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Development of Parthenogenetic Rat Embryos¹

^a Max-Delbrück Center for Molecular Medicine, D-13092 Berlin-Buch, Germany

	ABSTRACT

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In an effort to establish cloning technology for the rat, we tested several methods (electric stimulation, treatment with ethanol or strontium) for the parthenogenetic activation of rat oocytes. We observed marked individual differences among rats of the outbred Wistar strain in their ability to yield activatable oocytes. These differences were independent of the activation protocol and may be due to a genetic predisposition that is crucial for the parthenogenetic activation of oocytes. The activation of oocytes was dependent upon the time between superovulation of the donor animal and the collection of the embryos. Aged oocytes (derived about 24 h after superovulation) were more prone to activation by each method than were younger oocytes, and some even underwent spontaneous activation without treatment and exhibited pronuclear formation and blastocyst development. All activation methods were effective in generating parthenogenetic rat embryos, and rat parthenotes developed until implantation. However, in general, short-term (15 min) and long-term (2 h) strontium treatment was superior to stimulation by ethanol or electric pulse for parthenogenetic activation. These results will be helpful in achieving successful cloning in the rat.

implantation, oocyte development

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The rat is the prime animal model in cardiovascular research because it is larger than the mouse and because researchers have longstanding experience with experimental and genetic rat models for cardiovascular diseases. To study the function of genes in the cardiovascular system, transgenic technology has been established in the rat [1, 2]. In spite of the considerable efforts of several laboratories, germ-line competent embryonic stem cells that could be employed for gene targeting procedures are not yet available for the rat [3–5]. Therefore, we have focused our efforts on the generation of cloning technology for this species. Activation of the recipient oocyte is a crucial step in the cloning procedure. A variety of agents activate mammalian oocytes and have been applied when cloning sheep, goats, pigs, cattle, and mice. However, activation protocols must be optimized for use in each species.

The best criterion for evaluating the methods of activation is the ability of parthenogenetic embryos to undergo preimplantation and postimplantation development. Diploid parthenogenetic embryos of some mammals, such as the mouse [6, 7], sheep [8], pig [9], rabbit [10], and monkey [11], can implant after transfer to foster mothers and can even develop into live fetuses until the early heart-beating stage. However, rat oocytes have some important peculiarities. They are spontaneously activated after being released from the oviduct, but this spontaneous activation is incomplete, terminating at a metaphase III-like stage without pronuclear formation [12–14]. Spontaneous activation does not preclude preimplantation development because aged mouse oocytes, spontaneously activated without special treatment, can develop to blastocysts in vitro and activation of aged oocytes of other mammalian species is more efficient [15].

Nevertheless, the first attempts to obtain full preimplantation development of rat oocytes after parthenogenetic activation were unsuccessful. Some activating agents that produced good results with other species were not effective in the rat, and parthenogenetically activated rat oocytes only reached the four-cell stage [13].

Quite recently, a few reports have been published showing development until the blastocyst stage after parthenogenetic activation of rat oocytes by a combined treatment with ethanol or electrical stimulation and the protein phosphorylation inhibitor 6-dimethylaminopurine (DMAP) [16, 17].

Strontium is a very effective activation agent for mouse oocytes [15, 18–20], but results from attempts to activate rat oocytes by strontium are contradictory. Although effective activation of rat oocytes by strontium and development of the activated ova to the blastocyst stage in vitro has been reported [21], other researchers have been unsuccessful in obtaining activation of rat oocytes by strontium treatment [22].

In this study, we assessed the ability of three different agents, ethanol, strontium, and electric pulse, to activate rat oocytes. We also studied the effects of oocyte age on activation efficiency and the developmental capacity of spontaneously and artificially activated rat oocytes.

	MATERIALS AND METHODS

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Animals

Female Wistar outbred rats were obtained from a commercial animal breeder (Tierzucht Schönwalde, Schönwalde, Germany). The rats were kept at a temperature of 21°C ± 2°C on a 12L:12D cycle (lights-on 0600–1800 h) with a humidity of 65% ± 5%. All experimental protocols were performed in accordance with the guidelines for the human use of laboratory animals by the Max-Delbrück Center for Molecular Medicine and were approved by the local ethics committee.

Isolation of Oocytes and Zygotes

Immature 21- to 28-day-old female Wistar rats were induced to superovulation by i.p. injection of gonadotropins: 15 IU eCG (Intervet, Unterschleissheim, Germany) followed 45–50 h later by 30 IU hCG (Intervet). To obtain zygotes after hCG administration, female rats were mated overnight with males of the same strain. On the following morning, the rats were examined for the presence of vaginal plugs or spermatozoa in the vagina. Superovulated rats were killed by cervical dislocation at different time points after hCG injection. The oocytes or zygotes were recovered from the excised oviducts into warm M16 culture medium (Sigma, St. Louis, MO) immediately after death, and M16 medium containing 0.1% (w/v) hyaluronidase (Sigma) was used to remove cumulus cells.

Parthenogenetic Activation

Spontaneous activation The oocytes were placed into prewarmed and equilibrated M16 medium. In the first group, cumulus cells were removed immediately, and in the second group oocytes were removed 7–8 h after recovery from the oviduct.

Activation by electrical stimulation For electroactivation, the same protocol was used as described previously for electrofusion [23]. The oocytes were preequilibrated in fusion medium consisting of 0.3 M mannitol solution containing 0.1 mM MgCl₂ and 0.1 mM CaCl₂ (1–2 min) and then placed between the electrodes of a fusion chamber (GI-2; Institute for Instrumentation in Biology, Pushkino, Russia) in fusion medium. An alternating current field (8 V, 500 kHz, 10 sec) was used in the first step, and activation was induced with the aid of a pulse generator (GI-2; Institute for Instrumentation in Biology). Two direct current pulses (60 V, 20 µsec) were applied, with 100 msec between the pulses. The treated oocytes were carefully washed.

Chemical activation For strontium activation, the oocytes were incubated for different lengths of time (15 min or 2 h) in Ca²⁺-free and Mg²⁺-free M16 medium containing 2 mM Sr²⁺ at 37°C in a CO₂ incubator. After treatment, the oocytes were carefully washed. For ethanol activation, the oocytes were exposed directly to 8% (v/v) ethanol for 7 min and then rinsed three times.

Diploidization To obtain diploid parthenogenetic embryos, the oocytes from all experimental groups were cultured 7–8 h in the presence of 5 µg/ml cytochalasin B (Sigma).

Analysis The efficiency of oocyte activation was analyzed 10–12 h after treatment. The oocytes were observed under an inverted microscope with Nomarski optics. Those oocytes that formed visible pronuclei were recorded as activated.

Parthenogenetic and In Vivo-Fertilized Embryo Culture

Zygotes or treated oocytes after activation were washed in M16 medium and then cultured in the same medium under 5% CO₂ in air at 37°C for about 10 h. Not all activated oocytes with pronuclei were used for further cultivation.

Selected oocytes were washed in mR1ECM medium [24], transferred (10–20 embryos) into 700 µl of the same medium in four-well culture dishes (Nunc, Roskilde, Denmark), and cultured under 5% CO₂ in the air at 37°C. The culture medium had previously been equilibrated with the gas phase and temperature in the CO₂ incubator for 2–3 h. Ova showing one or more divisions and blastocyst cavity formation were classified as cleaving embryos and blastocysts, respectively. Blastocysts developed from parthenogenetically activated oocytes and in vivo-fertilized zygotes were assessed for cell number using an air-drying technique as described previously [23].

Oviduct Transfer of Activated Oocytes

Sprague-Dawley females 4–5 mo old (200–280 g body weight) were used for transplantation of the activated oocytes. For pseudopregnancy induction, females had been mated with vasectomized males of the same strain. On the day after activation, the oocytes were transferred into the oviducts (10–15 per oviduct) of recipients on Day 1 of pseudopregnancy (the day of vaginal plug detection). For oviduct transfer, the recipients were anesthetized with a mixture of 0.25 ml Ketavet (100 mg/ml; Pharmacia & Upjohn GmbH, Erlangen, Germany) and 0.05 ml Rompun (2%; Bayer AG, Leverkusen, Germany) per animal. The recipient rats were killed on Day 11 of gestation to count the number of implantation sites.

Statistical Analysis

The efficiencies of activation and development were compared using the chi-square test and the Student t-test.

	RESULTS

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In the first experiments, the time of ovulation after hormone treatment was determined. Oviducts from nine females were investigated 9–10 h after hCG injection, and none of them contained oocytes. The first oocytes were collected at 12–14 h after superovulation. In subsequent studies, oocytes collected at different time points after superovulation were compared starting with young oocytes (isolated at 12–14 h after hCG).

There was marked variation in the efficiency of activation among individual rats. Therefore, we defined active animals as those with efficiencies of oocyte activation of greater than approximately 40% and nonactive animals as those with an activation efficiency of not more than approximately 20% (see Fig. 1 and the ranges of efficiencies of pronuclear formation shown in the tables).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Percentage of oocytes activated by ethanol out of all oocytes isolated from an animal at different times after hCG injection. Two groups of animals are clearly distinguishable: active animals, with >40% activated oocytes, and nonactive animals, with <20% activated oocytes

The use of ethanol for parthenogenetic activation was highly effective only for aged oocytes (22–24 h after hCG) (Fig. 1). The proportion of active animals and the incidence of pronuclear formation and cleavage was reasonably high (Table 1).

Electrical stimulation alone was also effective for parthenogenetic activation. However, the efficiency of activation and cleavage in active animals was significantly lower in young oocytes (Table 2).

Short-term (15 min) treatment with Sr²⁺ was sufficient for oocyte activation. Although the proportion of active animals was somewhat lower for oocytes collected 16–18 h after hCG injection, the efficiencies of activation and cleavage were equal for all ages of oocytes (Table 3).

Two hours of incubation with Sr²⁺ resulted in a significantly increased proportion of active animals with increased time after hCG injection. The efficiencies of activation and cleavage were similarly high for oocytes of all ages, but the cleavage was observed slightly more often in young (12–14 h) oocytes than in older oocytes (Table 4).

In a separate group of oocytes, activation by Sr²⁺ was studied without diploidization. Increased duration of exposure of 18- to 20-h oocytes to Sr²⁺ led to the appearance of parthenotes with two pronuclei. After 15 min of exposure, only 3.4% (4/118) of the parthenotes contained two pronuclei; however, after 2 h the proportion had significantly increased to 21.2% (38/179) (P < 0.01).

Rat oocytes also were subject to spontaneous activation with pronuclear formation and cleavage without any treatment and without hyaluronidase (Fig. 2). However, the ability to form a pronucleus and the efficiency of activation depends on the age of the oocytes starting 22–24 h after hCG injection and becoming higher in very old oocytes (25–27 h after hCG) (Table 5).

View larger version (134K): [in this window] [in a new window]   FIG. 2. Rat oocytes with two visible pronuclei after spontaneous parthenogenetic activation and diploidization by cytochalasin B

The results of experiments in which activated oocytes developed to the blastocyst stage are shown in Table 6. There were no significant differences in the efficiency of cleavage (Fig. 3a) and blastocyst formation (Fig. 3b) between all experimental and control groups. However, the efficiency of blastocyst formation in Sr²⁺-activated 12- to 14-h oocytes was slightly lower than that of the controls. The mean number of cells in 31 blastocysts developed from oocytes activated by Sr²⁺ was 24.9 ± 1.4. This finding was not significantly different from the mean cell number in 11 control blastocysts developed in vitro from in vivo-fertilized zygotes (27.7 ± 3.3). When artificially and spontaneously activated oocytes were transferred to foster mothers, the efficiency of implantation was the same for all experimental groups (Table 7), but no live fetuses could be detected at Day 11 of pregnancy (Fig. 4).

View larger version (111K): [in this window] [in a new window]   FIG. 3. Development of rat oocytes parthenogenetically activated by strontium. a) Parthenogenetic two-cell embryos. b) Parthenogenetic blastocysts.

View larger version (79K): [in this window] [in a new window]   FIG. 4. Rat conceptuses at Day 11 of pregnancy. a) Normal conceptus from fertilized egg. b) Parthenogenetic conceptuses recovered from implantation sites.

	DISCUSSION

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One of the most striking findings of our work is the dramatic variation among individual rats in the efficiency of parthenogenetic activation of oocytes and the developmental ability of the resulting embryos. The proportion of activation ranged from 39.1% to 100% for active animals and from 0% to 22.2% for nonactive animals.

Because oocytes from different rats ovulate approximately simultaneously 10 h after hCG injection (consistent with a previous report [12]), differences in oocyte age cannot explain this individual variation. However, this effect may be explained by genetic differences among animals of this outbred Wistar strain, suggesting an influence of the maternal genetic background on the capability of parthenogenetic activation of oocytes. Such genetic differences may be more important when working with young oocytes. The ability of oocytes to undergo parthenogenetic activation depends upon age, and aged oocytes can be more efficiently activated. Therefore, individual differences are less noticeable and the proportion of active animals increases with time after hCG injection.

Parthenogenetic activation in mice is dependent upon genetic background [25, 26]. The efficiency of nuclear transfer in cattle also depends upon the maternal lineage of oocyte donors [27]. These effects of the maternal genotype on parthenogenetic activation and embryo development are thought to be linked to imprinting of the maternal genome or to differences in ooplasm composition that have long-term effects on development [28, 29].

Electrostimulation was the first method used for parthenogenetic activation. Without any additional activators, this method has been very effective for the activation of pig [9] and rabbit [10] oocytes. After diploidization by cytochalasin B, these oocytes develop until early fetal stages. In one study, development in rat oocytes stopped at the two-cell stage when only electrostimulation was used; however, the investigators omitted the diploidization step after stimulation [17]. The diploidization step seems to be critical to development; diploid mouse embryos develop better than do haploid mouse embryos [30–32]. Differences in the activation parameters may also play a role in the developmental performance of porcine [33, 34] and rabbit [10] parthenogenetic embryos, which depends upon parameters of the electric field. Nevertheless, development to the blastocyst stage was reported after combined treatment of rat oocytes with electric field and DMAP [16, 17]. DMAP may induce diploidization in rats; treatment of bovine oocytes with DMAP after electroactivation suppresses the second reduction division. Thus, karyokinesis does not occur and oocytes enter interphase of the first mitotic cycle as uniform diploid parthenotes [35]. In our experiments, we obtained development of diploid parthenotes after electrostimulation and diploidization by cytochalasin B. We used cytochalasin B for diploidization because it inhibits the extrusion of the second polar body, resulting in diploid zygotes with two pronuclei [36, 37].

Ethanol is a frequently employed activation agent for mammalian oocytes. However, ethanol alone cannot induce parthenogenesis of rat oocytes [13]. Recently, parthenogenetic activation was obtained after combined treatment of rat oocytes with ethanol and cycloheximide [22], and development to the blastocyst stage occurred after combined treatment with ethanol and DMAP [17]. Ethanol alone activated rat oocytes, but parthenotes did not develop beyond the two-cell stage when the diploidization step was omitted [17]. In our experiments, we obtained activation and further development until the blastocyst stage in vitro and even implantation in vivo with ethanol. However, aged oocytes that were diploidized after stimulation were used.

Strontium is a very popular agent for parthenogenetic activation of mouse oocytes. Brief exposure of mouse oocytes to medium supplemented with 1.6 mM strontium chloride for 2–10 min induced a high incidence of parthenogenesis. When oocytes were incubated in strontium for 20–60 min, a lower incidence of activation and a significant degree of oocyte degeneration was observed [18]. Nevertheless, different researchers have published various protocols for strontium treatment of mouse oocytes: 4.6 or 9.2 mM Sr²⁺ for 30–40 min [38]; 1.71 mM Sr²⁺ for 1 h [39], 10 mM Sr²⁺ for 1.5 h [40], 10 mM Sr²⁺ for up to 24 h [41], 10 mM Sr²⁺ for 6 h [20], and 25 mM Sr²⁺ for 1 h [15]. Postimplantation development was modestly improved by extending the time of exposure to Sr²⁺-containing medium. The parthenotes implanted, but only about 2% of the transferred embryos developed into fetuses [41]. Strontium activation was also successfully used for the cloning of mice [19, 42].

Activation by strontium may depend on the species involved; Chinese hamster oocytes are refractory to activation by strontium [43]. In the rat, different results have been obtained with strontium. In one report, oocytes arrested at a metaphase III stage after activation by strontium [22]. In contrast, others have reported successful activation and development of rat oocytes to blastocysts after treatment with 2.5–5 mM Sr²⁺ for 1.5–3 h [21]. In our experiments, 2 mM Sr²⁺ was a very effective agent for parthenogenetic activation of rat oocytes. Even increasing the exposure time from 15 min to 2 h did not decrease the efficiency of activation nor did it result in degeneration. This increase, however, resulted in parthenotes with two pronuclei, as has been observed in mice [18].

After Sr²⁺ activation, the mean cell number in parthenogenetic blastocysts (24.9) was the same as that reported for blastocysts derived from oocytes after electrical stimulation and DMAP treatment (26.9) [17].

Our results indicate that of the three methods evaluated Sr²⁺ ion treatment was the most effective method for parthenogenetic activation of rat oocytes. This method was suitable for oocytes of all ages and provided the highest proportions of active animals and pronuclear formation.

In our experiments, some rat oocytes developed after spontaneous activation without special treatment. In previous studies, spontaneously activated rat oocytes did not even form pronuclei [12–14]. However, in those studies, younger oocytes (up to 20 h after hCG injection) were used. In agreement with our results, mouse oocytes recovered 18 h after hCG injection developed to the blastocyst stage after spontaneous activation [15].

The aged rat oocytes collected 22–27 h after hCG underwent spontaneous activation and developed to blastocysts in vitro and implanted in vivo into the uterine wall. However, it remains unclear exactly how this activation was induced. In the mouse, hyaluronidase may trigger parthenogenetic activation [44–46]. However, in our experiments aged rat oocytes isolated without hyaluronidase treatment were also spontaneously activated. Cooling at the time of ovum recovery may explain the spontaneous activation. In previous studies, brief cooling of rat oocytes induced Ca²⁺ transients and resumption of the second meiotic division [13, 47].

The ability of oocytes to activate parthenogenetically depends upon age; aged oocytes can be more efficiently activated, as has been demonstrated previously for mouse [15], cow [35, 48], and rabbit oocytes [49] and now for rat oocytes. In bovine oocytes, this phenomenon may be associated with differences in activity of the maturation promoting factor and mitogen-activating protein kinases in young and aged oocytes [50]. However, the time lapse after hCG injection increased both the activation efficiency of oocytes of individual rats and the proportion of active animals. In contrast, the activation efficiency of oocytes from a nonactive animal did not increase with age of the oocyte.

Aged rat oocytes, after spontaneous activation and diploidization, can form pronuclei and can develop parthenogenetically. Aged rat oocytes can even implant after transfer, similarly to young oocytes, even if live fetuses are not obtained. In contrast, the developmental ability of activated bovine oocytes declines with the age of the oocytes [35]. However, aged and young porcine oocytes developed to the same degree [51].

We generated parthenogenetic rat embryos using various methods. These embryos were able to develop to the blastocyst stage in vitro and to implant into the uterine wall but were unable to develop normally. As a peculiarity of the rat, parthenogenetic activation occurred spontaneously without treatment, particularly in oocytes that were collected a considerable time after ovulation. These results will be helpful for achieving successful cloning in the rat.

	ACKNOWLEDGMENTS

 
We thank Liselotte Winkler, Reika Langanki, and Rosemarie Barnow for skillful technical assistance and Ora E. Lockley-Jones for help in the preparation of the manuscript.

	FOOTNOTES

 
¹ I.Z. was supported by a fellowship of the Berlin Program to Support Equal Opportunities for Females in Research and Teaching.

² Correspondence: Michael Bader, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin-Buch, Germany. FAX: 49 30 9406 2110; e-mail: mbader{at}mdc-berlin.de

Received: 16 April 2002.

First decision: 7 May 2002.

Accepted: 16 September 2002.

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