Hum. Reprod. Advance Access originally published online on January 31, 2006
Human Reproduction 2006 21(6):1359-1367; doi:10.1093/humrep/dei498
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Human fetal ovarian culture permits meiotic progression and chromosome pairing process
1 Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Bellaterra, Spain and 2 Follicle Biology Laboratory, Academisch Ziekenhuis van de Vrije Universiteit Brussel, Brussels, Belgium
3 To whom correspondence should be addressed at: Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. E-mail: montserrat.garcia.caldes{at}uab.es
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: The female meiotic process seems to be crucial for aneuploidy in humans. The first stages of mammalian female meiosis take place during the fetal period. Therefore, only little is known about female meiosis. The goal of this study was to develop a culture technique that permits human oocytes to progress through meiotic prophase, to provide a system to study human female meiosis. METHOD: Fetal ovaries from four cases were cultured up to 35 days in
-minimal essential medium, 2% human serum albumin, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium and 100 IU/ml penicillin–100 µg/ml streptomycin. RESULTS AND CONCLUSIONS: Although ovarian response to culture conditions varied, human oocytes survived in vitro up to 5 weeks. In three cases, we observed significant variation in stages of meiosis among the cultures. The homologous chromosome pairing process was studied for the first time in cultured oocytes, and the results suggested that the pairing process was completed following the same features described previously for euploid oocytes, as followed by the chromosome-13 pairing process and synaptonemal complex formation. Although a higher proportion of degenerated oocytes were observed as culture time increased, we also observed oogonial entrance to meiotic prophase.
Key words: fetal development/human oocytes/meiosis/ovarian culture/synapsis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Aneuploidy is a major cause of spontaneous abortions and severe anomalies among humans, and it is mainly due to errors in female gametogenesis (Hassold and Hunt, 2001
). As the first stages of mammalian female meiosis take place during the fetal time period and since human fetal ovarian tissue is especially difficult to obtain, few studies exist on this topic.
Meiosis is a reductional division of the genome that produces haploid gametes to ensure diploidy at fertilization. During meiotic prophase, homologous chromosomes pair and exchange genetic material. The meiotic prophase is classically subdivided into stages. At leptotene, chromosomes start condensation. At late leptotene, in human oocytes (Roig et al., 2004), all-chromosome telomeres cluster at a limited portion of the nuclear envelope (known as bouquet) (Harper et al., 2004) to promote pairing of homologous chromosomes and recombination. At zygotene stage, homologue pairing and synapsis takes place. During pachytene stage, recombination ends. Finally, at diplotene stage, homologues separate, only remaining joined by the points where recombination has occurred (chiasmata). At this stage, mammalian oocytes block and meiosis arrest until sexual maturity is achieved.
Because of the low sample availability, important efforts have been made to establish the critical conditions that promote oogonia to go through the first meiotic stages in vitro, in order to provide an approach to study the first stages of the meiotic process in females. Reports of fetal ovarian cultures in mouse (Lyrakou et al., 2002) and human (Blandau, 1969; Baker and Neal, 1974; Zhang et al., 1995; Hartshorne, 1996; Hartshorne et al., 1999; Sadeu et al., in press) assessed meiotic progression in vitro, and some of these papers also reported initiation of meiosis in vitro (Baker and Neal, 1974; Hartshorne et al., 1999). Similarly, histological analysis of long-term cultures of frozen/thawed, second-trimester human fetal ovarian pieces has suggested meiotic prophase progression of the female germ cells (R.C. and J.S., unpublished results). Moreover, Lyrakou et al. (2002) reported the influence of the culture medium in the evolution of meiotic recombination. However, there is no knowledge regarding how culture conditions may affect the homologue pairing process.
Thus, the aim of our study was to develop a new culture technique which facilitates the study of human female meiosis, permitting human fetal oocytes to progress through meiotic prophase, following the same dynamics known to happen in vivo. As the homologue pairing process of the human oocyte has been completely characterized (Roig et al., 2004, 2005a,b), we choose to monitor chromosome-13 pairing, synaptonemal complex (SC) formation and bouquet formation in cultured oocytes and compare them with the topological features of human oocytes developing in vivo.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Biological material
Ovaries from four fetuses (Table I) obtained after legal interruption of pregnancy, according to the Ethical Committee of the Hospital de la Vall d’Hebron, Barcelona, Spain, were used in this study. Fetus age was deduced from last menstrual period and foot length when available. Three fetuses (F3, V80 and V84) had euploid karyotypes. F15 was found to have trisomy 21 after prenatal diagnosis.
|
Ovarian culture
F3 and F15 ovaries were collected in phosphate-buffered saline (PBS). After removing adjacent tissues and repeated rinsing of fetal ovarian tissues, tissues were further dissected. To ensure equal inclusion of cortical and central ovarian regions in each ovary piece, ovaries were cut transversely obtaining 10 equal-sized pieces of approximately 15 x 20 x 20 mm (width x depth x height) from each ovary.
Ovarian fragments were equilibrated for 30 min on ice in freezing medium (Leibovitz L-15 medium) with 0.4% human serum albumin (HSA) (Grifols) and 1.5 M dimethylsulphoxide before transfer into precooled cryovials (one fragment/vial) containing 500 µl of freezing medium. The slow freezing was performed in a controlled-rate freezing machine (Kryo 10, series 10–20, Planer Biomed, Sunbury-on-Thames, UK) using the following programme: (i) cooled from 4°C to 7°C at a rate of 2°C/min; (ii) held for 10 min at 7°C; (iii) seeded manually; (iv) cooled to 40°C at a rate of 0.3°C/min; (v) cooled to 110°C at a rate of 10°C/min and (vi) plunged into liquid nitrogen tanks at 196°C and stored. For thawing, the vials were immersed in a 37°C water bath and agitated for a few minutes. The content of the melted vials was expelled into a Petri dish with Leibovitz L-15 medium at room temperature (approximately 20°C). The cryoprotectant was removed from the thawing solution by washing three times for 10 min. After thawing, the ovarian pieces were first equilibrated overnight in -minimal essential medium (Gibco BRL), supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2. After washing the ovarian pieces in freshly prepared and prewarmed medium, the pieces were distributed and cultured in tubes (two tubes per culture time) containing 3 ml -minimal essential medium, 2% HSA, 5 µg/ml insulin (Gibco BRL), 5 µg/ml transferrin (Gibco BRL), 5 ng/ml selenium (Gibco BRL) and 100 IU/ml penicillin–100 µg/ml streptomycin (Gibco BRL). For cases F3 and F15, ovarian cultures lasted 4 and 5 weeks, respectively. Samples were labelled as T0 after the overnight culture and depending on the extraction time as T1, T2, T3, T4 and T5, each label indicating the weeks of culture. All cultured tissues were fixed in methanol:acetic acid.
Two more cases, V80 and V84, were cultured to avoid freezing/thawing of the sample. For this purpose, ovaries were collected in sterile PBS 1% penicillin–streptomycin (Gibco BRL) at the hospital within 2 h of delivery. Ovarian pieces were cultured for 4 weeks following the culture protocol described. Samples were labelled as mentioned above (T0, T1, T2, T3 and T4), indicating the week of sample fixation.
For both frozen/thawed and fresh cultured ovarian pieces, at each culture week, tissues from two culture tubes were processed for analysis of meiotic progression by applying different techniques. At least one ovarian piece per culture time was processed immediately to obtain methanol:acetic acid spreads, and the rest of the pieces were kept at –80°C until use. Each week, 2.5 ml of culture medium per tube was changed for fresh prewarmed medium containing 3 ml of -minimal essential medium, 2% HSA, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium and 100 IU/ml penicillin–100 µg/ml streptomycin.
Oocyte preparations
To analyse the meiotic progression of the oocytes, spreads, preparations with preserved 3D structure and methanol:acetic acid spreads were performed as described elsewhere (Martínez-Flores et al., 2003; Roig et al., 2004, 2005b), once oocytes had been obtained by mechanical disgregation of the ovarian pieces.
Fluorescence in-situ hybridization on methanol:acetic acid preparations
Fluorescence in-situ hybridization (FISH) on methanol:acetic acid preparations was performed as described previously (Roig et al., 2005b) with slight modifications. Pepsin digestion was applied to preparations from culture times T1, T2, T3 and T4 to increase the efficiency of the hybridization. Slides were post-fixed in a formaldehyde solution and denatured in 70% formamide in 2x saline sodium citrate (SSC) at 69°C for 4 min. Probe denaturation was performed as described by the manufacturer, and the denatured probe was applied after slide dehydration. Oocytes were hybridized with a whole chromosome-13 probe labelled with Cy3 (Cambio, Cambridge, UK) and a dual locus-specific probe for chromosomes 13q14 [labelled with fluorescein isothiocyanate (FITC)] and 21q22 (labelled with tetramethylrhodamine isothiocyanate) (Appligene Oncor, Heidelberg, Germany). Three post-hybridization washes were performed in 50% formamide in 2x SSC, 2x SSC and 0.4x SSC, 0.05% Tween 20 at 45°C. Finally, DNA was counterstained applying an antifade solution (Vector Laboratories, Burlingame, CA, USA) containing 0.1 µg/ml of DAPI (4',6'-diamidino-2-phenylindole) (Sigma, Munich, Germany).
Oocyte staging was performed according to the morphological criteria previously described (Garcia et al., 1987; Roig et al., 2005b).
Immunostaining of structurally preserved preparations
Immunofluorescence (IF) against proteins of the SC [synaptonemal complex protein 3 (SYCP3), SYCP1 and cohesin REC8] and telomeric proteins (TRF2 and TIN2) was performed as described (Roig et al., 2004) with a minor modification. Oocytes from cultured ovarian pieces needed an extra permeation treatment with 0.05% triton X-100 (Sigma, Munich, Germany) in PBS for 30 min after fixation of the sample.
Identification of the SC proteins was performed using a rabbit polyclonal serum against the following antibodies: SYCP3 (Lammers et al., 1994), SYCP1 (Meuwissen et al., 1992) and cohesin REC8 (Eijpe et al., 2003) (all of them were kind gifts from Christa Heyting, Wageningen, The Netherlands). Chromosomal telomeres were detected using a mouse monoclonal antibody against telomeric protein TIN2 (Imgenex, San Diego, CA, USA) (Kim et al., 1999) and against TRF2 (Abcam, Cambridge, UK). IF staining was performed as described (Roig et al., 2004). Primary antibodies were diluted in PTBG (PBS, 0.2% bovine serum albumin, 0.2% gelatin, 0.05% Tween 20) and incubated overnight at 4°C in a humid chamber. After washing away unattached antibodies with PBTG, detection was performed using some of the following fluorochrome-conjugated secondary antibodies (all from Jackson ImmunoResearch Laboratories, West Grove, PA, USA, diluted in PTBG): goat anti-rabbit FITC antibody and a goat anti-mouse Cy3 antibody. Secondary antibodies were incubated for 1 h at 37°C in a humid chamber. After washing off excess secondary antibodies and fixation of the fluorescent signals with 1% formaldehyde in PBS, DNA was counterstained as mentioned above.
Analysis of degenerated oocytes (SYCP1-positive degenerated oocytes) was performed following the criteria for normal chromatin morphology.
Microscopy, image analysis and statistics
Preparations were evaluated using an Olympus BX70 fluorescence microscope (Olympus Optical Co, Hamburg, Germany). Images were captured and produced by Smart Capture software. Images were further processed using Adobe Photoshop to match the fluorescence intensity, as observed in the microscope.
Evaluation of the significance of the results was performed by applying different tests depending upon each case, and when two parameters were compared, a Chi-square test was used. To analyse meiotic progression, a lineal-by-lineal analysis was used to indicate variation of meiotic prophase-stage proportion with culture time. A recent article described the prevalence of each meiotic prophase stage with respect to fetal development using particular mathematical curves (Roig et al., 2005a), and regression of the in vitro data to these curves was used to relate variations obtained in vitro with those described for in vivo oocytes.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Presence of oocytes in culture
Oocytes at meiotic prophase were found at all culture times in all cultured ovarian pieces, except for a single ovarian piece from V80, which was cultured for 4 weeks. The total number of analysed oocytes differed in relation to culture time and tissue sample, ranging from 7 to 317, as reflected in Table II.
|
Oocyte survival and meiosis progression ‘in vitro’
Meiotic prophase progression was measured by analysing changes in the distribution of the oocytes in the meiotic prophase substages through culture times (Figure 1). Cases F3 and F15 were cultured as a first attempt to analyse meiosis progression in vitro. In F3 culture (Table III), only few oocytes were obtained from each culture time. Lineal-by-lineal association analysis revealed that the proportion of oocytes at meiotic prophase stage did not differ among culture times (P = 0.828), implying that no meiotic progression existed in this culture.
|
|
In F15 culture (Table IV and Figure 1), the number of oocytes found per culture time ranged from 11 to 67. In this case, a statistically significant variation of meiotic prophase-stage proportions was found as culture progressed (lineal-by-lineal association value = 6.171, P = 0.013). As a recent article adjusted the evolution of the proportion of each meiotic prophase stage with fetal development to particular mathematical curves (Roig et al., 2005a), we decided to study whether these changes observed in vitro followed the same dynamics described in fresh oocytes. Thus, results obtained for F15 culture were adjusted to the same curves described in the previous study (an inverse curve for the leptotene-stage proportion, a quadratic curve for the zygotene-stage proportion, a cubic curve for the pachytene-stage proportion and, lastly, a logarithmic curve for the diplotene-stage proportion). Regression of the data obtained in the F15 culture presented the same curves and described similar tendencies, as described in fresh oocytes (Figure 1A). Thus, leptotene-stage proportion decreases with culture time (F = 54.69, P = 0.001), zygotene-stage proportion increases slightly (F = 35.31, P = 0.001) and pachytene-stage proportion tends to increase until T4, from where it dramatically decreases (F = 1032.90, P = 0.001). No diplotene-stage oocytes were obtained in this culture; thus, no information regarding this meiotic prophase stage is presented. As in other studies (Hartshorne et al., 1999; Lyrakou et al., 2002), meiotic progression was assessed by the increase of the proportion of pachytene-stage oocytes. Thus, F15 culture showed a significant meiotic progression from T0 to T4 (Figure 1A).
|
In the V80 culture (Table V and Figure 1), lineal-by-lineal association analysis showed that there was a significant variation of the meiotic prophase stages in culture (lineal-by-lineal association value = 5.884, P = 0.015). In this case, data obtained from culture were processed as described, and again a significant agreement with the curves described for fresh oocytes was observed for all meiotic prophase stages with the exception of diplotene stage. However, V80 culture plots presented different tendencies to the F15 culture (Figure 1B): leptotene-stage proportion tends to increase with culture time (F = 33.03, P = 0.001), zygotene-stage proportion also tends to increase (F = 53.16, P = 0.001), pachytene-stage proportion oscillated with culture time, but there was a significant increase of pachytene-stage oocytes from the third to the fourth culture week (F = 95.37, P = 0.001). Diplotene-stage proportion did not adjust to a logarithmic curve as fresh oocytes (F = 0.02, P = 0.883), but these data adjust better to a quadratic curve (F = 1312.90, P = 0.001), showing that diplotene-stage proportion tends to increase until T2 and decrease from thereon. Thus, again a meiotic progression is observed in this culture between T3 and T4.
|
The V84 culture (Table VI and Figure 1C) showed a statistically significant variation of the meiotic prophase proportions throughout the culture time (lineal-by-lineal association value = 16.740, P = 0.001). Results obtained in this culture were processed as before, and they adjusted to the same curves as fresh oocytes. However, again these curves described different tendencies than the ones obtained in fresh oocytes. Leptotene-stage proportion increased following an inverse curve (F = 157.14, P = 0.001). Zygotene-stage proportion adjusted to a quadratic curve (F = 4.27, P = 0.016), but best adjustment was obtained following an inverse curve (F = 19.33, P = 0.001), showing its tendency to increase with culture time. Pachytene-stage proportion showed a decrease with culture time following a cubic curve (F = 868.4e + 15, P = 0.001). Finally, diplotene-stage oocytes adjusted to a logarithmic curve which showed an increase with culture time (F = 15.05, P = 0.001).
|
Dynamics of homologue pairing process in cultured oocytes
We analysed whether bouquet topology existed at V80 and V84 cultures and whether homologues paired in cultured oocytes following the same topological organization described previously in fresh oocytes (Roig et al., 2004).
In both cases, and in all culture times analysed, we commonly found oocytes at the bouquet stage showing no noticeable differences to the ones obtained from fresh and frozen samples (I.R. and M.G., data not published; Roig et al., 2004, 2005b). The proportion of bouquet-stage oocytes was counted for each culture time; figures did not differ statistically (Figure 2).
|
In both cultures (V80 T2–T3 and V84 T0–T1), there seems to be an increase of cells that do not present a bouquet topology (labelled as ‘no bouquet’ in Figure 2), when the prevalence of leptotene oocytes rises (Figure 2). This suggests either that cultured oocytes fail to start telomeric clustering at the leptotene stage or that early leptotene oocytes do proportionally increase in culture. [Early leptotene stages have been reported not to exhibit bouquet topology (Roig et al., 2004).]
To address these hypotheses, we investigated the pairing process of homologue chromosome-13 during meiotic prophase in vitro and also the SC formation in V80 and V84 cultured oocytes.
In both cases and at all culture times, chromosome-13 paired, following similar dynamics to fresh oocytes (Roig et al., 2005a). At almost all culture times, leptotene-stage oocytes displayed two separate chromosome-13s (Figure 3A), except for V80 T0, in which 3% of the analysed oocytes displayed an already-paired bivalent 13. In the V80 culture, the proportion of zygotene-stage oocytes with a chromosome-13 bivalent ranged from 66 to 78% (Table VII). In the V84 culture, the presence of paired bivalent 13 in zygotene oocytes ranged from 57 to 40% (Table VIII). The proportion of zygotene-stage oocytes with an already-paired bivalent 13 did not differ significantly among culture times. All pachytene-stage oocytes analysed had a paired bivalent 13 (Figure 3C). Similarly, all diplotene oocytes analysed displayed a desynapsing bivalent 13 (Figure 3D).
|
|
|
The data obtained for the chromosome-13 pairing process in V80 and V84 cultured oocytes were similar to those obtained from fresh samples (Roig et al., 2005a), suggesting that ovarian culture does not seem to affect the homologue-chromosome pairing process.
Moreover, the homologue pairing process was also analysed following SC formation. This was not found to differ between fresh sample (T0) and cultured oocytes. Despite finding degenerated oocytes (see below for more details about oocyte degeneration incidence), oocytes with apparently normal SCs were found at each culture time (Figure 2) after the evaluation of the SC proteins (SYCP3 and SYCP1) and cohesin REC8 localization. This again suggests that ovarian culture does not affect the homologue pairing process.
Oocyte degeneration in culture
As previous reports suggested the existence of degenerated oocytes in culture (Hartshorne et al., 1999; Sadeu et al., in press), especially at the pachytene stage (Baker and Neal, 1974), the percentage of oocytes in which the pairing process had started was analysed, followed by the presence of the SC central element protein SYCP1 and by displaying a nuclear DNA degeneration which is characteristic of apoptotic cells (Figure 2C), and these cells were scored as SYCP1-positive degenerated oocytes.
In the V80 culture, there was a low degeneration rate throughout the period studied (around 3%, see Table IX and Figure 2, A1), but it increased significantly at T3 to 46% (
2 = 21.662, P < 0.001). The degeneration rate decreased at the following culture time (T4) to 13%.
|
In the V84 culture, the percentage of oocyte degeneration significantly increased from 0% at T0 to 57% at T4 (2 = 13.416, P < 0.001) (Table X and Figure 2, B1).
|
In both cultures, the increase of the degeneration rate coincided in time with a rise in the proportion of oocytes that did not display telomeric clustering (no bouquet stage) and with an increase of the leptotene-stage oocytes (Figure 2).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
This article suggests that meiotic prophase could be maintained in oocytes grown in in vitro conditions. Results bring unequivocal evidences and demonstrate de novo initiation of meiosis in cultured human oocytes. Moreover, a complete analysis of the evolution of homologue-chromosome pairing process, chromosome dynamics (bouquet topology) and homologue synapsis is presented for the first time in cultured oocytes. The results suggest that the ovarian culture technique described here permits homologue pairing and synapsis progression with nearly similar patterns as those described in fresh oocytes (Roig et al., 2004
, 2005a).
Presence of oocytes in culture
Oocytes at meiotic prophase were found at all culture times in all cultured ovarian pieces, except for a single ovarian piece (V80 T4). However, a high variability in terms of number of oocytes per culture time was observed (Table II). This fact can be attributable to several factors. First, the variability in the number of oocytes may originate in the heterogeneous distribution of oocytes in the ovary, although this was diminished by the accurate division of the ovaries in similar size pieces, all of them containing both central and cortical parts of the ovary. However, because of this distribution of oocytes in the ovary, figures on the number of analysed oocytes were not considered as reliable markers for meiotic progression or degeneration analysis. Second, ovarian pieces may react differently to the same culture conditions. And, lastly, the intrinsic state of each ovarian piece when the culture was established could affect culture progression somehow.
‘In vitro’ culture can sustain meiosis
The techniques used in this study permit unequivocal identification of oocytes, thus finding meiocytes in almost all ovarian pieces. Analysis of the cultured tissue has demonstrated that meiosis can be maintained in vitro. Throughout all culture times, normal chromosome-13 pairing was found. The existence of normal telomeric dynamics and the apparently normal SC formation demonstrates that a normal homologue pairing process in human oocytes can be maintained in long-term culture. Other studies (Hartshorne et al., 1999) have also reported that oocytes could be maintained for long periods of time in vitro, but an analysis of homologue pairing was not described.
Surprisingly, and despite the different numbers of oocytes found, F15 and F3 cultures respond to the culture technique similar to that of V80 and V84 cultures, suggesting that freezing–thawing protocols do not disrupt viability of oocytes in culture.
In first attempts to stain cultured oocytes, it was noted that the original protocols for fresh samples did not perform as expected. Cultured oocytes needed a longer permeation for IF staining, and the hypotonic treatment did not reveal ‘clean’ chromosomes. An enzymatic digestion step was needed to remove residual cytoplasm before applying FISH.
Present observations suggest that ovarian culture conditions may alter the cell membrane, making it less reactive to classic protocols. However, these changes did not seem to affect the homologue-chromosome pairing process. Nevertheless, this observation should be kept in mind when discussing the results obtained in this study and other published studies, in which a defective meiotic recombination is observed in cultured mouse oocytes (Lyrakou et al., 2002).
Our results emphasize the existence of an important effect of differences between samples on response to the same culture technique. The sample developmental stage can also be the reason for a different response in terms of meiotic progression, as different fetal ages were used. Nevertheless, major patterns can be recognized in all cultures.
The individual particularities, shown during culture, have also been reported in previous studies performed in human fetal ovaries (Hartshorne et al., 1999) and may be attributable to ovary conditions when the sample was collected, time interval between sample collection and establishment of the culture, sample differences or to intrinsic inter-individual differences.
Meiosis starts ‘in vitro’
In the V80 and V84 cultures, an increase in number of oocytes that contained SYCP1 fibres and that showed degenerated DNA was observed as culture progressed. For V80, this was mainly observed at T3, and, for V84, there was a constant increase that led to a maximal degeneration rate at T4. At the same time, a decrease of the pachytene-stage oocytes was found (Figures 1 and 2), except for V84 T2 and T3 (most probably due to the low number of oocytes found in the ovarian pieces), suggesting either that late-zygotene-stage cells did not enter the pachytene stage or that most pachytene-stage oocytes were eliminated. In both cases, zygotene-stage proportion tended to increase as culture progressed (Figure 1), implying that most of the degenerated oocytes correspond to pachytene-stage oocytes. Concomitantly, the percentage of leptotene oocytes not displaying bouquet topology increased, suggesting that there exist an important entrance of oogonia into meiotic prophase, coinciding with observations made in previous works using other technical approaches (Blandau, 1969; Hartshorne et al., 1999).
Altogether, these data seem to suggest that a massive loss of oocytes accompanies oogonial entrance into meiotic prophase. These are the first observations in which the entrance of cells into meiotic prophase is related to a massive loss of meiotic cells.
During fetal ovarian development, a massive loss of oocytes takes place during the pachytene stage (Baker, 1963). The degeneration rate observed in our study could be either due to an ovarian internal ‘clock’ still working in culture or due to a deficient culture medium. In this sense, Hartshorne et al. (1999) also described a high rate of oocyte degeneration, but coinciding with the zygotene stage instead of pachytene-stage oocytes. Moreover, the pachytene stage is known to have a checkpoint that prevents abnormal meiocytes from progressing through meiosis (Roeder and Bailis, 2000), and thus it may be possible that the pachytene stage plays a role in the high loss of pachytene-stage oocytes observed in our cultures. More studies in fresh and cultured oocytes should be performed to better define the cause of this important degeneration rate and its relation to the entrance of oogonies into meiotic prophase.
Meiosis progress ‘in vitro’
Results obtained in the F15 culture suggest a meiotic progression in vitro. The proportion of pachytene-stage oocytes increased from 21 to 64% in culture. In the same way, in the V80 culture, meiotic prophase rates changed after T3, leading to a restoration of T0 values in only one culture week, showing a meiotic prophase progression which doubled the pachytene-stage proportion in a culture week. In both cases, regression of the pachytene-stage proportion indicates that it tended to increase with culture progression. The rapid transition through meiotic stages observed in the V80 culture agrees with the observations made in mouse fetal ovaries (Lyrakou et al., 2002) and in human fetal ovaries cultured in vitro (Hartshorne et al., 1999). However, these results contrasted with other studies made in human fetal ovaries, in which a longer period is needed to obtain a pachytene oocyte in vitro (Baker and Neal, 1974). This can be attributable to different developmental stages of the samples used in the different studies.
Meiotic progression observed for F15 is substantially longer than the one observed in the V80 culture. Again, this may be due to the different developmental stages of the sample. As F15 ovaries came from an 18-week-old fetus, the leptotene proportion at T0 is higher than that observed for V80 (48 versus 23%, respectively). This may suggest that the higher the proportion of leptotene-stage oocytes when the culture is established, the better response to the culture technique, in terms of meiotic progression, will be achieved. More studies should be done to test this hypothesis.
Nevertheless, the meiotic progression observed in the V80 culture seems to mainly originate from oocytes that started meiosis in vitro, as there is a massive loss of pachytene oocytes and a high entrance of oogonia into meiosis. However, results from the F15 culture suggest that oocytes that started the culture can continue meiosis in vitro. Currently, we have no evidence that meiotic progression observed in our study is derived from surviving oocytes or from de novo meiosis or from a combination of both.
Only meiotic progression observed in the F15 culture mirrors the situation previously described in vivo (Roig et al., 2005a). This suggests that the in vitro conditions used in this study may be optimal to promote meiotic progression for some cases, but unfortunately they are not for all the cases used.
Homologue pairing process is completed in cultured oocytes
This is the first study reporting the homologue pairing process in human cultured oocytes. Results show that the pairing process progresses following the same dynamics and topological patterns (bouquet topology) described in fresh euploid oocytes (Roig et al., 2004, 2005a). These results are in agreement with previous observations which assessed pairing process as an extremely reliable process that ensures the formation of a bivalent at pachytene with a low error rate (Roig et al., 2005a). This hypothesis has recently been confirmed in a study performed in human trisomic 18 oocytes (Roig et al., 2005b). Results presented here are another confirmation of pairing process fidelity, even while working at suboptimal conditions such as an in vitro culture.
In summary, results obtained in this study show that fetal oocytes can survive for long periods in vitro. Our data provide strong evidences that oogonia can start meiotic prophase in vitro. Moreover, meiotic prophase progression has been observed with the described culture technique, in some cases, following the same patterns observed in fresh human oocytes. Cultured oocytes preserved their capacity to form synapsis indistinguishable from fresh oocytes. Nevertheless, more studies should be carried out to refine the culture medium to increase the meiotic progression rate in culture and to analyse other meiotic processes such as recombination.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors thank Ms Isabelle Hellinckx and Ms Àngels Niubó for their technical assistance. We also thank Mr Miguel Martínez from the statistical laboratory of the School of Medicine (Universitat Autònoma de Barcelona, Spain) for his assistance with the analysis of the results. We would also like to thank Dr C. Heyting for kindly providing us with SYCP3, SYCP1 and REC8 antibodies. I.R. is the recipient of a fellowship from the Ministerio de Educación, Cultura y Deporte (AP2000-0992), R.G. has a fellowship from the Generalitat de Catalunya (2004FI 00953) and P.R. has a fellowship from a Ministerio de Sanidad grant (FIS 02/0297). This work was supported by a Ministerio de Sanidad grant, FIS 02/0297. The English language of this article has been revised by a native English-speaking instructor of English of this University.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Baker TG (1963) A quantitative and cytological study of germ cells in human ovaries. Proc R Soc Lond B Biol Sci 158,417–433.[Medline]
Baker TG and Neal P (1974) Oogenesis in human fetal ovaries maintained in organ culture. J Anat 117,591–604.[ISI][Medline]
Blandau RJ (1969) Observations on living oogonia and oocytes from human embryonic and fetal ovaries. Am J Obstet Gynecol 104,310–319.[ISI][Medline]
Eijpe M, Offenberg H, Jessberger R, Revenkova E and Heyting C (2003) Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SCM1
and SMC3. J Cell Biol 160,657–670.
Garcia M, Dietrich M, Freixa L, Vink ACG, Ponsa M and Egozcue J (1987) Development of the first meiotic prophase stages in human fetal oocytes observed by light microscopy. Hum Genet 77,223–232.[CrossRef][ISI][Medline]
Harper L, Golubovskaya I and Cande W (2004) A bouquet of chromosomes. J Cell Sci 117,4025–4032.
Hartshorne GM (1996) Fetal ovarian tissue in vitro. Assist Reprod Rev 6,71–82.
Hartshorne GM, Barlow AL, Child TJ, Barlow DH and Hulten MA (1999) Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture. Hum Reprod 14,172–182.
Hassold T and Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2,280–291.[CrossRef][ISI][Medline]
Kim S, Kaminker P and Campisi J (1999) TIN2, a new regulator of telomere length in human cells. Nat Genet 23,405–412.[CrossRef][ISI][Medline]
Lammers JH, Offenberg HH, van Aalderen M, Vink AC, Dietrich AJ and Heyting C (1994) 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.
Lyrakou S, Hulten MA and Hartshorne GM (2002) Growth factors promote meiosis in mouse fetal ovaries in vitro. Mol Hum Reprod 8,906–911.
Martínez-Flores I, Egozcue J and García M (2003) Synaptic process in rat (Rattus norvegicus): influence of the methodology on the results. Microsc Res Tech 60,450–457.[CrossRef][ISI][Medline]
Meuwissen RL, Offenberg HH, Dietrich AJ, Riesewijk A, van Iersel M and Heyting C (1992) A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes. Embo J 11,5091–5100.[ISI][Medline]
Roeder GS and Bailis JM (2000) The pachytene checkpoint. Trends Genet 16,395–403.[CrossRef][ISI][Medline]
Roig I, Liebe B, Egozcue J, Cabero L, Garcia M and Scherthan H (2004) Female-specific features of recombinational double-stranded DNA repair in relation to synapsis and telomere dynamics in human oocytes. Chromosoma 113,22–33.[ISI][Medline]
Roig I, Robles P, Garcia R, Martin M, Egozcue J, Cabero L, Barambio S and Garcia M (2005a) Evolution of the meiotic prophase and of the chromosome pairing process during human fetal ovarian development. Hum Reprod 20,2463–2469.
Roig I, Robles P, Garcia R, Martínez-Flores I, Egozcue J, Liebe B, Scherthan H and Garcia M (2005b) Chromosome 18 behaviour in human trisomic oocytes. Presence of an extra chromosome extends bouquet stage. Reproduction 129,565–575.
Sadeu JC, Cortvrindt R, Ron-El R, Kasterstein E and Smitz J Morphological and ultrastructural evaluation of cultured frozen-thawed human fetal ovarian tissue. Fertil Steril, in press.
Scherthan H (2001) A bouquet makes ends meet. Nat Rev Mol Cell Biol 2,621–627.[CrossRef][ISI][Medline]
Zhang J, Liu J, Xu KP, Liu B and DiMattina M (1995) Extracorporeal development and ultrarapid freezing of human fetal ova. J Assist Reprod Genet 12,361–368.[CrossRef][ISI][Medline]
Submitted on October 6, 2005; resubmitted on December 13, 2005; accepted on December 21, 2005.
Some of the data presented in this article have already been presented at the ESRHE workshop ‘Mammalian Oogenesis and Folliculogenesis. In vivo and in vitro Approaches’, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||