ISSN 10623590, Biology Bulletin, 2010, Vol. 37, No. 5, pp. 446–452. © Pleiades Publishing, Inc., 2010. Original Russian Text © G.P. Malenko, A.V. Komissarov, O.I. Stepanov, 2010, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2010, No. 5, pp. 527–534.

DEVELOPMENTAL BIOLOGY

In Vitro Development of the Reconstructed Bovine Embryos Activated at Various Time after Electrofusion G. P. Malenko, A. V. Komissarov, and O. I. Stepanov Biotechcenter, Afanasyev Research Institute for Breeding of FurBearing Animals and Rabbits, Russian Academy of Agricultural Sciences, Gorki Leninskie, 142712 Moscow Region, Russia email: [email protected] Received October 14, 2009

Abstract—The dynamics of in vitro development of reconstructed bovine embryos activated at various time after electrofusion was studied. The in vitro mature oocytes without zona pellucida enucleated using the blind method were taken as cytoplasts. Fetal fibroblasts were used as the nuclei source. Approximately 40% of embryos activated between 3 and 3.2 hours after electrofusion developed to blastocysts. The efficiency of in vitro development of cloned embryo of cloned embryo did not decrease when the time between electrofusion and activation was extended up to 4–5 hours. The pattern of more successful development of in vitro recon structed embryos was found using enucleated oocytes, extrusion the first polar body to 18 hours in compari son with oocytes matured in vitro afterwards, as cytoplasts. DOI: 10.1134/S106235901005002X

INTRODUCTION Animal cloning using the nuclei of somatic cells of embryos and adult animals is in the focus of attention of the world’s leading laboratories. The opportunity to produce healthy cloned organisms allows increasing the number of animals of high genetic potential. The high efficiency of the origination of transgenic farm animals with desired properties was confirmed using the method of nuclei transplantation of preliminarily in vitro transfected somatic cells into the cytoplasm of enucleated oocytes. The origination of transgenic ani mals could be a promising technique in biomedical investigations: for xenotransplantation of tissues and organs, both as models of human diseases for investi gations and as producers of recombinant pharmaceu tical proteins. However, the processing efficiency of somatic cloning is very low; it varies from 4 to 15% in cattle. In many cases a high frequency of fetal loss in the early postimplantation stages and abortion in the last trimester of pregnancy were revealed. In vitro matured oocytes enucleated at the metaphase II (MII) stage are commonly used as recipients of recon structed embryos of cattle. The factors making for reprogramming of nuclei of adult cells were found in the oocyte cytoplasm in metaphase II stage. As molec ular mechanisms of nucleus reprogramming are unknown, it has shown that the reprogramming starts right away after injection of the donor nucleus into the recipient cytoplasm and it continues after cleavage (Latham, 2004, 2005). The fused constructs should be activated to escape the initial stage of meiosis MII of cytoplastrecipients and the activation of embryogen esis. Herewith the activation terms can influence the

subsequent development of the reconstructed embryos. It was shown that the constructions activated after 2 hours developed better in comparison with those activated half an hour after electrofusion (Liu et al., 2001). According to data of other authors, the degree of development of the reconstructed embryos activated 2–2.5 hours after fusion was rather more than in embryos activated later (Choi et al., 2004; Aston et al., 2006). The crucial importance of the reprogramming stage in nuclei transplantation of somatic cells has specified the study purpose, i.e., investigation of the in vitro development of reconstructed bovine embryos up to the blastocyst stage depending on the activation time after electrofusion. MATERIAL AND METHODS Chemicals from the SigmaAldrich company (United States) were used in this study. TLPHEPES medium (Bavister, Yanagimachi, 1977) was used as a matrix of the medium for aerobic studies of oocytes, embryos, and somatic cells. TLPHEPES medium was supplied with 1 mM Lglutamine adjusted to pH = 7.2–7.4 and 270–280 mOSM (medium T0). The TLPHEPES medium contained 0.01% of polyvinil alcohol (PVA; medium TPVA) and 10% of blood serum (medium T10) or bovine albumin (BSA; medium TBSA) in amounts of 1 or 30 mg/ml for dif ferent cloning stages. The elecrofusion was carried out in the solution with 270 mM Dmannitol, 0.1 mM MgCl2, 0.5 mM HEPES, 0.05% BSA, pH 7.2–7.4. Mannitol solution, the TLPHEPES medium, and its varieties were prepared in the laboratory, sieved using a

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filter with pore size 0.22 μm, packed, and stored at – 20°С. The medium SOF was prepared for embryo growth (Tervit et al., 1972); it was sieved, stored at 4°С, and used within 2 weeks. The solutions and media were prepared using water purified by MilliQ (Millipore, United States) filtration. Estrous bovine serum (EBS) was prepared for the experiments. Oocyte and cytoplast preparation for cloning. All preparations of oocytes, cytoplasts, and reconstructed embryos were made in the laboratory without heating plate (except cases of definite temperature). Cow ova ries were obtained at an abattoir. The oocytes were iso lated from 2–8 mm antral follicles. Oocytes with homogenous cytoplasm covered with a multilayer cumulus were selected. The medium 199 was used with 10% EBS, 0.66 mM sodium pyruvate, 5.0 IU/ml human chorionic gonadotropin (Moscow endocrine factory), 5 μg/ml folliclestimulating hormone (FSH super, Agrobiomed, Russia), 1.0 μg/ml estradiol17β, and 50 μg/ml gentamicin for oocyte maturation. About 30–50 oocytes with homogenous cytoplasm and intact cumulus incubated in 0.5–0.6 ml maturing medium at 39°С and air conditions adding 5% СО2. The oocytes were cleared from the cumulus cells in 0.1% solution of hyaluronidase using Vortex (Heidolf, Germany) after 15 hours of maturation. The zona pel lucida was removed while oocytes were incubated in the solution of pronase (2 mg/ml) in T10 medium at 39°С for 2 min. The oocytes with a distinct polar body I (PB1) were used for enucleating. Other oocytes con tinued to mature; subsequent control and isolation with PB1 for enucleating were made at 30 min inter vals. PB1 extrusion was accepted as the in vitro oocyte maturation rate until stage MII. The blind method of oocyte enucleating was car ried out in the T10 medium using an inverted micro scope (Axiovert M35, OPTON, Germany) and micromanipulator (KM2, Russia). Polar body 1 was aspirated with a small adjacent part of the oocyte cyto plasm using a micropipette with a 25–30 μm diameter with a planecut edge. These cytoplasts were put back into the maturing medium, and they were incubated under the same conditions as before. In order to deter mine the enucleating efficiency, the part of oocytes assumed as cytoplasts and fragments, aspirated with PB1, was stained by 5 μg/ml Hoechst 33 342 in the medium TPVA. The chromosome location was determined using UVlight from the microscope (Zeiss, Germany). The diameters of cytoplasts and aspirated fragments of oocytes were measured. Fetal fibroblast donors of nuclei preparation. The fetal fibroblasts of cattle were used as donors of nuclei for construction design. After killing a cow at 45days pregnancy, the womb was isolated and delivered to the laboratory on ice within 30 min. Further procedures were carried out aseptically. The embryo was isolated from the womb, its head and organs were eliminated. Other tissues of the embryo were cut accurately and washed in phosphate saline buffer without calcium BIOLOGY BULLETIN

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(а)

(b) Fig. 1. The oocyte of the cattle at the MII stage without a zona pellucida. (a) Stained by Hoechst 33342, specimen by transmitted light; (b) stained by Hoechst 33342, speci men by UVlight (Figs. 1, 3). The maternal chromosomes of the oocytes are in immediate proximity to PBI. The arrow shows PBI. The objective: ×20.

and magnesium salts. The tissue was dispersed by the fractionary method in 0.25% tripsin solution at 39°С using a magnetic stirrer. Tripsin was inhibited by fetal calf serum (FCS); the cell suspension was centrifuged at 1000 turns/min for 5 min. To DMEM were added 10% FCS, 50 μg/ml gen tamicin, and 5.6 μg/ml amphotericin B. The cells were plated in the concentration 5–8 × 105 cells/ml and incubated at 39°С in an air conditioned medium with 5% СО2. The primary fibroblast culture of embryos of cattle developed into a monolayer in 4– 5 days. After 80% confluency, the fetal fibroblasts of the third and fourth passages were frozen in DMEM containing 15% FCS and 10% DMSO. After cryo preservation the unfrozen cells were plated and cul tured without change of plates and medium for 5–6 days. The monolayer was removed by 0.05% tripsin in 0.02% versene solution, suspended in T10 solution, and used as nuclei donors within 1–2 hours. Preparation of the constructs cytoplast–fetal fibro blast and electrofusion. The preparation of the pairs cytoplast–fetal fibroblast was made manually by pipette with a diameter of 200–250 μm under a stere omicroscope (NIKON, Japan) in the medium TPVA

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Fig. 2. The enucleated oocytes of cattle without a zona pel lucida. Stained by Hoechst 33342, specimen by UVlight. As a result of enucleation, the nuclear contents were com pletely removed. The objective: ×10.

with 50 μg/ml phytohemagglutinin for 5 min (Oback et al., 2003). The round cells of smallest diameter with an unruffled fine surface were selected from the sus pension as nuclei donors. The obtained constructions were removed into the mannitol solution and then to the electrofusion chamber with mannitol solution (the chamber with parallel wire electrodes for fusion, IBI RAS, Pushchino). The constructions were put into the chamber manually in such a way that the pane of the membrane junction was parallel to the electrodes. Electrofusion was carried out by the direct current 1800 V/cm for 20 μs. The constructions were removed from the chamber to a prepared microdrop of T10 medium covered with mineral oil. The results were checked 10–15 min later. Before activation the fused constructions put into the drops conditioned the maturing medium. Activation. The activation of the fused construc tions was carried out after 23–25 hours the oocytes started to mature. The constructions were incubated for 4 min at 39°С in TBSA medium (1 mg/ml) con taining 5 μM ionomycin, after which they were washed in TBSA with 30 mg/ml of albumin. Then they were incubated individually in drops of 5–10 μl of TALPFert medium with 2 mM 6DMAP covered with mineral oil for 4 hours at 39°С in an air condi tioned medium with 5% СО2 (SuskoParrish et al., 1994). Embryo cultivation. In order to cultivate the embryos without zona pellucida, the system WOW (the Well of the Well; Vajta et al., 2000) was prepared by making indentation microwells on the bottom of a 4well dish (Nunc, Roskilde, Denmark). Into the well was placed 0.4 ml of SOF medium with 5% EBS, then the medium was covered with 0.4 ml of mineral oil and

(b) Fig. 3. The cytoplasm parts removed together with polar bodies 1 during the bovine oocyte enucleation. The mater nal chromosomes are in the fragments of the oocytes removed with PBI. The arrows show PBI. The objective: ×10.

kept in an air conditioned medium with 5% СО2 at 39°С for several hours. The activated reconstructed embryos were put individually into microwells of the 4well dish, and the cultivation was carried out at 39°С in air with 5% СО2, 5% О2 and 90% N2. The embry onic cleavage was estimated to occur 42–48 h after the activation, the development until the blastocyst stage was checked after 8.5 days. The results were shown as the arithmetic mean ± standard error of mean. The data were analyzed by the ttest method. RESULTS The polar bodies I of the oocytes are distinct both under the stereomicroscope and under a microscope with transmitted light (Fig. 1a). The polar bodies I BIOLOGY BULLETIN

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Table 1. The in vitro development of reconstructed bovine embryos in depending of time between the electrofusion and the ac tivation The group

The number of embryos, n

The oocyte maturing, h

The oocyte maturing, h

1 2

104 152

20–24 ≤18

1–2 3–5.5

The development of embryos, n (%) ≥2 blastomeres 41 (42.3 ± 8.7)* 112 (80.0 ± 8.0)*

≥4 blastomeres

The blastocyst

20 (20.4 ± 4.0)* 15 (14.7 ± 4.5)* 93 (69.6 ± 10.1)* 59 (43.8 ± 7.5)*

* Among the columns the difference between the groups is reliable to within p < 0.01.

Table 2. In vitro development of the embryos of the cattle depending on terms of maturing of the oocyterecipients and activa tion time of the constructions The group 1 2 3

The interval The number The oocyte of embryos, n maturing, h before activation, h 109 340 313

18–20 ≤18 ≤18

3–3.7 3–3.2 4–5

without zona pellucida are kept on the surface of oocytes due to the connection with maternal chromo somes, which was shown according to their location (Fig. 1b). The efficiency of the blind method of enu cleating, when PB1 and a small part of the adjacent cytoplasm (approximately 3%) of the oocyte are removed, was 96.2% (180/187, 6 repeats; Figs. 2, 3). Using 50 μg/ml solution of phytohemagglutinin in the TPVA medium for pair preparation allowed the somatic ?ells to be kept on the surface of cytoplasts during further procedures. Most of the constructions fused in 10–15 min after the electrical impulse. Thus, in 12 experiments successfully fused 510 pairs of the somatic cellcytoplast out of 538 pairs, giving a success rate of 94.8%. The period between electrofusion and activation of the reconstructed embryos was from 1 to 2 hours (group 1) and more than 3 hours (group 2) in the first part of the experiments. Significantly more recon structed embryos cleaved and reached the blactocyst stage if the activation was carried out between 3 and 5.5 hours after electrofusion (Table 1). The isolated from ovaries and in vitro matured oocytes were taken from the same part for both groups in every day of the experiment (6 repeats). The age of the cytoplasts in both groups before activation was between 23 and 25 hours after the initiation of matura tion, and the activation of the constructions in both groups was carried out synchronously. The oocytes that finished in vitro maturing to 18 hours were used for cytoplast production in group 1 in comparison to oocytes of 20–24 hours in group 2. Thus, such a factor as the in vitro oocyte maturing pattern could influence the results. In the next experiment, in group 2 the cytoplasts were prepared from the oocytes extruded PB1 to BIOLOGY BULLETIN

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The development of embryos, n (%) ≥2 blastomeres

≥4 blastomeres

The blastocyst

73 (67.7 ± 4.7) 255 (76.4 ± 4.2) 222 (72.9 ± 4.8)

54 (49.1 ± 3.9) 201 (61.2 ± 4.5) 186 (62.8 ± 5.6)

34 (31.0 ± 5.1) 130 (40.2 ± 4.0) 136 (46.4 ± 5.0)

18 hours; in group 1 it was carried out later. In contrast to the first experiment, the period between the electro fusion and the activation of the constructions in both groups was nearly the same and it ranged from 3 to 3.7 hours. The rates of cleavage and development up to the blastocyst stage were higher in group 2 than in group 1 (Table 2). But these differences are not signif icant. Group 3 contained the embryos reconstructed from in vitro matured oocytes of 18 hours, but they were activated 4–5 hours after electrofusion. Of these, 46.4% of embryos developed to the blastocyst stage (Table 2; group 3). Thus, the prolongation of the period between the electrofusion and the activation had no effect on the development of the reconstructed embryos. DISCUSSION Oocyte meiosis in a great number of mammal spe cies is blocked at the MII stage because of the high activity of the maturationpromoting factor (MPF). The transplanted nucleus of the somatic cell under the influence of MPF of the recipient cytoplasm is char acterized by the nuclear envelope breakdown (NEBD) and the premature chromosome condensation (PCC). It was shown for mice and swine, in the majority of the nuclei transplanted in the cytoplasm of stage MII oocytes, that chromosome condensation was revealed after 1–2 hours (Czolowska et al., 1984; Wakayama et al., 1998; Lai et al., 2001). At the same time, if the nucleus was transplanted in a previously activated cytoplast with low activity of MPF, the nuclear mem brane remained intact (Campbell et al., 1993; Tani et al., 2001). If the activation did not precede the nucleus transplantation, but was carried out simulta neously or directly after the electrofusion, the NEBD

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and PCC were in the oocyte cytoplasm of rabbits (Col las, Robl, 1991) or cattle (Campbell et al., 1993; Tani et al., 2001; Shin et al., 2002) for 1–1.5 hours. How ever, according to data of Acagi et al. (2003a), the nuclear membrane remained there in most cases (26/28, 92.9%). MPF activity is regulated by phosphorylation level of protein kinase p34cdc2 and cyclin B. At the time of arrest of oocytes of the MII stage, the protein kinase p34cdc2 is in dephosphorylated activated form (Collas et al., 1993a). It is supposed that in the cytoplasm of matured oocytes of mammals there is a cluster of high labile proteins, such as the cytostatic factor (CSF), which support a high stable level of MPF activity due to prevention of cyclin B degradation. The increase of the concentration of intracellular Ca2+ as a conse quence of spermatozoon penetration or other chemi cal/physical influence has a destructive effect on CSF. It results in cyclin B degradation within 1 hour and kinase р34cdc2 phosphorilation that causes MPF inac tivation and meiosis renewal (Wu et al., 1997; Liu, Yang, 1999; Shen et al., 2008). The drastic increase in the intracellular calcium concentration in the oocyte cytoplasm could be caused by the electric impulse. The concentration growth of the intracellular Ca+2 is specified both by the parameters of the electric impulse and by the Ca+2 concentration in the medium (Sun et al., 1992). The effect of the electrical impulse of 1000 V/cm for 40 μs using a mannitol solution containing 0.1 mM Ca+2 caused the long increase of the intracellular Ca+2 con centration up to 1100 nM under a reference concen tration of about 55 nM (Collas et al., 1993b) in the oocytes of cattle. However, in the absence of Ca+2 in the medium, in spite of the appropriate parameters of the electric impulse, a growth of the concentration of intracellular Ca+2 in the oocyte cytoplasm of swine (Sun et al., 1992), cattle (Collas et al., 1993b), rabbits (Fissore, Robl, 1992), and mice (Rickords, White, 1992) was not revealed. According to Sun et al. (1992), the electrofusion between caryoplast and cytoplast could be successfully carried out in the Cafree medium without activation of the cytoplast. The authors came out with the sug gestion that this guaranteed the location of the trans planted nucleus in the conditions of the inactivated cytoplasm during the time while the nucleus was com pletely reprogrammed. According to Boquest et al. (2002) with use of in vivo matured oocytes of swine as recipientcytoplasts, the lack of Ca2+ in the medium during electrofusion was crucial for the prevention of the competitive activation of most of the reconstructed embryos. Based on these data with the purpose to prevent cytoplasts activation, Ca+2salts were removed from mannitol solution in our experiments during electro fusion. Approximately 95% of the constructions fused within 15 min after electrical impulse. Evidently, phy tohemagglutinin provided a tight joint in the contact

zone of plasma membranes of the fibroblast and cyto plast, and the lack of Ca+2 salts had no negative influ ence on the efficiency of the electrofusion. At the same time, it may be assumed that the nuclei of transplanted somatic cells were in the conditions of oocyterecipi ents of the MII stage before activation. Several laboratories used successfully the report of the “distant” activation, where the time between elec trofusion and activation of the constructions was 2– 4 hours (Cibelli et al., 1998; Galli et al., 2003; Vajta et al., 2006) or 5–6 hours (Schurmann et al., 2006). At the same time, high results both on the development of the reconstructed embryos up to the blastocyst stage and the appearance of young animals were obtained also during activation together with the fusion of the somatic cell and cytoplast (Wilmut et al., 1997; Kato et al., 1998, 2000; Goto et al., 1999; Kubota et al., 2000; Tani et al., 2000; Akagi et al., 2003b; Urakawa et al., 2004). During the comparative trials based on data of dif ferent authors, conflicting results were obtained. Thus, in mice (Wakayama et al., 1998) and cattle (Cibelli et al., 1998; Wells et al., 1998, 1999; Liu et al., 2001; Shin et al., 2001; Akagi et al., 2003b; Shen et al., 2008), the development of in vitro cloned embryos up to the blastocyst stage significantly increased during activation between 2 and 8 hours in comparison with synchronous fusion and activation. However, accord ing to other data, a prolongation period before activa tion of more than 2 hours caused a reliable decrease of the in vitro embryo development before the blastocysts stage in cattle (Choi et al., 2004; Aston et al., 2006; Sung et al., 2007). Ca+2 in the medium for electrofusion caused the concentration increase of the intracellular Ca2+ that initiated the first step of the activation. The oocyte cytoplasm was characterized by the transformations providing meiosis renewal with PBII extrusion (SuskoParrish et al., 1994; Soloy et al., 1997; Liu et al., 1998), but then the development of “young” oocytes was blocked going over to the MIII stage in 4– 5 hours (SuskoParrish et al., 1994). Probably, these mechanisms could assist in disorganization of the chromosome location in the caryoplast during its longterm existence in the conditions of partially acti vated cytoplasm of the oocyte. While we carried out the experiments with the period between the electro fusion and the activation in the range of 3–3.2 or 4– 5 hours, 40.2 and 46.4% of reconstructed embryos reached the blastocyst stage, respectively. Thus, when using mannitol solution without Ca2+ salts, the pro longation of the activation up to 4–5 hours after elec trofusion did not decrease of development efficiency of reconstructed bovine embryos up to the blastocyst stage in vitro. It was shown that two relatively compact groups of oocytes could be chosen according to the in vitro maturing time. Approximately 45% of the oocytes extruded PBI to 17 hours after the beginning of matu BIOLOGY BULLETIN

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ration, and about 25%, between 18 and 20 hours, but the maturing of other oocytes extended up to 24 hours (Malenko et al., 2009). It could be supposed that cer tain basic characteristics of the oocytes caused both such differences in terms of maturing and further effi ciency of the development of the reconstructed embryos on this basis. Thus, in the experiments on the cloning of swine, the oocyterecipients were used as usual after 40–44 hours of in vitro maturing, but 36% of swine oocytes matured after 24 hours. In their use significantly more of the swine reconstructed embryos developed up to the blastocyst stage in comparison with the oocytes of the later maturing time (Miyoshi et al., 2002). During our experiments reconstructed bovine embryos exhibited a tendency to more success ful in vitro development in the use of matured in vitro to 18 hours oocyterecipients if the time between electrofu sion and activation was 3–3.7 hours. CONCLUSION The prolongation of the period between the elec trofusion and the activation up to 4–5 hours did not lower the efficiency of the development of in vitro reconstructed embryos of the cattle when a mannitol solution without calcium salts was used. A pattern of more successful development of in vitro cloned embryos was found using the oocyte recipient reaching the MII stage to 18 hours of matur ing in comparison with ones that finished the nuclear maturing later. ACKNOWLEDGMENTS This study was supported by Bioline Pharmtorg, LLC. REFERENCES Akagi, S., Adachi, N., Matsukawa, K., et al., Developmen tal Potential of Bovine Nuclear Transfer Embryos and Post natal Survival Rate of Cloned Calves Produced by Two Dif ferent Timings of Fusion and Activation, Mol. Reprod. Dev., 2003a, vol. 66, no. 3, pp. 264–272. Akagi, S., Takahashi, S., Adachi, N., et al., In Vitro and in Vivo Developmental Potential of Nuclear Transfer Embryos using Bovine Cumulus Cells Prepared in Four Different Conditions, Cloning Stem Cells, 2003b, vol. 5, no. 2, pp. 101–108. Aston, K.I., Li, G.P., Hicks, B.A., et al., Effect of the Time Interval between Fusion and Activation on Nuclear State and Development in Vitro and in Vivo of Bovine Somatic Cell Nuclear Transfer Embryos, Reproduction, 2006, vol. 131, pp. 45–51. Bavister, B.D. and Yanagimachi, R., The Effects of Sperm Extracts and Energy Sources on the Motility and Acrosome Reaction of Hamster Spermatozoa in Vitro, Biol. Reprod., 1977, vol. 16, pp. 228–237. BIOLOGY BULLETIN

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BIOLOGY BULLETIN

Vol. 37

No. 5

2010