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Articles |
a Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo, Sassari, Italy
b University of Agriculture, Department of Animal Reproduction, Al. Mickiewicza 24/28, Krakòw, Poland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oogenesis begins in the fetus and ends weeks to months after birth. The growth phase of the oocyte is characterized by the progressive cytoplasmic accumulation of specific gene products that later regulate fertilization and early embryogenesis. Conventionally, oocyte maturation is split into cytoplasmic and nuclear maturation: cytoplasmic maturation refers to the process that prepares the oocyte for activation, pronuclear formation, and preimplantation development, while nuclear maturation refers to the ability of the oocyte to progress through meiosis. The developmental ability of oocytes from prepubertal animals has been the object of several studies. Some studies indicate a lower developmental competence [1, 2, 6] whereas others describe capacities similar to adult oocytes [7–9]. The rate of blastocyst formation from juvenile oocytes is variable, and survival to term after transfer of juvenile-derived embryos is generally low [1, 7, 10, 11]. The reasons for this high incidence of fetal loss are largely unknown.
Detailed studies have been performed on the development of the lamb ovary [12–14]. The ovary increases in weight 7-fold between birth and 4 wk of age; however, ovarian weight then decreases significantly and remains relatively constant to 33 wk of age. Peak numbers of growing follicles are seen 2 wk after birth, while peak numbers of vesicular follicles are reported at 4 wk of age, concomitant with the first signs of atresia. In the present study, 4-wk-old lambs were chosen as donors to take advantage of the maximal numbers of oocytes available.
Initially, the developmental competency to reach the blastocyst stage was compared for oocytes collected from adult and from prepubertal sheep. Secondly, the developmental competency of both fresh and cryopreserved blastocysts derived from adult and prepubertal oocytes was compared after transfer into recipients. Fetal development was monitored by high-performance ultrasound scanning and length of pregnancy. Our data show that hormonal stimulation and in vivo culture resulted in the highest proportion of blastocysts derived from prepubertal oocytes. However, in comparison to controls, development to term of embryos produced from prepubertal lambs was severely reduced, possibly as a consequence of incomplete imprinting in the female germline.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal experiments were performed in accordance with DPR 27/1/1992 (Animal Protection Regulations of Italy) in conformity with European Community regulation 86/609 and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.
The study was conducted on prepubertal (4–5 wk old) and sexually mature (3–6 yr old) ewes of the Sarda breed, during the breeding season of 1997–98. Forty-seven randomly chosen lambs with their mothers and 14 adult sheep were maintained under field conditions at the Institute of Zootecnics Research Farm, Bonassai, Sassari, Sardinia, Italy. Progesterone treatment was done by insertion of Norgestomet s.c. implants (Crestar, Intervet, Holland; Day 0), and lambs were then randomly divided into 3 groups and stimulated as shown on Table 1. Group 1 was primed with multiple doses of FSH, a treatment adopted from the protocol for stimulation of adult sheep [10]. Groups 2 and 3 were stimulated using modified methods for producing nonmatured and matured lamb oocytes, respectively [15, 16]. Follicular growth in adult ewes was primed with 4.8 mg of ovine FSH (Ovagen; ICP, Auckland, New Zealand) given in 6 equal doses, every 12 h on 10th, 11th, and 12th day of implant insertion [10].
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Oocyte Recovery
Animals were anesthetized with acepromazine maleate (0.05 mg/kg BW) and pentothal sodium (10 mg/kg BW) on the day after the final injection of hormones. Ovaries were exposed by midventral laparotomy, and follicular oocytes were aspirated with a 5-ml syringe fitted with a 20-gauge needle. The medium used for collection was tissue culture medium (TCM)-199 supplemented with 5% calf serum, 0.05 mg/ml heparin, and 0.05 mg/ml gentamicin sulfate. Oocytes from each donor were maintained separately throughout all in vitro procedures.
In Vitro Maturation (IVM), Fertilization, and Culture
Methods of in vitro embryo production were adopted from those previously described [17]. Aspirated oocytes were evaluated under the stereomicroscope, and only those surrounded by at least 2 layers of granulosa cells and with evenly granulated cytoplasm were selected for IVM. Maturation medium was bicarbonate-buffered TCM-199 (275 mOsm) containing 2 mM glutamine, 10% fetal bovine serum (FBS), 5 µg/ml FSH (Ovagen), 5 µg/ml LH, 1 µg/ml estradiol, 0.3 mM sodium pyruvate, and 100 µM cysteamine [18]. Oocytes from individual donors were incubated in 0.4 ml of medium in 4-well dishes (Nunclon, Nunc, Roskilde, Denmark) covered with mineral oil in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h.
All chemicals, unless indicated, were obtained from Sigma Chemical Co. (St. Louis, MO).
After maturation, oocytes were partially denuded of granulosa cells by gentle pipetting in Hepes-TCM-199 containing 300 IU/ml hyaluronidase. Fresh semen, obtained from a Sarda breed ram of proven fertility, was used throughout the experiments. Collected ejaculate was held at room temperature for up to 2 h, centrifuged twice at 200 x g for 5 min, and added directly to the fertilization medium. The IVF medium used was bicarbonate-buffered synthetic oviduct fluid (SOF) [19] enriched with 20% (v:v) heat-inactivated estrus sheep serum, 2.9 mM calcium lactate, and 16 µM isoproterenol [20]. Fertilization was carried out in 50-µl drops, using 1 x 106 sperm/ml and a maximum of 15 oocytes per drop, at 39°C in a humidified atmosphere of 5% CO2 in air for 20 h.
Presumptive zygotes were transferred to 20-µl culture drops consisting of SOF supplemented with 2% (v:v) basal medium Eagle (BME)-essential amino acids, 1% (v:v) minimum essential medium Eagle (MEM)-nonessential amino acids, 1 mM glutamine, and 8 mg/ml fatty acid-free BSA. Cultures were carried out in an atmosphere of 5% CO2, 7% O2, 88% N2 at 39°C with maximum humidity. At Day 3 and Day 5 of culture (Day 0 = day of fertilization), 5% charcoal-stripped FBS was added to the medium [21]. Cultures were maintained until 9 days postfertilization, at which point embryos that developed to the blastocyst stage were either transferred to synchronized recipients or vitrified.
In Vitro vs. In Vivo Culture
The oocytes collected from group 1 lamb donors resulted in the largest proportion of blastocysts produced in vitro. These oocytes were randomly distributed among two experimental groups: 1) in vitro culture (n = 15 animals), as described above, and 2) the remainder (n = 5 animals), cultured in vivo in the ligated oviducts of synchronized temporary recipients as described previously [22]. Embryos were embedded in agar and transferred into the oviduct of a synchronous sheep. A borosilicate glass capillary with nylon filter fitted inside was secured to the ampullary region of each oviduct to avoid embryo loss through the fimbria. After 6 days, the filters were removed, and the agar chips were collected by retrograde flushing of the oviduct.
Vitrification and Recovery
Dulbecco's PBS supplemented with 0.3 mM sodium pyruvate and 20% FBS was used as the basic vitrification solution (VT). Blastocysts were processed for vitrification at room temperature under the following regime: VT + 10% glycerol (G) for 5 min, followed by VT + 10% G + 20% ethylene glycol (EG) for 5 min. Embryos were finally transferred to VT + 25% G + 25% EG and loaded into the center of 0.25-ml straws. Subsequently, two columns of 0.5 M sucrose solution, separated from the embryos by air bubbles, were loaded into the straws. Sealed straws were plunged immediately into liquid N2. Embryos were recovered by immersing the straws into a water bath at 37°C and expelling the contents into an 800-µl drop of 0.5 M sucrose solution for 5 min. Embryos were then transferred into 0.25 M sucrose solution for 5 min and subsequently into 0.125 M sucrose solution for a further 5 min [23, 24]. Embryos were incubated in TCM-199 enriched with 20% FBS in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h. After incubation, only re-expanded blastocysts were transferred to synchronized recipient ewes.
Transfer to Recipients
Blastocysts were surgically transferred to recipient ewes 7 days after the onset of natural estrus. Pregnancy status was determined by ultrasonography at 40 and 80 days after transfer (Aloka, 7.5-MHz high-resolution linear probe), and pregnancies were allowed to develop to term.
Statistical Analysis
Statistical computations were performed using chi-square analysis of all data (SAS/STAT User's Guide, 6.03 Edition, SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovarian response was variable in all experimental groups (number of oocytes recovered per lamb ranged from 2 to 125). Some lambs did not respond to hormonal stimulation (5 in group 1, 4 in group 2, and 1 in group 3) and showed a limited number of small (0.3–1.0-mm) follicles per ovary. The number of oocytes obtained from each treatment and the number of embryos developing after in vitro culture are presented in Table 2. No blastocyst were obtained from donors with poor follicular response. Group 3 animals were stimulated in order to produce in vivo-matured oocytes, and in this group, out of 312 recovered oocytes only those with expanded cumulus cells (n = 93, 29.8%) were classified as mature and processed directly for IVF; the remainder, with compact cumulus cells, were matured in vitro. Only a few oocytes with expanded cumulus cells at recovery developed to blastocysts (4 out of 93, 4.3%), while 19 blastocysts were obtained from the IVM group (19 out of 81, 23.4%). The average number of oocytes recovered and cultured per lamb was not significantly different between groups; however, there were significant differences in blastocyst production between treatments and between adult and prepubertal material (see Table 2). In vitro, embryos derived from lambs developed to blastocysts 24–48 h later than embryos derived from adult donors (Fig. 1, A and B). Eleven blastocysts derived from juvenile oocytes (5 from group 1, 2 from group 2, and 4 from group 3), which were all late-developing embryos (8–9th day of culture), ceased development 24 h after formation of the blastocoele cavity.
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Vitrification
Survival rate of vitrified blastocysts was based on re-expansion of the blastocoele at 24 h postthaw (Table 3). From a total of 46 blastocysts, 4 from group 3 were not recovered at thawing while 17 (40%) re-expanded and were transferred to recipient sheep.
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Assessment of Fertilization and Embryo Culture In Vivo
Oocytes from lambs in group 1 responded best in terms of blastocyst production, and consequently a further 5 lambs were subjected to similar treatment. A number of the resulting oocytes were pooled and used to determine the rate of polyspermic fertilization. The remaining oocytes were cultured in vivo in the ligated oviducts of synchronous ewes. A total of 134 (80%) oocytes were recovered and, of these, 79 were selected for IVM/IVF. Thirty oocytes were fixed (ethanol:acetic acid 3:1) 12–15 h after insemination and stained with lacmoid staining 24 h later. Two pronuclei were detected in 22 oocytes (73%) whereas the remaining 8 eggs were polyspermic (27%). Forty-nine presumptive zygotes were transferred to the ligated oviduct of synchronized recipients 24–26 h after insemination. After 7 days, the oviducts were flushed, and 18 blastocysts were recovered (37%) (Fig. 1C).
Pregnancies and Lambing
In vivo-cultured lamb blastocysts were transferred to recipients in pairs. Because of the limited number of available recipients and the lower quality of fresh in vitro-cultured blastocysts, four ewes received three blastocysts each and six ewes received two blastocysts each. For similar reasons, vitrified blastocysts were transferred as follows: 2 recipients received 2 embryos each, 3 recipients received 3 embryos each, and 1 recipient received 4 embryos. Blastocysts produced in vitro from adult donors were transferred in pairs. Data for pregnant recipients and live births are presented in Table 4.
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Four lambs were born from in vitro-cultured lamb embryos, including one twin pregnancy (male, 2.8 kg; female, 4.0 kg) and 2 single pregnancies (males, 4.3 kg; 4.1 kg). Three embryos transferred after vitrification developed to term: one a single pregnancy (male, 3.7 kg) and 2 female twins (birth weight, 2.9 kg; 3.1 kg). In vivo-cultured lamb embryos resulted in 2 twin lambs (male, 3.4 kg, female, 1.5 kg) and a single female lamb (2.8 kg). Recipients carrying in vitro-derived embryos from adult donors produced 4 singleton pregnancies consisting of 2 male (4.5 kg; 5.0 kg) and 2 female offspring (3.9 kg; 4.1 kg), and 7 twin pregnancies produced 6 females (range of birth weight: 1.0–4.1 kg) and 8 males (range of birth weight: 2.4–6.0 kg). The birth weights of lambs were within the range for the Sarda breed; however, two cesarean sections were performed, as the recipient ewes failed to begin parturition by 154 days of pregnancy. Two recipients carrying twin pregnancies derived from in vitro-cultured lamb blastocysts delivered one normal lamb along with a mummified twin (died around third month of pregnancy). Similar mummified fetuses were observed in a pregnancy resulting from the transfer of vitrified embryos (2 normal lambs plus one mummified fetus) and in a pregnancy following the transfer of in vivo-cultured lamb-derived embryos (Fig. 2). Analyses performed on blood and on cervical smears of the recipients, in addition to samples obtained from fetuses, excluded all the common abortion agents (Salmonella abortus ovis, Brucella abortus ovis, Chlamydia psittaci, Campylobacter fetus, Mycoplasma agalactiae, Toxoplasma gondii, Listeria monocytogenes).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We observed an incomplete preparation of the cytoplasmic compartment in lamb oocytes as early as the postfertilization period. The frequent occurrence of polyspermy reported by others [2, 6, 27] was also noted here, affecting 27% of the zygotes, possibly as a consequence of the defective dispersion of cortical granules around the cortex [4]. Undoubtedly, differences between lamb- and adult-derived oocytes, such as the smaller size [28], and patterns of protein synthesis and energy metabolism [2, 28], contribute to the reduced competency of juvenile-derived embryos.
Blastocysts derived from lamb oocytes had a visible inner cell mass and did not differ morphologically from blastocysts derived from adult oocytes. However, one important difference observed in this and other studies [29] is the delayed embryonic development of lamb-derived embryos compared to adult-derived embryos. In fact, while the majority of blastocysts from adult-derived oocytes developed on Day 6 of culture, blastocysts from lamb-derived oocytes developed between one and two days later. As a delay in the development of lamb-derived embryos could be observed as early as the 4-cell stage, it is possible that a critical step in embryo development, such as zygotic genome activation, had been perturbed.
As anticipated, blastocysts produced in this study were morphologically normal and able to implant and to direct early fetal development; moreover, competency was not significantly impaired after vitrification. However, the ability of these blastocysts to develop into viable offspring after transfer to foster mothers was severely limited (see Table 4). The incidence of early embryonic loss in adult sheep after natural mating is generally 20–30% and occurs predominantly during the first 5 wk of pregnancy [30]. In vitro-produced embryos have a lower survival rate (40%), and embryo/fetal loss occurs at 30–35 days after transfer [31]. Embryo loss in the present study followed a similar pattern, albeit with notably higher mortality for lamb-derived embryos (80%) vs. adult sheep-derived ones (60%). The failure of established pregnancies occurred even after Day 80 in recipients carrying lamb-derived embryos, with only 6 of 12 (50%) surviving to term. Adverse genetic effects and seasonality cannot explain the pregnancy failures, since all embryos were produced using semen from one sire and transferred under identical conditions. Such pregnancy losses are a frequent feature following the transfer of juvenile-derived embryos in both sheep and cows [1, 7, 10, 11]. Hormonal treatments of prepubertal oocyte donors [29, 32] and growth factor addition during IVM of oocytes from calves have been reported to improve cleavage rate and blastocyst yield [26], and here we demonstrate a positive effect (P < 0.05) of culturing lamb-derived embryos in vivo. Despite these improvements, approximately two thirds of lamb-derived fetuses were lost in the first half of the pregnancy (Table 4), and a further 4 recipients delivered a normal lamb alongside a mummified twin (see Fig. 2). It is interesting to note that other authors have reported the occurrence of similar fetuses [33]. Blood and uterine samples collected from corresponding donors and analysis of the mummified fetuses excluded all of the common abortion agents that were analyzed.
Important clues to help explain juvenile-derived fetal losses may be drawn from the results of an elegant series of experiments investigating parthenogenetic development in mice [34]. This study describes the fusion of nongrowing oocytes from 1- and 13-day-old mice to fully grown germinal vesicle oocytes from which the chromosomes had been removed. The majority of reconstructed oocytes arrested at MII after IVM, and subsequently the chromosomes from these arrested oocytes were transferred into enucleated or intact metaphase II oocytes after ovulation. These reconstituted oocytes were able to support early embryonic development after either parthenogenetic activation or fertilization. Fertilized oocytes containing maternal alleles from nongrowing oocytes were unable to develop beyond Day 8 of embryogenesis [34]. The developmental failure of these reconstructed oocytes after fertilization indicated that epigenetic modifications established during oocyte growth have dramatic consequences for development, and even though such embryos are able to sustain preimplantation development, they do not survive beyond the first half of pregnancy [34].
The recent observations that genomic imprinting is not fully established in oocytes from neonatal mice [35] is suggestive of the mechanism underlying these previous findings [34]. Genomic imprinting is the epigenetic mechanism that makes parental genomes functionally nonequivalent during development, with the result that paternally expressed genes control extra-embryonic differentiation while maternally expressed genes regulate growth of the embryo proper [36]. More than 20 imprinted genes have been identified in mice and humans [37], and we have recently demonstrated that genomic imprinting is also evolutionarily conserved in sheep [38].
The results of Kono et al. [34] indicate that the imprinting of maternally expressed genes is not fully established in oocytes from young mice. The phenotype of the fetuses produced in our study strongly resembles the phenotype of mouse fetuses carrying a maternal genome derived from prepubertal mice [34], and in both instances fetal loss occurred in the first half of pregnancy.
The primary aim of this work was to exploit the dramatic increase in follicular growth that occurs at 4–5 wk of age in lambs and to increase the number of oocytes recovered from individual donors. This objective has been achieved as up to 125 oocytes were collected from individual donors; unfortunately, results in terms of fetal development were disappointing. This was possibly a result of defective imprinting in a significant proportion of oocytes from the ovaries of 1-mo-old lambs.
In our study, the number of recipient ewes carrying embryos derived from 4- to 5-wk-old lambs to term was around 20%, in contrast to reports of 38% pregnancy achieved with transferred embryos derived from oocytes from 10- to 12-wk-old animals [39]. An even higher pregnancy rate (60%) was obtained with embryos from much older (16–24-wk) lambs [27, 33]. In several studies dealing with juvenile sheep embryo production [6, 15, 16, 32], the pregnancy rate does not give a complete picture of the developmental capability of lamb oocytes. Indeed, the number of pregnancy failures that occurred in this study, particularly from those cultured in vivo, with only 2 offspring from 7 recipient sheep pregnant at 80 days, supports this idea. To our knowledge, only offspring derived from oocytes collected from substantially older (4–6 mo) lambs have been born [27, 33].
Here we report the production of normal offspring from the youngest ruminant oocytes donors employed to date (Fig. 3). Despite this success, the overall low efficiency and the demonstration that embryonic competence increases with oocyte age [29] leads to the recommendation that older lambs should be used as oocyte donors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Grazyna Ptak, Istituto Zootecnico e Caseario per la Sardegna, 18 Km strada Sassari Fertilia Fraz. Totubella, 07040 Olmedo (SS), Italy. FAX: 79 389450; gptak{at}tiscalinet.it
Accepted: July 28, 1999.
Received: April 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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