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Developmental Potential of Mouse Follicular Epithelial Cells and Cumulus Cells After Nuclear Transfer¹

^a Laboratory of Animal Reproduction, College of Agriculture, and Research Institute for Animal Developmental Biotechnology, ^b Kinki University, Nara 631–8505, Japan ^c Molecular Oncology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630–0101, Japan

	ABSTRACT

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The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 ± 5.9 µm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25°C to 60°C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.

	INTRODUCTION

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During follicular development, soon after oocytes are formed, oocytes become enclosed in a single layer of somatic pregranulosa cells, flattened epithelial cells arising from the rete ovarii or from the coelomic covering of the ovary. Concomitant with the initiation of oocyte growth, the surrounding pregranulosa cells start to proliferate, changing from a flattened to a cuboidal form, and form several layers around the oocytes. Fluid subsequently accumulates in spaces between the somatic cells, and an antrum forms. After proliferation of graafian follicles, the oocytes mature and ovulate. Throughout oogenesis, granulosa (cumulus) cells are coupled to oocytes and to each other by gap junctions. These highly specialized membrane connections mediate the transfer of cytoplasmic factors such as nutrients and maternal proteins from one cell to another. Granulosa cells, mural and cumulus cells, and oocytes form a gap junction-mediated syncytium [1, 2]. Thus, follicular epithelial cells are close relatives to oocytes in their cytoplasmic components. It has also been reported that cumulus cells in the human have telomerase activity [3], which is believed to prevent aging, and that this activity is maintained in stem cells but often disappears in somatic cells [4]. Recently, Wakayama et al. [5] demonstrated that cumulus cells surrounding ovulated oocytes have developmental totipotency after nuclear transfer. Moreover, we showed that enucleated oocytes receiving cumulus cells surrounding oocytes at the germinal vesicle stage, cultured for several passages in vitro, developed to normal calves at a high rate [6]. However, viability of cloned animals was limited in both cases. In the experiment in mice by Wakayama et al. [5], the rate of production of mice was low (2–2.8%), and some fetuses died during pregnancy or after birth. In our bovine experiment [6], only half of the calves survived. These results suggest that reprogramming of donor somatic nuclei after nuclear transfer was insufficient.

In nuclear transfer using differentiated cells in amphibians, pretreatment of donor cells with thermal stimulation or sperm factors before nuclear transfer effectively enhanced reprogramming of donor nuclei [7–9]. It has been unclear whether such pretreatment is effective in mammalian nuclear transfer.

In this study, we examined the developmental potency of mouse follicular epithelial cells cultured for several passages and of cumulus cells after nuclear transfer. Donor cumulus cells were treated with thermal stimulation before nuclear transfer to examine whether the stimulation was effective for reprogramming of donor cells in the mouse.

	MATERIALS AND METHODS

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Donor Cells

Follicular epithelial cells surrounding growing oocytes were obtained from ovaries of adult females of CD-1 strain mice, whose glucose phosphate isomerase (GPI) type, commonly used as a marker to check donor derivation, is GPI-1AA [10]. Cells were cultured in vitro until they attached to the bottom of a culture dish. The follicular stage was type 5 [11], judging by the size of oocytes (diameter, 62.6 ± 5.9 µm; n = 14) and the layers of epithelial cells surrounding the oocytes. Culture medium was modified DMEM (Dulbecco's modified Eagle's medium) for mouse embryonic stem cells, excluding leukemia inhibitory factor [12], supplemented with 10% fetal bovine serum (FBS). Attached and expanded cells were subcultured for several passages and subjected to serum starvation (0.5% FBS) for 2–9 days to synchronize the cell cycle at the G0 phase before nuclear transfer. Cumulus cells were also isolated from ovulated oocytes from the oviducts of matured CD-1 females at 17–19 h after hCG injection. Cells were rinsed with M2 medium [13] after dispersion with hyaluronidase (300 IU) and used as the donor cells in nuclear transfer. Only cells of middle size were selected as donor nuclei.

Thermal Stimulation

In an initial experiment, the effective duration for thermal stimulation was examined; cumulus cells were treated at 45°C or at -5°C for 5, 10, 30, and 60 min before nuclear transfer. Next, the effect of temperatures on the developmental potential of nuclear-transfer oocytes was examined. Cumulus cells were treated at -25, -20, -15, -10, -5, 1, 45, 50, 55, and 60°C for 10 min, or with drastic changes from 55°C to -20°C or from -20°C to 55°C for 10 min before nuclear transfer.

Nuclear Transfer and In Vitro Culture

Metaphase oocytes at the second meiosis were collected from F1 females (C57BL x CBA, GPI-1BB), except in the experiment on thermal stimulation described above in which CD-1 strain mice were used, at 17–19 h after hCG injection and were enucleated for use as recipient cytoplasm. A single donor cell was fused with an enucleated oocyte by inactivated Sendai virus and was activated with electrical stimulation (3 double DC pulses of 50–150 V/mm for 50 µsec at 20-min intervals). After activation, nuclear-transfer oocytes were cultured in vitro with M16 medium. When oocytes from CD-1 strain mice were used as recipient cytoplasm, 100 mM EDTA was used to supplement the culture medium to overcome the "2-cell block." Some oocytes were treated with medium supplemented with 3 µg/ml nocodazole overnight so that the chromosome number of donor nuclei could be checked by the air-dry method [14].

Serial Nuclear Transfer and Embryo Transfer

On the basis of results from previous reports, serial nuclear transfer at the 2-cell stage was performed [15, 16]. Briefly, another 2-cell embryo fertilized in vivo was used as the second recipient cytoplasm. After removal of nuclei from both blastomeres of fertilized 2-cell embryos, the karyoplasts of nuclear-transfer 2-cell embryos were fused, respectively. Reconstituted embryos were cultured in vitro for 3 days, and embryos that had developed into blastocysts were transferred to the oviducts of pregnant or pseudopregnant female mice. When pregnant females were used as recipients, females were mated with pigmented males to distinguish the albino nuclear-transfer fetuses from host fetuses. Pregnant females were killed on Day 19.5 of pregnancy, whereas pseudopregnant females were killed on Day 10.5, for examination of the viability of reconstituted embryos.

GPI Analysis

GPI analysis was used to identify the nuclear-transfer conceptuses according to Eicher and Washburn [17] and Mikami and Onishi [18].

Cell Cycle Analysis

The cell cycle was analyzed by 5-bromo-deoxyuridine (BrdU) incorporation and FACScan (Becton Dickinson, San Jose, CA) flow cytometry.

BrdU Incorporation

To confirm whether cells had proliferated, we used the thymidine analogue BrdU. The incorporation and detection of BrdU were analyzed with a Cell Proliferation Kit (Amersham Pharmacia Biotech, Piscataway, NJ; code RPN 20). BrdU detection was carried out according to the manufacturer's instructions. Briefly, cells were cultured overnight in modified DMEM with BrdU instead of thymidine. At the end of BrdU exposure, cells were washed and fixed. After immunocytochemical detection with reconstituted nuclease/anti-5-bromo-2'-deoxyuridine and peroxidase anti-mouse IgG2a, specimens were detected in diaminobenzidine solution with substrate/intensifier. To clarify the border of cells, 0.5% eosin for 2–3 min and Gill's hematoxylin (no. 1 or 3) for several seconds were used for counterstain. Nuclei progressing to the S-phase stained blue-black.

FACScan Flow Cytometry

To determine the cell cycle stages of donor cells, FACScan flow cytometry was used to measure the cells' DNA content [19]. Briefly, cells were suspended in a solution of 0.1% sodium citrate and 0.1% Triton X-100 containing 50 µg/ml propidium iodide and were treated for more than 10 min at room temperature or overnight at 4°C with 1 µg/ml of RNase. DNA fluorescence was measured with a FACScan flow cytometer, and the percentages of cells within the G0/1, S, and G2/M phase of the cell cycle were analyzed by CELLQuest and ModFit software (both Becton Dickinson).

	RESULTS

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Cell Cycle Analysis

Most follicular epithelial cells appeared to have entered into the G0 phase of the cell cycle after serum starvation for 2–9 days. When these cells were cultured with BrdU-supplemented medium overnight, BrdU incorporation rate was poor (9–23%). FACScan flow cytometry analysis revealed similar results (G0/1 phase, 84%; S-phase, 4%; G2-M phase, 11%). Cumulus cells after ovulation clearly stopped cycling at the G0/1 phase as evidenced by FACScan flow cytometry (G0/1 phase, 76–90%; S-phase, 2–4%; G2-M phase, 8–20%). Although there was a slight possibility of choosing cells whose cell cycle was not G0/1 phase at nuclear transfer, we regarded most cells as being in the "quiescent state."

Chromosome Analysis

In a preliminary experiment, we attempted to count the chromosome number in cumulus cells of ovulated oocytes by culturing them in vitro with modified DMEM for culture of mouse embryonic stem cells supplemented with 10% FBS medium. However, most cells remained quiescent under these conditions. We also failed to stimulate them with medium supplemented with serum from several species such as the horse and sheep and with that from the calf and the newborn calf, or with growth factors such as platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor at various concentrations (5–50, 100–1000, 50–500 µg/ml, respectively [20, 21]. Although a small population of cells progressed in the cycle, there were too few for analysis of the chromosome number. Thus, we evaluated the chromosome composition of the cumulus cells of ovulated oocytes after nuclear transfer. We found that 67% (38 of 57) of oocytes had a diploid chromosome composition. When the chromosome numbers of follicular epithelial cells or of oocytes reconstituted with follicular epithelial cells were analyzed, 59–79% were found to be diploid. Others were aneuploid.

Effect of Thermal Stimulation on Development of Oocytes Constituted with Cumulus Cells

The initial experiment suggested that duration of thermal stimulation did not influence the in vitro developmental rate of nuclear-transfer oocytes. When cumulus cells were treated at 45°C for 5, 10, 30, and 60 min before nuclear transfer, 52% (46 of 89), 69% (62 of 90), 73% (71 of 97), and 37% (41 of 111) of constituted oocytes fused; and 85% (39 of 46), 81% (50 of 62), 80% (57 of 71), and 85% (35 of 41) cleaved, respectively. Although 2% (1 of 46), 3% (2 of 62), 1% (1 of 71), and 0% developed to the 4-cell stage, none developed beyond the 4-cell stage. Similar observations were obtained in another group. When cells were treated at -5°C for the same duration, 50% (46 of 92), 56% (65 of 117), 61% (55 of 90), and 61% (76 of 125) fused; and 59% (27 of 46), 63% (41 of 65), 55% (30 of 55) and 50% (38 of 76) cleaved. Although 15% (7 of 46), 11% (7 of 65), 7% (4 of 55), and 13% (10 of 76) developed to 4-cells, all of them stopped development at this stage. From these results, we chose 10 min for the thermal stimulation of cumulus cells in the subsequent experiments. Table 1 demonstrates the effects of temperature on the in vitro development of constituted oocytes. When cells were exposed to severe temperatures such as -25°C or 60°C, almost all did not fuse with enucleated oocytes (0–1%). In the other groups, fusion and cleavage rates were not enhanced compared with those in a control group. Although a few oocytes developed to 4-cell and 8-cell stages, no blastocysts were obtained. When cells were exposed to a drastic change of temperature, such as from 55 to -20°C or from -20°C to 55°C, no cells fused with oocytes.

In Vitro Development of Constituted Oocytes After Serial Nuclear Transfer

Table 2 shows the in vitro development of nuclear-transfer oocytes whose nuclei were transferred again into enucleated fertilized 2-cell embryos (serial nuclear transfer). The fusion rate at the first nuclear transfer into oocytes was similar among groups (40–50%). The cleavage rate of nuclear-transfer oocytes was lower (P < 0.05) when follicular epithelial cells were used as donors (38 vs. 56–65%). Since inactivated Sendai virus was less effective for the fusion of follicular epithelial cells with enucleated oocytes, most nuclear transplants were fused by electrical stimulation. When follicular epithelial cells were fused at the first electrical pulse, the cleavage rate of reconstituted oocytes was significantly higher (P < 0.05) than for those fused later (30 of 50 [60%] vs. 44 of 120 [37%]). Moreover, reconstituted oocytes that fused later did not progress or were dramatically delayed in their cell cycle after fusion with donor cells. As evidenced by chromosome analysis of the first cell cycle, some oocytes still showed interphase nuclei, indicating that they had not undergone chromosome condensation after fusion with oocytes or that the first cell cycle was quite slow. Further development in such oocytes was not checked. After reconstitution at the 2-cell stage, reconstituted eggs developed into blastocysts in all groups except the 45°C-treated cumulus cells. The proportion of blastocysts that developed from eggs receiving follicular epithelium cells was higher than that for eggs receiving cumulus cells (34% vs. 20%).

In Vivo Development

Table 3 summarizes the in vivo development of blastocysts from nuclear-transfer oocytes after transfer to recipient females. When blastocysts that developed from oocytes constructed with cumulus cells of ovulated oocytes were transferred, no fetus was obtained on either Day 10.5 (0 of 15) or Day 19.5 (0 of 8) regardless of thermal stimulation. Only one small implantation site without a fetus was obtained from ovulated cumulus cells without thermal treatment. By contrast, when blastocysts that developed from oocytes reconstituted with follicular epithelial cells were transferred, 4 living fetuses on Day 10.5 and 1 dead female fetus of full size on Day 19.5 were obtained. Though fetuses on Day 10.5 were somewhat smaller than control fetuses, all appeared healthy. The fetus on Day 19.5 was also visually normal and showed the GPI-1AA isotype, supporting its nuclear donor origin. It was unclear why the fetus died before birth.

	DISCUSSION

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In this study, nuclear-transfer oocytes reconstituted with follicular epithelial cells, which were cultured in vitro for several passages and were in the quiescent state, developed to living fetuses on Day 10.5 and to a full-size female fetus on Day 19.5. In mice, this observation shows the most progress so far with respect to nuclear transfer with somatic cells cultured for several passages before nuclear transfer. Although Wakayama et al. [5] demonstrated the success of nuclear transfer of mouse cumulus cells into healthy cloned mice, the donor cells were not cultured in vitro. In contrast, we failed to obtain fetuses from cumulus cells of ovulated oocytes in this study. There would appear to be several possible explanations. First, the greatest difference between the work of Wakayama et al. [5] and ours appears to be whether donor nuclei were directly injected into recipient cytoplasm or fused with oocytes. In this study, donor cells were fused with oocytes by both inactivated Sendai virus and electrical pulses. Through use of the same nuclear-transfer system as in this study, normal mice had been obtained from the G1 phase of morula and inner cell mass cells [15]. Although in the previous work the donor cells were fused by Sendai virus, it was difficult to fuse cumulus cells with enucleated oocytes by Sendai virus alone, perhaps due to the smaller size of the donor cells. Therefore electrical stimulation was also used to facilitate fusion. This indicates that some oocytes had a chance to undergo activation by electric pulses before the fusion with donor cells. The cleavage rate of constituted oocytes fused at the first electrical pulse was significantly higher than for those fused later (60% vs. 37%). This suggests that the timing of fusion was critical for the later development of constituted oocytes. Some oocytes that fused later retained interphase nuclei and did not show chromosome condensation due to the low level of maturation promoting factor in the oocyte cytoplasm at fusion, because oocytes had been previously activated by electrical pulses. Wakayama et al. [5] also suggested that activation, which was induced by Sr^2- immediately after nucleus injection, led to significantly poor development in vitro.

A second reason may be that unknown changes might have occurred on the cytoplasm of recipient oocytes after fusion with somatic cells. When serial nuclear transfer was not performed, no blastocyst was obtained, and most constituted oocytes stopped at the 2-cell stage. Also, sometimes hardening of the cytoplasm of 2-cell embryos that developed from nuclear-transfer oocytes was observed. This means that cytoplasmic conditions changed, perhaps by incorporation of factors from cytoplasm of the somatic cells. Thirdly, it was demonstrated previously that the difference in mouse genetic background between donor cells and recipient cytoplasm was critically important for later development in androgenetic embryos [22] and pronuclei replacement [23]. In this study, the CD-1 strain was used for donor cells, and F1(C57BLxC3H) mice were used for recipient cytoplasm. Although this combination of mouse strains was appropriate to produce chimeric animals by nuclear transfer at the 2-cell stage [24], it was unclear whether the same combination was also suitable for nuclear transfer of somatic cells with oocytes.

In amphibians, pretreatment of donor cells with reduced temperatures and sperm proteins before nuclear transfer was effective for reprogramming of the donor nucleus [7, 8]. Such pretreatments enhanced the developmental capacity of Rana endodermal nuclei from late gastrula to early neurula or tailbud stages [7]. It was also reported that pretreatment with protamine and poly-arginine improved the developmental potential of nuclear transfers of anterior neural ectoderm and notochordal nuclei of Ambystoma mexicanum [8], although the mechanism was unclear. In Ambystoma mexicanum, spermine was not effective [8], suggesting that nuclei from different cell types react differently. In the current experiment, any thermal stimulation from -25°C to 60°C for 10 min to cumulus cells after ovulation did not improve the development of nuclear-transfer mouse oocytes.

In conclusion, it was clear in this study that at least some mouse follicular cells cultured in vitro for several passages had the ability to develop into normal fetuses after nuclear transfer. This suggests that some follicular cells have retained intact sets of genes for totipotency and that the developmental potency can be displayed by the appropriate nuclear transfer. If a nuclear-transfer system with mouse somatic cells cultured for several passages were established, it would be a powerful tool for much research on biological and medical issues. However, many problems, including low production or survival rate as demonstrated by other many studies [5, 6, 23–25], remain. Some questions will be resolved by clarifying the mechanism of reprogramming systems of somatic nuclei after fusion with oocytes.

	FOOTNOTES

 
¹ This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, and Grant-in-Aid for Encouragement of Young Scientists from The Ministry of Education, Science, Sports and Culture (09760259).

² Correspondence: Yoko Kato, Lab. of Animal Reproduction, College of Agriculture, Kinki University, 3327–204, Nakamachi, Nara 631–8505, Japan. FAX: 81 742 43 1155; yoko{at}nara.kindai.ac.jp

Accepted: May 12, 1999.

Received: July 9, 1998.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kato, Y. Articles by Tsunoda, Y. Search for Related Content PubMed PubMed Citation Articles by Kato, Y. Articles by Tsunoda, Y. Agricola Articles by Kato, Y. Articles by Tsunoda, Y.