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Regular Article |
a Nexia Biotechnologies, Inc., Vaudreuil-Dorion, Quebec, Canada J7V 8P5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, embryo, ovum, reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since Dolly's birth, a number of different laboratories have successfully used fetal and adult somatic cells from several species to produce cloned offspring [10–15]. Cloning with adult cells enables the replication of valuable animals, whether that value is due to pedigree (high genetic merit) or the presence of a transgene. In domestic animals, both skin fibroblasts and cumulus-granulosa cells (GCs) have been used successfully as donor cells [11, 12, 16, 17]. Granulosa cells may be a preferred source of cells for replication of females as they are easy to collect during ultrasound-guided or laparoscopic aspiration. In this study, we used GCs as a source of adult nuclei, along with a fetal fibroblast line that had been used previously to produce viable offspring [18]. Nuclear transfer procedures that had been optimized for the caprine system in an earlier study were used [18]. We demonstrate that adult somatic cells can be used effectively in NT using in vitro matured oocytes in the goat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless indicated otherwise, media and components used in the preparation of donor cells were obtained from Gibco (Canadian Life Technologies, Burlington, ON, Canada). Somatic donor cells were obtained from two different sources: 1) adult cumulus-granulosa cells and 2) fetal fibroblasts. Granulosa cells were isolated from follicular aspirants after laparoscopic oocyte pick-up (LOPU) of Breed Early Lactate Early (BELE) goats [19]. Two primary GC cultures (GC1, GC2) were obtained from two separate sets of follicular aspirants. In each case, aspirants were pooled from six donors to provide sufficient cell numbers for cryopreservation and subsequent propagation. These aspirants contained both mural granulosa and cumulus-granulosa cells. Aspirants were washed one to three times by centrifugation in fetal bovine serum (FBS) and then two times in PBS. Cells were frozen at 5 x 105 cells/ml in 10% DMSO and 90% FBS in small aliquots. Frozen aliquots of cells were thawed and cultured in M199 containing 10% FBS and 0.1% gentamicin for several days before preparation for NT. The second source of cells, frozen passage 0 fetal fibroblasts (FF4), had been isolated from a Day 30 fetus from a prolific dwarf breed of goat (BELE) and used successfully in NT, as previously described [18]. These proven cells were used primarily as a system control to ensure that all facets of the NT process were within expectations. Both the GCs and the FF4 cells were cryopreserved before the program (3 wk and 11 mo, respectively).
Frozen-thawed GCs and fetal fibroblasts (0.5–1 x 105) were plated into 24- or 96-well plates (Nunc, Invitrogen Canada Inc., Burlington, ON, Canada) and cultured in Dulbecco modified Eagle medium (DMEM) containing 10% FBS until they reached 100% confluency. The media was then replaced with low-serum media (DMEM containing 0.5% FBS and 0.1% gentamicin), and the cells were incubated at 38°C in 5% CO2 for 4 days [20]. The GCs and fetal fibroblasts were in passage 1 when used for NT. Just before cell transfer, the donor cells were collected by trypsinization using 0.05% trypsin-EDTA, washed twice, and resuspended in EmCare (Imuno-Chemical Products, Auckland, New Zealand) containing 1% FBS.
Donor and Recipient Animals
Oocyte recoveries and embryo transfers were performed during the month of July 1999. Intravaginal sponges containing 60 mg of medroxyprogesterone acetate were inserted into the vagina of donor goats (Alpine, Saanen, and Boer crossbred goats) and left in place for 10 days. An injection of 125 µg cloprostenol (Estrumate; Schering Canada, Inc., Pointe Claire, PQ, Canada) was given 36 h before sponge removal. Priming of the ovaries was achieved with gonadotrophin preparations including FSH and eCG. One dose equivalent to 70 mg NIH-FSH-P1 of Ovagen (Immuno-Chemical Products) was given together with 400 IU of eCG (Equinex; Wyeth-Ayerst Canada, St. Laurent, QC, Canada) 36 h before LOPU.
Recipients (Boer crossbred goats) were synchronized with intravaginal sponges as described previously for donor animals. Sponges were removed on Day 10, and an injection of 400 IU of eCG was given. Estrus was observed 24–48 h after sponge removal, and embryos were transferred 65–70 h after sponge removal.
LOPU and Embryo Transfer
The donor goats were fasted 24 h before laparoscopy. Anesthesia was induced with i.v. administration of diazepam (0.35 mg/kg body weight) and ketamine (5 mg/kg body weight) and was maintained with isoflurane via endotrachial intubation. Cumulus-oocyte complexes (COCs) were recovered by aspiration of follicular contents under laparoscopic observation [18, 19].
The recipient goats were fasted and anesthetized in the same manner as the donor goats. A laparoscopic exploration was performed to determine if the recipient had one or more recent ovulations or corpora lutea present on the ovaries. Eight to 13 NT-derived embryos (one-cell to four-cell stage) were transferred by means of a TomCat catheter (Sherwood, St. Louis, MO) threaded into the oviduct ipsilateral to ovulation(s). Donors and recipients were monitored after the surgical procedures, and antibiotics and analgesics were administered according to approved procedures.
Oocyte Maturation and Preparation
Unless indicated otherwise, chemicals used in the preparation of solutions for embryo manipulation and culture were obtained from Sigma-Aldrich Canada (ON, Canada). The COCs were cultured in 50-µl drops of maturation medium covered with an overlay of mineral oil and incubated at 38.5°C–39°C in 5% CO2. The maturation medium consisted of M199H (Gibco) supplemented with bLH (0.02 U/ml; Sioux Biochemicals, Sioux Center, IA), bFSH (0.02 U/ml; Sioux Biochemicals), estradiol-17ß (1 µg/ml), 0.2 mM sodium pyruvate, kanamycin (50 µg/ml), 100 µM cysteamine, and 10% heat-inactivated goat serum. After 23–24 h of maturation, the cumulus cells were removed from the matured oocytes by vortexing the COCs for 1–2 min in EmCare containing 1 mg/ml hyaluronidase. The denuded oocytes were washed in handling medium (EmCare supplemented with 1% BSA) and were returned to maturation medium. The enucleation process was initiated within 2 h of oocyte denuding. Before enucleation, the oocytes were incubated in handling medium containing 5 µg/ml Hoechst 33342 (Sigma) for 20–30 min at 30°C–33°C in an air atmosphere.
Enucleation
Oocytes were placed into manipulation drops (EmCare supplemented with 1% FBS) covered with an overlay of mineral oil. Oocytes stained with Hoechst were enucleated during a brief exposure of the cytoplasm to UV light (Zeiss Filter Set 01; Carl Zeiss Canada Ltd., Toronto, ON, Canada) to determine the location of the chromosomes. The stage of nuclear maturation was assessed and recorded during the enucleation process.
Donor Cell Transfer and Fusion
The enucleated oocytes and dispersed donor cells were manipulated in handling medium. Small (<20 µm) donor cells with smooth plasma membranes were picked up with a manipulation pipette and slipped into perivitelline space of the enucleated oocyte. Cell-cytoplast couplets were fused immediately after cell transfer. Couplets were manually aligned between the electrodes of a 500-µm gap fusion chamber (BTX, San Diego, CA) overlaid with sorbitol fusion medium (0.25 M sorbitol, 100 µM calcium acetate, 0.5 mM magnesium acetate, 0.1% BSA). A brief fusion pulse (15 µsec) at 2.39 kV/cm was administered by a BTX Electrocell Manipulator 200. After the couplets had been exposed to the fusion pulse, they were placed into 25-µl drops of medium overlaid with mineral oil. The postfusion culture medium consisted of M199H plus 10% FBS with or without 7.5 µg/ml cytochalasin B (CB). Fused couplets were incubated at 38.5°C–39°C in 5% CO2, 7% O2, and 88% N2. After 1 h, couplets were observed for fusion. Couplets that had not fused were administered a second fusion pulse as described previously.
Activation and Culture
Two to three hours after application of the first fusion pulse, the fused couplets were activated using the calcium ionomycin and 6-dimethylaminopurine (DMAP) method of Susko-Parrish et al. [21]. Briefly, couplets were incubated for 5 min in EmCare containing 5 µM calcium ionomycin, and then for 5 min in EmCare containing 30 mg/ml BSA. The activated couplets were cultured for 2.5–4 h in 2 mM DMAP, then washed in handling medium and placed into culture drops (25 µl in volume) consisting of G1.2 medium supplemented with 8 mg/ml BSA (Colorado Center for Reproductive Medicine, Englewood, CO; [22]) under an oil overlay. Embryos were cultured 12–18 h until embryo transfer. Nuclear transfer-derived embryos were transferred on Day 1 (Day 0 = day of fusion) into synchronized recipients on Day 1 of their cycle (Day 0 = estrus).
Determination of Embryonic Survival and Kidding
All recipient goats were examined by ultrasonography on Days 30 and 60 of gestation to record fetal development. All kids were delivered vaginally without induction. The number of cotyledons and birth weights were recorded. Neonates were observed for suckling behavior. All neonates were provided with colostrum by bottle to ensure an adequate intake. If a neonate demonstrated no or poor suckling, then the neonate was fed colostrum by intubation.
Cytogenetic Analysis and Genotyping
Chromosomal number was determined before cells were used in NT. Slides for cytogenetic analysis were prepared by standard techniques [23, 24]. Briefly, to increase the number of cells in mitotic metaphase, cells were cultured with colcemid (Gibco) in chamber slides (LabTek, Canadian Life Technologies). Spreads were stained with Giemsa (Harleco, EM Science, Gibbstown, NJ) and assessed for chromosome number.
Genomic DNA was isolated (QIAmp DNA Blood Mini Kit; Qiagen Inc., Mississauga, ON, Canada) from the donor cells and from blood samples collected from the GC clones, the surrogate dams, and the aspirant donor dams. DNA from each animal was analyzed by PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) to confirm that the clones were derived from the NT procedure. Briefly, a 286-base pair (bp) fragment of the goat major histocompatibility complex (MHC) class II DRB gene [25] was amplified using two primers (ACB0445: 5'-TATCCCGTCTCTGCAGCACATTTC-3'; ACB0446: 5'-ATCGCCGCTGCACACTGAAACTC-3'). The identity of the PCR product was confirmed by DNA sequencing analysis (MOBIX, Hamilton, Canada). The PCR products were analyzed by SSCP [26, 27] to confirm the genetic relationship between animals.
Animal Ethics
This project was approved by the McGill Animal Ethics and Animal Care Committees.
Statistical Analysis
The proportional data for cell fusion and pregnancy rates were analyzed by contingency tables using InStat (GraphPad). Birth weights and cotyledon number were analyzed using the Student t-test (GraphPad, San Diego, CA). Data were expressed as mean ± SEM.
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RESULTS |
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In two of the sessions, postfusion culture medium was supplemented with CB to reduce cytoplasmic blebbing [4]. The proportion of NT couplets fused did not appear to be affected by supplementation with CB (data not shown), whereas fragmentation was diminished in the presence of CB; however, additional replicates are required to confirm these observations.
A total of 91 GC NT embryos were transferred into eight recipients. Four recipients (50%) receiving female adult somatic cell NT embryos were confirmed pregnant on Days 30 and 60 by ultrasound (Table 1). All pregnancies were maintained through term (Table 2). One recipient delivered a female kid (2.2% of GC1 embryos transferred) derived from the GC1 cells. Three recipients delivered six female kids (13% of GC2 embryos transferred) derived from the GC2 cells (Fig. 1). One female twin died at birth. Birth weights ranged from 1.2 to 2.2 kg for the seven female clones. Birth weights of female kids (n = 10) born from naturally bred dwarf goats ranged from 0.95 to 1.6 kg. The average birth weight for the GC clones did not differ significantly from that of the female kids derived from natural breeding (1.7 ± 0.13 kg vs. 1.3 ± 0.06 kg, respectively). The number of cotyledons per recovered placenta for the GC clones was 40 ± 6.8 (n = 6), which represented an average of 60 ± 9.3 cotyledons per dam (n = 4; Table 2). The number and appearance of cotyledons were similar to those observed in our earlier study [18]. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients. One recipient (17%) receiving male fetal fibroblast NT embryos was confirmed pregnant. One recipient delivered two male kids (3.7% of FF4 embryos transferred) weighing 1.2 and 1.5 kg. The 1.5-kg male died during delivery. Half of the GC-derived neonates and the FF4 neonate initially showed a poor suckling reflex and were fed colostrum by intubation to ensure adequate and timely intake (Table 2). Good suckling behavior was usually demonstrated by Day 2. Because of the small number of recipients, there were no significant differences in the rates of pregnancy and NT efficiency (kids per NT embryo transferred) between GC and FF4 clones (50% vs. 17% and 7.7% vs. 3.7%, respectively).
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Mitotic spreads indicated that all cell lines contained a normal chromosome number consisting of 60 chromosomes (60XX or 60XY), although one cell culture, GC1, had a translocation in 30% of the spreads. Chromosomal analysis of the kid derived from GC1 indicated that she did not have the translocation in her cells.
The genetic identity of the NT-derived offspring was confirmed by PCR-SSCP analysis using the polymorphic MHC class II DRB gene. The analysis confirmed that all of the cloned kids were derived from the respective donor animals, whereas the surrogate dams carried different alleles of the DRB gene (Fig. 2). GC clone 1.1 (Fig. 2, gel A, lane 2) was derived from donor no. 402 or 403 (sibs: Fig. 2, gel B, lanes 8 and 9). GC clone 2.1 (Fig. 2, gel A, lane 1) was derived from donor no. 356 (Fig. 2, gel B, lane 3). Four of the GC clones (2.3, 2.4, 2.5, and 2.6) appeared phenotypically identical (Fig. 1). The PCR-SSCP analysis confirmed that they were identical clones (Fig. 2, gel A, lanes 3–6) and that they were derived from donor no. 358 (Fig. 2, gel B, lane 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Similar to the findings of our earlier study [18], prenatal loss after the Day 30 ultrasound scan was not observed in the present study. All pregnancies were full term, with recipients showing normal mammary gland development before parturition. Cotyledon numbers were comparable to those observed for naturally bred dwarf goats [18]. Birth weights were not significantly different from those of naturally derived kids. However, the numbers of kids per group were small, and the clones and natural births occurred within different herds, which may have affected results. Studies comparing cotyledon number and morphology and kid weights within a single herd are ongoing.
Neonatal loss was lower for these GC-derived clones (14%) than for the fetal fibroblast-derived clones (50%) reported in our earlier study [18]. In this study, only two kids were lost, one GC derived and one FF4 derived. Both died at the time of birth but otherwise appeared normal. This lower mortality rate for GC clones may be due in part to intensive management of neonates. However, it will be of interest to compare survival and maturation of clones derived from different cell sources and treatments. At this time, there is limited information available on the efficiencies of different cell types, much less on the long-term survival and production traits of the resulting offspring. In a large study from one group in Japan [29], 24 calves derived from nine different donor cell and tissue types (one fetal, three newborn, and five adult) were produced. The calving results from that study suggested that there can be differences in cell lines regarding developmental potential and in terms of both calf survival and birth weight. Further and even larger studies are needed to clarify the effect of donor cell type and treatment on success of nuclear transfer.
In this study we have demonstrated that adult somatic cells that have been cryopreserved can be used effectively in NT in the goat. An NT efficiency (kids per NT embryos transferred) of 7.7% was achieved with GC clones, which was similar to the 7.1% rate previously obtained with nontransfected fetal somatic cells [18]. Further studies are needed to determine the relative contributions of the source of the nuclei and the reprogramming capability of the recipient oocytes. It is clear that oocytes are capable of reprogramming a wide range of differentiated cells including fibroblasts, lymphocytes, granulosa, oviductal, mammary epithelial, immature Sertoli, and embryonic stem cells [9–11, 29–31]. It remains to be determined whether those failures occurring thus far with certain cell types (e.g., mature Sertoli cells and adult nerve cells [10]) are due to a truly irreversible alteration in DNA-chromatin structure or simply require more perseverance in fine-tuning the parameters of reprogramming. It may be that the process of reprogramming these cells is beyond the capabilities of oocytes but is amenable to more intensive treatments similar in concept to those used in the reprogramming of amphibian larval cells [32]. As we gain a better understanding of the processes involved in nuclear reprogramming of different cell types, we may be able to apply this knowledge to improving the overall efficiencies of NT.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 A preliminary report of this work was presented at the 33rd Annual Meeting of the Society for the Study of Reproduction, July 2000, Madison, WI. 
2 Correspondence: Carol L. Keefer, Nexia Biotechnologies, Inc., 1000 Avenue St. Charles, Bloc B, Vaudreuil-Dorion, QC, Canada J7V 8P5. FAX: 450 424 2096; ckeefer{at}nexiabiotech.com
Accepted: August 28, 2001.
Received: May 4, 2001.
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