Hum. Reprod. Advance Access originally published online on November 13, 2007
Human Reproduction 2008 23(1):54-61; doi:10.1093/humrep/dem334
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Mature oocytes derived from purified mouse fetal germ cells
1 Laboratory of Stem Cell and Regeneration Biology, College of Life Sciences, Peking University, Beijing 100871, People's Rebublic of China 2 Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking University, The University Town, Shenzhen, People's Rebublic of China
3 Correspondence address: Laboratory of Stem Cell and Regeneration Biology, College of Life Sciences, Peking University, Beijing 100871, People's Rebublic of China. Tel: +86-10-6275-6474; Fax: +86-10-6275-6954; E-mail: hongkui_deng{at}pku.edu.cn
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Abstract |
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BACKGROUND: Mouse fetal germ cells have been successfully purified from fetal gonads. However, there are no published reports describing a procedure for deriving mature oocytes from isolated fetal germ cells. The purpose of this present study is to explore whether purified fetal germ cells are able to differentiate into mature oocytes through an in vivo grafting procedure.
METHODS AND RESULTS: First, intact 11.5 and 12.5 days post-coitum (dpc) female gonads with or without the attached mesonephros and the reaggregated female gonad cells were transplanted into the recipient mice. The results demonstrate both the gonad accompanied by mesonephroi and the innate gonad structure are not absolutely required for 11.5 dpc and 12.5 dpc oogonia to generate mature oocytes. Next, oogonia were purified from female gonads, aggregated with different ovarian somatic cells and transplanted into the recipient mice. Purified 12.5 dpc oogonia were able to generate mature oocytes by aggregating with 12.5 dpc ovarian somatic cells, but not with 16.5 dpc or 0 days postpartum ovarian somatic cells. We also tested 12.5 dpc male germ cells but they were unable to undergo oogenesis.
CONCLUSIONS: Our study demonstrates that mature oocytes can be derived from purified fetal germ cells through an aggregation and transplantation procedure. It also suggests that the synchronized interactions between oogonia and gonadal somatic cells are important to ensure normal folliculogenesis.
Key words: fetal germ cell/mature oocyte/transplantation/folliculogenesis/oogonia
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Introduction |
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The precursors of gametes, primordial germ cells, are first distinguishable at the base of the allantois in gastrulating mouse embryos at 7.25 days post-coitum (dpc) (Ginsburg et al., 1990
; Tam and Zhou, 1996). After colonizing the female gonads, germ cells are referred to as oogonia. They begin to enter meiosis at 13.5 dpc (Hilscher et al., 1974; McLaren, 1994, 2003). The process of oogenesis involves the onset of the first meiotic prophase (transformation of oogonia to oocytes), formation of the primordial follicles (enclosure of the oocytes by pre-granulosa cells), progress from the primordial follicles to the antral follicles and eventually the generation of mature oocytes (fulfilment of first meiosis). Studies that closely examine the developmental process from oogonia have been hindered by the lack of an established in vitro system that can faithfully mimic the development process in vivo.
Several groups have successfully isolated germ cells from fetal gonads using various strategies (Pesce and De Felici, 1995; Yoshimizu et al., 1999). Isolated fetal germ cells are able to generate oogonia and enter the prophase of the first meiosis by addition of growth factors or by co-culturing with S1/S14 m220 feeder cells or fetal lung cells (McLaren and Southee, 1997; Richards et al., 1999; Chuma and Nakatsuji 2001; Maatouk Resnick, 2003; Farini et al., 2005). In recent years, mouse embryonic stem cells have also been demonstrated to be able to differentiate into oocytes in vitro (Huber et al., 2003). However, there are no published reports describing a procedure for deriving mature oocytes from in vivo isolated fetal germ cells. Recently, we reported that intact 12.5 dpc or 16.5 dpc female gonads were able to generate mature oocytes after transplantation of the gonads into the renal capsule of recipient mice or by using a combination of in vivo transplantation and in vitro culture (Shen et al., 2006a, b). Our results revealed that the entire process of oogenesis from the fetal female gonad to the mature oocyte could be carried out under the kidney capsule of recipient mice. However, whether or not purified fetal germ cells are able to generate mature oocytes through an in vivo grafting procedure has not been explored before.
In this study, we successfully differentiated purified 12.5 dpc oogonia into mature oocytes by aggregating them with fetal ovarian somatic cells and transplanting the aggregates into mice. The oocytes generated by our procedure were 
70 µm in diameter and were able to exclude the first polar body after in vitro maturation. To explore the possibility that ovarian somatic cells at different developmental stages are able to support oogonia development into oocytes, we used 12.5 dpc, 16.5 dpc, and 0 days postpartum (dpp) ovarian somatic cells during the differentiation procedure. We found that only 12.5 dpc somatic cells were able to support 12.5 dpc oogonia to differentiate into mature oocytes. We also used 12.5 dpc male germ cells during the differentiation procedure, but they were not able to undergo oogenesis.
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Materials and Methods |
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Mouse strains and mating procedures
All of the mice used were housed and bred in the Central Animal Facility at Peking University according to Chinese national standards and were maintained under controlled lighting conditions (12L:12D). Timed mating was produced by housing female mice with males overnight and then checking for vaginal plugs the next morning (0.5 dpc = noon of the day when a vaginal plug was found). The CAG/EGFP (chicken beta-actin promoter /enhanced green fluorescence proteins) transgenic mice line (Stock TgN(GFPU)5Nagy, ICR strain, Hadjantonakis et al., 1998
) was used to obtain cells carrying a ubiquitously expressed EGFP. Another transgenic mice line Oct4/EGFP [TgN(GOFGFP)11Imeg, C57BL/6J strain, RIKEN Bioresource Center, Japan, BRC No. 00 771], in which EGFP was driven by the 18kb Oct4 promoter, was used to harvest germ cells by sorting EGFP positive cells from the gonads using a fluorescence-activated cell sorter (FACS).
Transplantation of intact gonads and reaggregated gonads
Urogenital ridges were isolated from 11.5 dpc or 12.5 dpc embryos (ICR strains). The sex of 11.5 dpc embryos was determined using the Ube1 PCR method (Chuma and Nakatsuji, 2001), and 12.5 dpc embryos were sexed by their appearance. Gonads to be transplanted without the mesonephric region were isolated by cutting away the mesonephros with a fine needle. Two gonads from one female embryo were surgically implanted beneath the left renal capsules of bilaterally ovariectomized host female ICR mice (8–12 weeks old). There were four groups of transplanted mice as follows: group 1, 11.5 dpc gonad with mesonephroi, 18 mice transplanted; Group 2, 11.5 dpc gonad without mesonephroi, 16 mice transplanted; Group 3, 12.5 dpc gonad with mesonephroi, 20 mice transplanted and Group 4, 12.5 dpc gonad without mesonephroi, 20 mice transplanted.
To transplant reaggregated gonads, the gonads were first dispersed as single cells, then aggregated and transplanted. Briefly, pairs of 12.5 dpc gonads without mesonephros were dispersed with 0.25% trypsin (Gibco-BRL), 0.2% collagenase IV (Gibco-BRL) plus 0.02% DNase-I (Sigma Chemical Corp., St Louis, MO, USA) and maintained at 37°C for 15 min. Dissociation into a single-cell suspension was aided by repeated pipetting and any small aggregates were discarded. After adding culture medium M199 (Gibco-BRL) plus 10% fetal bovine serum (FBS), the cell suspension were resuspended in 100 µl prewarmed culture medium. Then phytohemagglutinin-P (Sigma) was added to a final concentration of 35 µg/ml and the mixture of cells was incubated at 37°C for 10 min. Samples were then centrifuged at 9000 g for 10 s and centrifuged again for 30 s after 180° rotation. Pellets of reaggregated cells were gently removed from the tubes and ‘organ’ cultured overnight with M199 medium plus 10% FBS. One aggregate, derived from eight female gonads, was implanted beneath the renal capsule of one bilaterally oophorectomized mouse (29 mice transplanted). In addition, female 12.5 dpc gonadal cells from 3 to 6 gonads of CAG/EGFP transgenic mice and 3–6 gonads of wild type mice were mixed, cultured overnight and implanted to one recipient mouse (eight mice transplanted).
Purification of germ cells, isolation of ovarian somatic cells and transplantation of the aggregates
Isolation of fetal germ cells from CAG/EGFP transgenic mouse embryos was performed according to the magnetic cell separation (MACS) protocol developed by Pesce and De Felici (1995) using the SSEA-1 marker. Briefly, pairs of gonads of 12.5 dpc were dispersed as described earlier. Then 2 µl of SSEA1 antibody [mouse immunoglobulin (Ig)M, Chemicon, Temecula, CA, USA, 1:100, a final concentration of 10 µg/µl] was added in 200 µl of DNaseI/phosphate-buffered saline (PBS) buffer. The cells were mixed well and were incubated on ice with shaking for 30 min. After washed with DNaseI/PBS twice, the cells were resuspended in 180 µl ice-cold DNaseI/PBS with 20 µl of rat antimouse IgM MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated on ice for another 30 min with shaking. Purification with the MiniMacs Separation Column (Miltenyi Biotec) was according to the manufacturer's instructions.
Germ cells from Oct4/EGFP transgenic mouse embryos were isolated by using FACS to collect EGFP-positive cells. The gonadal cell suspension was prepared as described earlier, then the cells were placed in a MoFlo High-Performance Cell Sorter (Dako Cytomation, Glostrup, Denmark) using Summit 4.0 Software (Dako Cytomation) for sorting.
Somatic cells from fetal or newborn mouse ovaries were isolated according to the method originally described by O and Baker (1978). This method takes advantage of the differences between germ cells and somatic cells in their ability to adhere to a culture dish after dissociation into single cells. Somatic cells adhere tightly to a culture dish whereas germ cells do not (Steinberger and Steinberger, 1966). Briefly, 12.5 dpc, 16.5 dpc or 0dpp ovaries were dispersed as described earlier and then cultured in tissue culture dishes with M199 plus 10% FBS. After culturing overnight, the dishes were washed, the cells were dispersed with trypsin and transferred to new dishes for an additional 6 h of culturing.
For various combinations of germ cells and somatic cells, one aggregate was composed of 104 CAG/EGFP oogonia derived from 20 gonads and 105 somatic cells of female gonads from wild-type mice at 12.5 dpc, 16.5 dpc or 0dpp. In addition, 104 male germ cells at 12.5 dpc were aggregated with 105 12.5 dpc ovarian somatic cells. Then aggregates were surgically implanted beneath the left renal capsules of bilaterally ovariectomized host female mice.
Histology
Grafts were removed on day 14, 20 or 28 following transplantation. They were fixed (Bousin solution), paraffin embedded, serially sectioned (7 µm), aligned in order on glass microscope slides and stained with hematoxylin and eosin. Images were captured with an Olympus phase contrast microscope (IX-71; Olympus, Tokyo, Japan).
Examination of estrous cycles of host mice by vaginal cytology
After transplantation for 28 days, the host mice were examined during the estrous cycle. These mice were monitored daily for 3 weeks. The vaginal wall of each recipient mouse was scraped gently and the cells were mixed with a drop of PBS on a clean glass slide. After fixation and staining with eosin, the slides were observed with an inverted microscope and the stage of the estrous cycle was determined from the different cell types observed. Finally, the grafts were dissected and used for histological processing as described earlier.
Immunocytochemistry analysis
Purified germ cells were fixed in 4% paraformaldehyde, blocked with 10% normal goat serum plus 0.2% Triton X-100 for 60 min at room temperature and then incubated with primary antibody to Oct4 (rabbit polyclonal IgG, Abcam, Cambridge, UK) or Mvh (rabbit polyclonal IgG, a kind gift from Dr Toshiaki Noce) overnight at 4°C. Further, incubation with antirabbit tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG (Santa Cruz Inc., Santa Cruz, CA, USA) was performed for 45 min at room temperature. Cells with only secondary antibody staining were used as negative controls. Nuclei were detected by 4',6-Diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) staining. Images were captured with an Olympus phase contrast microscope.
In vitro maturation of oocytes
Cumulus–oocyte - cell complexes (COCs) and nude oocytes were isolated from 28-day grafts or 21-day-old mice by puncturing the large antral follicles with a fine needle. The 21-day-old mice and host mice are not stimulated with gonadotropins. The diameters of oocytes were measured excluding the thickness of zona pellucida. Germinal vesicle (GV)-stage oocytes were matured for 16–18 h in minimum essential medium, 
medium (Gibco-BRL), supplemented with 0.23 mM sodium pyruvate, 5% FBS, 1 ng/ml epidemic growth factor (EGF, Sigma), and 0.1 IU/ml FSH (Sigma). GV breakdown (GVBD) oocytes were identified as when the GV had broken down, and metaphase II (M II) oocytes were identified as when the first polar body had been extruded. Some M II oocytes from unstimulated 21-day-old mice were found because of random activation and they were not used for the final calculation.
Statistics
The oocyte diameters, the percentage of GVBD or M II per total number of oocytes in each graft was considered as one replicate (n = 1). Values from at least three different grafts (n > 3) were used to obtain the mean ± SD. The percentages were compared between transplantation groups and control 21-day-old mice group by chi-square analysis using data pooled from all experiments. Oocyte diameters were compared between transplantation groups and control 21-day-old mice group using Student's t-test. When P 
0.05, the difference was considered as significant.
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Results |
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Ectopic differentiation of intact 11.5 dpc and 12.5 dpc female gonads with or without the accompanying mesonephros
To explore whether the accompanying mesonephroi is necessary for the gonad development, we transplanted 11.5 dpc and 12.5 dpc female gonads with or without their accompanying mesonephros into the renal capsule of bilaterally oophorectomized mice for 28 days.
Fourteen, 13, 15 and 14 grafts for Groups 1–4, respectively, were recovered after transplantation, showing high rates of recovery (77.8%, 81.3%, 75.0% and 70.0% for Group 1–4). Histological examination of the recovered grafts (four randomly selected grafts for each group) at day 20–25 revealed the presence of follicles at the primary stage as well as the second stage (Fig. 1). There was no significant difference in follicle generation observed among the four groups. Grafts from the groups with mesonephroi also generated fat, vesicles and other connective tissues (Fig. 1).
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We next isolated COCs and nude oocytes by mechanical dissection of the large antral follicles from the day 28 grafts (6, 5, 7 and 6 grafts were used for Group 1–4, respectively). These oocytes were used to calculate the diameters of oocytes and were cultured in a maturation medium. Oocytes recovered from Group 1 reached a diameter of 72.5 ± 1.9 µm (n = 131), 74.7 ± 2.5 µm (n = 126) from Group 2, 73.7 ± 1.8 µm (n = 173) from Group 3 and 74.6 ± 2.1 µm (n = 148) from Group 4. The diameters of the oocytes from the four groups were similar to those dissected from the control group of oocytes from 21-day-old mice (74.8 ± 2.0 µm, n = 80) (Figs. 1 and 2A). After maturation of the oocytes induced by FSH stimulation, the percentages of oocytes that underwent GVBD were 81.5%, 82.3%, 80.7% and 82.4% for Groups 1–4, respectively. Among these GVBD oocytes, 66.1%, 65.4%, 67.1% and 68.0% of them produced polar bodies for Groups 1–4, respectively. The ratio of GVBD and M II stage oocytes were also similar to oocytes dissected from the control 21-day-old mice (83.0% and 69.4%) (Figs. 1 and 2B–C).
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To explore whether the transplants are able to produce enough hormones to restore normal reproductive function of recipient mice, we examined the estrous cycles of recipient mice. Among the examined host mice, almost all of the mice where the transplants develop normally restored the estrous cycles of the host mice. These results indicated that both the germ cells and somatic cells within the 11.5 dpc or 12.5 dpc female gonads could develop normally after transplantation, independent of the accompanying mesonephroi.
Ectopic differentiation of reaggregated 12.5 dpc female gonads
To explore whether the innate structure of female gonads is necessary for the contained oogonia to generate mature oocytes, we first separated germ cells and somatic cells from each other and then allowed these cells to reaggregate before transplanting them into mice.
Grafts (n = 17) were recovered 14 days or 28 days after transplantation (58.6% recovery). Histological examination of the recovered grafts (n = 3 for 14-day grafts, n = 3 for 28-day grafts) revealed that the primary follicles formed after 14 days of transplantation and the antral follicles formed after 28 days of transplantation (Fig. 3A–B). Fully grown oocytes were also dissected from large antral follicles of grafts after 28 days and the diameter was 72.3 ± 3.5 µm (n = 48). After in vitro maturation using FSH, 48.6% of the oocytes proceeded to GVBD. We also observed the presence of oocytes that have excluded the first polar bodies (Fig. 3C).
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To ensure that germ cells and somatic cells were fully separated, female 12.5 dpc gonadal cells from CAG/EGFP transgenic mice and wild-type mice were mixed and implanted to recipient mice. As shown in Fig. 3D–I, chimeric follicles could be isolated from the grafts after 20 days of grafting and chimeric COCs were present after 28 days of grafting. These results proved that the reaggregated gonads could still generate mature oocytes after the innate structure of female gonads was destroyed.
Ectopic differentiation of purified fetal germ cells by aggregation with gonadal somatic cells
To differentiate purified fetal germ cells and to explore whether or not gonadal somatic cells at different developmental stages are able to support fetal germ cell development into oocytes, we aggregated purified fetal germ cells with different gonadal somatic cells and transplanted the aggregates into mice for 28 days.
Germ cells were purified by MACS from genital ridges of CAG/EGFP transgenic fetuses at 12.5 dpc. The identity of the germ cells was confirmed by the detection of alkaline phosphatase activity (>95%) and the expression of Oct4 and Mvh antigens (Fig. 4A). Germ cells were then combined with somatic cells at 12.5 dpc, 16.5 dpc or 0dpp and transplanted into mice. As shown in Table 1, after transplantation, only 12.5 dpc oogonia aggregated with gonadal somatic cells at 12.5 dpc were able to generate full-grown oocytes. We noted that the rate of recovery was low: six grafts from 29 transplanted mice derived oocytes. Follicles containing EGFP-positive oocytes and EGFP-negative granulosa cells were present after transplantation (Fig. 4B and C). Twenty-five fully grown EGFP oocytes were isolated from antral follicles of the grafts after 28 days. The diameter of oocytes was 70.5 ± 3.5 µm and eight of them excluded the first polar body after FSH stimulation (Fig. 4D and E). The further IVF and culture did not generate blastocysts probably because of the low number of cultured oocytes.
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We also used Oct4/EGFP mice to purify germ cells by FACS and to confirm the above results. EGFP-positive cells were nearly 100% alkaline phosphatase positive (data not shown). Eleven aggregates from 12.5 dpc Oct4/EGFP oogonia and 12.5 dpc wild-type somatic cells were grafted into recipient mice and two of them generated antral follicles after transplantation for 28 days. Eight full-grown oocytes were derived from the antral follicles and one of them excluded the first polar body after FSH stimulation (Fig. 5). Thus, our results showed that purified fetal germ cells could generate M II oocytes by aggregating them with gonadal somatic cells and grafting into mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAuthors' RolesAcknowledgementsReferences |
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This study demonstrates that germ cells purified from 12.5 dpc female mice were able to differentiate into mature oocytes after aggregating germ cells with fetal gonad somatic cells and then grafting them beneath the renal capsule of recipient mice. The derived oocytes were
70 µm in diameter and could exclude the first polar body after in vitro maturation. To our knowledge, this is the first report that mature oocytes can be derived from isolated fetal germ cells, instead of from intact fetal or adult ovary tissues.
In this study, we demonstrate that although the innate structure of 12.5 dpc female gonad is not necessary, mature oocytes were not derived when purified 12.5 dpc germ cells were aggregated with 16.5 dpc or 0dpp ovarian somatic cells. Only by aggregating oogonia with 12.5 dpc ovarian somatic cells, were mature oocytes able to be produced. Previous studies by McLaren et al. have suggested a critical role of somatic environment on oocyte development by in vitro culture of different reaggregates of germ cells and somatic cells (McLaren, 1995; McLaren and Southee, 1997; Adams and McLaren, 2002). Studies by Odor (Odor and Blandau, 1969) and Byskov (1978) showed that in the 14.5 dpc ovary, germ cell and somatic cell establish important interactions and these interactions are critical for the further folliculogenesis. Recent studies by Lei et al. (2006) reported that fetal oocytes from 13.5 dpc mice could reform follicles in vitro with ovarian somatic cells at 13.5 dpc but not with somatic cells at later stages; e.g. 17.5 dpc. Our results, as well as others (McLaren, 1995; McLaren and Southee, 1997; Adams and McLaren, 2002; Lei et al., 2006), suggest that the synchronized interactions between oogonia and gonadal somatic cells could be important to ensure normal folliculogenesis. Recently, results of microarray analysis of gene expression profiles at different developmental stages of fetal ovaries demonstrated that both germ cells and somatic cells undergo numerous gene expression changes during this process, especially from 11.5 dpc to 16.5 dpc (Wertz and Herrmann, 2000; McClive et al., 2003; Nef et al., 2005; Small et al., 2005; Beverdam and Koopman, 2006; Bouma et al., 2007). Several factors expressed by ovarian somatic cells have been demonstrated to play an important role in oogenesis at fetal stage, such as Fst, Wnt4 and Foxl2 (Feijen et al., 1994; Vainio et al., 1999; Kim et al., 2006; Schmidt et al., 2004; Yao et al., 2004; Ottolenghi et al., 2005, 2007). On the other hand, Eppig et al. (2002) showed that after pre-antral follicle formation, oocytes determine the rate of follicle development. By transferring oocytes of secondary follicles back into primordial follicles, they found the rate of follicle development doubled and evidence of accelerated granulosa cell differentiation. Therefore, these results altogether suggest that the interactions of germ–somatic cell during different developmental stages of oogenesis could be different: in the fetal ovary, the development of germ cells and somatic cells might be synchronized, whereas after pre-antral follicle formation, oocytes orchestrate the further follicle development.
We also demonstrate that mesonephroi are dispensable for the generation of mature oocytes from 11.5 dpc and 12.5 dpc female gonads by transplantation. Several previous studies have shown that during the development of female gonads, there is no mesonephric cell migration (Buehr et al., 1993; Martineau et al., 1997; Tilmann and Capel, 1999) and in vitro culture of 11.5 dpc female gonads separated from the mesonephros could result in normal-looking ovaries (McLaren and Buehr, 1990). Studies of in vitro culture of 10.5–12.5 dpc germ cells also demonstrate that germ cells enter into meiosis in a cell autonomous way (McLaren and Southee, 1997; Chuma and Nakatsuji, 2001; Adams and McLaren, 2002). On the other hand, Nielsen and Byskov (2001) reported that mesonephric cell also migrated into female gonad when in vitro culturing reassembled gonads. Furthermore, two recent reports showed that mesonephroi produced retinoic acid (RA) and then RA triggers the meiotic initiation of germ cells at 13.5 dpc in female fetus (Bowles et al., 2006; Koubova et al., 2006), highlighting an important role of mesonephroi during the development of female gonads in vivo. RA has been shown as an important regulator of embryonic patterning and development (Ross et al., 2000). It will be interesting to explore whether RA also played a role in our studies of the ectopic development of female gonads. It is possible that other tissues in the host mice, such as the kidney, secrete RA to promote the development of transplanted gonads.
Our results show that male 12.5 dpc germ cells were not able to generate oocytes after aggregation with 12.5 dpc ovarian somatic cells and transplantion into the host mice. Previous studies demonstrated that there was a temporal window for fetal germ cells to enter spermatogenesis or oogenesis (McLaren and Southee, 1997; Tilmann and Capel, 1999; Adams and McLaren, 2002; Yao et al., 2003). It was found that 11.5 dpc male germ cells are sexually dimorphic, whereas at 12.5 dpc, male germ cells became committed to spermatogenesis (McLaren and Southee, 1997; Adams and McLaren, 2002). Our results also indicate that at 12.5 dpc, male germ cells are no longer able to undergo oogenesis even by aggregating them with ovarian somatic cells and transplanting into host female mice.
Our results show that mature oocytes can be derived from purified fetal germ cells using the aggregation–transplantation procedure described in this report. However, the number of mature oocytes derived from aggregates is less than from intact gonads after transplantation. There are two possible reasons for the low yield of oocytes. First, the purification procedure required to obtain germ cells, as well as the aggregation with somatic cells in vitro, might reduce the viability of germ cells. Previous studies have shown that isolated germ cells from 11.5 dpc and 12.5 dpc gonads underwent rapid apoptotic degeneration when cultured without feeder layer (De Felici and McLaren, 1983; Pesce et al., 1993). Further improvement of our method will include the addition of anti-apoptosis factors (Farini et al., 2005) to the culture medium during the aggregation procedure. Second, the transplantation procedure might also cause germ cell apoptosis in the renal capsule. Acute follicle loss and apoptosis has been found in newborn mouse ovarian grafts after 2–12 h of transplantation and this has been considered partly due to the ischaemia–reperfusion injury in ovarian grafts (Nugent et al., 1998; Liu et al., 2002). Steps, such as chilling the aggregates before transplantation to minimize ischaemia, will be adopted in our future procedure to reduce the possibility of germ cell apoptosis after transplantation.
Differentiation of purified fetal germ cells into mature oocytes by transplantation will provide a new approach for studying the development of fetal germ cell. Fetal germ cells can now be first manipulated in vitro through the addition of growth factors to the culture medium or by vector transfection (Watanabe et al., 1997; De Miguel et al., 2002). Then these factors can be assessed for their effects on germ cell differentiation in vivo. These functional studies will be helpful to elucidate the molecular mechanisms that underlie the differentiation of fetal germ cells.
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Funding |
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National Nature Science Foundation of China for Creative Research Groups (30421004); National Basic Research Program of China (973 Program, 2007CB947900); Bill & Melinda Gates Foundation Grant (37 871); 111 project to H.D.
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Authors' Roles |
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Experimental design, acquisition of data, analysis of data and writing the article—T.Q.
Acquisition of data and analysis of data—H.L., W.W., X.Y., W.S., D.Z., Z.S. and W.Y.
Experimental design—M.D.
Experimental design and writing the article—H.D.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAuthors' RolesAcknowledgementsReferences |
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We thank Dr Toshiaki Noce for kindly providing the Mvh antibody. We thank Dr Matt Stremlau for critical reading of the manuscript. We also thank Zuoxiang Li, Zhaodai Bai, Jing Cheng, Han Qin, Jun Yong, Jiefang You, Yuhua Han, Zan Tong, Yetao Wu and other colleagues in our laboratory for technical assistance and advice during experiments. Finally, we thank Liying Du for providing the technical support needed for the flow cytometric analysis.
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Footnotes |
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These authors contributed equally to this article. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAuthors' RolesAcknowledgementsReferences |
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Adams IR, McLaren A. Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development (2002) 129:1155–1164.
Beverdam A, Koopman P. Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes. Hum Mol Genet (2006) 15:417–431.
Bouma GJ, AVourtit JP, Bult CJ, Eicher EM. Transcriptional profile of mouse pre-granulosa and Sertoli cells isolated from early-differentiated fetal gonads. Gene Expr Patterns (2007) 7:113–123.[CrossRef][Medline]
Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, et al. Retinoid signaling determines germ cell fate in mice. Science (2006) 312:596–600.
Buehr M, Gu SB, McLaren A. Mesonephric contribution to testis differentiation in the fetal mouse. Development (1993) 117:273–281.
Byskov AG. The anatomy and ultrastructure of the rete system in the fetal mouse ovary. Biol Reprod (1978) 19:720–735.[Abstract]
Chuma S, Nakatsuji N. Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling. Dev Biol (2001) 229:468–479.[CrossRef][Web of Science][Medline]
De Felici M, McLaren A. In vitro culture of mouse primordial germ cells. Exp Cell Res (1983) 144:417–427.[CrossRef][Web of Science][Medline]
De Miguel MT, Cheng L, Holland EC, Federspiel MJ, Donovan PJ. Dissection of the c-kit signaling patheway in mouse primordial germ cells by retroviral-mediated gene transfer. Proc Natl Acad Sci USA (2002) 99:10458–10463.
Eppig JJ, Wigglesworth K, Pendola FL. The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci USA (2002) 99:2890–2894.
Farini D, Scaldaferri ML, Iona S, La Sala G, De Felici M. Growth factors sustain primordial germ cell survival, proliferation and entering into meiosis in the absence of somatic cells. Dev Biol (2005) 285:49–56.[CrossRef][Web of Science][Medline]
Feijen A, Goumans MJ, van den Eijnden-van Raaij AJ. Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development (1994) 120:3621–3637.[Abstract]
Ginsburg M, Snow MH, McLaren A. Primordial germ cells in the mouse embryo during gastrulation. Development (1990) 110:521–528.
Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev (1998) 76:79–90.[CrossRef][Web of Science][Medline]
Hilscher B, Hilscher W, Bulthoff-Ohnolz B, Kramer U, Birke A, Pelzer H, Gauss G. Kinetics of gametogenesis. I. Comparative histological and autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue Res (1974) 154:443–470.[Web of Science][Medline]
Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF III, Boiani M, Scholer HR. Derivation of oocytes from mouse embryonic stem cells. Science (2003) 300:1251–1256.
Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, Chaboissier MC, Poulat F, Behringer RR, Lovell-Badge R, Capel B. Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. PLoS Biol (2006) 4:e187.[CrossRef][Medline]
Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci USA (2006) 103:2474–2479.
Lei L, Zhang H, Jin S, Wang F, Fu M, Wang H, Xia G. Stage-specific germ-somatic cell interaction directs the primordial folliculogenesis in mouse fetal ovaries. J Cell Physiol (2006) 208:640–647.[CrossRef][Web of Science][Medline]
Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod (2002) 17:605–611.
Maatouk DM, Resnick JL. Continuing primordial germ cell differentiation in the mouse embryo is a cell-intrinsic program sensitive to DNA methylation. Dev Biol (2003) 258:201–208.[CrossRef][Web of Science][Medline]
Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R, Capel B. Male-specific cell migration into the developing gonad. Curr Biol (1997) 7:958–968.[CrossRef][Web of Science][Medline]
McClive PJ, Hurley TM, Sarraj MA, Van den Bergen JA, Sinclair AH. Subtractive hybridisation screen identifies sexually dimorphic gene expression in the embryonic mouse gonad. Genesis (2003) 37:84–90.[CrossRef][Web of Science][Medline]
McLaren A. Germline and soma: Interactions during early mouse development. Semin Dev Biol (1994) 5:43–49.[CrossRef]
McLaren A. Germ cells and germ cell sex. Philos Trans R Soc Lond B Biol Sci (1995) 350:229–233.[Web of Science][Medline]
McLaren A. Primordial germ cells in the mouse. Dev Biol (2003) 262:1–15.[CrossRef][Web of Science][Medline]
McLaren A, Buehr M. Development of mouse germ cells in cultures of fetal gonads. Cell Differ Dev (1990) 31:185–195.[CrossRef][Web of Science][Medline]
McLaren A, Southee D. Entry of mouse embryonic germ cells into meiosis. Dev Biol (1997) 187:107–113.[CrossRef][Web of Science][Medline]
Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, Schaer G, Malki S, Dubois-Dauphin M, Boizet-Bonhoure B, Descombes P, et al. Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol (2005) 287:361–377.[Web of Science][Medline]
Nielsen M, Byskov AG. Somatic cell exchange occurs between mouse fetal gonads and mesonephroi during in vitro culture. Cells Tissues Organs (2001) 169:325–333.[CrossRef][Web of Science][Medline]
Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil (1998) 114:341–346.
Odor DL, Blandau RJ. Ultrastructural studies on fetal and early postnatal mouse ovaries. Am J Anat (1969) 124:163–186.[CrossRef][Web of Science][Medline]
Ottolenghi C, Omari S, Garcia-Ortiz JE, Uda M, Crisponi L, Forabosco A, Pilia G, Schlessinger D. Foxl2 is required for commitment to ovary differentiation. Hum Mol Genet (2005) 14:2053–2062.
Ottolenghi C, Pelosi E, Tran J, Colombino M, Douglass E, Nedorezov T, Cao A, Forabosco A, Schlessinger D. Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells. Hum Mol Genet (2007) August (Epub ahead of print).
O W-S, Baker TG. Germinal and somatic cell interrelationships in gonadal sex differentiation. Ann Biol Anim Biophys (1978) 18:351–357.[CrossRef]
Pesce M, De Felici M. Purification of mouse primordial germ cells by MiniMACS magnetic separation system. Dev Biol (1995) 170:722–725.[CrossRef][Web of Science][Medline]
Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M. Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development (1993) 118:1089–1094.[Abstract]
Richards AJ, Enders GC, Resnick JL. Differentiation of murine premigratory primordial germ cells in culture. Biol Reprod (1999) 61:1146–1151.
Ross SA, McCaffery PJ, Drager UC, De Luca LM. Retinoids in embryonal development. Physiol Rev (2000) 80:1021–1054.
Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier AC, Treier M. The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. Development (2004) 131:933–942.
Shen W, Zhang D, Qing T, Cheng J, Bai Z, Shi Y, Ding M, Deng H. Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells. Biol Reprod (2006) 75(a):615–623.
Shen W, Li L, Zhang D, Pan Q, Ding M, Deng H. Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization. Mol Rprod Dev (2006) 73(b):1312–1317.[CrossRef]
Small CL, Shima JE, Uzumcu M, Skinner MK, Griswold MD. Profiling gene expression during the differentiation and development of the murine embryonic gonad. Biol Reprod (2005) 72:492–501.
Steinberger A, Steinberger E. In vitro culture of rat testicular cells. Exp Cell Res (1966) 44:443–452.[CrossRef][Web of Science][Medline]
Tam PP, Zhou SX. The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Dev Biol (1996) 178:124–132.[CrossRef][Web of Science][Medline]
Tilmann C, Capel B. Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad. Development (1999) 126:2883–2890.[Abstract]
Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP. Female development in mammals is regulated by Wnt-4 signalling. Nature (1999) 397:405–409.[CrossRef][Medline]
Watanabe M, Shirayoshi Y, Koshimizu U, Hashimoto S, Yonehara S, Eguchi Y, Tsujimoto Y, Nakatsuji N. Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control. Exp Cell Res (1997) 230:76–83.[CrossRef][Web of Science][Medline]
Wertz K, Herrmann B. Large-scale screen for genes involved in gonad development. Mech Dev (2000) 98:51–70.[CrossRef][Web of Science][Medline]
Yao HH, DiNapoli L, Capel B. Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads. Development (2003) 130:5895–5902.
Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, Swain A, Capel B. Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. Dev Dyn (2004) 230:210–215.[CrossRef][Web of Science][Medline]
Yoshimizu T, Sugiyama N, De Felice M, Yeom YI, Ohbo K, Masuko K, Obinata M, Abe K, Scholer HR, Matsui Y. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ (1999) 41:675–684.[CrossRef][Web of Science][Medline]
Submitted on January 31, 2007; resubmitted on September 19, 2007; accepted on September 26, 2007.
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