Hum. Reprod. Advance Access originally published online on June 21, 2006
Human Reproduction 2006 21(9):2228-2237; doi:10.1093/humrep/del184
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Developmental and functional evidence of nuclear immaturity in prepubertal oocytes
1 Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy and 2 Department of Animal Reproduction, University of Agriculture, Krakow, Poland
3 To whom correspondence should be addressed at: Department of Comparative Biomedical Sciences, Piazza A. Moro, 45, Teramo 64100, Italy. E-mail: gptak{at}unite.it
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Abstract |
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BACKGROUND: The nuclear compartment has been proposed as responsible for the developmental arrest of prepubertal mouse oocytes while the studies on prepubertal sheep and cow oocyte model suggested the cytoplasm immaturity accounts for this failure. METHODS: The apparent disagreement on the causes of developmental defects between these two species prompted us to study: (i) follicular and oocyte growth allometry in lambs, (ii) oocyte compartment (nucleus versus cytoplasm) responsible for developmental failure by nucleus exchange between lamb and adult sheep oocytes, (iii) nucleolar features of prepubertal oocytes by ultrastructural observation and (iv) in vivo developmental survey of prepubertally derived embryos. RESULTS: The oocyte growth inside the follicle is asynchronous during prepuberty. The nuclear transfer revealed that the lamb nucleus was responsible for developmental failure. Immature fibrillogranular structure of the nucleolus has been revealed in small lamb oocytes and also in a few adult-size lamb oocytes. Studies in vivo revealed a high occurrence of developmental arrest of prepubertal derived fetuses, which we have attributed to the low genome-wide methylation detected in prepubertal oocytes. CONCLUSIONS: Our studies have indicated incomplete nuclear maturation of prepubertal gamete. The implication of this finding suggests caution when the strategy of rescue of prepubertal oocytes for assisted fertilization is considered such as in the case of therapeutic treatment which precludes the maintenance of fertility of sexually immature patients.
Key words: developmental arrest/methylation/nuclear immaturity/oocyte/prepuberty
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Introduction |
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During the late stages of antral follicle development, the nucleus of the mammalian oocyte undergoes maturational changes that are fundamental for the development of the embryo. In mice, the ability to support development occurs in a stepwise manner as oocyte diameter increases from 65 to 80 µm (Sorensen and Wassarman, 1976
; Eppig and Schroeder, 1989; Eppig et al., 1994). This indicates that developmental changes occurring in the oocyte during the final stages of growth are crucial for full developmental competence. However, it does not explain which compartment of the oocyte, nucleus or cytoplasm, is responsible for developmental progression. Importantly, besides well-confirmed cytoplasm immaturity in oocyte before the attainment of its final size (Schultz and Wassarman, 1977; Wassarman and Josefowicz, 1978), it was recently demonstrated by nucleus transfer experiments that the maternal chromatin is competent to support development to term only when it originates from adult mice oocytes 50–59 µm in diameter. Similar results were obtained when chromatin from bigger oocytes (diameter 60–69 µm) from juvenile mice were used (Bao et al., 2000). Thus, it appears that epigenetic modifications of the maternal genome necessary for full-term development are established during the later oocyte growth phase in the prepubertal ovary.
To date, very little information is available regarding oocyte maturation and growth in non-mouse species. Important genetic and economic advantages of the use of juvenile domestic animals in breeding programmes are ample justification to consider a prepubertal animal model as a potential oocyte donor. Despite the selection of genetic background and optimization of gonadotrophin stimulation of prepubertal females (Ptak et al., 2003), frequent developmental failures of embryos/fetuses derived from prepubertal oocytes persist. The lack of developmental capacity of juvenile animal oocytes was addressed in numerous studies on ruminant species (Revel et al., 1995; Damiani et al., 1996; Ledda et al., 1997; O’Brien et al., 1997; Presicce et al., 1997), and all these reports ascribed the developmental arrest of prepubertally derived embryos to various aspects of oocyte cytoplasm deficiency. Obviously, considering that oocytes collected from prepubertal calves and lambs are usually smaller in diameter (Biensen et al., 1998) compared with those from sexually mature females, it seems to be entirely feasible that the composition of their cytoplasm will vary on a morphological and/or metabolic level from the mature adult female gamete.
The seminal studies of re-establishment of imprints during oogenesis, by Kono et al. (1996), together with the unusual occurrence of late gestation failure following transfer of prepubertal sheep embryos (Ptak et al., 1999), especially its particular resemblance to the phenotype of mouse fetuses, where the epigenetic immaturity of oocytes derived from neonatal females was confirmed, prompted us to study the developmental preparedness of sheep oocyte in early prepuberty, focusing on unravelling nuclear aspects. Our aims were (i) to establish ovarian morphologies and growth allometry between follicle and its oocyte in female lamb, (ii) to identify the oocyte compartment responsible for developmental failure by direct nucleus exchanging between lamb and adult sheep oocytes, (iii) to outline nucleolar and nuclear features in prepubertal oocytes and (iv) to assess the large-scale in vivo development to terms of prepubertally derived embryos.
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Oocyte collection and animal treatment
For all the experiments, oocytes were collected from 4-week-old Sarda breed lambs and adult sheep. The studies involving morphology and morphometry of the ovarian follicles, immunocytochemistry and nuclear transfer were performed on ovaries/oocytes obtained from regularly slaughtered donors.
For the large-scale developmental survey on the in vivo survival of embryos produced prepubertally and as a control, from adult sheep, hormonal stimulation of oocyte donors was carried out to enhance the developmental potential of in vitro produced embryos destined for transfer to synchronized recipient sheep. Two hundred and forty-six lamb donors with their mothers and 75 adult donors were maintained under field conditions. Progesterone treatment was done by the insertion of Norgestomet implants s.c. (Crestar, Intervet, Holland; day 0), and follicular growth was primed with 2.7 and 4.8 mg of ovine FSH (Ovagen, ICP, Auckland, New Zealand), given in six equal doses every 12 h on the 10th, 11th and 12th days of implant insertion, for prepubertal and adult ewes, respectively. Animals were anaesthetized with acepromazine maleate (0.05 mg/kg bodyweight) and pentothal sodium (10 mg/kg bodyweight) on the day after the final injection of hormones. Ovaries were exposed by midventral laparotomy, and follicular oocytes were aspirated. To avoid any adhesions, we extensively washed the reproductive organs with saline after the puncture of follicles and covered the surface of the ovary with hydrocortisone acetate ointment (Cortison Chemicetina; Pharmacia & Upjohn, Milano, Italy) (Ptak et al., 2003). All animal experiments were performed in accordance with DPR 27/1/1992 (Animal Protection Regulations of Italy) and in conformity with European Community regulation 86/609.
Morphometric examination of follicles
Formaldehyde (10%) fixed ovaries were serially sectioned at 8 µm and stained with haematoxylin and eosin. The diameter of all follicles (>500 µm) was measured microscopically, using the mean of two measurements taken at right angles. A mean oocyte diameter (without zona pellucida) was calculated from equatorial sections. Follicles at the advanced stages of atresia (Brand and De Jong, 1973) were ignored. Follicles and oocytes were measured with a computerized image analysis system (AxioVision 3.1; Carl Zeiss, Oberkochen, Germany) connected to Zeiss-Axioscope 2 Plus microscope and television monitor.
In vitro production of embryos
All chemicals, unless otherwise indicated, were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Methods of in vitro embryo production were adapted from those previously described (Ptak et al., 2003). Briefly, oocytes were matured in vitro in bicarbonate-buffered TCM-199 containing 2 mM glutamine, 100 µM cysteamine, 0.3 mM sodium pyruvate, 10% fetal bovine serum, 5 µg/ml FSH (Ovagen), 5 µg/ml LH and 1 µg/ml estradiol (E2). IVM and IVF were carried out in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h.
The IVF medium used was bicarbonate-buffered synthetic oviduct fluid (SOF) enriched with 20% (v:v) heat-inactivated estrus sheep serum, 2.9 mM calcium lactate and 16 µM isoproterenol. Frozen-thawed semen, obtained from rams of proven fertility, was used throughout the study. Fertilization was carried out at a concentration of 1 x 106 sperm/ml for 20 h.
Presumptive zygotes were cultured in SOF enriched with 1% (v:v) basal medium Eagle (56)-essential amino acids, 1% (v:v) minimum essential medium (MEM) non-essential amino acids, 1 mM glutamine and 8 mg/ml fatty acid-free bovine serum albumin (BSA). Cultures were carried out in a humidified atmosphere of 5% CO2, 7% O2 and 88% N2 at 39°C. On day 3 and day 5 of culture (day 0 = day of fertilization), the medium was renewed.
Nucleus exchange between prepubertal and adult sheep oocytes
All manipulations were carried out with a Narishige micromanipulator fitted to a Nikon inverted microscope equipped with a warm stage, as previously described (Loi et al., 2001). Following a complete denudation (by gentle pipetting in HEPES-199-TCM with 300 IU/ml hyaluronidase), in vitro matured oocytes collected from 4-week-old lamb and adult sheep were incubated for 15 min in HEPES-199-TCM plus 4 mg/ml BSA containing 7.5 µg/ml Cytochalasin B and 10 µg/ml Hoechst 33342. Lamb oocytes were divided into two groups: similar in size to their adult counterparts (diameter 120 µm) and small lamb oocytes (Biensen et al., 1998). One lamb oocyte and one sheep oocyte were immobilized with two holding pipettes and the metaphase plate located after a short exposure (=2 s) to UV light. The portion of cytoplasm with the metaphase chromosomes was aspirated with a bevelled pipette and exchanged between two oocytes. The reconstructed oocytes were allowed to recover from Cytochalasin B and fertilized by ICSI. Briefly, an aliquot of semen used for IVF was diluted with a drop of 7% polyvinylpyrrolidone (PVP) (MW: 36 000) in 150 mM KCl and 2 mM PIPES (piperazine-N,N' bis (2-ethanesulfonic acid)), and a single sperm cell was injected into the oocyte. Manipulated oocytes were allowed to recover from ICSI and than placed between two electrode wires of a fusion chamber filled with 0.3 M mannitol plus 4 mg/ml BSA solution. Fusion was accomplished by exposing them to DC pulse of 1.25 kV/cm for 80 µs (Biojet CF 50; Braun Technology, Germany). The absence of Ca++ ions in the fusion medium prevented the activation of the oocytes which was instead induced with a more effective treatment with ionomycin (5 µM) for 5 min followed by cycloheximide (10 µg/ml) for 3–4 h. Reconstructed oocytes were cultured in vitro according to methods described above.
Oocyte processing for transmission electron microscopy
Oocytes were fixed in 2.5% glutaraldehyde (pH 7.2) for 3 h and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer for 60 min. The samples were subsequently dehydrated through an ethanol series, rinsed in propylene oxide and embedded in epoxy resin (Durcupan® ACM Fluka). One-micron sections were stained with toluidine blue and examined at the light microscopic level. Oocytes were ultrathin sectioned (70 nm), and grids were stained with uranyl acetate and lead citrate. Electron micrographs were taken on a Zeiss-EM900 electron microscope. Unless otherwise stated, all chemicals were purchased from Electron Microscopy Sciences (Fort Washington, PA, USA).
Indirect Immunofluorescence
The labelling with anti-methylcytosine antibody (Eurogentec, Seraing, Belgium) was performed as previously described (Dean et al., 2001) with minor modification. Immature oocytes were washed in phosphate-buffered saline (PBS), fixed for 15 min in 2% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After permeabilization, oocytes were treated with 4 N HCl at room temperature for 10 min and subsequently neutralized for 15 min with 100 mM Tris/HCl buffer (pH 8.5). All samples were blocked overnight at 4°C in 1% BSA/0.05% Tween-20 in PBS. The primary antibody was detected by a rabbit anti-mouse secondary antibody coupled with fluorescein isothiocyanate (FITC) (Chemicon, Temecula, CA, USA). The oocytes were stained by Hoechst 33342 (Fluka, Buchs, Switzerland). Then they were inspected under Nikon fluorescent microscope. Control of immunostaining specificity was carried out by omitting primary antibody.
Embryo transfer and follow-up of pregnancies
In vitro produced blastocysts were surgically transferred in pairs to the recipient ewes 7 days after the onset of natural estrus. The pregnancies were confirmed by ultrasonography (7.5 MHz high-resolution linear probe; Aloka, Assago, Italy). Parturition was induced at 146th day of pregnancy by a single injection of E2 benzoate (2 mg) (Estradiolo; AMSA, Milano, Italy) followed by four i.m. injections of betamethasone (Bentelan Glaxo Welcome, Alges, Portugal) at 12-h intervals (1 mg/10 kg bodyweight) as described previously (Ptak et al., 2002).
Statistical and image analysis
The relationship between oocyte diameter and follicular size was estimated by correlation coefficient (r). All data regarding development of embryos were compared by Chi-square test. Fisher’s exact probability test was used when the expected value for any parameter was <5. The methylation status of the chromatin was measured using ImagePro® software (Media Cybernetics, Inc.) based on the intensity of the fluorescent staining for total chromatin and the specific staining for methyl groups. The chromatin areas were compared with corresponding methylation areas, and the percentage of chromatin methylation was calculated. The data analysis was made by PROC GLM of SAS (SAS/STAT User guide; SAS Institute, Inc., Cary, NC, USA).
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Morphology of lamb ovaries and allometry of follicles and oocytes
Regardless of breed and age uniformity, there were substantial differences in the morphology of collected lamb ovaries (Figure 1). The majority of lamb donors (60.3%, 226/375) had ovaries containing numerous follicles (40–130) of similar size, ranging from 0.5 to 3 mm depending on donor (Figure 1B). Another type of ovary consisted of limited number of follicles (=5); alternatively no follicles were macroscopically visible on the surface of the ovary (Figure 1A). Owing to low number and size of follicles, such ovaries were not used in the experiments. This type of ovary was found in 17.3% (65/375) of collected reproductive organs.
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The remaining 22.4% (84/375) of lambs had ovaries with mixed population of follicles, 0.5–3.5 mm, and lower follicle numbers (10–25) (Figure 1C). Lamb and sheep oocytes were collected approximately from 0.5 to 4 mm follicles. The diameter of oocytes recovered from adult sheep ovaries ranged from 98 to 146 µm, whereas the lamb oocytes had variable diameter of 95–137 µm.
To elucidate the relationship between the relative growth of follicles and their oocytes, we dissected a total of 327 follicles. Figure 2(A) demonstrates substantial heterogeneity between follicle and oocyte growth in 4-week-old lamb ovaries (r = 0.11). In all lamb ovaries examined, follicle size and oocyte growth showed no correlation (Figure 2A). In adult sheep, as expected, follicle size and oocyte growth showed a positive correlation (r = 0.61) (Figure 2B).
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Nucleolar activity in prepubertal sheep oocytes
The results obtained by means of transmission electron microscopy (TEM) are presented in Figure 3. In small lamb oocytes, nucleoli appeared to be composed of aggregations of electron-dense granular material embedded in a reticulum and one or two large vacuoles surrounded by smaller ones. The fibrillar centres are located at the periphery of the nucleolus (Figure 3A). This nucleolar morphology is classified as superficial fibrillogranular nucleolus (Fair et al., 1996
). Adult-size lamb oocyte category showed a compact nucleolus (Figure 3B): the nucleolar vacuoles disappeared and nucleolus with diameter 1–2 µm consists of an electron-dense fibrillar sphere with a fibrillar centre attached to it in the form of a halo (dense fibrillar—dF—nucleolus). This nucleolar morphology is present also in all sheep oocytes examined (Figure 3C).
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Development of reconstructed oocytes
Figure 4 presents the results of reciprocal nucleus exchange between adult oocytes and both size groups of lamb oocytes. The efficiency of micromanipulation in terms of fusion was high for all groups of reconstructed oocytes (mean 94%) as well as the rate of activation, which varied between groups from 83 to 100%. When lamb oocytes smaller than those from adult sheep were used for nuclear transfer, the development of oocytes reconstructed with either sheep (n = 43) or lamb nuclei (n = 42) was arrested before blastocyst stage, demonstrating that small lamb oocytes lack both cytoplasmic and nuclear competence. Surprisingly, the developmental progress of oocytes reconstructed from nucleus exchange between similar-sized oocytes from adult sheep (n = 60) and lambs (n = 62) was significantly different for sheep and lamb nuclei derived from embryos (18 versus 0% blastocysts, respectively), indicating that oocytes reaching similar to adult diameter still lack nuclear competence.
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Control groups were represented by exchanging MII chromosomes between two similar-size small lamb oocytes, (Biensen et al., 1998
) (n = 31), between two adult-size lamb oocytes (=120 µm) (n = 34) and between those from adult sheep (n = 44), and the blastocyst rate was 0, 0 and 23% respectively. Furthermore, a proportion of lamb and sheep oocytes were left intact and fertilized by ICSI (small lamb oocytes: n = 39; adult-size lamb oocytes: n = 53; adult sheep oocytes: n = 55); their development to blastocyst was 3, 15 and 31%, respectively.
Methylation level in prepubertal oocyte nucleus
Genome-wide methylation can be reliably evaluated by immunostaining with a 5-methyl cytosine antibody. The incorporation of anti-5-methyl cytosine antibody was observed in all the germinal vesicle stage nuclei of adult sheep oocytes (n = 58) confirming an established genome methylation (Figure 5B). On the contrary, an absence of methylation was registered in more than half of small lamb oocytes (34/62); alternatively, a very low immunofluorescent signal was noted in the rest of them (28/62) (Figure 5F) as well as in all adult-size lamb oocytes (n = 65, Figure 5D). The genome-wide methylation status, expressed as the percentage of methylated versus total DNA, was significantly lower in adult-size lamb oocytes than in those from adult sheep (P < 0.02).
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Survival of embryos derived by IVF of prepubertal sheep oocytes
The development of embryos derived from 4-week-old lambs was evaluated in vitro and in vivo. Prepubertal embryos can develop into blastocysts in vitro, although at lower rates compared with adult-derived oocytes. The distinguished fate of prepubertal embryos is evidenced by their post-implantation development. In fact, the survival rate of embryos derived from prepubertal oocytes was significantly lower (P
0.01) than that of embryos derived from adult oocytes (Table I). The fate of prepubertal embryos is more underlined by their post-implantation development. Here, the pregnancy is severely interrupted between 40 and 60 days of fetus development (P = 0.001), and even those that survive until the first trimester often arrest their developmental programme before full gestation (Figure 6). Interestingly, eight fetuses that arrested their development at around 80–100 days were delivered together with normal lambs at term of the pregnancy (150 days).
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Our study on prepubertal oocyte immaturity has allowed us to investigate the different causes responsible for the developmental failure of embryos derived from prepubertal oocytes. The pattern of follicle distribution in the lamb ovary and the unexpected discordance in follicle and its corresponding oocyte growth are likely contributing factors to further intrinsic causes of immaturity of prepubertal gamete. Granulosa cell activity is required for normal oocyte growth and development (Eppig, 1979
; Brower and Schultz, 1982); therefore, differences in the proliferation index of these cells supporting the oocyte (Hirshfield, 1986) can be responsible for the asynchrony between follicular and oocyte growth during prepuberty. Previously, some heterogeneity between follicle and oocyte growth has been proposed in young gilts (Lucas et al., 2003), suggesting that more synchrony between follicle and oocyte growth is acquired only after puberty. In the adult ovary, it has been demonstrated that the developmental potential of the oocyte is reflected by its corresponding follicle size (Pavlok et al., 1992; Lonergan et al., 1994). Furthermore, the positive correlation between follicle and oocyte growth in the cyclic ovary (Motlik, 1989) established the criteria for selecting the follicles for oocyte retrieval. The demonstration of asynchrony between follicular and oocyte growth in prepubertal ovary implies, however, that follicular size is not a universal indicator of the developmental capacity of the oocyte.
Two events seem necessary to allow the mammalian oocytes to progress through meiosis: the compaction of the nucleolus into inactive electron-dense fibrillar spheres and the cessation of ribosome synthesis (Crozet et al., 1981; Crozet et al., 1986; de Smedt et al., 1994). The down-regulation of the rRNA transcription in oocytes is accompanied by the separation of proteins of rDNA transcription (Roussel et al., 1993) and pre-rDNA processing (fibrillarin) complex. Upon the silencing of rRNA synthesis in fully grown oocytes, fibrillarin is visible as small spot (Roussel et al., 1993; Baran et al., 2004). Nucleolar compaction has been described during oogenesis, although not at the same stages in all species. For several mammals, it has been demonstrated that the nucleolar inactivation occurred in the late stage of oocyte development (Tesarik et al., 1983; Takeuchi et al., 1984; Antoine et al., 1987; Sutovsky et al., 1993; Fair et al., 1996). Our expectation that the prepubertal sheep oocyte (not >110 µm) still displays transcriptional activity was confirmed by ultrastructural inquiries. This is the first report revealing transcription in prepubertal oocyte in sheep; however, similar data were reported before for mice (Chouinard, 1971). In addition, less pronounced differences in the fibrillar structure of prepubertal and adult bovine nucleus were observed (Damiani et al., 1996). The lack of proof of RNA transcription in that previous study can be due to older animals, species difference and hormonal stimulation of oocyte donors.
The lack of information regarding nuclear function of prepubertal oocyte was probably due to the inadequate classical definition of nuclear competence, i.e. its ability to complete meiosis, as prepubertal antral oocytes of ruminant species progress through meiosis at nearly adult rates (Revel et al., 1995; Ledda et al., 1997; O’Brien et al., 1997). Some differences in dynamics of meiosis have been demonstrated in prepubertal cattle oocytes (Khatir et al., 1998). The ability of the oocyte to complete meiosis does not predispose its competence to be fertilized and, furthermore, to complete embryonic development. Our nuclear transfer experiments showed that in growing oocytes, both nuclear and ooplasmic compartments are severely lacking the competence to progress through first stages of preimplantation development. This demonstrates the nuclear immaturity of prepubertal oocytes and, on the contrary, reinforces previous reports about aspects of immaturity of the cytoplasm (Levesque and Sirard, 1994; Damiani et al., 1996).
Although the diameter of oocytes in prepuberty is generally smaller than that of adult oocytes (Gandolfi et al., 1998), we were able to select lamb oocytes with diameter similar to that of adult sheep oocyte for the nucleus exchange between prepubertal and adult sheep oocytes. In this variant, the development of reconstructed oocytes revealed the lack of competence of lamb nuclei, but not the cytoplasm. In contrast, previously reported data suggested that the cytoplasm, but not the nucleus, was responsible for the developmental arrest of prepubertal bovine oocyte (Salamone et al., 2001). Although comparing different species (bovine versus sheep) and different experimental approaches (hormonally stimulated oocyte donors versus no stimulated; parthenogenetic activation versus ICSI) these discrepancies are probably due to the low number of successfully reconstructed oocytes and the lack of statistically relevant differences between the embryo rate (Salamone et al., 2001).
To date, only the cytoplasmic inadequacies have been identified describing the morpho-functional deficiencies of prepubertal oocytes (Damiani et al., 1996; Gandolfi et al., 1998; Salamone et al., 2001; Oropeza et al., 2004). In these reports, the observation of development, if provided, was limited to early embryo stages. Although it is not our intention to contest the cytoplasmic deficiencies in prepubertal oocytes, a remarkably high proportion of fetal loss observed in this and other experiments [bovine (Presicce et al., 1997; Taneja et al., 2000); ovine (O’Brien et al., 1997)] at far advanced developmental stages cannot be attributed exclusively to cytoplasmic immaturity of the oocyte. Furthermore, in studies on the development of lamb-derived embryos, the fetal number during the pregnancy progression, rather than offspring rate, is often reported (Ledda et al., 1997; O’Brien et al., 1997), whereas the occurrence of fetuses which arrested their development around 80–100 days of gestation noted in our previous and present study (an important feature of pregnancies derived from prepubertal oocyte) precludes the correct calculation of offspring, based on an advanced pregnancy diagnosis (Ptak et al., 1999). Our large-scale trial with 628 blastocysts derived from 1-month-old female lambs transferred to synchronized adult sheep recipients (n = 314) resulted in a low proportion of embryos (6%) able to progress to term gestation. Previous experiments on in vitro grown prepubertal mice oocytes have shown that the acquisition of the size similar to adult mice oocyte and even the acquisition of comparable to adult oocyte capability to be fertilized and to cleave did not ensure adequate survival of embryos transferred to synchronized foster mothers, giving only few offspring (5%) from oocytes isolated from 12-day-old mice (Eppig and Schroeder, 1989). Further studies by Eppig et al. (1992) demonstrated that the first embryos capable of full-term development are derived from the mice oocytes just before reaching puberty, whereas those from younger oocyte donors acquire the developmental competence gradually. Our previous study on oocytes collected from various age prepubertal sheep have shown that the embryogenic competence increases with age (Ptak et al., 2003), a finding confirmed in prepubertally derived bovine embryos (Taneja et al., 2000). Mouse embryos containing a maternal genome from a growing oocyte extended their development gradually, and the first embryos capable of full-term development contained maternal genomes from juvenile oocytes, where the primary imprinting was fully established (Bao et al., 2000). Gene expression analysis showed that imprinting signals for each gene is acquired in a specific time window throughout the oocyte growth (Obata and Kono, 2002), and the progressive stage-related increase in the methylation of the imprinted genes was further confirmed by bisulphite-sequencing analysis (Lucifero et al., 2004). A rapid demethylation that occurs in primordial germ cells followed by de novo methylation in the last stages of oocyte growth can be indicative of epigenetic maturity of the oocyte (Surani, 1998). The loss of methylation corresponding to the loss of allele-specific expression of certain imprinted genes by the oocyte [induced by the deletion of oocyte-specific DNA methyltransferase-1 (Dnmt1o)] causes the death of majority of mice fetuses in the last third of gestation and very few live born offspring from homozygous mutant females (Howell et al., 2001). This implied that the establishment of methylation patterns during oogenesis is crucial for later fetal phenotype, without causing developmental abnormalities in earlier stages. Interestingly, similar fetal phenotypes were present in our developmental survey on prepubertal lamb-derived embryos. From the studies in mouse, it appears that maternal imprints are established relatively late in oogenesis and more importantly, methylation imprint establishment is related to oocyte diameter (Lucifero et al., 2004). This last observation may explain why some of prepubertal sheep oocytes are able to complete the developmental programme and healthy offspring can be obtained alongside the majority of embryos that are not able to progress beyond the second half of gestation. A recessive lethal phenotype with no fetus surviving past mid-gestation was also associated with a substantial reduction of the level of 5-methyl cytosine in the DNA in mutant mice embryos (Li et al., 1992). Accordingly, the lack of incorporation of 5-methyl cytosine antibody in lamb oocytes suggests an epigenetically immature phase that may result in the failure to start the developmental programme. Moreover, the late developmental arrest of prepubertally derived fetus further indicates the epigenetic nature of gestational failure, as any obvious morphological abnormalities were noted in those fetuses. A growing amount of evidence suggests a role for genomic imprinting in an aberrant resource and nutrient acquisition through placenta (Constancia et al., 2004), the organ likely responsible for growth arrest of prepubertally derived sheep fetuses.
Epigenetic regulation has been studied extensively in murine and human oocytes but not in sheep oocytes. We have recently demonstrated that the DNA methylation pattern in sheep embryo may be different from the majority of species studied (Beaujean et al., 2004). The lethal phenotype of embryos derived from prepubertal lamb oocyte strongly resembles that from mice with confirmed maternal imprinting disruption (Kono et al., 1996; Obata et al., 1998; Howell et al., 2001). Our group demonstrated that genomic imprinting is conserved in sheep and can be perturbed by embryo technologies (Feil et al., 1998; Young et al., 2003). However, the genome-wide demethylation in prepubertal female gamete demonstrated here suggests that gene regulation that relies on DNA methylation, especially imprinted genes, is not established in prepubertal oocytes. Therefore, the most direct approach for opening this exciting area of investigation would be to study the genomic imprinting in ovine gametogenesis. However, it is not feasible at present because insufficient genomic data are available on imprinted loci in ovine.
Our observations on nuclear immaturity of prepubertal oocytes have challenged the idea of reproductive utilization of germ cells collected before puberty. Oocytes isolated before puberty represent a wasted source of germ plasm as a substantial portion is destined to be lost during the lifetime due to natural follicular atresia. Consequently, it is an attractive prospect to harvest these fated gametes from animal population under threat of extinction or in human circumstances before sexual maturity is attained, particularly in cases when therapeutic treatment precludes fertility maintenance. For these cases, the preservation of gametes or ovarian tissues collected before puberty for their future use in assisted reproduction has been indicated. However, the outcome of this study casts doubt on the virtues of such strategies suggesting that care should be taken in proper selection of oocytes for reproductive purposes due to asymmetry between oocyte and follicle growth before puberty. More importantly, even when the prospect of obtaining a fully grown ooplast is entertained, it will not ensure nuclear maturity of that oocyte. The genome-wide demethylation in oocytes collected from prepubertal female and their developmental feature during pregnancies imply the occurrence of epigenetic failure.
These studies provide a thorough testing of the approach used in animal assisted reproduction. Such testing should precede the introduction of new embryo technologies into the clinic. It is highly required to include similar-to-human animal model to provide the susceptibilities of the condition considered as a new tool in reproductive technology. Our investigation extends the body of knowledge about prepubertal oocyte nucleus immaturity for the first time in a species other than mouse. The classical view of oocyte nuclear competence, i.e. its meiotic progression and even successful preimplantation development, is not an axiom of completed preparation of this compartment, and reproductive applications of prepubertally derived oocytes should be aware of this limitation.
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Special thanks to Dr Wendy Dean (Babraham Institute, Cambridge) and Dr Anna Grazul-Bilska (North Dakota State University) for critical suggestion on the manuscript. We are also indebted to Dr Luisella Bogliolo (University of Sassari), Mrs Federica Lopes and Mrs Margherita Yayoi Turco (University of Teramo) for the preparation of samples for immunocytochemistry and Dr Pawel Borowicz (North Dakota State University) for image analysis. This work was supported by FIRB (RBNE01HPMX), MIUR 2003073943-002, British Council/MIUR/CRUI Programme 2004 and University of Teramo grant 2005–2007.
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Submitted on February 27, 2006; resubmitted on April 27, 2006; accepted on May 3, 2006.
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