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Embryo |
Department of Clinical Veterinary Medicine,3 University of Cambridge, Equine Fertility Unit, Mertoun Paddocks, Newmarket, Suffolk CB8 9BH, United Kingdom
Department of Development and Genetics,4 The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, gamete biology, growth factors, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In general, two basic factors of oocyte maturation, in both the nucleus and the cytoplasm, and G0/G1 regulation of the donor cell nuclear cycle are believed to enhance nuclear reprogramming and the subsequent development of the reconstructed oocytes following nuclear transfer [1]. Previous studies on horse oocytes in an in vitro culture system have indicated that around 50%–80% of nuclei mature to the metaphase II stage (MII), which is similar to the rates achieved in other species [10–16]. Nevertheless, addition of IGF-I to an in vitro maturation (IVM) medium containing hormones acts positively on cytoplasmic maturation of equine oocytes, as measured by increased rates of parthenogenetic cleavage [17].
There is only limited information available about the first cell cycle organization of horse oocytes reconstructed by nuclear transfer and the influence of culture conditions on nuclear remodeling of such reconstructed oocytes. This study was undertaken to investigate the influence of IGF-I on nuclear and cytoplasmic maturation of horse oocytes during culture in vitro. In addition, the organization of chromatin and microtubules during the first cell cycle was compared in enucleated horse oocytes reconstructed by nuclear transfer and oocytes stimulated to divide parthenogenetically.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Horse ovaries were obtained from a commercial abattoir and cumulus oocyte complexes (COCs) recovered from them by scraping the walls of follicles 0.5–3.0 cm in diameter. Groups of 20–30 COCs were matured in vitro for 28–30 h at 38°C in 5% CO2 in air in TCM199 (Sigma, St. Louis, MO) supplemented with 10% v:v heat-inactivated fetal bovine serum (Sigma), 10 µg/ml FSH (Sigma), 5 µg/ml LH (Sigma), and 1 µg/ml estradiol (Sigma), with or without 200 ng/ml added IGF-I (Sigma) [17]. The same medium was used to culture the reconstructed oocytes after nuclear transfer.
Western Blot Analysis of MPF and MAP Kinase During Oocyte Maturation
The influence of IGF-I on cytoplasmic maturation was assessed by analyzing the changes in MPF (cdc2 and cyclin B1) and MAPK (ERK1 and ERK2). Four groups of oocytes were prepared to be at different stages of the meiotic cycle. Namely, they were used directly after collection of the COCs, or they were cultured for 12, 24, or 28–30 h, with and without 200 ng/ml added IGF-I. Fifteen denuded oocytes were used for each treatment, after which their proteins were extracted with SDS electrophoresis sample buffer [18] and then heated to 100°C for 5 min, before being frozen at –80°C. Western blot analysis was then performed as reported previously by Dai et al. [19]. Briefly, the samples were run on 8%–15% linear gradient SDS-polyacrylamide gels, after which the proteins were transferred onto an Immobilin-P membrane using a Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories, Hercules, CA) for 1 h at 2.5 mA/cm in transfer buffer.
The filters containing the proteins produced by the oocytes at selected stages of meiosis were first incubated for 1 h in a medium of 15% fetal calf serum in PBS-Tween. Then, for a further 2 h at 20°C on each occasion, they were incubated, respectively, with rabbit antiphosphorylated cdc2, cyclin B, and MAPK antibodies (Upstate, Milton Keynes, UK). After three 15-min washes in PBS-Tween, all the filters were incubated for 1.5 h in a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobin (Dako, Bucks, UK). Finally, the membranes were washed extensively in PBS-Tween and then processed using the enhanced chemiluminescence detection system (Amersham Life Science, UK) and x-ray film (Kodak, Hemel Hempstead, UK).
Nuclear Transfer and Parthenogenetic Treatment
The nuclear transfer procedure was performed as described by Li et al. [5, 6], and all the micromanipulations were performed in EBSS containing 20% v:v FBS (EBSS/FBS). After 28–30 h of in vitro maturation, MII oocytes were placed for 10 min in EBSS/FBS containing 5 µg/ml Hoechst 33342 (Sigma). They were then enucleated in EBSS/FBS containing 5 µg/ml cytochalasin B (CCB; Sigma), and only those oocytes in which removal of both the polar body and the MII nucleus was confirmed by observation under ultraviolet light were included in the study. Adult skin fibroblast cells (AFC) were cultured from subdermal biopsies recovered from a 4-yr-old Pony mare. After two or three passages, lines of cultured AFC cells were either frozen in liquid nitrogen or used for nuclear transfer after starvation culture for 24–30 h in DMEM containing 0.5% v:v FBS and 15 µM roscovitine [20]. The fibroblast-cytoplasm couplets were aligned manually in 0.28 M mannitol fusion buffer in a 1.0-mm fusion chamber and subjected to two DC pulses, each of 2.2–2.5 kV/cm for 30 µsec and delivered by an ECM830 Electro Square Porator (BTX; San Diego, CA). Fibroblast-cytoplasm couplets that had fused successfully were then activated chemically by immersing them in PBS medium containing 5 µM ionomycin for 5 min followed by culture for 4 h in TCM199 medium containing 5 µg/ml CCB and 10 µg/ml cycloheximide (Sigma).
For parthenogenesis, MII oocytes were subjected to the same conditions applied to activate the reconstructed oocytes. Groups of 5–10 reconstructed or parthenogenetic oocytes were then cultured in 500-µl drops of development medium at 38°C in an atmosphere of 5% CO2 in air. The rates of two-cell and blastocyst stage development were counted 28–30 h after activation and at Days 7 and 8, respectively, in both the nuclear transfer and the parthenogenesis groups.
Chromatin and Microtubule Analysis at the First Cell Cycle
Oocytes submitted to either nuclear transfer or parthenogenesis were used to analyze the degree of organization in the first cell cycle at 12–15 h after cell fusion or parthenogenetic treatment, as previously described [6, 21]. Briefly, oocytes were initially fixed by immersing them for 1 h at 38°C in M medium [22] followed by 30 min in 2.5% paraformaldehyde. The microtubules were labeled by incubating the fixed oocytes for 90 min at 38°C in a 1:250 dilution of a monoclonal anti-
-tubulin antibody (Sigma), followed by incubation for 1 h in a blocking solution. Next, the oocytes were exposed for 1 h at 38°C to a goat anti-mouse gamma globulin (Sigma) conjugated to fluorescein isothiocyanate (FITC) and diluted 1:250 in PBS containing 0.5% Triton X-100 and 0.5% BSA. DNA was detected fluorescently by placing the oocytes for 10 min in PBS containing 5 µg/ ml propidium iodide (PI; Sigma). The stained oocytes were mounted under a coverslip in antifade medium (Vector Lab, Burlingame, CA) and analyzed by confocal laser scanning microscopy (Leica TCS MP, Heidelberg, Germany).
Statistical Analysis
The results were evaluated by z-test analysis (SigmaStat 2.03). Differences between the groups were considered statistically significant when P < 0.05.
All the animals used in these experiments were licensed (Project no. PPL 80/1442) and maintained (Certificate of Designation no. PCD 80/ 9044) under the provision of the Animals (Scientific Procedures) Act 1986 and under the supervision of the Home Office Experimental Animals Inspectorate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rates of oocyte maturation and embryonic development after nuclear transfer and parthenogenetic stimulation are summarized in Table 1. Following maturation culture and nuclear transfer, the rates of MII stage oocyte (47% vs. 45%), two-cell (26% vs. 23%), and blastocyst stage development (11% vs. 3%) were not significantly different between the two groups matured in medium that did or did not contain added IGF-I, respectively. Similar rates of two-cell development were exhibited by the two groups of MII stage oocytes stimulated to undergo parthenogenesis, whether or not IGF-I was added to the medium (51% vs. 35%). However, these rates of parthenogenetic cleavage were significantly higher than the rates of cleavage obtained following nuclear transfer, when the oocytes were matured in medium containing IGF-I (P < 0.05).
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Changes of MPF and MAP Kinase During Oocyte Maturation
Changes in the components of MPF (cdc2 and cyclin B1) and MAPK (ERK1 and ERK2) during oocyte maturation are shown in Figure 1. A 34-KDa signal of phosphorylated cdc2 was found in all the groups of treated oocytes cultured in vitro for different periods of time between 0 and 30 h, and the signal tended to increase during meiotic progression. IGF-I supplementation of the maturation medium did not clearly increase the quantity of cdc2 kinase activation for any of these periods (12, 24, or 28–30 h). Cyclin B was observed in the oocytes at the beginning of the culture period, and it accumulated only sparsely during 30 h of culture. Two subunits of MAPK, ERK1 (44 kDa) and ERK2 (42 kDa), were likewise present in all the treated oocytes throughout the 30 h of culture. More phosphorylated ERK2 than ERK1 was present in the column that was activated following oocyte maturation. IGF-I stimulated more accumulation of MAPK, in both subunits of ERK1 and ERK2 in the cytoplasm, than that obtained from the oocytes matured in medium without added IGF-I.
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First Cell Cycle Organization
Nuclear transfer oocytes As shown in Table 2 and Figure 2, two types of nuclear remodeling at first cell cycle organization were observed in the reconstructed oocytes following nuclear transfer. In the first (Type I), the donor nucleus of the reconstructed oocytes underwent morphological changes of NEBD and PCC resulting in normal cleavage of a two-cell stage embryo. In the second (Type II), either the nucleus from the donor cell formed a polyploid two-cell embryo or the embryo fragmented. The total rate of nuclear remodeling in the reconstructed oocytes did not differ between the oocyte maturation media that did or did not contain IGF-I (83% vs. 88%, respectively). In the oocytes matured in medium containing IGF-I, similar proportions showed Type I and Type II nuclear remodeling (45% vs. 38%, respectively). However, when IGF-I was removed from the maturation medium, the rate of Type II remodeling became significantly higher than the rate of Type I (60% vs. 28%, respectively, P < 0.05).
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Parthenogenetic oocytes The states of the chromatin and microtubules at the end of the first cell cycle in the parthenogenetically stimulated oocytes are summarized in Table 2 and Figure 3. Organization of the microtubules was clearly normal after this treatment, compared to the picture obtained after nuclear transfer. Some 74%–84% of the treated oocytes were activated, and 40%–50% proceeded through to the metaphase stage of first mitotic cell cycle including twin nuclei formation following 12–15 h of culture after activation treatment. These rates were much higher and the changes had occurred faster than those exhibited by oocytes that had undergone nuclear transfer. However, blastocyst stage embryos were not obtained in either the oocytes cultured with or those cultured without IGF-I in the maturation medium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth factors have a regulatory role in ovarian function, and IGF-I is a potent mitogen for granulosa cells [24, 25]. It has also been shown to exert pleiotropic effects on the maturation of bovine oocytes and on early embryonic development in this species [26–28]. In the horse, Carneiro et al. [17] indicated that the addition of IGF-I to an IVM medium containing hormones and FCS did not increase the rate of nuclear maturation to the MII stage but did exert a positive effect on cytoplasmic maturation, as measured by parthenogenetic cleavage. To avoid any interference from growth hormone or other growth factors that might be present in the FBS, we used only FBS that was known to contain low levels of LH and FSH and no growth factor. Under these conditions, the results indicated no difference in the rates of nuclear maturation in oocytes cultured in vitro with or without IGF-I supplementation (47% vs. 45%, respectively). However, IGF-I did induce an increase in the two-cell cleavage rate, from 35% to 51%, when the MII oocytes were induced to develop parthenogenetically. But when the oocytes were used for nuclear transfer, no increase in the two-cell cleavage rate was achieved by adding IGF-I to the culture medium (26% vs. 23%). This is clearly a very interesting physiological phenomenon with regard to the activation mechanisms employed by horse oocytes that requires further investigation.
A report of nuclear transfer in bovine oocytes indicated that as many as 83% of fused donor nuclei showed PCC by 1 h after cell fusion, which resulted in 67% of the oocytes exhibiting two nuclei by 3–6 h and 20% reaching blastocyst stages of development during subsequent in vitro culture [4]. In the present study, our rate of total nuclear remodeling following 12–15 h culture after oocyte activation (combination of the various nuclear stages from NEBD to two nuclei) had increased to 83%–88%, which is much closer to the bovine figures. Furthermore, there was no significant difference between the oocytes matured with or without IGF-I in the medium, although addition of IGF-I did significantly increase the rate of Type I normal nuclear remodeling (from 28% to 45%, P < 0.05), and the number of reconstructed oocytes that continued development to the blastocyst stage also increased from 3% to 11%.
The meiotic cell cycle is controlled by a network of protein kinases and phosphatases. Studies have demonstrated that the MII arrest of in vitro matured mammalian oocytes is maintained by MPF and MAPK [19, 29–31]. MPF is a complex of two subunits, the catalytic subunit P34cdc2 protein kinase (cdc2 or cdk1) and regulatory subunit cyclin B [29], which is believed to be the main regulator of all the major morphological changes for the resumption of meiosis in oocytes, such as chromosome condensation, breakdown of the nuclear envelope, and rearrangement of the microtubules. MPF activation requires new protein synthesis; phosphorylation of cdc2 at T14, Y15, and T161; and subsequent dephosphorylation of cdc2 at T14 and Y15 [29– 31]. Interference in any of those processes will inhibit MPF activation, prevent the resumption of meiosis, and hold the oocyte at the GV stage. Goudet et al. [32] reported the presence of MPF and MAPK in equine oocytes whatever the nuclear stage during oocyte maturation. They also noted that the incompetence of the equine oocyte to resume and complete meiosis is not due to the absence of MPF and MAPK but is probably caused by a deficiency of regulators of MPF and/or an inability to phosphorylate MAPK.
In our study, cdc2 was found in all groups of treated oocytes, and it showed a tendency to increase in quantity by the end of 30 h of culture, although IGF-I supplementation gave no additional response (Fig. 1). We observed cyclin B in horse oocytes at the beginning of culture, which did not increase during 30 subsequent hours of maturation culture. In general, our results suggest that cdc2 does, but cyclin B does not, relate directly to the resumption of meiosis during the maturation of horse oocytes and that IGF-I does not improve cytoplasmic MPF synthesis (cdc2 and cyclin B) in this species.
Two subunits of MAPK, ERK1 and ERK2, became phosphorylated and activated when oocyte maturation was initiated. These enzymes are maintained at a high level of activity throughout the maturation period in many mammalian species [33, 34]. The importance of MAPK activity has also been demonstrated for the resumption of second meiosis in mouse and porcine oocytes [35, 36]. In the present experiment, ERK1 and ERK2 were found in the treated horse oocytes throughout the 30-h period of culture, which is in agreement with the previous finding of Goudet et al. [32]. However, we were also able to show that IGF-I can stimulate more accumulation of both ERK1 and ERK2 in the cytoplasm of the oocyte (Fig. 3), which hints at the value of IGF-I in being able to induce more normal embryonic development in the reconstructed horse oocytes at the time of first cell cycle organization (Table 2 and Fig. 2). Our results also indicated that the high concentration of IGF-I that is normally present in estrous follicular fluid may also improve the rate of blastocyst development (i.e., from 3% to 11%; Table 1) as has already been reported in bovine embryos [28]. The IGF system comprises a highly complex network of growth factors, and further studies are clearly required to determine if other members of the IGF family, such as IGF-II or the IGF binding proteins, might also exert a beneficial effect on oocyte maturation and embryonic development in vitro, as has been indicated in other species [25].
In summary, our experiments demonstrated that IGF-I may improve the cytoplasmic maturation of horse oocytes by inducing an increased accumulation of MAPK. This, in turn, will help stimulate horse oocytes reconstructed by nuclear transfer to complete nuclear remodeling and go on to develop normally as embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: W.R. Allen, TBA Equine Fertility Unit, Mertoun Paddocks, Woodditton Rd., Newmarket, Suffolk CB8 9BH, United Kingdom. FAX: 01638 667207; efu{at}tesco.net
Received: 3 March 2004.
First decision: 31 March 2004.
Accepted: 8 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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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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X. Li, Y Qin, S. Wilsher, and W R Allen Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer. Reproduction, April 1, 2006; 131(4): 661 - 667. [Abstract] [Full Text] [PDF]
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