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RESEARCH |
1 Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, via Porcellasco 7/f, 26100 Cremona, Italy and 2 Dipartimento Clinico Veterinario, Universitàdi Bologna, via Tolara di Sopra, 50-40064 Ozzano Emilia (Bologna), Italy
Correspondence should be addressed to C Galli, Laboratorio di Tecnologie della Riproduzione, Istituto Spermentale Italiano Lazzaro Spallanzani, CIZ srl, via Porcellasco 7/f, 26100 Cremona, Italy; Email: cesaregalli{at}ltr.191.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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This relatively late and limited application of somatic cell nuclear transfer is a consequence of the limited information available in the literature on assisted reproductive techniques in Equids, in particular oocyte maturation and in vitro culture of early pre-implantation embryos. Indeed, the work that led to the cloning of mules (Wood et al. 2003) relied on in vivo matured oocytes that were transferred to the oviducts of recipient mares immediately after nuclear transfer and activation. Only the work of Galli et al. (2003b) has been carried out completely in vitro up to the blastocyst stage.
One reason for the limited development of assisted reproduction techniques in Equids is the limited interest demonstrated by the horse industry, contrary to the cattle industry that still plays a very active role in the development and application of reproductive biotechnologies in ruminants. In general, few studies are available in the literature on equine in vitro embryo production (see reviews by Squires et al. 2003, Westhusin et al. 2003), and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation, fertilization by intra-cytoplasmic sperm injection (ICSI) and in vitro culture (Galli et al. 2002a, Lazzari et al. 2002a, Choi et al. 2004). These latter reports demonstrate the real possibility of applying a completely in vitro procedure for obtaining horse ICSI blastocysts able to establish a pregnancy (Galli et al. 2001a, 2002b) and develop to live term offspring (present authors, unpublished data).
In horse nuclear transfer, the availability of horse oocytes is a limiting factor due to the anatomy and physiology of the mare’s ovary which makes this species a poor oocyte donor as compared with other large domestic species. Oocytes can be harvested from the ovaries of slaughtered mares (Zhang et al. 1989) or from ovum pick up (OPU) of live donors. On average it is possible to recover 3–4 immature oocytes per ovary from the ovaries of slaughtered animals (Galli & Lazzari 2001, Lazzari et al. 2002a); oocyte retrieval from live donors by OPU is slightly more variable and can be from 3 to 6 oocytes per session of OPU (Galli et al. 2002a, Lorenzo et al. 2002). The maturation rate of horse oocytes is also quite variable, averaging between 25 and 70% in published studies (Hinrichs & Williams 1997, Carneiro et al. 2001, Dell’Aquila et al. 2001, 2003, Bogh et al. 2002, Choi et al. 2002, Lorenzo et al. 2002). Interestingly, in the horse the recovered cumulus–oocyte complexes are a mixed population with either a compact or an expanded cumulus. While expanded cumulus–oocyte complexes are often discarded for in vitro embryo production in cattle and pigs, they represent about 40% (2060/5127, our observations) of the total population of recovered oocytes in the horse and seem to have some developmental competence for maturation (Dell’Aquila et al. 2003), cleavage (Choi et al. 2002) and blastocyst formation (Zhang et al. 1989). Another peculiar aspect specifically relevant to horse nuclear transfer is the reported (Choi et al. 2002, Li et al. 2002) low ability of oocytes to fuse with donor cells and the limited developmental competence of nuclear transfer (NT)-embryos in vitro. Even high DC voltage pulses of 2.2–2.5 kV/cm were able to fuse less than 60% of couplets, the cleavage rate was 35–50% and only about 2.5% of NT-embryos reached the blastocyst stage (Li et al. 2002).
The refinement of the in vitro culture conditions suitable for equine oocyte maturation and embryo development (Galli et al. 2002a, Lazzari et al. 2002a), the development of an adequate horse oocyte activation protocol (Lazzari et al. 2002b) and the application of zona-free manipulation for embryo reconstruction (Booth et al. 2001, Oback et al. 2003, Vajta et al. 2003), are all fundamental steps for the development of a successful in vitro procedure for somatic cell nuclear transfer in the horse. In this paper we describe, in detail, all the technical aspects involved in the in vitro production of cloned blastocysts in the horse. The objectives of our research concentrated on the optimization of the nuclear transfer procedure to make efficient use of the limited number of oocytes available in this species. Both expanded and compact cumulus–oocyte complexes were used as well as a modified zona-free method for embryo reconstruction that is known to increase the fusion rate in other species (Oback et al. 2003). Different sources of donor somatic cells were compared in various experiments for their ability to develop to the blastocyst stage and to term.
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Materials and Methods |
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Oocyte collection and in vitro maturation
Ovaries of slaughtered mares of mixed breeds were collected during the breeding season at 45° latitude (Italy, northern hemisphere). Cumulus–oocyte complexes (COCs) were recovered by a combination of scraping and washing of the inner wall of follicles that were between 0.5 and 3.0 cm in diameter with a Jacobson curette and a syringe filled with PBS. The fluid was recovered into Petri dishes containing Hepes-buffered TCM 199 supplemented with 1 mg/ml BSA and 10 µg/ml heparin. COCs were classified as either compact or expanded depending on cumulus and granulosa cell morphology (Fig. 1a,b
), and transferred separately to maturation medium (Galli et al. 2001a, 2002a, 2002b). Briefly, the maturation medium was TCM 199 supplemented with 10% fetal calf serum (FCS), 1 µl/ml insulin, transferin, sodium selenite (ITS), 1 mM sodium pyruvate, 50 ng/ml long-epidermal growth factor, 100 ng/ml long-insulin-like growth factor-I and 0.1 IU/ml each of follicle-stimulating hormone and luteinizing hormone (Pergovet, Serono, Italy). COCs were matured for 22–24 h in 4-well plates at 38.5 °C in an atmosphere of 5% CO2 in air.
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Adult fibroblasts were produced by organ culture of minced tissue from chest biopsies of adult animals of the Haflinger and Arabian breeds. Fetal fibroblasts were produced as described for adult fibroblasts from an aborted 6-month-old fetus cloned from an adult fibroblast cell line. Both adult and fetal fibroblasts were cultured and stored as described for granulosa cells. Cells were used at passage number 5 to 15.
Before using the cells in NT experiments, granulosa cells and fibroblasts were induced into quiescence by either serum starvation (0.5% FCS) for 1–3 days or by growth to confluence. A cell suspension was prepared by trypsinization of cell culture 30 min before nuclear transfer, washed and resuspended in SOF-Hepes.
Preparation of enucleated oocytes (cytoplasts) and NT-embryo construction
After 22–24 h of maturation the oocytes were denuded of cumulus cells by pipetting first in the presence of hyaluronidase, then placed in 0.25% trypsin for 1.5 min and afterwards in SOF-Hepes with 10% FCS where the cumulus cells were completely removed by pipetting. Finally, the oocytes were returned to maturation medium. For reconstruction of NT-embryos we used both the zona-enclosed and zona-free methods (Oback et al. 2003). For the zona-enclosed method, oocytes with an extruded polar body were stained with Hoechst 33342 in the presence of cytochalasin B (5 µg/ml). Enucleation was performed by the aspiration of the polar body and metaphase II plate in a minimal volume of ooplasm under UV light. All manipulations were in SOF-Hepes with 6 mg/ml BSA (Gardner et al. 1994), except fusion. Individual nuclear donor cells prepared as described above (cumulus cells 22–24 h of maturation or adult fibroblasts) were transferred into the perivitelline space of zona-enclosed cytoplasts with the enucleation pipette. The cytoplast–karyoplast couplets were transferred in 0.3 M mannitol (50 µM Ca and 100 µM Mg) solution and fused one or two times within a 15 to 30 min interval by a double DC pulse of 1.8–2.4 kV/cm applied for 30 µs at 26–27 h of maturation. For the zona-free method, the zona pellucida of oocytes with extruded polar bodies was digested with 0.5% pronase in PBS. Zona-free oocytes were enucleated under UV light with a blunt micropipette (with perpendicular break). All manipulations were performed in SOF-Hepes with 10% FCS. Subsequently, zona-free cytoplasts were individually washed for a few seconds in 300 µg/ml phytohemagglutinin P in TCM 199-Hepes and then quickly dropped over a single donor cell (Vajta et al. 2003) settled to the bottom of a drop of TCM 199 with 0.5% FCS. Formed cell couplets were washed in 0.3 M mannitol (50 µM Ca and 100 µM Mg) solution and fused one or two times at 15 to 30 min intervals by a single DC pulse of 1.2 kV/cm applied for 30 µs at 26–27 h of maturation.
Activation
Oocytes and NT-embryos were activated 1–2 h after fusion at 27–28 h of maturation. Oocytes and NT-embryos were treated with 8.7 µM ionomycin in SOF-Hepes for 4 min followed by 4 h culture in a combination of 1 mM 6-(dimethylamino) purine (6-DMAP) and 5 µg/ml cycloheximide in SOF supplemented with MEM essential and non essential amino acids, 1 mM glutamine, and 4 mg/ml BSA (m-SOFaa) (Lazzari et al. 2002b).
Embryo culture
Zona-enclosed and zona-free embryos were cultured, respectively, in 20 µl culture drops and individually in 3 µl drops of m-SOFaa with 4 mg/ml BSA in 5% CO2 and 5% O2 in humidified air at 38.5 °C under mineral oil. Half of the medium was renewed on Day 3 with fresh m-SOFaa and on Day 5 with TCM 199 with 5% FCS and 5% Serum Replacement (Knockout SR, Gibco BRL) (Day 0 was the day of fusion and activation). Cleavage was assessed 48 h after activation, the rate of blastocyst formation was recorded at Day 7 and Day 8 when the embryos were either frozen or transferred to synchronized recipients (Fig. 1c,d
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Embryo transfer and foaling
Embryo transfer to recipients was performed over 3 breeding seasons (2002–2004). Haflinger mares were examined 2–3 times a week by ultrasound to determine the day of ovulation. Three to seven days after ovulation the mares received one to four Day 7 or Day 8 blastocysts (fresh or after thawing) by nonsurgical transfer. On Day 17 after ovulation the animals were examined (5 MHz linear probe, Sonovet 600, Medison) for pregnancy diagnosis and thereafter were examined weekly throughout the first trimester of pregnancy and later at monthly intervals until foaling.
All the experiments that involved the use of recipient animals were carried out under veterinary supervision in accordance with Decreto Legislativo 116/92 that regulates the use of animal experimentation in Italy.
Statistical analysis
Differences between the experimental groups were verified using the Chi-square test or Student’s t-test. P < 0.05 was considered significant.
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Results |
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. In this experiment only compact oocyte–cumulus complexes were used. The results indicate that more oocytes were fused and the cleavage rate was higher in the zona-free system (P < 0.05). The rate of blastocyst formation of the fused couplets was generally low (1.3–2.9% for cumulus cells and 4.2–6.4% for fibroblasts on Day 8) and did not differ significantly between groups. However, when comparing the efficiency of the two systems by calculating the percentage of enucleated oocytes subjected to fusion that developed into Day 8 blastocysts (Table 1, blastocysts on Day 8, percent of oocytes), we found that the zona-free method is about 3.2 times more effective than the zona-enclosed method in the case of cumulus-derived NT-embryos and 2.3 times more effective in the case of fibroblast-derived NT-embryos.
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Developmental competence of horse NT-embryos derived from different cell types
We tested the developmental ability of NT-embryos derived from cultured granulosa cells, cumulus cells, and adult and fetal fibroblasts from various donor animals and in parallel with the developmental ability of parthenogenetically activated matured (MetII) oocytes (Table 2). There was no difference in the rate of cleavage between parthenogenetic and NT-embryos derived from different donor cell types obtained by the zona-free method. The developmental capacity of NT-embryos to form blastocysts on Day 8 differed among donor cells (from 1.3% to 16%, P < 0.05). NT-embryos derived from adult fibroblasts of male B and from fetal fibroblasts (cloned fetus from male C) were better able to support development to blastocysts and this rate was similar to the rate of parthenogenetic development (Table 2).
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)) on the umbilical cord with a cystic structure filled with allantoic fluid; this cyst was likely to be a dilatation of the uracus and was the only abnormality observed in otherwise normal fetuses and placentae at necroscopy. The placenta of the fetus originating from the fetal cells presented an area without villi (see Fig. 2a). The fetus was aborted at 180 days (Fig. 2c) and was apparently normal at necroscopy. Two pregnancies developed to term and delivered two live foals from the same male genotype (B). The first foal was delivered after 344 days of pregnancy; it weighed 49 kg while the placenta was 4.6 kg. This foal was apparently normal, stood up on its own, and nursed within 2 h after birth. However, 24 h after birth he developed a massive diarrhea that rapidly caused the death of the foal 40 h after birth. Laboratory analysis and anatomo-pathological examination confirmed that the foal developed a hemorrhagic enteritis that evolved into septicemia, while no other gross morphological abnormalities were reported at necroscopy. The second foal was delivered after 329 days of gestation; it weighed 42 kg and the placenta was 4.1 kg. Like the previous foal, this one stood up on its own, nursed within 2 h after birth and developed well without any sort of problems (Fig. 3). At present he is 2 months old and growing very well.
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Discussion |
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In our experience in the horse, contrary to other species, expanded cumulus–oocyte complexes maintain some developmental competence after in vitro fertilization by ICSI, although oocytes with a compacted cumulus give rise to more embryos (Galli et al. 2000). However, the classification between compacted and expanded cumulus is not well defined in the literature and therefore the selection can be influenced by subjective criteria applied by different people. This may explain why, in another study, no difference in development was observed between compact and expanded oocyte–cumulus complexes (Tremoleda et al. 2003). In nuclear transfer embryos, as shown in this study, no differences were observed between the two types of cumulus–oocyte complexes and therefore the only reason to grade oocytes before maturation is the fact that expanded oocytes mature 2–4 h earlier than compact oocytes so they can be manipulated earlier.
Amongst the several types of cells tested in the zona-free method (cumulus cells, adult and fetal fibroblasts) there were no observed differences in the cleavage rate nor were there observed differences with parthenogenetic activation in contrast to what has been reported by others (Choi et al. 2003, Li et al. 2003). Subsequent embryo development to the blastocyst stage was, however, much lower ranging from 0 to 16% on average.
As in mice (Wakayama & Yanagimachi 2001), pigs (Kühholzer et al. 2001, Yin et al. 2002, Lee et al. 2003) and cattle (Kato et al. 2000, Servely et al. 2003) we have found a significant difference in the developmental potential of NT-embryos derived from different cell cultures. Blastocyst formation rates with some donors were low and comparable to those found by Li et al.(2002). However, with one adult and one fetal cell line the development to the blastocyst stage was comparable to the parthenogenetic controls (15.8 and 16% vs 13% respectively) and comparable with in vivo data on development of cloned mule blastocysts on Day 14 (12%; Woods et al. 2003). These data indicate that the source of the donor nuclei for nuclear transfer is a major source of variability in Equids as in other species.
Interestingly, the only fetus derived from fetal fibroblasts was aborted at the same time as the fetus derived from the genetically identical adult fibroblasts and had the same type of abnormality on the umbilical cord (see Fig. 2). The number of transfers with cumulus cell-derived embryos was small in this study and did not result in any pregnancies. However, we believe that this cell type can successfully serve as a nuclear donor in horse cloning as in cattle (Wells et al. 1998, Kato et al. 2000), mice (Wakayama & Yanagimachi 2001), rabbits (Chesne et al. 2002), goats (Keefer et al. 2002) and in mules where NT-embryos derived from cumulus cells were able to implant in recipient mares and to survive to Day 80 of pregnancy (Vanderwall et al. 2004).
Pregnancy losses reported here are typical of cloned pregnancies in other species (Heyman et al. 2002, Chavatte Palmer et al. 2004) as well as those reported by Woods et al.(2003) with cloned mule embryos. Fifty-seven percent (4/7) of NT-pregnancies from adult fibroblasts were lost between Days 17 and 35, in comparison with less than 15% of early embryonic death in normal breeding (Villahoz et al. 1985, Ball 1993). Two pregnancies resulted in the natural birth of two male foals. The average development to term was 5.4% and depended on donor cell culture as in other cloned animals (see Wilmut et al. 2002 for a review).
The first ever cloned foal (Galli et al. 2003b) that was born from a Haflinger female adult fibroblast NT-embryo is now about 20 months old. Incidentally, she was born to the recipient that served as the nuclear donor for the nuclear transfer experiment and therefore she is the genetic copy of the surrogate mother. Interestingly, the outcome of this experiment is contrary to the idea that the maintenance of gestation depends on adequate immunological recognition of fetal antigens by the mother (Szekeres-Bartho 2002).
The number of observations made on advanced pregnancies that aborted is limited; however, the pattern is similar to that of other species whereby abnormalities have a higher incidence in the extraembryonic tissues. In our case, the umbilical cord was abnormal, which was the same observation reported by us for a previous abortion (Galli et al. 2003b). One placenta had an area without villi, which is compatible with normal pregnancies; appearance and thickness were normal. Also the placentae of the pregnancies that went to term were normal and the weight was in the normal range (LeBlanc 1997). The foal that died perinatally developed septicemia. This pathological situation is described in horse neonatology and can be a cause of death for the foal. It is difficult to correlate this event to the fact that it was a cloned offspring; however, it is known that perinatal mortality of cloned offspring in other species is higher than normal (Chavatte-Palmer et al. 2002, Cibelli et al. 2002).
In conclusion, we have investigated the developmental capacity of NT-embryos derived from cumulus cells and from adult and fetal fibroblasts to develop in vitro and of embryo and fetal development in vivo following embryo transfer into recipients. We have shown that different cell cultures possess different capacities for supporting the pre-implantation development of horse NT-embryos and that blastocyst formation rate does not correlate with or predict future pregnancy fate. The zona-free method for embryo reconstruction proved very efficient in increasing the fusion rate and the efficient use of oocytes. In this study, oocytes with expanded and compact cumulus had equal ability to support the preimplantation development of NT-embryos. Finally, horse embryos cloned from adult fibroblasts were able to implant and develop to term.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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  K Hinrichs, Y H Choi, D D Varner, and D L Hartman Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin Reproduction, August 1, 2007; 134(2): 319 - 325. [Abstract] [Full Text] [PDF]
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K Hinrichs, Y H Choi, C C Love, Y G Chung, and D D Varner Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. Reproduction, June 1, 2006; 131(6): 1063 - 1072. [Abstract] [Full Text] [PDF]
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4.
