| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
a South Australian Research and Development Institute (SARDI) Reproduction Laboratory, Turretfield Research Centre, Rosedale, SA 5350, Australia
b South Australian Research and Development Institute (SARDI) Molecular Biology Laboratory, Glenside, SA 5065, Australia
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
assisted reproductive technology, developmental biology, embryo, oocyte development, pregnancy
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nutritional status and dietary intake of the animal are known to influence reproductive performance through a network of complex relationships and interactions [8]. Importantly, recent studies have indicated a link between nutrition and oocyte quality as assessed by in vitro developmental capacity [9] and oocyte morphology [10, 11]. Collectively, these studies indicate that, under appropriate experimental conditions, the viability of the oocyte can be improved in association with the changes in mRNA processing abilities. Furthermore, the effects of nutrition during the pre- and periovulatory period can have far-reaching effects, not just on early embryo differentiation [12] but also on fetal development and the fitness of any offspring produced [13]. It is therefore hypothesized that nutrition during the preovulatory period might also influence the molecular events associated with nuclear reprogramming in the reconstructed embryo.
The aim of this study was to determine if the nutrition of the oocyte donor ewe influences the outcomes of somatic cell cloning. Addressed outcomes included the efficiency of reconstructed embryo production, the ability of embryos to establish pregnancies and to develop to term, and the normality of offspring.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nutrition and Superovulation of Ewes
All experimental procedures involving the use of animals were conducted according to guidelines of the Australian Code of Practice for the Use of Animals for Scientific Purposes [14]. All protocols were approved by a Primary Industries and Resources South Australia Animal Ethics Committee. Mature (age, 4–6 yr) Merino ewes were randomly allocated to three groups after stratification by live weight. Each group was fed a pelleted diet of cereal/legume grain and roughage (dry matter 907 g/kg, metabolized energy 9.5 mJ/kg, and crude protein 120 g/kg) in a 0.7-ha feedlot paddock with water provided ad libitum. Two groups, comprised of donor ewes, were fed at either 0.7x maintenance level [15] (i.e., low group) or 1.3x maintenance level (i.e., high group) three times weekly for a 3- to 5-mo period before imposition of superovulation treatments. The third group, comprised of recipient ewes, was fed at the maintenance level.
Superovulation treatment consisted of an intravaginal progestogen pessary (45 mg of flugestone acetate; Laboratorie Pharmaceutique Porges, Paris, France) being inserted on Day 0. Each ewe received a dose of FSH (Folltropin; Vetrepharm Canada, Belleville, ON, Canada) equivalent to 180 mg of NIH-FSH-P1 standard. Six injections were given over Days 11, 12, and 13 (two injections per day). Ewes also received 500 IU of eCG per ewe (Pregnecol; Horizon Technology, North Ryde, NSW, Australia) at the time of the first FSH injection. Pessaries were removed on the afternoon of Day 13, and 0.3 ml of GnRH (Fertagyl; Intervet International, Boxmeer, Holland) was administered on the afternoon of Day 14. Oocytes were collected by laparotomy 24–27 h after GnRH treatment. The exposed oviducts were retrograde flushed using PBS supplemented with 5% heat-inactivated sheep serum and immediately transferred to Ca2+- and Mg2+-free culture medium (synthetic oviduct fluid [SOF]-HCO3 with essential and nonessential amino acids and 4 g/L of BSA; Gibco Life Technology, Auckland, New Zealand) [16]. The oocytes were kept in this medium in 5% CO2 in humidified air at 38.5°C until manipulation within 2 h of collection.
Donor Cell Treatment
Donor granulosa cells were obtained from an adult Merino ewe and cultured for two to three passages in Glasgow medium supplemented with sodium pyruvate, L-glutamine, Dulbecco modified Eagle medium, penicillin, streptomycin, and 5% fetal calf serum (FCS) (CSL, Parkville, VIC, Australia). For nuclear transfer, cells were serum starved for 4 days in the presence of 0.5% FCS before freezing in Nunc cryotubes (Nunc, Roskilde, Denmark) in 10% dimethyl sulfoxide and 20% FCS in Glasgow medium. On the day of nuclear transfer, one tube was thawed in a 40°C water bath, diluted with fresh medium, and centrifuged at 200 x g for 5 min to remove cryoprotectants. Thawed cells, suspended in Glasgow medium + 5% FCS, were kept in an incubator until used for cloning within 2–4 h.
Nuclear Transfer Procedures
The initial handling and incubation of oocytes occurred in Ca2+- and Mg2+-free media, but after the micromanipulations, media containing Ca2+ and Mg2+ were used. Before manipulations, oocytes were preincubated for 5–10 min in handling medium containing 7.5 µg/ml of cytochalasin B and 5 µg/ml of Hoechst 33342. Oocyte enucleation and injection of donor cells into the perivitelline space were performed concurrently in batches of 10–20 oocytes in a handling medium supplemented with cytochalasin B. A fresh aliquot of donor cells in a fresh manipulation drop was used for each batch. The exact location of the metaphase spindle was established with less than a 1-sec exposure to ultraviolet (UV) illumination, after which the first polar body and the spindle were removed by aspiration. The success of enucleation was then confirmed by viewing the contents of the pipette under UV illumination. Immediately after enucleation, a donor cell was selected from the chamber area not exposed to UV illumination and injected into the perivitelline space of the oocyte through the enucleation hole in the zona pellucida. Cytoplast-donor cell couplets were then incubated in culture medium until the time of fusion. After oocyte manipulation, the couplets were fused in mannitol fusion medium (0.3 M mannitol, 0.05 mM CaCl2, and 0.1 mM MgSO4) using a Genaust fusion machine (Genetics Australia, Bacchus Marsh, VIC, Australia) and a manipulation chamber that consisted of two parallel, 0.1-mm-diameter platinum wires separated by 0.2 mm. An AC-alignment pulse of 400 kHz and 10 V was followed by two AC/DC fusion pulses of 1.25 kV/cm and 80 µsec. The fusion success was evaluated within 30 min, and unfused but intact couplets were pulsed again. Couplets that remained unfused after the second attempt were discarded. Successfully fused couplets were incubated within 1 h of pulsing in 10 µM Ca2+ ionophore in protein-free SOF-Hepes for 5 min, followed by 2-h incubation in 2 mM 6-dimethylaminopurine (6-DMAP) in culture medium. Finally, the reconstructed embryos were transferred to 500 µl of culture medium covered with 400 µl of mineral oil in Nunc four-well dishes and cultured in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C. Embryo cleavage and development to blastocysts were recorded on Days 2 and 6 of culture, respectively (nuclear transfer = Day 0). The incidence of embryo fragmentation, in which uncontrolled cytoplasmic cleavage without accompanying normal karyokinesis results in the formation of anucleate multicellular structures resembling morulae, was also recorded. On Day 6, all grade I blastocysts and some grade I compact morulae were either immediately transferred to recipient ewes or, on a few occasions (6 of 44 transfers) when insufficient recipients were available, vitrified using the open pulled straw method [17]. These embryos were subsequently transferred to the first available recipients after warming using the open pulled straw method [17] and after 1–2 h in culture.
Embryo Transfer, Pregnancy Monitoring, and Production of Offspring
Recipient ewes were synchronized with intravaginal progestogen pessaries (45 mg of flugestone acetate) inserted for 12 days, followed by an i.m. injection of 400 IU of eCG (Pregnecol). Compact morulae and blastocysts were transferred to recipient ewes approximately 6 days after the expected median time of ovulation. Two, and occasionally one or three, embryos were transferred laparoscopically to the uterine horn ipsilateral to a corpus luteum. After embryo transfer, ewes were grazed on annual grass/clover pastures to maintain a body condition score of 3.0 [18] throughout pregnancy. Pregnancies were first monitored by ultrasonography between Days 40 and 60 of pregnancy and then once a month until term. During the last week of pregnancy, recipients were given an i.m. injection of 3 ml of Dexafort and 3 ml of Dexadreson (Intervet International BV, Boxmeer, Holland) a few days apart or 3 ml Dexadreson alone to assist fetal lung maturation.
Microsatellite Analysis
A small skin biopsy specimen was collected from all lambs born in this study, excluding some cases in which a dead newborn was found too late for a proper sample to be obtained. Genomic DNA was prepared from these samples, from the original donor granulosa cell line, from four other somatic lines being used as donor lines in parallel nuclear transfer experiments in the laboratory, and as a comparison, from lambs produced by natural mating, all according to the procedure of Sambrook et al. [19] (proteinase K digestion followed by multiple phenol extractions then ethanol precipitation). One hundred nanograms were used as the template DNA in each microsatellite analysis. Microsatellite band patterns were generated following two rounds of polymerase chain reaction (PCR) and size fractionation of reaction products in denaturing 6% polyacrylamide gels (7 M urea/19:1 [w/w] acrylamide:bis-acrylamide; SequaGel-6; National Diagnostics, Atlanta, GA). In the first round of PCR, samples were held at 95°C for 11.5 min, then cycled 50 times (Stratagene Robocycler, Stratagene, La Jolla, CA) at 95°C for 40 sec, 60°C for 40 sec, 72°C for 40 sec, and finally held at 72°C for 10 min. Amplification reactions used 0.5 U of Amplitaq Gold enzyme (Perkin-Elmer, Boston, MA) and were carried out in 25 µl at 1 mM MgCl2/1x Amplitaq buffer with 200 µM dNTPs and 0.4 µM of each microsatellite-specific oligonucleotide primer pair (see [20] for the sequences for OarFCB11-, OarFCB128-, and MAF209-specific oligonucleotide primers). One microliter from the first-round PCR reaction was then taken into a second, asymmetric PCR reaction including the same amount of Amplitaq Gold enzyme, cold dNTPs, Amplitaq buffer, and MgCl2, but with only one oligonucleotide primer at 0.2 nM and radioactively labeled by phosphorylation using Phage T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [
-33P]ATP (3000 Ci/mmol; Perkin-Elmer). The PCR samples were cycled as described above, then approximately one-tenth of each radiolabeled mix was used in gel fractionations. Sequencing gels were washed in 12% acetic acid/20% ethanol to "fix" the DNA, transferred to Whatman 3M (Whatman, Maidstone, U.K.) paper, and dried under vacuum at 80°C, then microsatellite bands were detected by autoradiography at room temperature overnight.
Statistical Analysis
The effects of treatment on ewe live weight, number of ovulations, and number of retrieved oocytes were analyzed using the general linear model in SAS [21]. Differences in embryo development rates between the two nutritional groups were analyzed by unpaired t-test after arcsin transformation of percentage data using GraphPad Software (San Diego, CA). The procedure CATMOD in SAS [22] was used to test the effect of nutritional treatment on pregnancy rates.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The mean live weight of the two treatment groups at the beginning of the experiment did not differ significantly (59.2 vs. 58.3 kg for high vs. low, respectively). However, at the end of the treatment, ewes in the high group were, on average, 5.6 kg heavier than their initial weight, and ewes in the low group were, on average, 3.2 kg lighter than their initial weight (P = 0.0001).
Oocytes were collected from 44 ewes (22 per nutrition group) in six replicates. The mean numbers of ovulations, estimated per the number of corpora lutea, were 16.2 and 15.6 in the high and low groups, respectively. The mean numbers of recovered oocytes were 11.2 and 9.4 in the high and low groups, respectively (Table 1). However, because of a variable proportion of oocytes being arrested at the metaphase I stage at the time of collection, only 183 and 165 oocytes could be used for cloning (74.4% and 79.7% of collected oocytes, respectively) in the high and low groups, respectively. None of these values was significantly different between the groups.
|
In Vitro Development of Cloned Embryos
No significant differences were observed between the groups in cleavage rates or in blastocyst development rates (Table 2). Blastocyst development was 40.4% and 35.8% for the high and low groups, respectively. Significant differences also were not observed in the incidences of fragmented embryos. Excluding noncleaved and fragmented embryos, the blastocyst development rate was 51.8% and 50.0% in the high and low groups, respectively (P = 0.9815).
|
Pregnancies
Pregnancy data from the transfer of fresh or vitrified embryos were pooled, because we found no significant differences in pregnancy rates between the two. Significantly (P = 0.0498) more pregnancies were observed following the transfer of embryos derived from the high compared with the low group (69.2% vs. 38.9%) (Fig. 1). However, by Day 90 of pregnancy, the difference was only approaching significance (P = 0.0587) (Fig. 1), and by Day 130 and at term, no significant differences were observed. In the high group, 50.0% (9 of 18) of established pregnancies went to term, as opposed to only 28.6% (2 of 7) in the low group, but this difference was not statistically significant (Fig. 1). When calculated on an individual-embryo basis, again, significantly (P = 0.0219) more transferred embryos in the high group were observed to have implanted at the time of the first ultrasonography: 22 fetuses from 51 embryos in the high group (43.1%) but only 7 fetuses from 37 embryos in the low group (18.9%). Three sets of twin pregnancies were observed in the high group, as opposed to none in the low group. At the end of gestation, 17.6% (9 of 51) and 5.4% (2 of 37) of transferred embryos survived in the high and low groups, respectively (P = 0.1047) (Fig. 1), although no twin births were observed.
|
Deliveries and Offspring
Of 11 pregnancies going to term, 7 were delivered by cesarean section, and 4 were born spontaneously before the planned surgery. All spontaneous deliveries were without supervision, and in two cases, the lambs were found dead. Subsequent autopsy revealed that one had breathed but that the other had not. One of these lambs had an undershot jaw, and both had a mild case of hydronephrosis. However, no other gross abnormalities were observed. One of seven cesarean deliveries yielded a lamb that appeared to have died approximately 1 wk before, whereas all other lambs were born alive. Autopsy of the dead lamb was not conclusive because of advanced autolysis.
Of eight lambs born alive, four died within minutes or hours of birth because of breathing difficulties. Of the remaining four lambs, one had severe joint abnormalities and was killed, another was killed at 10 days of age because of serious umbilical inflammation and septic polyarthritis, and a third succumbed at 29 days of age to enteritis, peritonitis, and pluritis. Autopsies of nine dead lambs revealed some internal abnormalities, mainly in the urogenital system (Table 3). The fourth remaining lamb, derived from the high nutrition group, survived the critical perinatal period and, as of this writing, is thriving without health problems at 15 mo of age.
|
Microsatellite Analysis
Microsatellite analyses proved, without a doubt, that the lambs born in this study were clones of the used donor cell line. In comparison to the band patterns generated by analysis of DNA from each of the other somatic cell lines, the microsatellite marker bands diagnostic for loci OarFCB11, OarFCB128, and MAF209 contained in the donor cell line were found to be identical in DNA samples from each of the lambs produced in this experiment. The DNA microsatellite band sizes determined for each of these loci in lambs from natural matings were invariably different from each other and from the microsatellite bands shared by the donor cell line and the experimental lamb group (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The effect of nutrition on reproductive performance is a long-recognized and comprehensively studied area of animal physiology. These effects can be manifested at different levels, including the hypothalamus and pituitary gland to modify gonadotropin secretion, the ovary to alter oocyte quality and steroid production, and the uterus to modify the milieu of developing embryos and fetuses (reviewed in [8]). The relationship between nutrition and reproduction in ruminants is complex, and responses are often variable and inconsistent, explaining why much of the literature regarding nutrition effects on the ewe may appear, at first, to be confusing and contradictory. Prolonged undernutrition in the sheep has long-term effects by reducing ovulation rates and reproductive performance despite animals subsequently returning to adequate live weight [24]. On the other hand, increased energy intake for a relatively short period will increase the ovulation rate, although continued exposure to excess energy can decrease embryo quality and overall reproductive performance, and this effect has been suggested to be mediated at the follicle and oocyte level [8]. Indeed, the optimum nutritional requirements for follicle growth and ovulation may be different from those required for optimum embryo development. Nevertheless, the beneficial effects of short-term nutrition restrictions on superovulation response and embryo yield have been observed [25]. Our reason for choosing a relatively long-term nutritional treatment was to ensure that most of the follicle and oocyte growth occurred during the feeding period. In the ewe, the process of follicle development from primordial follicle to ovulation takes approximately 6 mo [26], with the majority of this time (
4 mo) being spent in preantral stages of development. The follicle growth period is characterized by high levels of synthetic activity, as indicated by the presence of one or more nucleoli, RNA polymerase activity, and continuous uptake of amino acids and ribonucleosides [27]. During this time, the foundation of subsequent oocyte functionality is laid. Despite significant changes in live weight in ewes receiving the different feeding regimens of the present study, no significant differences were observed in the mean number of ovulations per ewe or in the mean number of oocytes recovered per ewe. These observations indicate that cellular events implicated in nuclear reprogramming are more sensitive to changes in nutrition than are the events involved in follicle growth and ovulation. Alternatively, these events may be independent of each other.
Few studies have looked specifically at the effect of nutrition on oocyte quality. McEvoy et al. [9] reported that superovulated ewes on a low-nutrition diet had a higher proportion of viable oocytes than ewes on a high-nutrition diet. The viability was assessed by the in vitro culture of Day 2 embryos to the blastocyst stage. However, the nutritional treatment in this case was relatively short-term (duration, 18 days). Those authors concluded that excessive feeding during follicular recruitment and the final stages of oocyte maturation resulted in retarded embryo development. Yaakub et al. [10] observed abnormalities in ultrastructural morphology of oocytes obtained from unsuperovulated ewes receiving a low level of nutrition (0.5x maintenance level for 28 days). However, O'Callaghan et al. [11] did not find correlations between oocyte morphological abnormalities and the diet (0.5x, 1.0x, or 2.0x maintenance level for 32 days), although they did find differences between unsuperovulated and superovulated oocytes. We have previously found differences in development rates of embryos derived from ewes on a high, medium, or low level of nutrition (1.5x, 1.0x, and 0.5x maintenance level, respectively) for 4 wk [12], especially in blastocyst cell number and cell lineage differentiation. However, in this model, the oocyte effect can not be separated from the embryo development effect, and furthermore, the duration of nutritional treatment was only short term. Although the concept of developmental quality of an oocyte is not well defined [23], in summary, it represents the functional properties acquired during the growth and final steps of oocyte maturation. Usually, the oocyte quality and subsequent developmental competence is characterized by its ability to produce viable and fertile offspring after fertilization [23]. Nevertheless, several other factors, such as genomic contributions from spermatozoa or oocyte, in vitro culture conditions, or developmental milieu as represented by hormonal and nutritional characteristics of the gestational mother, affect this outcome. Hence, we attempted in the present study to more objectively evaluate oocyte cytoplasmic quality by using a "standardized" genomic component (i.e., donor cell line) combined with standardized culture conditions. Given the observations that high nutrition can adversely affect oocyte quality [9, 10], one question that emanates from the present study is why the pregnancy rate was significantly higher in the high group compared with the low group. Such an inconsistency with published data might result from differences in the feeding regimen (amount and duration of feeding); alternatively, cellular events associated with nuclear reprogramming might be independent of those associated with oocyte morphology and early embryo development.
The differences in the pregnancy results between the two treatment groups were observed throughout gestation, although the difference was no longer significant after midgestation. The majority of the established pregnancies in both groups were lost early, with 88% and 80% of the losses in the high and low groups, respectively, occurring before or around Day 60 of pregnancy, as opposed to only 12% and 20%, respectively, thereafter. This differs from the results of other studies, in which even though more than 50% commonly are lost early (as in sheep [1] and cattle [2, 28]), high rates of abortion are also observed during late gestation (as in sheep [1, 29, 30] and cattle [2, 28, 31]). Early losses might be associated with placental deficiencies during implantation, as reported by De Sousa et al. [32].
Microsatellite analysis of DNA from each of the newborn lambs confirmed their identity as bona fide somatic cell nuclear transfer clones derived from the donor granulosa cell line. The microsatellite sequences chosen, including OarFCB11, OarFCB128, and MAF209, are unlinked autosomal loci known to have multiple alleles in the Australian Merino and have previously been used for such analyses [33, 34]. Most offspring produced in the present study exhibited some of the typical anomalies associated with cloned offspring: inadequate lung function, urogenital abnormalities, and skeletal abnormalities, including undershot jaws and contracted forelegs [35]. The majority of the lambs that died during the perinatal period exhibited breathing problems associated with immature lung development. This problem may be related to insufficient fetal production of cortisol and inadequate hormonal signaling between the fetus, placenta, and mother, which is also suspected to result in absent or delayed parturition in cloned pregnancies. However, in a study comparing endocrine characteristics of cloned lambs produced in the present and related studies with control lambs produced by natural matings, plasma ACTH and cortisol concentrations were observed to be elevated after birth and during the first 4 wk of life in cloned lambs [36]. Thus, problems with the fetal production of cortisol appear to be unlikely. The loss of two lambs to infection is consistent with previous observations regarding susceptibility of cloned calves to infections [35] and may be an indication of their immune system not functioning optimally, as reported for cloned cattle [37] and mice [38]. The incidences of any clinical problems, however, were not influenced by the high- and low-nutritional treatments.
In conclusion, the present study found no differences in preimplantation development rates of cloned embryos derived from oocytes obtained from ewes fed either high or low levels of nutrition for several months. However, a significant difference was observed in the initial pregnancy rate, and proportionally more pregnancies went to term and resulted in offspring in the high-nutrition group. Although not conclusive, these results suggest that more research into the effect of nutrition on oocyte quality and its subsequent effect on cloning outcomes is warranted.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 61 8 8524 9088; peura.teija{at}saugov.sa.gov.au
Received: 14 May 2002.
First decision: 4 June 2002.
Accepted: 12 July 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. Borowczyk, J. S. Caton, D. A. Redmer, J. J. Bilski, R. M. Weigl, K. A. Vonnahme, P. P. Borowicz, J. D. Kirsch, K. C. Kraft, L. P. Reynolds, et al. Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep J Anim Sci, June 1, 2006; 84(6): 1593 - 1599. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
