| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
Department of BioScience,3 Tokyo University of Agriculture, Tokyo 156-8502, Japan
Bio-oriented Technology Research Advancement Institution (BRAIN),4 Tokyo 105-0001, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, embryo, gamete biology, gene regulation, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nuclear transfer studies have shown that nuclei from nongrowing (ng) oocytes have already been competent to mature into MII stage when transferred into fully grown (fg) germinal vesicle-stage oocytes [4]. However, the resultant oocytes lack developmental competence, and nuclei from oocytes more than 65 µm in diameter first become competent to support term development after fertilization in vitro [5]. To acquire competence to support development, the germ cells need to undergo epigenetic modifications during gametogenesis, by which parental-specific expression of imprinted genes is established [6, 7]. However, this epigenetic modification is a barrier to parthenogenetic development in mammals. Therefore, mouse parthenotes that consist of two sets of only maternal genomes or androgenotes that consist of two sets of only paternal genomes result in embryonic death before 10 days of gestation [8, 9]. However, parthenogenetic embryos (ng/fg PE) that carry two sets of haploid genomes from ng and fg oocytes are able to develop to 13.5 days of gestation [4]. The morphology of the ng/fg PE is normal with developed organs [4, 10], including ovaries, which contain oocytes.
On the other hand, it has been recently reported that oocytes produced by an in vitro organ-culture system commencing with ovaries of 12.5-day-old fetuses contained competent genomes that support normal development after oocyte reconstruction by nuclear transfer and in vitro fertilization [11]. This finding provided us an opportunity to examine whether genomes of oocytes derived from ng/fg PE are able to acquire the epigenetic modification necessary to support normal development in vitro.
In the present study, therefore, we conducted several lines of experiments to clarify the feature of the oocyte genome derived from the ng/fg PE by in vitro organ culture and nuclear transfer (Fig 1). The results reveal that the ovary genomes derived from the ng/fg PE are competent to support normal development after fertilization. DNA methylation analysis showed that the regulation regions of imprinted genes are normally methylated in these genomes, suggesting that the mechanisms required for epigenetic modification in maternal genomes function in the oocytes derived from parthenogenetic embryos.
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All procedures described within were reviewed and approved by the Tokyo University of Agriculture Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Wild-type B6D2F1 (C57BL/6NJcl x DBA/ 2Jcl) hybrid mice were used for all experiments. The eCG (5 IU Serotropin; Teikoku Hormone MFG, Tokyo, Japan) and hCG (5 IU Puberogen; Sankyo Lifetech, Tokyo, Japan) were injected into adult females at 48-h intervals, and then ovulated oocytes at the metaphase of the second meiosis (MII) were collected from the oviducts 14–16 h after the hCG injection. Nongrowing oocytes at the diplotene stage of the first meiosis were collected from the ovaries of 1- to 3-day-old females. These oocytes were washed and placed in M2 medium [12]. Fully grown germinal vesicle (GV)-stage oocytes were collected from ovaries 44–48 h after eCG injection of 5 IU. To prevent germinal vesicle breakdown (GVBD), GV-stage oocytes were collected and manipulated in 
-MEM medium (Gibco) containing 240 µM dibutyrl cAMP (dbcAMP; Sigma) and 5% fetal bovine serum (FBS; Gibco). PGCs were collected from the fetal ovaries at 12.5 days of gestation. The ovaries were treated with 0.25% trypsin (Nacalai Tesque, Kyoto, Japan) for 15 min at 37°C, and the morphology of the isolated cells was observed under an inverted microscope (Diaphot-TMD; Nikon, Tokyo, Japan) so as to select only PGCs.
Oocyte Reconstruction by Nuclear Transfer and Production of ng/fg PE
Nuclear transfer was carried out according to our previous reports [4, 10]. Oocytes containing genomes from ng oocytes and another genomes from ovulated MII oocytes were constructed by serial nuclear transfer. Briefly, fusion of ng oocytes with enucleated GV-stage oocytes was induced with inactivated Sendai virus (HVJ; 2700 hemagglutinating activity units/ml). The reconstructed oocytes were cultured for 14 h in -MEM medium containing 5% FBS after the fusion. The ng/fg oocytes were reconstructed by transplantation of a set of MII chromosomes into ovulated MII oocytes and were artificially activated with 10 mM SrCl2 in Ca2+-free M16 medium for 2 h [13]. The resultant btastocysts were transferred to the uterine horns of CD-1 females at 2.5 days of pseudopregnancy. The parthenogenetic fetuses at Day 12.5 of gestation were recovered, from which ovaries containing PGCs were removed and used in organ culture to derive in vitro-grown oocytes.
Nuclei of in vitro-grown oocytes, which were more than 50 µm in diameter and derived from ng/fg PE at 12.5 days of gestation, were fused to enucleated GV-stage oocytes by a first nuclear transfer instead of to the above-mentioned ng oocytes. After the reconstructed oocytes were cultured and the set of chromosomes was transplanted into enucleated ovulated MII oocytes by a second nuclear transfer, the resultant oocytes were used for in vitro fertilization.
In Vitro Fertilization, In Vitro Culture, and Embryo Transfer
Sperm were collected from fertile B6D2F1 males and capacitated for 1 h in T6 medium containing 3 mg/ml BSA at a concentration of 1 x 106–107 sperm/ml. The reconstructed oocytes were incubated with capacitated sperm from B6D2F1 males in IVF medium containing 3 mg/ml BSA [14]. Reconstructed oocytes after fertilization were cultured for 4 days in a drop of M16 medium [15] in 5% CO2, 5% O2, and 90% N2 at 37°C. Blastocysts developed from the reconstructed oocytes were transferred to the uterine horns of CD-1 females at 2.5 days of pseudopregnancy. To assess postimplantation development, the embryos containing genomes of in vitro-grown oocytes derived from ng/fg PE were dissected at 9.5 and 19.5 days of gestation.
Organ Culture
Organ culture was carried out according to the methods of Obata et al. [11]. Ovaries attached to mesonephroi were dissected from ng/fg PE at 12.5 days of gestation. The fetal ovaries were cultured in -MEM medium containing 10% FBS and 0.01 IU/ml follicle-stimulating hormone (FSH; Sigma) on Coaster Transwell-COL membrane (Corning) in 5% CO2 and 95% air at 37°C. The mesonephroi were removed from the ovaries at 7 days of culture. Secondary follicles were mechanically isolated from the ovaries at 17 days and cultured in -MEM containing 5% FBS, 0.1 IU/ ml FSH, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 µg/ml selenium (Sigma) on Coaster Transwell-COL membrane. The oocytes were isolated from the follicles at 28 days, and their diameters were measured using an ocular micrometer (U-OSM; Olympus, Tokyo, Japan). They were then used for DNA methylation analysis of the imprinted genes and also used for nuclear transfer experiments, as noted before. Similarly, ovaries at 12.5 days of gestation from B6D2F1 mice were cultured, and the oocytes were collected as a control.
DNA Methylation Analysis
Methylation of imprinted genes: insulin-like growth factor 2 receptor (Igf2r) [16], long QT intronic transcript 1 (Lit1) [17, 18], and H19 (H19) [19] were analyzed on 30CpG, 17CpG, and 20CpG sites in differentially methylated regions (DMRs), respectively. The bisulfite genomic-sequencing technique using the CpGenome DNA Modification Kit (Intergen) according to previously described methods [20] was performed on three samples: 1) PGCs collected from the ovaries of ng/fg PE and from controls at 12.5 days of gestation; 2) in vitro-grown oocytes, which were more than 50 µm in diameter and derived from ng/fg PE and from controls at 12.5 days of gestation; and 3) 9.5-day-old fetuses developed from reconstructed oocytes derived from ng/fg PE and from controls. PCR products amplified by PCR were cloned using the TA Cloning kit (pGEM-T vectorTA; Promega). Primers were generated to match the DMRs of imprinted genes. The primers used were Igf2r, 5'-TAGAGGATTTTAGTATAATTTTAA-3' and 5'-CACTTTTAAACTTACCTCTCTTAC-3' for first Nested PCR; 5'- GAGGTTAAGGGTGAAAAGTTGTAT-3' and 5'-CACTTTTAAACTTACCTCTCTTAC-3' for second Nested PCR; Lit1, 5'-TAAGGTGAGTGGTTTAGGAT-3' and 5'-AATCCCCCACACCTAAATTC-3' for first Nested PCR; 5'- TAAGGTGAGTGGTTTAGGAT-3' and 5'-CCACTATAAACCCACACATA-3' for second Nested PCR; H19, 5'-TTTGGGTAGTTTTTTTAGTT-3' and 5'-AACCCCAACCTCTACTTTTA-3' for first Nested PCR; 5'-TCCTAATCTCTAATCTCAAC-3' and 5'-AACCCCAACCTCTACTTTTA-3' for second Nested PCR. Clones were isolated using the Flexi Prep Kit (Amersham Pharmacia Biotech) and sequenced by using the Applied Biosystems sequencing system (ABI PRISM 3100).
Statistical Analysis
For comparison of oocyte size, a t-test was used. Frequencies of development to various stages of embryogenesis were compared with 
2 analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Reconstructed ng/fg oocytes produced by nuclear transfer developed into live fetuses at 12.5 days of gestation at a rate of 12.2% (live fetuses/embryos transferred to recipient females: 52/426). The weight of the fetuses (70.7 ± 3.13 mg, n = 12) was about 30% lighter than that of B6D2F1 as a control (100.9 ± 2.96 mg, n = 14); however, the morphology of the fetuses was normal (Fig. 2, A and B).
|
Ovaries with mesonephroi from ng/fg PE were introduced into the culture medium (Fig. 2, C and D) and grew as well as those from the controls with the days of culture. After the removal of mesonephroi on Day 7, the ovaries were attached to the collagen-coated membrane, and they then started folliculogenesis. Many secondary follicles were formed in the ovaries by Day 17 of the culture (Fig. 2, E and F). These follicles, which were mechanically isolated from the ovaries, grew to vesicular follicles with follicular antrums in the following culture (Fig. 2, G and H). At the end of the culture, Day 28, the oocytes reached 54.7 ± 0.43 µm (37.5–74.5 µm, n = 257) in diameter, and no differences were found in comparison with the controls (Fig. 2, I and J). The growth process of the ovaries during the culture and the morphology of the oocytes obtained were not different from those of the controls, 53.2 ± 0.27 µm (38.5–69.5 µm, n = 384) in diameter. The size distribution of the oocytes did not differ from that of the controls (Fig. 3). The results demonstrated that oocytes derived from ng/fg PE can proceed with normal folliculogenesis in vitro.
|
Development of Reconstructed Oocytes after Fertilization In Vitro
To investigate the features of the genomes of the nuclei of in vitro-produced oocytes, we reconstructed the oocytes, which contain a haploid set of genomes that originated from ng/fg PE oocytes and then attempted to produce live pups. A total of 44 ovaries from ng/fg PE at 12.5 days of gestation were cultured in vitro. Nuclear transfer was performed using 234 oocytes that were more than 50 µm in diameter after a 28-day culture period. The reconstructed oocytes were matured and reached the MII stage at a high frequency (223/234, 95.3%) (Table 1). After the second nuclear transfer into in vivo-matured oocytes and in vitro fertilization (IVF), 88.3% of fertilized oocytes developed to the blastocyst stage in vitro (Table 1; Fig. 4). To determine the level of postimplantation development, autopsies were carried out at 9.5 and 19.5 days of gestation. The results showed that 4 of 21 embryos (19.0%) were developed to 9.5 days of gestation, one of which (4.8%), with 28 somites, was morphologically normal. The others, however, with 9, 20, and 25 somites, were behind in development and morphologically abnormal. The abnormalities included congestion in the extraembryonic tissue, the neural tube remaining open in the 9-somite-embryo, bleeding in the yolk sac, and congestion in the body cavity in the 20- and 25-somite embryos. To produce live pups, we transferred 79 blastocysts into 11 recipients and obtained two normal pups (2.5%) (Table 1; Fig. 4). This transfer revealed that oocyte genomes derived from ng/fg PE received appropriate epigenetic modification during in vitro growth. Of the two live male pups, one pup grew up to be a normal adult with reproductive ability (Fig. 4), but the other was rejected by a foster mother.
|
|
Methylation Status of Imprinted Genes
Evaluation of epigenetic modification imposed during oocyte growth in vitro provides further information for the competence. Therefore, we analyzed the DNA methylation status in the DMRs of maternally expressed imprinted genes, Igf2r, Lit1, and H19. The Igf2r and Lit1 genes were methylated at the CpG sites of the DMRs during oogenesis, while the H19 gene was methylated during spermatogenesis. First, DNA methylation status was analyzed in PGCs collected from the ovaries of ng/fg PE at 12.5 days of gestation before the culture. PGCs from both the ng/fg PE and the controls were almost completely unmethylated at all of the CpG sites for analyzed imprinted genes (Fig. 5). This finding confirmed the erasure of genomic imprinting markers, which had been imposed in the parental generation, in PGCs from ng/fg PE at 12.5 days of gestation.
|
In the oocytes collected from the antral follicles at the end of the in vitro culture, the H19 gene was completely unmethylated at all CpG sites, the same as the methylation status in the in vitro-produced control GV and in vivo MII oocytes (Fig. 6). The analysis of the Igf2r and Lit1 genes brought slightly different results from the controls, namely, the presence of many DNA clones with low methylation status (Fig. 5). In the in vitro-grown oocytes derived from ng/fg PE, the percentage of DNA clones with methylated sites at more than 70% of all CpG sites was 33% (4/12) in the Igf2r gene and 54% (7/13) in the Lit1 gene. However, this figure shows 67% (12/18) in the Igf2r gene and 92% (11/12) in the Lit1 gene in the in vitro-grown control oocytes, and the Igf2r and the Lit1 genes were almost completely methylated at all CpG sites in the in vivo MII oocytes. These findings indicated that there were fewer DNA clones with high methylation status in in vitro-grown oocytes derived from ng/fg PE, which were oocytes that underwent genomic imprinting, than in in vivo controls. Thus, it was suggested that genomic imprinting memories were erased in PGCs from ovaries of ng/fg PE at 12.5 days of gestation and then was reestablished in some oocytes during oogenesis by in vitro culture.
|
Three of four fetuses at 9.5 days of gestation obtained from reconstructed oocytes containing genomes originated from ng/fg PE were morphologically abnormal. To determine the reason for their abnormal development, we then analyzed the DNA methylation status in the DMRs of Igf2r, Lit1, and H19 genes in the 28-somite embryo that was morphologically normal and in the 9- and 25-somite embryos that were morphologically abnormal. In the 28-somite embryo, DNA clones with high and low methylation status, which were probably derived from oocytes and sperm, respectively, were found in a nearly one-to-one ratio for all of the analyzed genes, the same as in the controls (Fig. 7). In 9- and 25-somite embryos, the DNA clones for Igf2r and H19 showed almost normal methylation status; however, DNA clones with methylated CpG sites for Lit1 were detected in low amounts (Fig. 6), suggesting that DMRs of Lit1 remained unmethylated in genomes of in vitro-grown oocytes derived from ng/fg PE. These results suggested that the disrupted methylation status of imprinted genes established in the oocyte genomes was maintained in the embryos and was correlated with the developmental delays and abnormalities of the embryos.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Generally, in vitro-grown oocytes have rarely developed to live pups when directly applied in IVF without oocyte reconstruction by nuclear transfer [11, 26]. Recently, however, O'Brien et al. have dramatically improved the developmental competence of oocytes from primordial follicles of newborn mice using a revised protocol for in vitro organ culture [27]. This suggests that oocytes originated from parthenogenetic embryos would normally grow up with superior developmental competence as a result of an improved oocyte culture system.
The incomplete methylation status of in vitro-produced parthenogenetic oocytes may be responsible for developmental abnormalities in some of the 9.5-day-old fetuses, which were developed from the reconstructed oocytes containing genomes derived from ng/fg PE. For example, the methylation of the DMRs of the Lit1 gene was hypomethylated and disrupted in these fetuses with developmental deficiencies. The imprinted gene, Lit1, is located in an imprinting control region in the human KvLQT1 locus, and it has been reported that disruption of the imprinting of the Lit1 gene is associated with Beckwith-Wiedemann syndrome [17, 18, 28].
Although it has been reported that the cells of parthenogenetic embryos differentiate into the germ line in chimeras, the orientation by cells from fertilized embryos is necessary [29–31]. The present results, however, reveal that germ line cells of ng/fg PE possess a latent competence for contributing as female genomes on ontogeny, suggesting that maternal epigenetic modification during oogenesis does not require paternal contribution. Further, the possibility that ng/fg PE can be produced in other species suggests that this novel procedure contributes to animal breeding.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Tomohiro Kono, Department of BioScience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. FAX: 81 3 5477 2543; tomohiro{at}nodai.ac.jp
Received: 15 April 2004.
First decision: 7 May 2004.
Accepted: 9 June 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. Shen, L. Li, Z. Bai, Q. Pan, M. Ding, and H. Deng In vitro development of mouse fetal germ cells into mature oocytes Reproduction, August 1, 2007; 134(2): 223 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Shen, D. Zhang, T. Qing, J. Cheng, Z. Bai, Y. Shi, M. Ding, and H. Deng Live Offspring Produced by Mouse Oocytes Derived from Premeiotic Fetal Germ Cells Biol Reprod, October 1, 2006; 75(4): 615 - 623. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
