure of mouse oocytes to culture media supplemented with barium, strontium, or manganese ions has also been observed to induce parthenogenesis, but the proportion-
Received May 1, 1991; accepted June 12, 1991. Address reprint requests to Dr. G.T. O’Neill, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin EH25 9PS, Scotland.
STRONTIUM-INDUCED PARTHENOGENESIS ate incidence of the various classes of parthenogenone usually observed several hours after activation, their chromosome constitution, and their developmental potential in vitro were not investigated (Whittingham and Siracusa, 1978). The incubation of sperm in strontium-supplemented media has not been found to interfere with the processes associated with capacitation in vitro but was observed to inhibit fertilisation as exposure to strontium ions had induced both the resumption of meiosis and cortical granule release in freshly ovulated oocytes prior to sperm penetration (Fraser, 1987). The study described here examined the ability of strontium-supplemented embryo culture medium to induce the activation of ovulated mouse oocytes and analysed both the developmental potential and the chromosome constitution of the resultant parthenogenones. We have found that strontium-induced parthenogenesis can be more efficient and versatile than other methods currently used to induce oocyte activation.
MATERIALS AND METHODS Parthenogenetic Activation Eight- to twelve-week-old (C57B1 x CBA)Fl hybrid female mice were administered a n intraperitoneal injection (i.p.) of 5 IU pregnant mares serum gonadotrophin (PMSG) followed 46 h r later with a n i.p. injection of human chorionic gonadotrophin (HCG) to induce superovulation. At 18 h r after HCG injection, the female mice were killed by cervical disslocation and their oviducts were removed to either 1 ml standard M16 embryo culture medium (Whittingham, 19711, calcium-free M16 medium (Ca2+-freemedium; NaCl adjusted to 6.437 g/liter), or calcium-free M16 medium supplemented with 1.6 mM of SrCl, (M16 Sr2+)in a n embryological watchglass. The cumulus masses were then released from the swollen ampullary region of the oviduct and exposed to M16 Sr2+medium for 2, 5, 10, 30, or 60 min. Cumulus masses were also incubated in calcium-free M16 medium and standard M16 medium for 5 min or 30 rnin as controls to M16 Sr2+exposure. The cumulus masses were washed three times in M16 medium and then transferred to microdrops of M16 medium under liquid paraffin at 37°C in an atmosphere of 5% COz in air for 6 hr. At the end of this period the microdrops containing the cumulus masses were fused to adjacent microdrops of M16 medium supplemented with 1mg/ml of hyaluronidase to remove the adhering cumulus cells. Activated oocytes at this stage of development exhibited pronuclei. The majority of activated oocytes had developed a single haploid pronucleus following the extrusion of the second polar body (1PN). Other parthenogenones either exhibited two haploid pronuclei (2PN) or a single diplid pronucleus (1PND) as second polar body extrusion had failed to occur. A small proportion had undergone immediate cleavage (IC), in which each blastomere exhibited a single haploid pronucleus. 2PN parthenogenones were transferred to fresh microdrops of M16 medium to permit development to the blastocyst stage
215
Metaphase Chromosome Preparations of 1PN Embryos 1PN parthenogenones were transferred to M16 medium supplemented with 1kg/ml of colcemid at 8-10 hr after activation. This treatment arrested development at metaphase of the first cleavage mitosis. Metaphase chromosome spreads were prepared early the next morning using the air-drying technique described by Tarkowski (1966) and were stained with 3% Giemsa’s stain for 10 min at pH 6.8. In Vitro and In Vivo Development of Fertilised One-Cell Embryos Two groups of F1 hybrid female mice were superovulated as described above and caged individually with F1 hybrid male mice shortly after the injection of HCG. The female mice were then checked early the next morning for the presence of a vaginal plug. This was taken as evidence that mating had occurred and was designated to be day 1 of pregnancy. The female mice from the first group were killed by cervical dislocation early on the morning of the first day of pregnancy, and the cumulus masses were isolated as described above and placed in microdrops of M16 medium supplemented with 1 mg/ml of hyaluronidase to remove the adhering cumulus cells. Fertilised oocytes were observed to exhibit a second polar body and two pronuclei. The one-cell fertilised embryos were then washed in two changes of M16 medium and transferred to fresh microdrops of M16 to permit development to the blastocyst stage. At specific times during this period of development, embryos were removed from culture, and cell number counts were determined from air-dried, fixed preparations using the technique described above. A second group of pregnant mice was used to examine the in vivo development of fertilised one-cell embryos. At specific times during in vivo development the female mice were killed, a s described above, and the embryos were flushed either from the oviduct or, in the later stages of preimplantation development, from the uterine horns. Air-dried spreads of these embryos were prepared, as described above, to determine their cell number. The mean number of cell divisions per embryo at specific times during embryo development was calculated from the formula:
where n = embryo cell number, N = No. of embryos per sample, and A = No. of cell divisions per embryo.
RESULTS Spontaneous activation was not observed following the exposure of cumulus masses (total 65 oocytes) at HCG + 18 h r to M16 medium for 5 or 30 min. The exposure of ovulated mouse oocytes to M16 S?’ medium at HCG + 18 h r for 2-60 min was observed to
216
G.T. O’NEILL ET AL.
TABLE 1. Proportionate Incidence of Parthenogenetic Activation Following the Exposure of Ovulated Mouse (C57B1 X CBA) F1 Hybrid Oocytes t o M 1 6 Medium Supplemented With Sr2+at HCG 18 hr Class of parthenogenone Activation Exposure No. of No. No. nonNo. (min) oocytes dead activated activated 1PN 2PN IC lPND frequency(%) 51.5 0 1 37 170 0 196 208 2 404 (81.7)a 94 10 11 85.2 221 336 0 78 5 414 (65.7) 42 14 87.2 121 129 41 306 3 10 351
+
(42.2)
20
267
67
50
150
30
137
97
11
29
40
53
45
0
12
60
52
45
0
7
75 (50.0) 12 (41.3) 10 (83.3) 7 (100.0)
60
9
6
56.2
12
0
5
21.2
1
1
0
22.6
0
0
0
13.5
aPercent activated oocytes that develop as 1PN.
induce parthenogenetic activation. The duration of exposure to M16 Sr2+ medium was found to have a significant effect on the incidence of oocyte activation (Table 1).The highest frequencies of activation, 85.2% and 87.2%, were observed when oocytes were exposed to M16 Sr2+medium for 5 min and 10 min, respectively. The incidence of activation induced by the exposure of oocytes to M16 Sr2+medium for 2 rnin (51.5%) was significantly lower (x2 = 16.4; P > 0.001). The incidence of activation following the exposure of oocytes to M16 Sr2+ medium for 20 rnin was not significantly different from that observed in the former group (x2 = 0.42; P = 0.50). However, following this treatment, the incidence of activation was low, because 25.1% of the treated oocytes died a s a result of the prolonged exposure of cumulus masses to M16 Sr2+ medium. Incubation of the isolated oocytes in M16 Sr2+ media for periods greater than 20 min induced a significant increase in cell death. The duration of exposure to M16 Sr2+media also influenced the proportionate incidence of the four standard classes of parthenogenone. The proportionate incidence of the 1PN class decreased significantly from 81.7% to 42.2% as the duration of incubation was increased from 2 min to 10 min. This was related to a n increase in the proportionate incidence of the 2PN, IC, and 1PND classes as the duration of exposure increased from 2 to 30 min. The majority of the parthenogenones observed following exposure to M16 Sr2+medium for 40 or 60 min were of the 1PN class. However, under these conditions, the majority of the exposed oocytes were observed to be dead when the cumulus cells were removed a t 5-6 h r after activation. The incidence of oocyte activation and the proportionate incidence of the four classes of parthenogenone induced by the exposure of ovulated oocytes to Ca2+free M16 medium at HCG + 18 h r is presented in Table 2. In contrast to the events observed following M16 Sr2+ exposure, the incidence of activation induced by the
incubation of oocytes in calcium-free medium for 5 min (17.6%) was low. The proportion of activated oocytes was not observed to be greater than the nonactivated group of oocytes until incubation in calcium-free medium was increased to 30 min (59.2%). The majority of the activated oocytes developed as 1PN parthenogenones following exposure to Ca2+-free M16 medium for either 5 min (86.6%) or 30 min (80.6%). Incubation of ovulated oocytes in calcium-free M16 a t HCG + 18 h r for either 5 min or 30 min did not induce the cell lysis. Chromosome Constitution of M16 S?+-Induced 1 P N Parthenogenones The chromosome constitution of M16 Sr2’-induced 1PN parthenogenones a t metaphase of the first cleavage is presented in Table 3. The nonanalysable preparations represent those t h a t were overtly hypohaploid due to chromosome scattering and those in which overlapping chromosomes hindered analysis of the chromosome number. The total ratio of hypohaploidy to hyperhaploidy was 4.5:l. The adjusted frequency of aneuploidy was derived from the observed incidence of (No. hyperhaploid preparations) (total NO. preparations)
and accounts for hypohaploidy induced by the spreading technique. The adjusted incidence of aneuploidy following exposure to M16 Sr2+for 5 min and 10 min was 4.7% and O.O.%, respectively. This agent induced a low incidence of aneuploidy that did not differ significantly from that previously observed (O’Neill and Kaufman, 1988) in hyaluronidase-induced parthenogenones (x2 = 0.48: P = 0.50-0.40). The two hyperhaploid spreads were recovered from one group of cumulus masses and indicate t h a t they were derived from one female mouse.
STRONTIUM-INDUCED PARTHENOGENESIS
2 17
TABLE 2. Proportionate Incidence of Parthenogenetic Activation Following the Exposure of Ovulated (C57B1 X CBA) F1 Hybrid Oocytes at HCG + 18 hr to Calcium-Free M16 Embryo Culture Medium Exposure (min) 5
No. of oocytes
No. dead
No. nonactivated
No. activated
170
0
140
30
Class of parthenogenone 1PN 2PN IC lPND 26
Activation frequency(%)
4
0
0
17.64
6
0
0
59.21
(86.6)a
30
76
0
45
31
25
(80.6)
aPercentage activated oocytes that develop as 1PN.
TABLE 3. Chromosome Constitution of Sr2+-Induced1PN Parthenogenones at Metaphase of the First Cleavage Division* c2+ Chromosome constitutiona Aneuploidy (%) No. nonexposure Total no. analysable 19 20 21 Observ. Adj. analysed (min) U I
4 86 2 6.9 4.7 5 101 9 10 104 5 5 94 0 5.3 0.0 *Adjusted incidence = 2 X (No. hyperhaploid preparations/total no. of preparations). aThe normal haploid complement of the mouse (n) is 20.
Developmental Potential of One-Cell Fertilised Embryos and S?-Induced 2PN Parthenogenones The analysis of the in vitro developmental potential of the M16 Sr2+-induced2PN parthenogenones and F2 fertilised mouse embryos retained both in vitro and in vivo, a s determined from the mean number of cell divisions per embryo (X.CD/embryo), analysed a t intervals between 55 and 121 h r postactivation or postfertilisation, is presented in Table 4. The analysis of the X.CD/embryo for the in vivo group of F2 embryos showed that by 93 h r postfertilisation the one-cell embryo had undergone 6.46 cell divisions, with a mean number of 86.0 t 2.8 cells. Subsequent analyses of cell number were unobtainable; hatching from the zona pellucida occurred after this time period. The analysis of the group of one-cell F2 embryos that were retained in vitro found that they had undergone a mean of 5.53 cleavage divisions at 93 h r postfertilisation. At 113 h r postfertilisation they were found to have undergone a mean of 5.68 cell divisions per embryo. Their mean cell number a t 93 h r postfertilisation was 51.4 2.98, and this increased to 85.3 & 4.93 when air-dried spreads were prepared at 113 h r postfertilisation. Hatching from the zona pellucida occurred in all embryos prior to their analysis at 121h r postfertilisation. In comparison to the fertilised F2 embryos retained in vitro and in vivo, the M16 Sr2+-induced2PN parthenogenones were found to exhibit a lower X.CD/embryo value of 4.48 at 93 hr. In these embryos, hatching from the zona pellucida was not observed until after 121 h r postactivation. At 113 and 121 h r postactivation, the X.CD/ embryo values were 6.32 and 6.63, respectively, and the mean cell numbers were 79.3 2 4.33 and 99.3 5.24, respectively.
*
DISCUSSION
This study has clearly demonstrated that the brief exposure of mouse oocytes to embryo culture medium in which CaCl, has been replaced with a n equimolar concentration of SrC1, has the potential to induce the resumption of meiosis and stimulate parthenogenetic development. Previous analyses of mammalian parthenogenetic development have established that both the conditions under which activation occurs and the stimulus employed can significantly influence the incidence of parthenogenesis a s well as the proportionate incidence of the four main classes of parthenogenone (Kaufman, 1983).The significant differences observed between the potentials of M16 Sr2+-and Ca2+-free media to induce parthenogenesis indicate that different mechanisms of oocyte activation were induced following exposure to these media. Previous studies have also reported that maximum levels of activation were observed following the exposure of oocytes to calciumdeficient media when the duration of exposure was in excess of 1 hr, particularly when exposure occurred a t HCG + 20-22 hr. The majority of the activated oocytes were also reported to develop as 2PN parthenogenones (Whittingham and Siracusa, 1978; Whittingham et al., 1978; Kaufman, 1983). In this study the exposure of oocytes to Ca2+-freemedium at HCG + 18 h r for either 5 or 30 min induced only a relatively low incidence of activation, and the majority of activated oocytes developed a s 1PN parthenogenones. Several aspects of the postactivation response of the M16 Sr2+-inducedparthenogenones indicate that this agent is a n efficient parthenogenetic agent. The incidence of activation was high, in the range of 85%, following the exposure of mouse oocytes to this medium
218
G.T. O’NEILL ET AL. TABLE 4. Mean Number of Cleavage Divisions per Embryo of Sr2+ Activated F1 Oocytes Compared W i t h In Vivo- and In Vitro-Maintained Fertilized Embryos Derived From F1 X F1 Matings (Sr2+-induced2PN embrvos derived from arouns 2 a n d 3 of Table 1) Mean No. of cell divisons per embryo F2 embryo F2 embryo Sr2+-induced2PN parthenogenones in vivo in vitro 3.19 2.30 3.49 2.97 5.54 3.37 3.61 5.99 4.76 4.48 6.46 5.53 6.32 5.68 121 6.63 Mean group size 10.0 51.0 31.2 aFertilization assumed to occur at 13 hr after the injection of HCG, activation a t 18 hr after the injection of HCG.
Hours post fertilization/ activationa 55 72 79 93 113
for only 5-10 min. In addition, the majority of the activated oocytes also developed a s 1PN parthenogenones. The ability to extrude the second polar body and develop a s 1PN parthenogenones indicates t h a t the activating stimulus does not interfere significantly with the reorganisation of the cytoskeletal elements of the oocyte during the completion of the second meiotic division. Furthermore, analysis of the chromosome constitution of the 1PN parthenogenones showed that the overall incidence of chromosome segregation errors induced during the completion of the second meiotic division in M16 Sr2+-induced 1PN parthenogenones did not differ from that previously observed in hyaluronidase-activated mouse oocytes. Analysis of the developmental potential of the M16 Sr2+-induced 2PN parthenogenones revealed that the rate of cell division, as determined from the mean number of cell divisions per embryo, was less than that observed in the F2 fertilised embryos that had also been retained in vitro from the one-cell stage. Modifications to culture media to identify the optimal molar concentrations of both strontium chloride and calcium chloride may enhance the rates of both activation and subsequent embryonic development. The ability of divalent ions, such as strontium, barium, and manganese, to induce activation is believed to be related directly to their ability to displace calcium ions from intracellular stores (Fraser, 1987), such as the mitochondria and endoplasmic reticulum closely associated with the spindle apparatus (see Van Blerkom and Runner, 1984; Eisen and Reynolds, 19851, which subsequently leads to a n increase in the intracellular concentration of free calcium ions. The increase in the intracellular concentration of free calcium ions in both ethanol- and sperm-activated mouse oocytes has been observed as a transient series of photoemissions in aequorin-permeated mouse oocytes (Cuthbertson et al., 1981). The resultant nonspecific increase in the intracellular concentration of free calcium ions induces electophysiological changes to occur within the oocyte, which initiate a cycle of calcium and
inositol 1,4,5-trisphosphate release similar to that believed to arise during the induction of oocyte activation at fertilisation (Miyazaki, 1988). However, the electrophysiological response of mouse oocytes to parthenogenetic stimuli, as recorded by the transient calcium ion induced photoemissions from aqueorin-permeated mouse oocytes and analyses of the membrane hyperpolarising response in both fertilised and parthenogenetically activated rodent oocytes (Eusebi and Siracusa, 1983) have all indicated that the activation response in parthenogenetically activated oocytes differs from that recorded from those activated by sperm penetration. It has been reported by Fraser (1987) that ovulated mouse oocytes at HCG + 14-15 h r can initiate parthenogenesis when exposed to calcium-free modified Tyrode’s medium supplemented with strontium ions. The ability of strontium ions to induce recently matured secondary oocytes to initiate parthenogenesis provides new opportunities to improve the efficiency of nuclear transfer (embryo cloning) procedures in ruminant species (Willadsen, 1986; Prather et al., 1987; Rob1 et al., 1987; Smith and Wilmut, 1989). Present techniques use electric pulses of microsecond durations to induce both the cell-oocyte fusion and the parthenogenetic activation of the enucleated recipient oocyte. However, the incidence of reconstituted embryo development following cell-oocyte fusion, oocyte activation, and the pronuclear-like remodelling of the donor nucleus using electric pulse stimulation indicates that modifications to transfer and activation procedures would improve the efficiency of nuclear transfer techniques. Two principal factors that influence the success of nuclear transfer procedures are recognised to be the cell cycle compatibility of the donor nucleus to remodelling within the activated oocyte and the use of recipient oocytes prior to the onset of degenerative changes associated with postovulatory aging (Smith and Wilmut, 1990). Other studies have indicated that the success of nuclear transfer procedures using in vitromatured oocytes (IVM; see Younis et al., 1989) would be improved if recipient oocytes were used prior to the
STRONTIUM-INDUCED PARTHENOGENESIS degenerative changes associated with in vitro postmaturation (“postovulatory”) aging (see Collas and Robl, 1990). This study found th at the removal of metaphase I1 chromosomes from rabbit oocytes was achieved with greater efficiency if these procedures were done prior to postovulatory aging. The frequencies of both cell-oocyte fusion and development to the blastocyst stage of reconstituted rabbit embryos were also found to be greater when recently ovulated oocytes were used as recipient cytoplasm. Controlled modifications to the activating conditions th at induce a transient release of calcium ions similar to th at observed during fertilisation have also been found to enhance both the pre- and the postimplantation developmental potential of electric pulse-induced diploid rabbit parthenogenones (Ozil, 1990). These studies have indicated that the response of a n oocyte to activating stimuli is not a uniform event; differing electroactivation treatments produced a marked effect upon subsequent development. These observations strongly indicate th a t the effects of the activation stimulus and its response are not limited to a specific period of meiotic resumption or early embryogensesis. The exposure of recently ovulated oocytes to strontium-supplemented media has been observed to induce the activation of recently ovulated mouse oocytes (Fraser, 1987) and has been observed in this study to readily activate oocytes at HCG + 18 hr. The brief exposure of bovine oocytes and transferred nuclei th a t have undergone electric pulse stimulation to induce cell-oocyte fusion (and activation) to strontium-supplemented medium may provide opportunities to enhance the incidence of parthenogenetic activation in this species. Studies are currently in progress to evaluate the ability of strontium to induce the activation of in vitro-matured bovine oocytes.
ACKNOWLEDGMENTS The present work was supported by grants to G.T.0”. from the AFRC, and to M.H.K. from the CRC and the Scottish Home and Health Dept. L.R.R. was supported by a University of Edinburgh Medical Faculty Undergraduate Vacation Scholarship REFERENCES Austin CR (1970): Ageing and reproduction: Postovulatory deterioration of the egg. J Reprod Fertil 12[Suppll:39-53. Collas P, Robl JM (1990): Factors affecting the efficiency of nuclear transplantation in the rabbit embryo. Biol Reprod 43:877-884. Cuthbertson KSR, Whittingham DG, Cobbold PH (1981). Free Ca2’ increases in exponential phases during mouse oocyte activation. Nature 294:75&757. Eisen A, Reynolds GT (1985): Source and sinks for the calcium released during fertilization of single sea urchin eggs. J Cell Biol 100:1522-1527. Eusebi F, Siracusa G (1983): An electophysiological study of parthenogenetic activation in mammalian oocytes. Dev Biol 96:386-395.
219
Eusebi F, Siracusa G (1983): An electophysiological study of parthenogenetic activation in mammalian oocytes. Dev Biol 96:386395. Fraser LR (1987): Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res 18:363-374. Kaufman MH (1973):Parthenogenesis in the mouse. Nature 242:475476. Kaufman MH (1975):The experimental induction of parthenogenesis in the mouse. In M Balls, AE Wild (eds): “The Early Development of Mammals.” Cambridge: Cambridge University Press, pp 25-44. Kaufman MH (1982): The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment. J Embryol Exp Morphol 71:139-154. Kaufman MH (1983): “Early Mammalian Development: Parthenogenetic Studies.” Cambridge: Cambridge University Press. Kaufman MH, Surani MAH (1974): The effect of osmolarity on mouse parthenogenesis. J Embryol Exp Morphol31:513-526. Miyazaki SI (1988): Inositol 1,4,5,trisphosphate-induced calcium release and nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs. J Cell Biol 106:345353. ONeill GT, Kaufman MH (1988):Influence of postovulatory aging on chromosome segregation during the second meiotic division in mouse oocytes: A parthenogenetic analysis. J Exp Zool 248:125-131. ONeill GT, Kaufman MH (1989): Cytogenetic analysis of ethanolinduced parthenogenesis. J Exp Zool 249:182-192. ONeill GT, McDougall RD, Kaufman MH (1989): Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones. Gamete Res 22:285299. Ozil J P (1990): The parthenogenptic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109:117-127. Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH, First NL (1987): Nuclear transplantation in the bovine embryo: Assessment of donor nuclei and recipient oocyte. Biol Reprod 37:859-866. Robl JM, Prather R, Barnes F, Eyestone W, Northey D, Gilligan B, First NL (1987): Nuclear transplantation in bovine embryos. J Anim Sci 64:642-647. Smith AL, Lodge J R (1987): Interactions of aged gametes: In vitro fertilisation using in vitro-aged sperm and in vivo-aged ova in the mouse. Gamete Res 16:47-56. Smith LC, Wilmut I (1989): Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. Biol Reprod 40:1027-1035. Smith LC, Wilmut I (1990):Factors affecting the viability of nuclear transplanted embryos. Theriogenology 33:153-164. Surani MAH, Kaufman MH (1977): Influence of extracellular Ca2’ and Mg2’ ions on the second meiotic division of mouse oocytes: Relevance to obtaining haploid and diploid parthenogenetic embryos. Dev Biol 59:8690. Tarkowski AK (1966): An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:39&400. Van Blerkom J, Runner MN (1984): Mitochondria1 reorganization during resumption of meiosis in the mouse oocyte. Am J Anat 171:335-3 55. Whittingham DG (1971): Culture of mouse ova. J Reprod Fertil 141Suppl1:7-21. Whittingham DG, Siracusa G (1978): The involvement of calcium in the activation of mammalian oocytes. Exp Cell Res 113:311-317. Whittingham DG, Siracusa G, Fulton B (1978):Ionic activation of the mammalian egg. Biol Cell 32:149-154. Willadsen SM (1986): Nuclear transfer in sheep embryos. Nature 320:63-65. Younis AI, Brackett BG, Fayer-Hosken RA (1989):Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 23:189-201.
Developmental Potential and Chromosome Constitution of Strontium-InducedMouse Parthenogenones G.T. O’NEILL,l L.R. ROLFE? AND M.H. KAUFMAN2 ‘Institute of Animal Physiology and Genetics Research, Roslin, Scotland; ‘Department of Anatomy, University Medical School, Edinburgh, Scotland The brief exposure of recently ovuABSTRACT lated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr”) for 2-10 min was observed t o induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure t o M16 Sr2+ was increased from 2 t o 30 min. Under these conditions a greater proportion of the activated oocytes developed as twopronuclear diploid parthenogenones, due t o failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes t o calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr2+-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr2+induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities t o improve the efficiency of embryo cloning techniques with the ruminant species.
Key Words: Sr2+-induced parthenogenesis, Oocyte, Preimplantation embryo
INTRODUCTION Ovulated mouse oocytes can be induced to initiate parthenogenetic development following their in vitro exposure to a variety of physical or chemical agents (Kaufman, 1983). Two agents t h a t are recognised to be efficient parthenogenetic stimuli are hyaluronidase (Kaufman, 1973; O’Neill and Kaufman, 1988) and dilute solutions of ethanol (Kaufman, 1982; O’Neill and
0 1991 WILEY-LISS, INC.
Kaufman, 1989). A high incidence of hyaluronidaseinduced parthenogenesis is observed only after ovulated oocytes have aged in vivo for at least 8-10 h r prior to their exposure to this agent. Although postovulatory aging prior to either fertilisation or parthenogenetic activation is recognised to interfere with polar body extrusion (Austin, 1970; Kaufman, 1975), hyaluronidase-induced activation has not been found to induce chromosome segregation errors, i.e., aneuploidy, during the completion of the second meiotic division (O’Neill and Kaufman, 1988), Postovulatory aging has also been reported to reduce the potential of mouse embryos to progress through the cleavage stages of early development (Smith and Lodge, 1987). Exposure to dilute solutions of ethanol can induce the activation of more recently ovulated oocytes but its use as a parthenogenetic stimulus may be disadvantageous in some studies, in that a significant proportion of the resultant parthenogenones are recognised to carry numerical chromosomal anomalies (Kaufman, 1982; ONeill and Kaufman, 1989; O’Neill et al., 1989). The majority of activated oocytes that initiate development following exposure to this stimulus develop a single haploid pronucleus following the extrusion of the second polar body. Several studies have also reported that postovulatory aged oocytes incubated in media of low osmolarity (Kaufman and Surani, 1974) or deficient in calcium ions (Surani and Kaufman, 1977; Whittingham and Siracusa, 1978; Whittingham et al., 1978) may also initiate parthenogenetic development. These studies showed that a significant proportion of the activated oocytes developed into two-pronuclear diploid parthenogenones a s their incubation in culture media with a reduced concentration of calcium ions for 1-2 h r interfered with the modifications to the oocyte cytoskeleton required for second polar body extrusion. The exposure of mouse oocytes to culture media supplemented with barium, strontium, or manganese ions has also been observed to induce parthenogenesis, but the proportion-
Received May 1, 1991; accepted June 12, 1991. Address reprint requests to Dr. G.T. O’Neill, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin EH25 9PS, Scotland.
STRONTIUM-INDUCED PARTHENOGENESIS ate incidence of the various classes of parthenogenone usually observed several hours after activation, their chromosome constitution, and their developmental potential in vitro were not investigated (Whittingham and Siracusa, 1978). The incubation of sperm in strontium-supplemented media has not been found to interfere with the processes associated with capacitation in vitro but was observed to inhibit fertilisation as exposure to strontium ions had induced both the resumption of meiosis and cortical granule release in freshly ovulated oocytes prior to sperm penetration (Fraser, 1987). The study described here examined the ability of strontium-supplemented embryo culture medium to induce the activation of ovulated mouse oocytes and analysed both the developmental potential and the chromosome constitution of the resultant parthenogenones. We have found that strontium-induced parthenogenesis can be more efficient and versatile than other methods currently used to induce oocyte activation.
MATERIALS AND METHODS Parthenogenetic Activation Eight- to twelve-week-old (C57B1 x CBA)Fl hybrid female mice were administered a n intraperitoneal injection (i.p.) of 5 IU pregnant mares serum gonadotrophin (PMSG) followed 46 h r later with a n i.p. injection of human chorionic gonadotrophin (HCG) to induce superovulation. At 18 h r after HCG injection, the female mice were killed by cervical disslocation and their oviducts were removed to either 1 ml standard M16 embryo culture medium (Whittingham, 19711, calcium-free M16 medium (Ca2+-freemedium; NaCl adjusted to 6.437 g/liter), or calcium-free M16 medium supplemented with 1.6 mM of SrCl, (M16 Sr2+)in a n embryological watchglass. The cumulus masses were then released from the swollen ampullary region of the oviduct and exposed to M16 Sr2+medium for 2, 5, 10, 30, or 60 min. Cumulus masses were also incubated in calcium-free M16 medium and standard M16 medium for 5 min or 30 rnin as controls to M16 Sr2+exposure. The cumulus masses were washed three times in M16 medium and then transferred to microdrops of M16 medium under liquid paraffin at 37°C in an atmosphere of 5% COz in air for 6 hr. At the end of this period the microdrops containing the cumulus masses were fused to adjacent microdrops of M16 medium supplemented with 1mg/ml of hyaluronidase to remove the adhering cumulus cells. Activated oocytes at this stage of development exhibited pronuclei. The majority of activated oocytes had developed a single haploid pronucleus following the extrusion of the second polar body (1PN). Other parthenogenones either exhibited two haploid pronuclei (2PN) or a single diplid pronucleus (1PND) as second polar body extrusion had failed to occur. A small proportion had undergone immediate cleavage (IC), in which each blastomere exhibited a single haploid pronucleus. 2PN parthenogenones were transferred to fresh microdrops of M16 medium to permit development to the blastocyst stage
215
Metaphase Chromosome Preparations of 1PN Embryos 1PN parthenogenones were transferred to M16 medium supplemented with 1kg/ml of colcemid at 8-10 hr after activation. This treatment arrested development at metaphase of the first cleavage mitosis. Metaphase chromosome spreads were prepared early the next morning using the air-drying technique described by Tarkowski (1966) and were stained with 3% Giemsa’s stain for 10 min at pH 6.8. In Vitro and In Vivo Development of Fertilised One-Cell Embryos Two groups of F1 hybrid female mice were superovulated as described above and caged individually with F1 hybrid male mice shortly after the injection of HCG. The female mice were then checked early the next morning for the presence of a vaginal plug. This was taken as evidence that mating had occurred and was designated to be day 1 of pregnancy. The female mice from the first group were killed by cervical dislocation early on the morning of the first day of pregnancy, and the cumulus masses were isolated as described above and placed in microdrops of M16 medium supplemented with 1 mg/ml of hyaluronidase to remove the adhering cumulus cells. Fertilised oocytes were observed to exhibit a second polar body and two pronuclei. The one-cell fertilised embryos were then washed in two changes of M16 medium and transferred to fresh microdrops of M16 to permit development to the blastocyst stage. At specific times during this period of development, embryos were removed from culture, and cell number counts were determined from air-dried, fixed preparations using the technique described above. A second group of pregnant mice was used to examine the in vivo development of fertilised one-cell embryos. At specific times during in vivo development the female mice were killed, a s described above, and the embryos were flushed either from the oviduct or, in the later stages of preimplantation development, from the uterine horns. Air-dried spreads of these embryos were prepared, as described above, to determine their cell number. The mean number of cell divisions per embryo at specific times during embryo development was calculated from the formula:
where n = embryo cell number, N = No. of embryos per sample, and A = No. of cell divisions per embryo.
RESULTS Spontaneous activation was not observed following the exposure of cumulus masses (total 65 oocytes) at HCG + 18 h r to M16 medium for 5 or 30 min. The exposure of ovulated mouse oocytes to M16 S?’ medium at HCG + 18 h r for 2-60 min was observed to
216
G.T. O’NEILL ET AL.
TABLE 1. Proportionate Incidence of Parthenogenetic Activation Following the Exposure of Ovulated Mouse (C57B1 X CBA) F1 Hybrid Oocytes t o M 1 6 Medium Supplemented With Sr2+at HCG 18 hr Class of parthenogenone Activation Exposure No. of No. No. nonNo. (min) oocytes dead activated activated 1PN 2PN IC lPND frequency(%) 51.5 0 1 37 170 0 196 208 2 404 (81.7)a 94 10 11 85.2 221 336 0 78 5 414 (65.7) 42 14 87.2 121 129 41 306 3 10 351
+
(42.2)
20
267
67
50
150
30
137
97
11
29
40
53
45
0
12
60
52
45
0
7
75 (50.0) 12 (41.3) 10 (83.3) 7 (100.0)
60
9
6
56.2
12
0
5
21.2
1
1
0
22.6
0
0
0
13.5
aPercent activated oocytes that develop as 1PN.
induce parthenogenetic activation. The duration of exposure to M16 Sr2+ medium was found to have a significant effect on the incidence of oocyte activation (Table 1).The highest frequencies of activation, 85.2% and 87.2%, were observed when oocytes were exposed to M16 Sr2+medium for 5 min and 10 min, respectively. The incidence of activation induced by the exposure of oocytes to M16 Sr2+medium for 2 rnin (51.5%) was significantly lower (x2 = 16.4; P > 0.001). The incidence of activation following the exposure of oocytes to M16 Sr2+ medium for 20 rnin was not significantly different from that observed in the former group (x2 = 0.42; P = 0.50). However, following this treatment, the incidence of activation was low, because 25.1% of the treated oocytes died a s a result of the prolonged exposure of cumulus masses to M16 Sr2+ medium. Incubation of the isolated oocytes in M16 Sr2+ media for periods greater than 20 min induced a significant increase in cell death. The duration of exposure to M16 Sr2+media also influenced the proportionate incidence of the four standard classes of parthenogenone. The proportionate incidence of the 1PN class decreased significantly from 81.7% to 42.2% as the duration of incubation was increased from 2 min to 10 min. This was related to a n increase in the proportionate incidence of the 2PN, IC, and 1PND classes as the duration of exposure increased from 2 to 30 min. The majority of the parthenogenones observed following exposure to M16 Sr2+medium for 40 or 60 min were of the 1PN class. However, under these conditions, the majority of the exposed oocytes were observed to be dead when the cumulus cells were removed a t 5-6 h r after activation. The incidence of oocyte activation and the proportionate incidence of the four classes of parthenogenone induced by the exposure of ovulated oocytes to Ca2+free M16 medium at HCG + 18 h r is presented in Table 2. In contrast to the events observed following M16 Sr2+ exposure, the incidence of activation induced by the
incubation of oocytes in calcium-free medium for 5 min (17.6%) was low. The proportion of activated oocytes was not observed to be greater than the nonactivated group of oocytes until incubation in calcium-free medium was increased to 30 min (59.2%). The majority of the activated oocytes developed as 1PN parthenogenones following exposure to Ca2+-free M16 medium for either 5 min (86.6%) or 30 min (80.6%). Incubation of ovulated oocytes in calcium-free M16 a t HCG + 18 h r for either 5 min or 30 min did not induce the cell lysis. Chromosome Constitution of M16 S?+-Induced 1 P N Parthenogenones The chromosome constitution of M16 Sr2’-induced 1PN parthenogenones a t metaphase of the first cleavage is presented in Table 3. The nonanalysable preparations represent those t h a t were overtly hypohaploid due to chromosome scattering and those in which overlapping chromosomes hindered analysis of the chromosome number. The total ratio of hypohaploidy to hyperhaploidy was 4.5:l. The adjusted frequency of aneuploidy was derived from the observed incidence of (No. hyperhaploid preparations) (total NO. preparations)
and accounts for hypohaploidy induced by the spreading technique. The adjusted incidence of aneuploidy following exposure to M16 Sr2+for 5 min and 10 min was 4.7% and O.O.%, respectively. This agent induced a low incidence of aneuploidy that did not differ significantly from that previously observed (O’Neill and Kaufman, 1988) in hyaluronidase-induced parthenogenones (x2 = 0.48: P = 0.50-0.40). The two hyperhaploid spreads were recovered from one group of cumulus masses and indicate t h a t they were derived from one female mouse.
STRONTIUM-INDUCED PARTHENOGENESIS
2 17
TABLE 2. Proportionate Incidence of Parthenogenetic Activation Following the Exposure of Ovulated (C57B1 X CBA) F1 Hybrid Oocytes at HCG + 18 hr to Calcium-Free M16 Embryo Culture Medium Exposure (min) 5
No. of oocytes
No. dead
No. nonactivated
No. activated
170
0
140
30
Class of parthenogenone 1PN 2PN IC lPND 26
Activation frequency(%)
4
0
0
17.64
6
0
0
59.21
(86.6)a
30
76
0
45
31
25
(80.6)
aPercentage activated oocytes that develop as 1PN.
TABLE 3. Chromosome Constitution of Sr2+-Induced1PN Parthenogenones at Metaphase of the First Cleavage Division* c2+ Chromosome constitutiona Aneuploidy (%) No. nonexposure Total no. analysable 19 20 21 Observ. Adj. analysed (min) U I
4 86 2 6.9 4.7 5 101 9 10 104 5 5 94 0 5.3 0.0 *Adjusted incidence = 2 X (No. hyperhaploid preparations/total no. of preparations). aThe normal haploid complement of the mouse (n) is 20.
Developmental Potential of One-Cell Fertilised Embryos and S?-Induced 2PN Parthenogenones The analysis of the in vitro developmental potential of the M16 Sr2+-induced2PN parthenogenones and F2 fertilised mouse embryos retained both in vitro and in vivo, a s determined from the mean number of cell divisions per embryo (X.CD/embryo), analysed a t intervals between 55 and 121 h r postactivation or postfertilisation, is presented in Table 4. The analysis of the X.CD/embryo for the in vivo group of F2 embryos showed that by 93 h r postfertilisation the one-cell embryo had undergone 6.46 cell divisions, with a mean number of 86.0 t 2.8 cells. Subsequent analyses of cell number were unobtainable; hatching from the zona pellucida occurred after this time period. The analysis of the group of one-cell F2 embryos that were retained in vitro found that they had undergone a mean of 5.53 cleavage divisions at 93 h r postfertilisation. At 113 h r postfertilisation they were found to have undergone a mean of 5.68 cell divisions per embryo. Their mean cell number a t 93 h r postfertilisation was 51.4 2.98, and this increased to 85.3 & 4.93 when air-dried spreads were prepared at 113 h r postfertilisation. Hatching from the zona pellucida occurred in all embryos prior to their analysis at 121h r postfertilisation. In comparison to the fertilised F2 embryos retained in vitro and in vivo, the M16 Sr2+-induced2PN parthenogenones were found to exhibit a lower X.CD/embryo value of 4.48 at 93 hr. In these embryos, hatching from the zona pellucida was not observed until after 121 h r postactivation. At 113 and 121 h r postactivation, the X.CD/ embryo values were 6.32 and 6.63, respectively, and the mean cell numbers were 79.3 2 4.33 and 99.3 5.24, respectively.
*
DISCUSSION
This study has clearly demonstrated that the brief exposure of mouse oocytes to embryo culture medium in which CaCl, has been replaced with a n equimolar concentration of SrC1, has the potential to induce the resumption of meiosis and stimulate parthenogenetic development. Previous analyses of mammalian parthenogenetic development have established that both the conditions under which activation occurs and the stimulus employed can significantly influence the incidence of parthenogenesis a s well as the proportionate incidence of the four main classes of parthenogenone (Kaufman, 1983).The significant differences observed between the potentials of M16 Sr2+-and Ca2+-free media to induce parthenogenesis indicate that different mechanisms of oocyte activation were induced following exposure to these media. Previous studies have also reported that maximum levels of activation were observed following the exposure of oocytes to calciumdeficient media when the duration of exposure was in excess of 1 hr, particularly when exposure occurred a t HCG + 20-22 hr. The majority of the activated oocytes were also reported to develop as 2PN parthenogenones (Whittingham and Siracusa, 1978; Whittingham et al., 1978; Kaufman, 1983). In this study the exposure of oocytes to Ca2+-freemedium at HCG + 18 h r for either 5 or 30 min induced only a relatively low incidence of activation, and the majority of activated oocytes developed a s 1PN parthenogenones. Several aspects of the postactivation response of the M16 Sr2+-inducedparthenogenones indicate that this agent is a n efficient parthenogenetic agent. The incidence of activation was high, in the range of 85%, following the exposure of mouse oocytes to this medium
218
G.T. O’NEILL ET AL. TABLE 4. Mean Number of Cleavage Divisions per Embryo of Sr2+ Activated F1 Oocytes Compared W i t h In Vivo- and In Vitro-Maintained Fertilized Embryos Derived From F1 X F1 Matings (Sr2+-induced2PN embrvos derived from arouns 2 a n d 3 of Table 1) Mean No. of cell divisons per embryo F2 embryo F2 embryo Sr2+-induced2PN parthenogenones in vivo in vitro 3.19 2.30 3.49 2.97 5.54 3.37 3.61 5.99 4.76 4.48 6.46 5.53 6.32 5.68 121 6.63 Mean group size 10.0 51.0 31.2 aFertilization assumed to occur at 13 hr after the injection of HCG, activation a t 18 hr after the injection of HCG.
Hours post fertilization/ activationa 55 72 79 93 113
for only 5-10 min. In addition, the majority of the activated oocytes also developed a s 1PN parthenogenones. The ability to extrude the second polar body and develop a s 1PN parthenogenones indicates t h a t the activating stimulus does not interfere significantly with the reorganisation of the cytoskeletal elements of the oocyte during the completion of the second meiotic division. Furthermore, analysis of the chromosome constitution of the 1PN parthenogenones showed that the overall incidence of chromosome segregation errors induced during the completion of the second meiotic division in M16 Sr2+-induced 1PN parthenogenones did not differ from that previously observed in hyaluronidase-activated mouse oocytes. Analysis of the developmental potential of the M16 Sr2+-induced 2PN parthenogenones revealed that the rate of cell division, as determined from the mean number of cell divisions per embryo, was less than that observed in the F2 fertilised embryos that had also been retained in vitro from the one-cell stage. Modifications to culture media to identify the optimal molar concentrations of both strontium chloride and calcium chloride may enhance the rates of both activation and subsequent embryonic development. The ability of divalent ions, such as strontium, barium, and manganese, to induce activation is believed to be related directly to their ability to displace calcium ions from intracellular stores (Fraser, 1987), such as the mitochondria and endoplasmic reticulum closely associated with the spindle apparatus (see Van Blerkom and Runner, 1984; Eisen and Reynolds, 19851, which subsequently leads to a n increase in the intracellular concentration of free calcium ions. The increase in the intracellular concentration of free calcium ions in both ethanol- and sperm-activated mouse oocytes has been observed as a transient series of photoemissions in aequorin-permeated mouse oocytes (Cuthbertson et al., 1981). The resultant nonspecific increase in the intracellular concentration of free calcium ions induces electophysiological changes to occur within the oocyte, which initiate a cycle of calcium and
inositol 1,4,5-trisphosphate release similar to that believed to arise during the induction of oocyte activation at fertilisation (Miyazaki, 1988). However, the electrophysiological response of mouse oocytes to parthenogenetic stimuli, as recorded by the transient calcium ion induced photoemissions from aqueorin-permeated mouse oocytes and analyses of the membrane hyperpolarising response in both fertilised and parthenogenetically activated rodent oocytes (Eusebi and Siracusa, 1983) have all indicated that the activation response in parthenogenetically activated oocytes differs from that recorded from those activated by sperm penetration. It has been reported by Fraser (1987) that ovulated mouse oocytes at HCG + 14-15 h r can initiate parthenogenesis when exposed to calcium-free modified Tyrode’s medium supplemented with strontium ions. The ability of strontium ions to induce recently matured secondary oocytes to initiate parthenogenesis provides new opportunities to improve the efficiency of nuclear transfer (embryo cloning) procedures in ruminant species (Willadsen, 1986; Prather et al., 1987; Rob1 et al., 1987; Smith and Wilmut, 1989). Present techniques use electric pulses of microsecond durations to induce both the cell-oocyte fusion and the parthenogenetic activation of the enucleated recipient oocyte. However, the incidence of reconstituted embryo development following cell-oocyte fusion, oocyte activation, and the pronuclear-like remodelling of the donor nucleus using electric pulse stimulation indicates that modifications to transfer and activation procedures would improve the efficiency of nuclear transfer techniques. Two principal factors that influence the success of nuclear transfer procedures are recognised to be the cell cycle compatibility of the donor nucleus to remodelling within the activated oocyte and the use of recipient oocytes prior to the onset of degenerative changes associated with postovulatory aging (Smith and Wilmut, 1990). Other studies have indicated that the success of nuclear transfer procedures using in vitromatured oocytes (IVM; see Younis et al., 1989) would be improved if recipient oocytes were used prior to the
STRONTIUM-INDUCED PARTHENOGENESIS degenerative changes associated with in vitro postmaturation (“postovulatory”) aging (see Collas and Robl, 1990). This study found th at the removal of metaphase I1 chromosomes from rabbit oocytes was achieved with greater efficiency if these procedures were done prior to postovulatory aging. The frequencies of both cell-oocyte fusion and development to the blastocyst stage of reconstituted rabbit embryos were also found to be greater when recently ovulated oocytes were used as recipient cytoplasm. Controlled modifications to the activating conditions th at induce a transient release of calcium ions similar to th at observed during fertilisation have also been found to enhance both the pre- and the postimplantation developmental potential of electric pulse-induced diploid rabbit parthenogenones (Ozil, 1990). These studies have indicated that the response of a n oocyte to activating stimuli is not a uniform event; differing electroactivation treatments produced a marked effect upon subsequent development. These observations strongly indicate th a t the effects of the activation stimulus and its response are not limited to a specific period of meiotic resumption or early embryogensesis. The exposure of recently ovulated oocytes to strontium-supplemented media has been observed to induce the activation of recently ovulated mouse oocytes (Fraser, 1987) and has been observed in this study to readily activate oocytes at HCG + 18 hr. The brief exposure of bovine oocytes and transferred nuclei th a t have undergone electric pulse stimulation to induce cell-oocyte fusion (and activation) to strontium-supplemented medium may provide opportunities to enhance the incidence of parthenogenetic activation in this species. Studies are currently in progress to evaluate the ability of strontium to induce the activation of in vitro-matured bovine oocytes.
ACKNOWLEDGMENTS The present work was supported by grants to G.T.0”. from the AFRC, and to M.H.K. from the CRC and the Scottish Home and Health Dept. L.R.R. was supported by a University of Edinburgh Medical Faculty Undergraduate Vacation Scholarship REFERENCES Austin CR (1970): Ageing and reproduction: Postovulatory deterioration of the egg. J Reprod Fertil 12[Suppll:39-53. Collas P, Robl JM (1990): Factors affecting the efficiency of nuclear transplantation in the rabbit embryo. Biol Reprod 43:877-884. Cuthbertson KSR, Whittingham DG, Cobbold PH (1981). Free Ca2’ increases in exponential phases during mouse oocyte activation. Nature 294:75&757. Eisen A, Reynolds GT (1985): Source and sinks for the calcium released during fertilization of single sea urchin eggs. J Cell Biol 100:1522-1527. Eusebi F, Siracusa G (1983): An electophysiological study of parthenogenetic activation in mammalian oocytes. Dev Biol 96:386-395.
219
Eusebi F, Siracusa G (1983): An electophysiological study of parthenogenetic activation in mammalian oocytes. Dev Biol 96:386395. Fraser LR (1987): Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res 18:363-374. Kaufman MH (1973):Parthenogenesis in the mouse. Nature 242:475476. Kaufman MH (1975):The experimental induction of parthenogenesis in the mouse. In M Balls, AE Wild (eds): “The Early Development of Mammals.” Cambridge: Cambridge University Press, pp 25-44. Kaufman MH (1982): The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment. J Embryol Exp Morphol 71:139-154. Kaufman MH (1983): “Early Mammalian Development: Parthenogenetic Studies.” Cambridge: Cambridge University Press. Kaufman MH, Surani MAH (1974): The effect of osmolarity on mouse parthenogenesis. J Embryol Exp Morphol31:513-526. Miyazaki SI (1988): Inositol 1,4,5,trisphosphate-induced calcium release and nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs. J Cell Biol 106:345353. ONeill GT, Kaufman MH (1988):Influence of postovulatory aging on chromosome segregation during the second meiotic division in mouse oocytes: A parthenogenetic analysis. J Exp Zool 248:125-131. ONeill GT, Kaufman MH (1989): Cytogenetic analysis of ethanolinduced parthenogenesis. J Exp Zool 249:182-192. ONeill GT, McDougall RD, Kaufman MH (1989): Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones. Gamete Res 22:285299. Ozil J P (1990): The parthenogenptic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109:117-127. Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH, First NL (1987): Nuclear transplantation in the bovine embryo: Assessment of donor nuclei and recipient oocyte. Biol Reprod 37:859-866. Robl JM, Prather R, Barnes F, Eyestone W, Northey D, Gilligan B, First NL (1987): Nuclear transplantation in bovine embryos. J Anim Sci 64:642-647. Smith AL, Lodge J R (1987): Interactions of aged gametes: In vitro fertilisation using in vitro-aged sperm and in vivo-aged ova in the mouse. Gamete Res 16:47-56. Smith LC, Wilmut I (1989): Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. Biol Reprod 40:1027-1035. Smith LC, Wilmut I (1990):Factors affecting the viability of nuclear transplanted embryos. Theriogenology 33:153-164. Surani MAH, Kaufman MH (1977): Influence of extracellular Ca2’ and Mg2’ ions on the second meiotic division of mouse oocytes: Relevance to obtaining haploid and diploid parthenogenetic embryos. Dev Biol 59:8690. Tarkowski AK (1966): An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:39&400. Van Blerkom J, Runner MN (1984): Mitochondria1 reorganization during resumption of meiosis in the mouse oocyte. Am J Anat 171:335-3 55. Whittingham DG (1971): Culture of mouse ova. J Reprod Fertil 141Suppl1:7-21. Whittingham DG, Siracusa G (1978): The involvement of calcium in the activation of mammalian oocytes. Exp Cell Res 113:311-317. Whittingham DG, Siracusa G, Fulton B (1978):Ionic activation of the mammalian egg. Biol Cell 32:149-154. Willadsen SM (1986): Nuclear transfer in sheep embryos. Nature 320:63-65. Younis AI, Brackett BG, Fayer-Hosken RA (1989):Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 23:189-201.
