Effect of Cytoplasmic Volume on Developmental Competence of Buffalo ( Bubalus bubalis ) Embryos Produced Through Hand-Made Cloning

Abstract

This study examined the effects of cytoplasmic volume on the developmental competence of hand-made cloned buffalo embryos. Two different cell types, that is, buffalo fetal fibroblast (BFF) and buffalo embryonic stem (ES) cell-like cells were taken as donor cell and fused with one, two, or three demicytoplasts to generate embryos with decreased, normal (control), and increased cytoplasmic volume. Using BFF as a nuclear donor, the cleavage rate was similar in all the groups (p > 0.05), but the blastocysts rate was significantly lower (p < 0.05) for embryos generated with decreased cytoplasmic volume. Using ES cell-like cells, the cleavage and blastocyst rate with increased cytoplasmic volume was significantly higher (p < 0.05) compared that with reduced cytoplasmic volume. Blastocysts produced from embryos having increased cytoplasmic volume had significantly higher (p < 0.05) cell number than normal (control) embryos in both BFF and ES cell-like cells groups. Pregnancies were established in all the groups except for the embryos reconstructed with decreased cytoplasmic volume. The pregnancy rate was almost double for embryos reconstructed using increased cytoplasmic volume compared to that with the controls. Most of the pregnancies aborted in the first trimester and one live calf was delivered through Caesarean, which died 4 h after birth.

Introduction

During oogenesis in mammals, the developing oocytes accumulate proteins, mRNA, and other biomolecules in their cytoplasm, which are needed during fertilization and early embryonic development before the major embryonic genome activation occurs. In bovine (Barnes and Eyestone, 1990) and bubaline species, this genome activation occurs at the 8- to 16-cell stage. As the quality of the recipient oocyte is an important factor in successful nuclear transfer, the amount of maternally derived mRNA and proteins in the cytoplasm is an important determinant in successful cloning. It has been observed that metaphase II arrested oocytes and those derived from large follicles (diameter 6–8 mm) develop better after nuclear transfer (Barnes et al., 1993). With the advancements in the in vitro maturation protocols of oocytes, enucleated in vitro matured oocytes are now commonly used for fusion with donor cells as the results are comparable with oocytes matured in vivo (Yang et al., 1993).

The quantity and quality of yet unknown reprogramming factors in oocyte determines the overall reprogramming efficiency of the somatic nuclei transplanted in the oocyte, as such an increase or decrease in cytoplasmic volume may affect reprogramming. Whereas micromanipulator-assisted enucleation results in the removal of 5 to 50% of the cytoplasm with the meiotic metaphase plate (Westhusin et al., 1992, 1996), Hand-made cloning (HMC) results in almost 15–50% loss in cytoplasmic volume using protrusion cone guided oocyte bisection method (personal observation). Both Greising et al. (1994) and Zakhartchenko et al. (1997) reported a decreased blastocyst development rate in nuclear transfer embryos when cytoplasmic volume of the recipient oocyte was sufficiently reduced. Although no difference was observed in the blastocyst production rate, a significant reduction in cell number of blastocysts was observed when the cytoplasmic volume was reduced from 5–50% (Westhusin et al., 1996). Peura et al. (1998) also reported no significant difference in the cleavage and blastocysts rate of cloned embryos generated using an increased or decreased cytoplasmic volume, but the total cell number of blastocysts was found to be significantly higher for embryos reconstructed using increased cytoplasmic volume. Recently, Bowles et al. (2008) and Ribeiro et al. (2009) reported that increase in cytoplasmic volume either by fusion or aggregation, increased the cleavage rate, blastocyst rate, and cell density of bovine HMC embryos.

The present study was carried out to study the effect of cytoplasmic volume on the developmental potential of buffalo HMC embryos using buffalo fetal fibroblasts (BFF) and embryonic stem cell (ES) like-cells as donor cells.

Materials and Methods

All chemicals and media were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and disposable plastic wares from Nunc (Roskilde, Denmark) unless otherwise mentioned.

Preparation of donor cells

Primary cell culture of BFF was derived as described previously (Shah et al., 2009). Cells forming a confluent monolayer (passages 9–10) were allowed to grow further for 3 days to achieve over confluence in order to attain a higher proportion of cells in G1 phase of cell cycle. The cells were trypsinised into single cell suspension for use as nucleus donors.

Buffalo ES cell-like cells derived from in vitro produced embryos according to the procedure described earlier (Verma et al., 2007) were used in the present study. The ES cell-like cells had been characterized using transcription-based markers like OCT-4, NANOG, SOX-2, and surface markers like alkaline phosphatase, SSEA-3, and SSEA-4 (data not shown). ES cell-like cells between passage 20 and 30 were cultured in ES culture medium consisting of Knockout-DMEM + 15% Knockout Serum Replacer + 5 ng/mL bFGF + 1000 IU/mL mLIF. For HMC, two to three colonies of ES cell-like cells were mechanically separated from the feeder layer using a microblade (MicroBlades, MTB-05; Micromanipulator Microscope Company, Inc., USA) and incubated in trypsin for 2–3 min to remove the feeder layer cells. The colonies were then quickly transferred to T2 [where T denotes HEPES modified M-199 supplemented with 2.0 mM L-Glutamine, 0.2 mM sodium pyruvate, 50 μgmL⁻¹ gentamicin and the following number denotes fetal bovine serum (FBS) in percent v/v] and subjected to vigorous pipetting to make a single cell suspension, which were used for HMC.

Hand-made cloning

The recipient cytoplast maturation and processing was performed as described previously (Chauhan et al., 1998; Shah et al., 2009). Briefly, in vitro matured cumulus oocyte complexes with expanded cumulus were stripped off their cumulus investment and zona pellucida using Hyaluronidase (0.5 mgmL⁻¹ in T2) and Pronase (2.0 mgmL⁻¹ in T containing 10% FBS), respectively. Oocytes with completely digested zona pellucida were transferred to T20 (T containing 20% FBS) and incubated at 38.5°C for 10–15 min or until a prominent protrusion cone was easily visible. Protrusion cone guided bisection was performed using microblade in T20 with 2.5 μgmL⁻¹ Cytochalasin-B. The larger demicytoplasts without protrusion cone were transferred to T20 and incubated for 10–15 min at 38.5°C to enable them to regain spherical shape. The enucleated demicytoplasts were immersed in Phytohemagglutinin (0.5 mgmL⁻¹ in T2) for 3–4 sec and transferred to T2 containing the donor cells. Each demicytoplast was then allowed to attach to a single, rounded, and medium-sized fetal fibroblast or ES cell-like cell by gently rolling the demicytoplast over it. The couplets (single demicytoplast donor cell pairs) were transferred to the fusion medium (0.3 M D-mannitol, 0.1 mM MgCl₂, 0.05 mM CaCl₂, and 1 mgmL⁻¹ polyvinyl alcohol) for equilibration. A single-step fusion protocol was followed wherein a demicytoplast and a couplet were picked using a fine pulled capillary pipette (Unopette^® Becton Dickinson, Franklin Lakes, NJ, USA) having an internal diameter of 100–150 μm. Initially, the couplet was expelled and aligned with an A.C. pulse (4 Volts) using BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA), so that the somatic cell faced the negative electrode, and immediately after alignment, another demicytoplast was introduced into the fusion chamber (BTX microslide 0.5 mm gap, model 450; BTX, San Diego, CA, USA) close to the somatic cell. As soon as the somatic cell was sandwiched between the demicytoplasts, single D.C. pulse (3.36 kVcm⁻¹ for 4 μsec) was applied to produce normal reconstructed embryo (Fig. 1B). For construction of embryos with decreased (Fig. 1A) or increased (Fig. 1C) cytoplasmic volume, either only single couplets or couplets with two demicytoplasts were allowed to fuse, as described above. Reconstructed embryos were then incubated in T20 (for rounding up) for 6 h at 38.5°C. The embryos were activated by incubating in T20 containing 5 μM calcimycin A23187 for 5 min at 38.5°C. After washing thrice with T20, the oocytes were incubated individually in 5 μL droplets of T20 containing 2 mM 6-dimethylamino purine, covered with mineral oil, and kept in incubator at 38.5°C for 4 h. The activated embryos were cultured in 400 μL of Research Vitro Cleave medium (K-RVCL-50, Cook^® Australia, Queensland, Australia) supplemented with 1% fatty acid-free (FAF) BSA in a four-well dish (15–20 embryos per well), covered with mineral oil, and kept undisturbed in a CO₂ incubator (5% CO₂ in air) for 8 days.

FIG. 1.

Reconstruction of embryos with decreased (A), normal (B), and increased cytoplasmic volume (C), as observed just after fusion.

Determination of total cell number

The effect of cytoplasmic volume on the total cell number of blastocysts was determined by differential staining of day 8 blastocysts generated using BFF or ES cell-like cells, with Hoechst 33342 (10 μg/mL for 30 min) followed by propidium iodide (25 μg/mL for 1 min). Trophoectoderm and inner cell mass nuclei, which were stained purple and blue, respectively, were manually counted using an epifluorescence microscope.

Synchronization of recipients and embryo transfer

Cycling buffaloes possessing a functional corpus luteum were treated with a PGF_2α analogue (Cloprostenol sodium, 500 μg) intramuscularly. Those exhibiting estrus around 72 h after the treatment were selected as recipients. Day 8 blastocysts were transferred nonsurgically to synchronized recipients after confirming the presence of corpus luteum. Two embryos were transferred into ipsilateral uterine horn, whereas a single embryo was transferred into the contralateral horn. Pregnancy diagnosis was carried out by transrectal ultrasonography at day 40 after transfer.

Experimental design

In the present study, two experiments using two different cell types were carried out for determining the effect of increased and decreased cytoplasmic volume on developmental competence of HMC embryos. In Experiment 1, a single BFF cell was fused to one, two, or three demicytoplasts using a D.C. pulse to produce HMC embryos with variable cytoplasmic volume, whereas in Experiment 2, an ES cell-like cell was used in place of BFF cell. Subsequently, the in vitro developmental competence of HMC embryos was studied followed by determination of total cell number of blastocysts. The embryos from each group were either transferred to synchronized recipients or cryopreserved.

Statistical analysis

The data were analyzed using SYSTAT 7.0 (SPSS Inc., IL, USA) and all the values are presented as mean ± SEM unless indicated otherwise. Differences among means were analyzed by one-way ANOVA after arc-sine transformation of the percentage data of in vitro embryo development. For data on total cell number, the difference among means was compared using Student's t-test. The differences were considered significant at p < 0.05.

Results

Effect of cytoplasmic volume on development of HMC embryos using BFF as a donor cell

No significant difference was observed in the cleavage rate among different cytoplasmic volume groups (85.6 ± 1.7 vs. 86.4 ± 6.1 vs. 92.4 ± 1.7). However, the blastocyst rate for embryos reconstructed using decreased cytoplasmic volume (6.5 ± 3.4) was significantly lower (p < 0.05) than that of the control group (38.0 ± 6.8) and the group where embryos were generated using increased cytoplasmic volume (46.0 ± 1.5; Table 1). As expected, most of the embryos with reduced cytoplasmic volume arrested at the 8–16 cell stage, where embryonic genome activation takes place in buffalo.

Table 1.

Developmental Competence of HMC Embryos Produced by Distinct Cytoplasmic Volume Using Fetal Fibroblast as a Nuclear Donor

Cytoplasmic Volume	Embryos (n)	Cleaved^ (*n)	Blastocyst^ (*n)	Total Cell Number^*	Embryo Transferred (n)/Recipient (n)	Pregnant (n)
Decreased	100	85.6 ± 1.7^a (86)	6.5 ± 3.4^a (6)	43.0 ± 4.2 (3)	3/1	0
Control	104	86.4 ± 6.1^a (90)	38.0 ± 6.8^b (41)	149.7 ± 2.5^a (20)	16/5	1
Increased	101	92.4 ± 1.7^a (93)	46.0 ± 1.5^b (47)	352.4 ± 3.0^b(20)	13/5	2

Figures quoted as mean percentages ± SEM.

Values in same columns with different superscripts differ significantly at p < 0.05.

Postimplantation development of cloned embryos was evaluated by transfer of day 8 blastocysts to synchronized recipients and the pregnancies were confirmed at day 40 after embryo transfer using transrectal ultrasonography. Following transfer of control HMC embryos to five recipients, one became pregnant and delivered a calf through Caesarean operation. The calf, with a body weight of 28 kg, died 4 h after birth due to respiratory distress and multiple organ failure. HMC blastocysts generated using increased cytoplasmic volume, when transferred to five recipients, resulted in two pregnancies. One fetus was found to be aborted at day 90 and another one at day 220 of gestation. Most of the blastocysts generated using decreased cytoplasmic volume, were not of transferable grade, and upon transfer of three embryos to a recipient no pregnancy was established.

Blastocysts (n = 20) reconstructed using increased or normal cytoplasmic volume, generated by BFF were used for determination of total cell number (Fig. 2A–C). Average total cell numbers for blastocysts generated using increased cytoplasmic volume (352.4 ± 3.0) was significantly higher (p < 0.05) than control ones (149.7 ± 2.5; Table 1). Only six blastocysts were generated using decreased cytoplasmic volume, as such the group was not included for determination of cell number.

FIG. 2.

Blastocyst with decreased (A, D), normal (B, E) and increased (C, F) cytoplasmic volume generated using BFF (A, B, C), and ES cell like cell (D, E, F) as a nuclear donor.

Effect of cytoplasmic volume on development of HMC embryos using ES cell-like cells as a donor cell

When ES cell-like cells were used as nuclear donors for the construction of HMC embryos, the cleavage rate was significantly lower (p < 0.05) for embryos with reduced cytoplasmic volume (58.1 ± 10.8) compared to the controls (87.4 ± 3.0) or those with increased cytoplasmic volume (89.2 ± 10.7). The blastocysts rate, which was significantly different (p < 0.05) among the three groups, was the lowest in the decreased cytoplasmic volume group (1.8 ± 1.0) and highest in the controls (30.7 ± 1.4; Table 2).

Table 2.

Developmental Competence of HMC Embryos Produced by Distinct Cytoplasmic Volume Using ES Cell Like Cells as a Nuclear Donor

Cytoplasmic Volume	Embryos (n)	Cleaved^ (*n)	Blastocyst^ (*n)	Total Cell Number ^*	Embryo Transferred (n)/Recipient (n)	Pregnant (n)
Decreased	105	58.1 ± 10.8^a (61)	1.8 ± 1.0^a (2)	73.5 ± 6.5 (2)	0	0
Control	138	87.4 ± 3.0^b (121)	30.7 ± 1.4^b (42)	227.5 ± 5.2^a (25)	13/4	1
Increased	107	89.2 ± 10.7^b (97)	16.3 ± 4.0^c (17)	454.2 ± 10.7^b(7)	10/4	2

Figures quoted as mean percentages ± SEM.

Values in same columns with different superscripts differ significantly at p < 0.05.

Transfer of HMC blastocysts of control group, generated using ES cell-like cells to four recipients resulted in one pregnancy that was found to be aborted at day 120 of gestation, whereas after transfer of blastocysts reconstructed with increased cytoplasmic volume, two out of four recipients got pregnant (Table 2). One of them was found to be aborted around day 60 of gestation, whereas the other one was found to be aborted around day 90 of gestation. Blastocysts reconstructed using decreased cytoplasmic volumes were not of transferable quality.

Blastocysts (n = 7) with increased or normal cytoplasmic volume generated using ES cell-like cells were used for the determination of total cell number (Fig. 2D–F). Total cell number for blastocysts reconstructed with increased cytoplasmic volume (454.2 ± 10.7) was significantly higher (p < 0.05) than that for controls (227.5 ± 5.2; Table 2).

Discussion

Developmental abnormalities in cloned mammals are mainly due to aberrant epigenetic reprogramming of the donor genome. Accurate nuclear reprogramming of somatic cell genome by the recipient cytoplast is a prerequisite for successful somatic cell nuclear transfer. The source of biological material has a great impact on the outcome of cloning experiments (Vajta et al., 2005). In the case of farm animals, most of the cloning experiments rely on slaughterhouse ovaries for isolation of recipient oocytes; as such, the developmental competence of the oocytes is not known. The lower developmental potential of some of the oocytes may be improved by fusing cytoplast from two or more oocytes. But this approach will result in an increased mitochondrial heteroplasmy (Bowles et al., 2008), which may compromise cloning efficiency in terms of development up to term or postnatal survival (Vajta et al., 2005). The present strategy of nuclear transfer (i.e., HMC) also alters the nucleocytoplasmic ratio to a greater extent from the normal ratio, which is present in in vivo or in vitro fertilized embryos. The present work was thus carried out to determine the effect of cytoplasmic volume on the developmental competence of HMC embryos.

In our study, the poor developmental competence of HMC embryos generated using decreased cytoplasmic volume may have been due to the reduced amount of cytoplasmic factors required for proper embryo development or factors required for efficient epigenetic reprogramming of somatic cell genome. Greising et al. (1994) reported that replacement of the amount of cytoplasm removed from the recipient oocytes during enucleation procedure with an equal amount of cytoplasm obtained from another oocyte resulted in an increase from 32 to 60% in cleavage rate and from 14 to 38% in morula and blastocysts rate in bovine nuclear transfer experiments. Zakhartchenko et al. (1997) also reported an increase in the blastocysts rate when an increased amount of cytoplasm was retained by the oocyte during enucleation procedure. Peura et al. (1998) reported an increase in blastocyst rate with an increased amount of cytoplasmic volume in HMC embryos, when a single fusion pulse was used. Bowles et al. (2008) reported that the cleavage and blastocyst rate for bovine embryos reconstructed with use of two or three cytoplasts was significantly higher than when one cytoplast was used for nuclear transfer. However, Westhusin et al. (1996) observed no significant differences in the blastocyst rate of nuclear transfer embryos generated with different cytoplasmic volumes, although they reported a significant increase in the cell number of blastocysts derived from 5% reduced cytoplasmic volumes compared to those derived from 50% reduced cytoplasmic volumes. Experiments have also been carried out to assess the developmental capacity of preimplantation embryos reconstructed with reduced embryonic volume by culturing isolated blastomeres from two-, four-, or eight-cell stage embryos (Eckert et al., 1997; Johnson et al., 1995; Saito and Niemann, 1991). Although development up to the blastocysts stage was achieved with reduced cytoplasmic volumes, the total cell number and development to full term were significantly reduced (Johnson et al., 1995; Saito and Niemann, 1991).

Microsurgical procedures like ooplasm transfer, in which the donor ooplasm is microinjected along with the sperm, have been developed for clinical treatment of infertility in the human (Cohen et al., 1998; Zhang et al., 1999). It would be interesting to apply such procedures to HMC for improving the developmental potential of ooplasm obtained from an oocyte of low developmental competence. Another approach that has been used to improve the quality of cloned embryos is to increase the cell number by preparing clone–clone aggregates from two or more genetically identical clones at the four-cell stage (Boiani et al., 2003). The normal gene expression and higher rates of fetal and postnatal development associated with these clone aggregates were believed to be due to the compensation of noncell-autonomous defects that are epigenetic in nature, by combining two epigenetically different clones leading to development of whole embryo even if one of the two contributing embryos may not have been viable alone. Moreover, if cells within one clone do not have all the same transcriptional activity or developmental potential due to genetic differences, aggregation of two may increase the number of normal cells with correct gene expression above a threshold required for further development.

As the expression profile of ES cells is quite similar to embryonic stage blastomeres, they give higher cloning efficiency in terms of live offspring rate and are comparatively easier to be reprogrammed (Wakayama et al., 1999). Therefore, we used ES cell-like cells as nuclear donor to determine whether the low developmental potential of HMC embryos reconstructed using decreased cytoplasmic volume could be rescued. The degree of differentiation of the donor cell markedly influences cloning efficiency. Compared to somatic cells, murine ES cells give higher cloning efficiency in terms of live offspring (Wakayama et al., 1999). The use of ES cells has also been envisaged for increasing cloning efficiency in farm animals (Wells et al., 2003). ES cells have been used for reproductive cloning (Saito et al., 2003) and transgenesis (Cibelli et al., 1998) of cattle but when used to produce chimeric bovine offsprings they did not contribute to germline of the offspring (Iwasaki et al., 2000).

Although the cleavage rate of embryos with increased cytoplasmic volume was similar to that for the controls for both somatic cell and ES cell-like cell groups (Tables 1 and 2), the blastocysts rate for embryos with increased cytoplasmic volume, derived using ES cell-like cells was significantly lower than that for the controls. The overall embryo development rate was found to be lower for embryos derived from ES cell-like cells compared to those derived from somatic cells, but the total cell number of blastocysts was higher for embryos reconstructed using ES cell-like cells for all the groups compared. Although the embryo transfer data is too small to draw a reasonable conclusion, the pregnancy rate obtained was almost double for embryos generated with increased cytoplasmic volume compared to the control ones for both the cell types.

In conclusion, no significant difference was observed in the developmental potential of embryos generated with increased cytoplasmic volume compared to the control ones when BFF was used as donor nuclei, whereas for ES cell-like cells, it was higher for control embryos. Embryos reconstructed with reduced cytoplasmic volume may have the potential to support early cleavage division, activation of embryonic genome, and even cavitation, but as the cytoplasmic volume does not increase during the first few rounds of embryonic cell division, the total cell number tends to be reduced. On the other hand, embryos reconstructed with increased cytoplasmic volume have increased cell density in blastocysts, but whether it results in increased embryo developmental potential or fetus survival is still not clear. A significant increase in the total cell number of blastocysts was observed for embryos with increased cytoplasmic volume compared to the control ones for both the cell types.

Footnotes

Acknowledgments

The authors are thankful to the past and present members of Embryo Biotechnology Lab for their assistance and helpful discussion. The present work was funded by National Agriculture Innovative Project (NAIP) Grant to MSC (C-2067 and 075) and SKS (C 2-1-(5)/2007).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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