Molecular Human Reproduction, Vol. 8, No. 10, 906-911,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Growth factors promote meiosis in mouse fetal ovaries in vitro
Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK
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Abstract |
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Mouse fetal ovaries were cultured to investigate germ cell development in the presence of a combination of the growth factors (GFs) stem cell factor, insulin-like growth factor-1 and leukaemia inhibitory factor. Ovaries were isolated from fetal mice at 13 and 14 days post-coitum (dpc) and cultured to the equivalent of 17 dpc. Culture conditions comprised minimal essential medium-
plus 5% fetal calf serum, with or without GFs. Oocytes were assessed using immunofluorescence to illustrate synaptonemal complexes and recombination foci. The proportions of pachytene cells in freshly isolated 13, 14 and 17 dpc ovaries were 0, 8 and 74% respectively. There was a significant (P < 0.0001) increase in the number of pachytene cells after 4 days culture with GFs, with 24% of germ cells from 13 dpc ovaries reaching pachytene. In contrast, no pachytene cells were detected in cultures of 13 dpc ovaries without GFs. After 3 days in culture with GFs, 38% of germ cells from 14 dpc ovaries were at pachytene compared with 19% without GFs. In conclusion, we have demonstrated positive effects of GFs upon oocyte formation by meiosis in vitro. The observed results could be explained by an increased survival of premeiotic oogonia entering meiosis, or by effects on oocytes already in early meiosis. growth factors/meiosis/MLH1/mouse/oocyte
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Introduction |
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There is a major excess of female germ cells produced in many species, including mice and humans, yet the factors controlling oocyte selection for the ovarian reserve are poorly understood. The ovarian reserve is the population of oocytes, embedded in primordial follicles, which provides the supply of gametes for the whole of the female’s reproductive life. In humans, the menopause occurs when the number of oocytes in the ovarian reserve has diminished below a certain threshold, and the age at which menopause occurs is believed to relate to the magnitude of the initial ovarian reserve of oocytes, established before birth. In humans, gaining control of the age of menopause, potentially possible through manipulating the rate of loss of follicles from the reserve, would offer new options for fertility therapy and control. However, it is crucial to understand the potential risks of interference with oocyte selection for the ovarian reserve, in terms of both meiosis (a major source of inheritable genetic anomalies) and epigenetic factors during oogenesis (such as imprinting and growth control) before such ideas could be considered. Information on this area is extremely difficult to obtain in humans, and so our research programme includes studies of both humans and mice in order to learn about the earliest stages of oocyte formation.
In mice, oocyte formation is initiated during fetal development. On day 13.5 of embryonic life, 
25 000 oogonia are present per ovary (Tam and Snow, 1981
). However, most oocytes fail to complete their prefollicular development and a minority form the primary oocytes in the primordial follicles that comprise the ovarian reserve. Most oocytes are lost through apoptotic cell death (Morita and Tilly, 1999; review), although other mechanisms may also contribute (Motta et al., 1997; McLaren, 2000). Interference with apoptosis by genetic manipulation of key genes results in an alteration in the size of the ovarian reserve. For example, Bcl2 knockout mice were found to possess a significantly reduced number of primordial follicles compared with age-matched wild-type females (Ratts et al., 1995). Nevertheless, our understanding of the inter-relations between the risk of apoptosis in individual oocytes and overall size of the ovarian reserve remains at a very preliminary stage.
We have been developing an in-vitro model of oocyte development in fetal ovaries (Hartshorne et al., 1999) to provide a means of studying the mechanisms controlling the formation of the ovarian reserve. It is possible that the death of a particular oocyte is linked to some abnormal meiotic chromosome behaviour, such as anomalous homologous chromosome pairing during early prophase I (Speed, 1988). The principal stage at which oocyte death is manifested varies in different species; for example, zygotene in rat (Beaumont and Mandl, 1962), pachytene in calf, mouse (Borum, 1961; Erickson, 1966), pig (Black and Erickson, 1968), man (Baker, 1963) and marsupia (Alcorn and Robinson, 1983), or diplotene in guinea pig (Ioannou, 1964).
Certain growth factors (GFs) promote the survival of primordial germ cells (PGCs) in vitro (McLaren and Buehr, 1990; Matsui et al., 1991; Pesce et al., 1993; McLaren and Southee, 1997; Hara et al., 1998; Morita et al., 1999), possibly through anti-apoptotic activities. Among those which have been demonstrated to promote germ cell survival are stem cell factor (SCF, kit ligand), leukaemia inhibitory factor (LIF) and insulin-like growth factor (IGF)-1. Genetic analyses have revealed gonadal malformation and sterility in male and female mice lacking functional expression of SCF or its receptor c-kit (Mitz and Russel, 1957). This report, together with other investigations, indicates that SCF is a primary survival and GF for PGCs (De Felici and Dolci, 1991; Matsui et al., 1991; Pesce et al., 1993; Morita et al., 1999). IGF-1 has been identified as an anti-apoptotic agent and a potent survival factor for murine PGCs (Morita et al., 1999). LIF stimulates proliferation of PGCs (Matsui, 1991; Pesce et al., 1993; Morita et al., 1999). LIF knock-out mice do not show impaired gonadal development or fertilization problems; however, the uterus fails to respond normally to the presence of the embryo (Chen et al., 2000).
To date, some effects of GFs upon PGCs in culture have been reported, but few investigations have been performed on any resulting oocytes. It is known that organ cultures can produce enlarged oocytes in a variety of conditions (McLaren and Buehr, 1990; Byskov et al., 1997; McLaren and Southee, 1997). However, it is not clear whether any such oocytes are developing normally with respect to meiosis-specific features such as homologous chromosome pairing and recombination. The potential of GFs to inhibit apoptosis might impact upon the normal development of oocytes. Moreover, a full understanding of the meiotic process in vitro is lacking.
Our previous work has demonstrated that in-vitro conditions may affect the survival or meiotic progression of cultured human oocytes (Hartshorne et al., 1999). We performed the present study to test the hypothesis that GFs known to act as survival factors for primordial germ cells may influence the chances of oocytes undergoing meiosis in vitro. To this end, we have exposed mouse fetal ovaries to a cocktail of GFs in vitro, at a stage when meiosis in most oocytes is imminent [13 days post-coitum (dpc)] or at an early stage of progression (14 dpc). The results of exposure to the culture conditions were assessed by counting the proportions of oocytes which had reached the pachytene stage of meiotic prophase I, in cultures with or without GFs in comparison with freshly collected fetal oocytes of a similar age, or those remaining in vivo for a similar time. Oocytes which had reached the pachytene stage were identified in cell spreads using an antibody specific for SCP3, a protein constituent of the synaptonemal complex (SC) (Lammers et al., 1997; Hultén et al., 2001). The SC forms only during meiosis and is therefore an unambiguous marker of oocytes, being absent from PGCs or oogonia. The structure of the SC, highlighted by the antibody, also allows the stage of meiotic prophase I to be identified. This method therefore enabled the progression of meiosis in vitro to be assessed quantitatively.
In addition, in order to test whether meiotic recombination would occur similarly under in-vitro and in-vivo conditions, we applied antibodies to MutL homologue 1 (MLH1), a protein which forms discrete foci at sites of meiotic recombination (Baker et al., 1996; Barlow and Hultén, 1998). Meiotic recombination foci are usually most evident at the pachytene stage of meiotic prophase I; however, our previous experiments with human oocytes have suggested that MLH1 may be considerably affected by conditions in vitro (unpublished observations).
Our results confirm our previous observation (Hartshorne et al., 1999) that the progression of oocytes through meiosis in vitro is affected by exposure to culture conditions. The presence of the GF cocktail promoted meiotic progression, up to and including the pachytene stage, in vitro; however, meiotic recombination, as assessed by MLH1 foci, was absent in vitro.
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Materials and methods |
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Collection of fetal ovaries
Mice of the TO strain (University of Warwick breeding colony) were used for this investigation. The mice were housed in a 12:12 h light:dark environment at a temperature of 23°C with food and water ad libitum. One female mouse was caged with one male overnight. A vaginal plug was considered evidence of mating. The morning on which the plug was seen was set as day 0. Pregnant females were killed at 13, 14 or 17 dpc, their uteri were removed and gonads were dissected from individual embryos. Ovaries were identified by their characteristic appearance. Both ovaries and their associated mesonephric regions were removed and placed initially into Ham’s F10 medium (Gibco, Paisley, UK) supplemented with 100 mIU/ml penicillin (Sigma, Poole, UK) and 50 µg/ml streptomycin (Sigma).
A total of 162 ovaries from 81 mouse fetuses at 13, 14 and 17 dpc were used. Some of these ovaries (30/162) were dissociated from the mesonephroi and stained for alkaline phosphatase (AP), a cytoplasmic marker which is specific for germ cells, including PGCs, oogonia and early meiotic stages in oocytes (Chiquoine, 1954). Most of the remaining ovaries (120/132) were from 13 and 14 dpc mice and were cultured until the equivalent of 17 dpc for comparison with freshly collected 17 dpc ovaries. Testes from 16 adult male mice were used as positive controls for the evaluation of prophase staging by SC and MLH1 analysis.
Culture system
Ovarian explants were cultured individually in 1 ml minimum essential medium- (MEM-; Gibco) plus 5% fetal calf serum (suitable for embryo stem cells, ES-FCS; Gibco), penicillin (100 mIU/ml) and streptomycin (50 µg/ml) in 12-well clusters (Corning Costar Co., Cambridge, UK). Half of the wells were further supplemented with a combination of 100 ng/ml SCF (Sigma), 50 ng/ml IGF-1 (Sigma) and 100 ng/ml LIF (Sigma). The control wells contained an equivalent concentration of the vehicle used [phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin]. These doses were based upon those shown by Morita et al. to be effective in promoting ovarian germ cell survival (Morita et al., 1999). The cultures were maintained at 37°C in a humidified incubator under 5% CO2 in air for 3 or 4 days (for 14 and 13 dpc specimens respectively) with the culture medium changed after 2 days. For each experiment 12 wells were prepared, each containing one whole ovary, and the experiments were repeated 10 times to obtain means and standard errors, with a total of 120 fetal ovaries being analysed.
Germ cell identification by AP staining
Germ cells were identified before and after culture in 13 and 14 dpc samples, and in uncultured 17 dpc ovaries, using histochemical staining for AP activity according to a previously described method (De Felici and McLaren, 1982). Individual ovaries were removed from the wells, disaggregated manually and air-dried onto precleaned microscope slides before staining. Germ cells stained red, and somatic cells remained unstained.
Immunofluorescence of meiotic chromosomes
Immunocytogenetic analysis was used to determine the various stages of meiotic prophase I (Barlow and Hultén, 1998; Hartshorne et al., 1999; Hultén et al., 2001) in testicular and ovarian preparations. Ovarian tissue from 13, 14 and 17 dpc mice was dissected free from the mesonephros in unsupplemented Ham’s F10 medium (Gibco) and placed on a clean slide within a small drop of medium. The ovary was teased apart using sterile needles and the cells were dispersed in the drop. Three drops of 0.2 mol/l sucrose solution were added and the cell suspension was left to settle at room temperature for 30 min. Ten drops of fixative (2% ultrapure formaldehyde; TAAB, Aldermaston, UK) and 0.02% sodium dodecyl sulphate (Sigma) were then added (both sucrose solution and fixative had been adjusted to pH 8.4 with borate buffer). After 20 min, the slides were rinsed in distilled water and washed in three changes of PBS–0.1% Tween 20 (PBT) for 30 min. Immunostaining was performed as previously described (Barlow and Hultén, 1998; Hultén et al., 2001) using a mouse monoclonal antibody to human MLH1 (Pharmingen, San Diego, USA) at 1:500 dilution and a rabbit antibody to rat SCP3 (a component of SC lateral elements, a gift from Professor C.Heyting) at 1:2500 dilution.
Following primary antibody incubation overnight, slides were washed three times in PBT. The same process was followed for the SC and MLH1 analysis of pachytene cells in adult male mice. Fluorescein isothiocyanate (Sigma)-labelled anti-mouse secondary antibodies were used at 1:200 dilution and incubated for 30 min at 37°C. Nuclei were stained with DAPI and slides were mounted with Vectarshield after the final washing steps. The slides were viewed directly or stored at –70°C until required. Cells were analysed using a Zeiss fluorescence microscope. Electronic imaging and recording of cells was achieved using a cooled charged-coupled device camera and Vysis QUIPS software.
Germ cells were identified by their DAPI-stained nuclei, which were visibly larger than those of somatic cells in the spread preparations. Individual cells in prophase I of meiosis were scored using the criteria set out by Barlow and Hulten (Barlow and Hulten, 1998). Nuclei were classified as in pachytene if the SCs were fully formed, i.e. the cells contained 20 fully synapsed and distinct bivalents. The numbers of oocytes at the pachytene stage were counted under different conditions of culture and presented as a fraction of the total number of enlarged nuclei present (Speed, 1982). The proportions of pachytene cells were compared at different stages of fetal ovary development (13, 14 or 17 dpc) and after exposure to different conditions in vitro.
Statistics
Comparison between the numbers of pachytene oocytes observed in different culture conditions and in fresh specimens was made using two-way analysis of variance. P < 0.05 was considered a significant result.
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Results |
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Fresh tissue from fetuses at 13–17 days gestation stained positively for AP, demonstrating the presence of germ cells (Buehr and McLaren, 1993
). In vitro, explants tended to round-up and attach to the surface of the vessel, remaining healthy in appearance (pale colour, translucent) for up to 4 days. Figure 1 shows the appearance of 13 and 14 dpc explants after culture for 2 days.
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Qualitative assessment of AP staining indicated a similar prevalence and intensity of staining of germ cells in fresh and GF-supplemented cultures after 3 and 4 days, whilst control cultures without GFs had fewer cells staining positive for AP.
SC analysis of oocytes
Pachytene cells were clearly and unequivocally identified following SC analyses. In 13, 14 and 17 dpc fresh preparations, 0, 8 and 74% of germ cells respectively had fully developed SCs and were therefore considered to be in the pachytene stage. The numbers of enlarged nuclei per ovary, after preparation for SC analysis, ranged between 145 and 186. Following culture of the 13 dpc specimens for 4 days, no meiotic cells were observed in the absence of GFs. On the other hand, those in the presence of GFs had identifiable pachytene cells at a frequency of 24%. After culture of 14 dpc specimens for 3 days, pachytene cells, as identified by the presence of fully formed SCs, were detected in both GF-free and GF-supplemented conditions. However, the latter resulted in a significantly higher proportion of pachytene cells; 38% compared with 19% (P < 0.0001). Yet this proportion remained at approximately half of the in-vivo pachytene rate, 74%, at 17 dpc. These results are summarized in Figure 2 and Table I.
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MLH1 foci were present on SCs in pachytene oocytes from freshly prepared 17 dpc fetal mouse ovaries (Figure 3a
). In total, 63 ± 3% of pachytene nuclei in fresh 17 dpc ovaries were clearly labelled with MLH1 foci. On the other hand, MLH1 foci were never observed in 13 or 14 dpc fresh ovaries or after culture for 4 or 3 days respectively (Figure 3b).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The present study demonstrates that exposure to a combination of GFs (SCF, LIF and IGF-1) promotes mouse germ cell progression in vitro. This combination of GFs promoted meiosis of 13 and 14 dpc ovarian germ cells after in-vitro culture to the equivalent of 17 dpc in vivo. Nevertheless, it is unclear from our experiments whether the effect is mediated principally by promoting the survival of premeiotic oogonia entering meiosis, or by an effect on oocytes which have already entered the early meiotic stages.
We applied AP staining because we were interested to know whether the pool of germ cells from which the oocytes would arise was surviving in our cultures. AP staining is a simple technique which specifically marks the cytoplasm of PGCs, oogonia and oocytes in early meiosis, but its intensity reduces once meiosis has begun. Our results seemed to indicate, in a qualitative manner, that the cellular precursors of oocytes survived better in GF-supplemented medium than under control conditions. However, because it is also known that AP expression in vitro may be spurious (McLaren and Buehr, 1990), we have not ascribed high importance to this observation, and have focused instead on quantitative assessment of oocytes in meiosis.
We have concentrated on the identification of pachytene cells since this is a critical stage of meiosis, when chromosome synapsis is completed and recombination occurs (Zickler and Kleckner, 1999; review). It is also the stage at which a major reduction in germ cells occurs in mice due to apoptosis (Borum, 1961). Our observations are in accord with those of Speed who reported that at 13 days of mouse gestation, fetal PGCs are in the preleptotene stage and SC formation has not occurred (Speed, 1982). Speed reported that on 14 and 17 dpc, 5 and 67% of germ cells were at the pachytene stage in comparison with 8 and 74% respectively in the present study.
We have applied an organ culture method, since various types of organ cultures (e.g. ovary fragment culture or mixed organ culture) are known to support mouse female meiosis in vitro (McLaren and Buehr, 1990; McLaren and Southee, 1997). McLaren and colleagues showed that prolonged germ cell culture (>12 days) produced growing (enlarging) oocytes, but although the ovaries appeared histologically normal, they were small and contained fewer oocytes than normal (McLaren and Southee, 1997). No further investigation regarding the oocytes was reported. Byskov et al. related the numbers and growth of mouse oocytes in vitro to the cortex–medulla organization of the developing ovary which occurs around the time of meiosis initiation (Byskov et al., 1997). Such organization required a sufficient number of oocytes, and could still occur in vitro, despite the loss of many oocytes under in-vitro conditions.
In this study, we have used GFs to explore their effects on oogenesis in vitro. Previous investigations of GFs affecting the survival and proliferation of germ cells have shown that SCF, LIF and IGF1 (De Felici and Dolci, 1991; Pesce et al., 1993, Morita et al., 1999) support the survival and proliferation of PGCs, which would otherwise undergo apoptosis. In these studies, PGCs have been identified using mainly haematoxylin and eosin staining and immunohistochemistry for the presence of apoptotic factors. Therefore, the exact stage of the cells surviving is uncertain (Hartshorne, 1996; review).
We have assessed the stage of female mouse meiosis following a period of culture, using immunofluorescence analysis of SCs and MLH1 recombination foci. There was no visible difference in SC morphology in cells from the in-vivo or in-vitro treatment groups, although a lower proportion of pachytene cells was observed in vitro compared with a similar age in vivo. This might be related to the previously reported lower survival of germ cells or oocytes in vitro (Prepin et al., 1985; Byskov et al., 1997), or to a delay in development caused by suboptimal conditions of culture. However, no recombination foci were detected following culture, suggesting that this crucial aspect of meiosis may be compromised by the culture conditions applied. Thus, while GFs promoted the survival of AP-positive germ cells, and also germ cell progression to the pachytene stage of meiotic prophase I, these pachytene cells do not seem to be able to complete the process of crossing-over. The reason for the absence of MLH1 foci in oocytes cultured to the pachytene stage of meiotic prophase I is uncertain at present and warrants further investigation. The possibility of a connection between the stage of meiotic prophase I and the point at which oocyte degeneration is most prevalent, previously suggested to be pachytene in the mouse (Borum, 1961), is intriguing. The suggestion of a pachytene recombination checkpoint (Baker et al., 1996; Edelmann et al., 1996; Baarends et al., 2001) may be elucidated by further studies.
In summary, we have described a tissue culture system for mouse fetal ovaries that promotes meiotic progression in vitro, and possibly entry of germ cells into meiosis. We investigated the formation of the meiosis-specific proteinaceous pairing structure, the SC, as well as the formation of recombination foci of cultured mouse oocytes. We have demonstrated positive quantifiable effects of a combination of GFs upon survival and progression of fetal oocytes in this in-vitro system, although such oocytes did not show evidence of being able to support meiotic recombination. This model could be usefully applied in the future in order to understand mechanisms controlling oocyte formation and also to provide a basis for testing factors which may control the ovarian reserve. Such studies would be particularly valuable in humans where an in-vitro model would be an asset in conducting research which would otherwise be impossible to perform for ethical reasons.
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Acknowledgements |
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We are grateful to Dr Charles Tease for his assistance with various aspects of immunofluorescence, and to the technical staff of the Department of Biological Sciences. Professor Christa Heyting is gratefully acknowledged for her kind provision of the anti-SCP3 antibody. The work was funded in part by grants from the Royal Society (G.H.), from Wellbeing (H1/98; G.H., M.A.H.), the Wellcome Trust (M.A.H.) and from the British Federation of Women Graduates (S.L.).
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1 To whom correspondence should be addressed at: 23 Butterworth Drive, Coventry, CV4 8JL (until 1.11.02), and the address above thereafter. E-mail : ghartshorne{at}bio.warwick.ac.uk
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References |
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Alcorn, G.T. and Robinson, E.S. (1983) Germ cell development in female pouch young of the tammar wallaby (Macropus eugeni). J. Reprod. Fertil., 67, 319–325.[Abstract]
Baarends, W.M., van Der Laan, R. and Grootegoed, J.A. (2001) DNA repair mechanisms and gametogenesis. Reproduction, 121, 31–39.[Abstract]
Baker, T.G. (1963) Cytology of the female germ cells in mammals with particular reference to man. The time sequence of the chromosome situation in ova in relation to age of females, ovulation and menstruation. Br. J. Radiol., 41, 715.
Baker, S.M., Plug, A.W., Proll, T.A., Bronner, C.E., Harris, A.C., Yao, X., Christie, D.M., Monell, C., Arnheim, N., Bradley, A. et al. (1996) Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nature Genet., 13, 336–342.[ISI][Medline]
Barlow, A.L. and Hultén, M.A. (1998) Crossing over analysis at pachytene in man. Eur. J. Hum. Genet., 6, 350–358.[ISI][Medline]
Beaumont, H.M. and Mandl, A.M. (1962) A quantitative study of oogonia and oocyte in the fetal and neonatal rat. Proc. R. Soc. B., 155, 557–579.
Black, J.L. and Erickson, B.H. (1968) Oogenesis and ovarian development in the prenatal pig. Anat. Rec., 161, 45–56.[Medline]
Borum, K. (1961) Oogenesis in the mouse: a study of the meiotic prophase. Exp. Cell Res., 24, 495–507.[ISI][Medline]
Buehr, M. and McLaren, A. (1993) Isolation and culture of primordial germ cells. Meth. Enzyol., 225, 55–77.
Byskov, A.G., Guoliang, X. and Yding Andersen, C. (1997) The cortex-medulla oocyte growth pattern is organized during fetal life: an in-vitro study of the mouse ovary. Mol. Hum. Reprod., 3, 795–800.
Chen, J., Cheng, J., Shatzer, T., Sewell, L., Hernandez, L. and Stewart, C. (2000) Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology, 141, 4365–4372.
Chiquoine, A.D. (1954) The identification, origin and migration of the primordial germ cells of the mouse embryo. Anat. Rec., 118, 135–146.[Medline]
De Felici, M. and McLaren, A. (1982) Isolation of mouse primordial germ cells. Exp. Cell Res., 142, 476–482.[ISI][Medline]
De Felici, M. and Dolci, S. (1991) Leukaemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM4 feeder layers. Dev. Biol., 147, 281–284.[ISI][Medline]
Edelmann, W., Cohen, P.E., Kane, M., Lau, K., Morrow, B., Bennett, S., Umar, A., Kunkel, T., Cattoretti, G., Chaganti, R. et al. (1996) Meiotic pachytene arrest in MLH1-deficient mice. Cell, 85, 1125–1134.[ISI][Medline]
Erickson, B.H. (1966) Development and radioresponse of the prenatal bovine ovary. J. Reprod. Fertil., 11, 97–105.
Hara, T., Tamura, K., de Miguel, M.P., Mukouyama, Y., Kim, H., Kogo, H., Donovan, P.J. and Miyajima, A. (1998) Distinct roles of oncostatin M and leukaemia inhibitory factor in the development of primordial germ cells and Sertoli cells in mice. Dev. Biol., 201, 144–153.[ISI][Medline]
Hartshorne, G.M. (1996) Fetal ovarian tissue in vitro. Ass. Reprod. Rev., 6, 71–82.
Hartshorne, G.M., Barlow, A.L., Child, T.J., Barlow, D.H. and Hultén, M.A. (1999) Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture. Hum. Reprod., 14, 172–182.
Hultén, M., Barlow, A. and Tease, C. (2001) Meiotic studies in humans. In Rooney, D.E. (Ed.) Human Cytogenetics. A Practical Approach. Oxford University Press, Oxford, New York, pp. 211–236.
Ioannou, J. (1964) Oogenesis in the guinea pig. J. Embryol. Exp. Morph., 12, 673–691.[Medline]
Lammers, J., Offenberg, H., van Aalderen, M., Vink, A., Dietrich, A. and Heyting, C. (1997) The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes. Mol. Cell Biol., 14, 1137–1146.
McLaren, A. (2000) Germ and somatic cell lineages in the developing gonad. Mol. Cell. Endocrinol., 25, 3–9.
McLaren, A. and Buehr, M. (1990) Development of mouse germ cells in cultures of fetal gonads. Cell Differ. Dev., 31, 185–195.[ISI][Medline]
McLaren, A. and Southee, D. (1997) Entry of mouse embryonic germ cells into meiosis. Dev. Biol., 187, 107–113.[ISI][Medline]
Matsui, Y., Toksoz, D., Nishikawa, S., Nishikawa, S., Williams, D., Zsebo, K. and Hogan, B.L. (1991) Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature, 353, 750–752.[Medline]
Mitz, B. and Russel, E. (1957) Gene-induced embryological modification of primordial germ cells in the mouse. J. Exp. Zool., 134, 207–230.[ISI][Medline]
Morita, Y. and Tilly, J.L. (1999) Oocyte apoptosis: like sand through an hourglass. Dev. Biol., 213, 1–17.[ISI][Medline]
Morita, Y., Manganaro, T.F., Tao, X.J., Martimbeau, S., Donahoe, P.K. and Tilly J.L. (1999) Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse. Endocrinology, 140, 941–949.
Motta, P.M., Makabe, S. and Nottola, S.A. (1997) The ultrastructure of human reproduction. I. The natural history of the female germ cell: origin, migration and differentiation inside the developing ovary. Hum. Reprod. Update, 3, 281–295.
Prepin J., Gibello-Kervran, G., Charpentier, G. and Jost A. (1985) Number of germ cells and meiotic prophase stages in fetal rat ovaries cultured in vitro. J. Reprod. Fertil., 73, 579–583.[Abstract]
Pesce, M., Farrace, M.G., Piacentini, M., Dolci, S. and De Felici, M. (1993) Stem cell factor and leukaemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development, 118, 1089–1094.[Abstract]
Ratts, V., Flaws, J.A., Kolp, R., Sorenson, C.M. and Tilly, J. (1995) Ablation of bcl-2 expression decreases the number of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology, 136, 3665–3668.[Abstract]
Speed, R.M. (1982) Meiosis in the fetal mouse ovary. Chromosoma, 55, 426–437.
Speed, R.M. (1988) The possible role of meiotic pairing anomalies in the atresia of human fetal oocytes. Hum. Genet., 78, 260–266.[ISI][Medline]
Tam, P.P. and Snow, M.H.L. (1981) Proliferation of primordial germ cells during compensatory growth in mouse embryos. J. Embryol. Exp. Morph., 64, 133–147.[ISI][Medline]
Zickler, D. and Kleckner, N. (1999 Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet., 33, 603–754.[ISI][Medline]
Submitted on September 26, 2001; resubmitted on April 4, 2002; accepted on June 26, 2002.
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