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Gamete Biology |
Urology Research Laboratory, Department of Surgery,3
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Department of Biology,4 McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, meiosis, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a cause of oocyte loss, both environmental and genetic factors have been considered. The first hypothesis states that a limited amount of trophic factors is produced and hence can support only a fraction of the germ cells. Leukemia inhibitory factor (LIF), c-Kit ligand stem cell factor (SCF), retinoic acid, and unidentified serum factors have been reported to promote survival of germ cells in vitro [6, 7]. However, it has not been proven whether these factors play roles in preventing oocyte death in vivo. The second hypothesis predicts a genetic basis for elimination of oocytes [3, 8]. Any germ cells with compromised genetic material might contain an internal trigger to self-destruct. The genetic defect may be inherent or imposed during active cell proliferation in otherwise normal germ cells. Yet, this hypothesis hardly explains massive loss of oocytes in the normal XX ovary.
As a mechanism of oocyte loss, involvement of apoptosis has been supported by compelling evidence [9]. Fluorescence-activated cell sorting and DNA fragmentation assays have demonstrated the oocytes that undergo apoptosis in the normal fetal ovary [10, 11]. Furthermore, the number of primordial follicles is greater in the postnatal mouse ovary deficient in caspase-2 or Bax, which has been implicated in execution of apoptosis [12, 13]. It remains to be determined whether apoptosis accounts for oocyte death at all stages and by all causes [14].
An interesting model for studying female meiosis is the B6.YTIR sex-reversed female mouse [15, 16]. We have previously reported that the germ cells enter meiosis in the absence of testicular differentiation in the B6.YTIR fetal gonad, but their X and Y chromosomes do not pair during the meiotic prophase [17, 18]. Furthermore, all oocytes in the medullary region undergo degeneration by the end of fetal life [17]. It remains to be determined if the excessive oocyte death is associated with X-Y pairing failure. Although oocytes in the cortical region survive and continue to develop, they cannot develop properly after fertilization [15, 17–19]. The developmental incompetence appears to be intrinsic to the oocytes of the B6.YTIR genotype [20]. The present study addressed whether the early stages of meiotic progress are defective in the oocytes of the B6.YTIR fetal ovary.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal procedures were performed in accordance with the Canadian Council on Animal Care and approved by the McGill University Animal Care Committee. B6.YTIR male mice (N30-32 backcross generations) carrying a B6 genetic background and the Y chromosome originated in a Mus musculus domesticus house mouse (caught in Tirano, Italy) were prepared as previously described [16] and maintained in our mouse colony. B6.YTIR progeny were produced by caging B6.YTIR males with B6 females (Jackson Laboratory, Bar Harbor, ME). We assumed that copulation occurred around 0200 h on the day when copulation plugs were identified, and this time was defined as 0 day post coitum (dpc).
Histological Examination of Fetal and Neonatal Ovaries In Vivo
Fetuses were removed from pregnant females at every gestation day from 13.7 to 20.7 dpc, and their gonads were dissected out in a minimum essential medium (MEM) containing Hanks salts (GIBCO/BRL, Grand Island, NY) and 25 mM Hepes buffer (MEM-H), fixed in a fresh mixture of glacial acetic acid and ethanol at a 1:3 ratio at room temperature for 1 h, and stored in 70% ethanol at 4°C. At the same time, a piece of liver was removed from each fetus and stored at -20°C. The liver was used to determine the chromosomal sex by polymerase chain reaction (PCR) amplification of the Y-specific Zfy sequence using the primers and conditions designed by Nagamine et al. [21]. Examples of PCR amplification are shown in Fig. 1. The fixed ovaries were embedded in paraffin, sectioned at 5 µm thickness, and stained with Harris hematoxylin and eosin according to the standard protocol.
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Bromo-deoxyuridine Incorporation and Culture of Fetal Ovaries
Fetuses were removed from pregnant females either in the morning (0800–1000 h) or evening (1600-1800 h) at 12.7–18.7 dpc as described above. Each ovary with (12.7–15.3 dpc) or without (15.7–18.7 dpc) the adjacent mesonephros was incubated in 0.5 ml of MEM containing Earle salts, 10% heat-inactivated horse serum, 25 mM Hepes, 50 U/ml of penicillin G sodium, and 50 µg/ml of streptomycin sulfate (MEM-E, all reagents from GIBCO/BRL), supplemented with 5 µM bromo-deoxyuridine (BrdU; Boehringer Mannheim, Indianapolis, IN) in a 4.5-ml culture tube with rotation at 37°C for 4 h under an atmosphere of 5% CO2 in air. Then, the ovaries were washed in MEM-E three times and cultured on Nuclepore membranes, floating each on 0.5 ml of MEM-E for up to 6.5 days. The medium was refreshed at the third day of culture. At the end of culture, the ovaries were fixed and stored until paraffin embedding as described above.
Immunocytochemical Detection of BrdU in Histological Sections
Serial paraffin sections were cut at 5-µm thickness and placed on histology slides. Four sections at the mid area of each ovary were deparaffinized through toluene and gradient concentrations of ethanol, rinsed in PBS, and treated with 1% periodic acid in PBS for 30 min at 50°C. The slides were then processed for standard immunocytochemical staining using a monoclonal antibody against BrdU (Boehringer Mannheim) at a 1:30 dilution, a secondary antibody against mouse IgG conjugated with biotin (Pierce, Rockford, IL) at a 1:500 dilution, and the reagents of the ABC kit (Vectastain; Vector Laboratories, Burlingame, CA). The slides were counterstained with twice-diluted Harris hematoxylin, dehydrated with ethanol and toluene, and mounted in Histoclad (Clay Adams, Franklin Lakes, NJ). Photographs were taken from five representative areas (144 x 96 µm2 each, two in the central region and three in the peripheral region) of each ovarian section under a light microscope (Axiophot; Zeiss, Oberkochen, Germany). The oocytes between the zygotene (Z) and diplotene (D) stages with or without BrdU incorporation were counted in each photograph. Five areas together covered 25%–50% (depending on the developmental stage) of the whole ovarian section.
Cytological Analysis of Meiotic Prophase
The ovaries after culture were dissociated into single cells as described previously [18]. The cell suspensions were applied to droplets of hypotonic solution on Plus-coated histology slides (Fisher Scientific Canada, Montreal, QC, Canada), fixed in 2% paraformaldehyde (pH 8.2), washed with 0.4% Kodak Photo-Flo 200 (Kodak Canada, Toronto, ON, Canada), and air-dried. The slides were incubated first with a rabbit polyclonal antibody against synaptonemal complex (SC) synaptic proteins and a human autoantibody against centromere components (CREST) both at a 1:1000 dilution overnight and then with the monoclonal antibody against BrdU at a 1:30 dilution for 30 min. The binding of primary antibodies was detected by incubation with an anti-rabbit IgG conjugated with TMR-X, an anti-human IgG conjugated with rhodamine, and an anti-mouse IgG conjugated with biotin (all at a 1:1000 dilution), followed by avidin conjugated with fluorescein isothiocyanate at a 1:500 dilution. The slides were mounted in Prolong Antifade (Molecular Probe, Eugene, OR) containing 0.4 µg/ml of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI; Boehringer Mannheim) and examined under a fluorescence microscope (Axiophot; Zeiss). The antibody CREST and the antibody against SC synaptic proteins were gifts from P. Moens (York University, Toronto, ON, Canada), and their specificity was described previously [22]. SC staining indicates the pairing core between homologous chromosomes and precisely defines the stage of meiotic prophase, whereas the CREST antibody cross-reacts with mouse centromeres and indicates the end of individual chromosomes.
Statistical Analyses
Unless specified, data were collected from four ovaries dissected from two to four fetuses in each category (i.e., XX or XY ovary dissected at a developmental stage), and statistical differences were assessed by ANOVA followed by Tukey test or 
2 test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphological changes in germ cells during development were examined by standard histological staining of the XX and XY ovaries dissected at 13.7–20.7 dpc (Fig. 2). In both types of ovaries at 13.7 dpc, most germ cells were in either the mitotic cell cycle or early meiotic prophase because none were seen with characteristic meiotic chromosomes. At 15.7 and 16.7 dpc, many oocytes were seen with condensed chromosomes at the zygoten (Z) stage of meiotic prophase whereas a few had reached the pachytene (P) stage. At 17.7 dpc and later, morphological differences between the XX ovary and the XY ovary began to emerge. In the XX ovary at 17.7 dpc, many oocytes had reached the P stage whereas most were still at the Z stage. In the XY ovary at 17.7 dpc, fewer oocytes were seen at the P stage compared with the XX ovary. In the XX ovary at 18.7 dpc, a few oocytes had reached the D stage, whereas others were seen at the Z and P stages. In the XY ovary at 18.7 dpc, no oocyte was seen at the D stage and only a few oocytes were seen at the P stage. In the XX ovary at 19.7 dpc (i.e., the day of delivery), many oocytes were seen at the D stage, particularly concentrated in the central region. In the XY ovary at 19.7 dpc, most oocytes were seen at the Z stage, concentrated in the peripheral region. Few oocytes were seen at either the D or P stage. In the XX ovary at 20.7 dpc, most oocytes had reached the D stage in the central region whereas some oocytes were seen at the Z stage in the peripheral region. In the XY ovary at 20.7 dpc, many oocytes were seen at the Z stage in the peripheral region whereas no oocyte was seen in the central region.
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Onset and Progress of Meiotic Prophase In Vitro
To determine the onset of meiosis, i.e., the premeiotic S phase, fetal ovaries were dissected out at varying developmental stages between 13.3 and 18.7 dpc and incubated with BrdU for 4 h. Incubation for 4 h was shorter than the reported premeiotic S phase (10.5–12 h) [23] and therefore must have covered only a cycle of DNA replication. To differentiate the labeling during premeiotic S phase from that during mitotic S phase, the ovarian explants were further cultured in BrdU-free medium for 4.5 days. At the end of culture, oocytes at varying stages of meiotic prophase were found either intensely labeled (evenly or partially), faintly labeled, or unlabeled (Fig. 3). The oocyte in the premeiotic S phase at the time of BrdU incorporation was expected to remain intensely labeled during culture. Partial BrdU labeling was most likely caused by partial overlapping with the premeiotic S phase and not by DNA repair during meiotic prophase. The oocyte at the mitotic S phase at the time of BrdU incorporation was also expected to be labeled, but the labeling intensity must have diminished because of at least one more cycle of DNA replication during culture. The oocyte excluded from mitotic S phase or already in meiotic prophase at the time of BrdU incorporation was expected to remain unlabeled until the end of culture. Meiotic stages were identified by the appearance of chromosomes in oocytes after hematoxylin counterstaining. In addition, numerous numbers of somatic cells were seen labeled with BrdU after culture. They were distinguished from germ cells by their smaller and irregular nuclei. BrdU-labeled somatic cells were widely distributed in both the XX and XY ovaries dissected at 13.3 dpc and cultured for 4.5 days (Fig. 3a and e). The labeling became limited to the stromal cells between clusters of oocytes in the XX ovaries dissected at later developmental stages (Fig. 3b–d). BrdU-labeled somatic cells were abundantly seen in the central region of the XY ovaries dissected at 15.7 dpc and cultred for 4.5 days (Fig. 3g) but became scarce in the XY ovaries dissected at 18.7 dpc (Fig. 3h).
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The total number of oocytes counted in each XX ovary after culture ranged from 106 to 161 in average regardless of the developmental stages at dissection (Table 1). The total number of oocytes counted in each XY ovary was also consistent at varying developmental stages, ranging from 77 to 121 in average. The number in the XY ovary was lower than that in the XX ovary at most developmental stages examined, although significant difference was not found. The percentage of BrdU-labeled oocytes was at the peak (74%–76%) in both XX and XY ovaries dissected at 13.3 dpc and decreased in the ovaries dissected at later developmental stages. There was a slight but significant decrease to 60% in the XX ovary dissected at 14.3 dpc (P < 0.05) and a further decrease to 31% at 15.3 dpc (P < 0.001), whereas there was a significant decrease to 45% in the XY ovary dissected at 15.3 dpc (P < 0.001). The labeling frequency remained higher in the XY ovary than in the XX ovary between 15.3 and 18.7 dpc, although significant differences were found only at 17.7 and 18.7 dpc (P < 0.05 and 0.001, respectively).
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The meiotic stages of BrdU-labeled oocytes after culture indicate how far the oocytes progressed from the premeiotic S phase within 4.5 days in vitro (Fig. 4). Most labeled oocytes reached the late Z to early P transitional (Z/P) stage in both the XX and XY ovaries dissected at 13.3–15.7 dpc. The rest of oocytes were distributed mainly at mid Z and P stages. The number of oocytes at the Z/P stage was significantly smaller in the XY ovaries than in the XX ovaries dissected at 13.7 and 14.7 dpc (P < 0.05). In the XY ovaries dissected at 16.7 and 17.7 dpc, BrdU-labeled oocytes were still seen with a peak at the Z/P stage, fewer at the P stage, and almost none at the Z stage. In comparison, much fewer BrdU-labeled oocytes were seen in the XX ovaries of corresponding stages (P < 0.001–0.05). The data obtained from the ovaries dissected at 12.7 dpc were not included in these analyses. In these early ovarian explants, many faintly labeled cells were seen, but few reached the Z/P stage after culture (data not shown).
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The BrdU-unlabeled oocytes represent the population that was excluded from the S phase during BrdU incorporation (Fig. 5). Of this population, the oocytes that had already entered meiosis at the time of BrdU incorporation were expected to have progressed beyond the Z/P stage because most BrdU-labeled oocytes had reached the Z/P stage by the end of culture. Such oocyte populations, at either the late P to D transitional (P/D) or D stage, began to appear in the XX ovaries dissected at 15.3 dpc and cultured for 4.5 days and rapidly increased in the ovaries dissected at later developmental stages. For comparison, a small population of unlabeled oocytes at the D stage began to appear in the XY ovaries dissected at 15.7 dpc and cultured for 4.5 days, and the number slowly increased in the ovaries dissected at later developmental stages but never reached the levels in the XX ovaries of corresponding ages. Significant differences in the population of D oocytes were found between the XX ovaries and the XY ovaries dissected at 15.3 to 18.5 dpc (P < 0.001).
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The average number of BrdU-labeled oocytes at each meiotic prophase stage was added for XX and XY ovaries dissected at 13.3–18.7 dpc (Table 2). No difference was found at all stages between the two types of ovaries. When similar analyses were made for BrdU-unlabeled oocytes, no difference was found at mid P stage or earlier; however, significant deficit was seen at the P/D and D stages in XY ovaries compared with XX ovaries (P < 0.001).
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Exact staging of meiotic prophase was difficult in histological sections. In particular, late Z stage could not be distinguished from early P stage and late P stage could not be distinguished from early D stage. Furthermore, the Z oocyte was characterized by a cluster of condensed chromosomes, which might have been seen in the oocyte undergoing degeneration. To confirm our criteria of meiotic stages in histological sections, chromosome spreading preparations from four ovarian explants (dissected at 14.7 dpc and cultured for 4.5 days) were immunostained for BrdU incorporation, SC proteins, and centromere components (Fig. 6). Continual SC staining indicated the extension of homologous pairing, whereas centromere staining indicated the end of individual chromosomes. BrdU incorporation was seen in fine spots, probably along the DNA loops around the pairing cores (Fig. 6b). Such heavy BrdU labeling was mainly seen in the oocytes at the Z/P stage. Much less staining was seen in the oocytes at mid Z or mid P stage (Fig. 6c and d). Interestingly, in these partially BrdU-labeled oocytes at the P stage, BrdU incorporation was restricted to centromere regions, overlapped with the CREST staining. This observation is consistent with the hypothesis that DNA replication is delayed in heterochromatin regions such as centromeres.
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Progress of Meiotic Prophase After a Prolonged Culture
In the above experiments, the duration of culture for 4.5 days was chosen to monitor the meiotic progress of the oocytes with BrdU incorporation without a major loss, which was assumed to occur during the P stage. However, we were concerned that the oocytes with BrdU incorporation might have been unable to progress beyond the P stage, and therefore most BrdU-labeled oocytes were consistently seen at the Z/P stage. Accordingly, five XX and four XY ovaries were dissected at 13.7 dpc and cultured for 6.5 days (Fig. 7a and b). Some D oocytes with or without BrdU labeling were seen in the XX ovarian explants, whereas such oocytes were rare in the XY ovarian explants. In contrast, BrdU labeling was seen in a few D oocytes among many unlabeled D oocytes in the XY ovaries dissected at 15.7 dpc and cultured for 6.5 days (six ovaries were examined) (Fig. 7d). All oocytes were unlabeled in the XX ovaries of corresponding age (four ovaries were examined) (Fig. 7c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Onset of Premeiotic S Phase Determined by In Vitro BrdU Labeling
Our results indicate that the germ cells began to enter meiosis by 13.3 dpc in both XX and XY ovaries. The germ cells in the ovaries dissected at 12.7 dpc entered meiosis during culture but did not reach the same stages as the ovaries dissected at later developmental stages. We speculate that most germ cells were still in mitotic cell cycle in the ovary at 12.7 dpc. Our observation agrees with the hypothesis that the germ cells enter meiosis autonomously regardless of their genotype unless they are enclosed in the testis cords [26, 27]. However, we have observed a clear difference in the last onset of meiosis between XX and XY ovaries. Very few germ cells entered meiosis in the XX ovaries dissected at 15.7 dpc or later, whereas a considerable number of germ cells continually entered meiosis in the XY ovaries dissected at as late as 18.7 dpc. This observation is difficult to explain if we assume that all germ cells are destined to enter meiosis autonomously. One explanation is that once a certain number of oocytes reached a critical stage of meiotic prophase, the remaining germ cells were eliminated from entering into meiosis in the normal XX ovary. It is intriguing that the oocytes at the Z/P stage were significantly fewer in the XY ovaries than the XX ovaries dissected at 13.7–14.7 dpc and cultured for 4.5 days. By contrast, significantly larger numbers of germ cells entered meiosis and reached the Z/P stage in the XY ovaries dissected at 16.7–18.7 dpc. When the average number of BrdU-labeled oocytes at each meiotic prophase stage was added from 13.3 to 18.7 dpc, no difference was found between XX and XY ovaries. These observations postulate a mechanism that recognizes a defect or excessive loss in the oocyte population at early meiotic prophase and recruits more germ cells to enter meiosis.
Time Required for the Progress of Meiotic Prophase In Vitro
By culturing the ovaries after BrdU incorporation, we could assess the time required for meiotic progress in vitro. Most oocytes progressed from the premeiotic S phase to the Z/P stage in 4.5 days. The results were consistent using histology sections and chromosome spreadings. Moreover, similar meiotic progress was observed among the ovaries dissected at varying developmental stages. We conclude that meiotic prophase progressed at a normal rate up to the late Z stage in the XY ovary. However, the deficit in the BrdU-labeled oocytes at the Z/P stage in the XY ovaries dissected at 13.7 or 14.7 dpc and cultured for 4.5 days may indicate an early defect in the XY oocyte. Since no difference was found in the number of oocytes at the Z stage, we speculate that this deficit resulted from loss of oocytes during the Z/P stage rather than a delay in the onset of meiosis in the XY ovary. Because of the major loss of oocytes beyond the Z/P stage, the meiotic progress at later meiotic stages in the XY ovary could not be compared with that in the XX ovary.
Factors That Influence Survival of Oocytes During Meiotic Prophase
Onset of meiosis Our results suggest the presence of two critical factors that influence the chance of an oocyte to survive through the meiotic prophase. First, the developmental stage at which a germ cell enters meiosis appears to influence its chance of survival. This trend was observed in the XX ovary and exacerbated in the XY ovary. A large number of germ cells constantly entered meiosis as evident by BrdU labeling in both XX and XY ovaries at 13.3–14.7 dpc, and most proceeded to the Z/P stage within 4.5 days in culture. This oocyte population would have been already in the meiotic prophase if the ovaries were dissected out 1 day later in development (i.e., 14.3–15.7 dpc), and therefore they would have been unlabeled during BrdU incorporation. Then, they must have progressed beyond the Z/P stage after 4.5 days in culture, because the oocyte population that had been in the premeiotic S phase during BrdU incorporation reached the Z/P stage at the end of culture. Such BrdU-unlabeled oocytes were scarce in the ovaries dissected at 14.3 and 14.7 dpc and cultured for 4.5 days (Fig. 5). This absence of unlabeled oocytes at advanced stages was not due to the culture conditions, since unlabeled oocytes at the D stage appeared when the XX ovaries were dissected at 15.3 dpc and cultured for 4.5 days. Furthermore, the number of unlabeled oocytes at the D stage increased rapidly in the ovaries dissected at later developmental stages. If there were a delay in the progress from the Z/P stage to the D stage, we would have anticipated accumulation of unlabeled oocytes at the Z/P stage. This was not the case. Therefore, we speculate that most oocytes with early entry into meiosis were eliminated during the P stage. Speed has proposed a hypothesis that oocytes that lag behind in development become atretic and degenerate at the P stage [8]. This hypothesis explained his observation that although the oocytes at the Z and P stages were found at a wide range of developmental stages, most oocytes reached the D stage in a more synchronized manner. Our hypothesis that oocytes that have precociously entered meiosis become eliminated can equally well explain the observation by Speed.
Sex chromosomal composition Second, the XY genotype of oocytes reduced the chance of survival beyond the late Z stage. We observed fewer BrdU-labeled oocytes at the Z/P stage in the XY ovaries than the XX ovaries dissected at 13.7 or 14.7 dpc and cultured for 4.5 days. This difference was exacerbated after culture for 6.5 days, since BrdU-labeled oocytes at the D stage were seen only in the XX ovaries. Furthermore, BrdU-unlabeled oocytes at the D stage appeared in the XY ovaries dissected at much later developmental stages, and the number remained much smaller at all developmental stages examined compared with the XX ovaries. These observations indicate that an excessive number of oocytes began to disappear during the Z/P stage in the XY ovary. It is conceivable that the failure in X-Y pairing may lead to degeneration of oocytes. However, the deficit in the oocyte population at the Z/P stage is too early to result from a P checkpoint mechanism of homologous pairing. Furthermore, the X-Y pairing failure alone cannot explain the observation that many oocytes survive and contribute to folliculogenesis in the XY ovary [17, 19]. We speculate that the germ cells that enter meiosis at early developmental stages already have less chance to complete the meiotic prophase and the additional defect due to the XY genotype further reduces the number of oocytes that survive in the XY ovary. As a net result, the central region of XY ovaries becomes devoid of oocytes [17, this study], since the germ cells begin to enter meiosis in this region.
It is informative to compare oogenesis in XY and XO females. The XO female mouse is also known to lose an excessive number of oocytes during meiotic prophase [3]. This oocyte loss has been attributed to the lack of pairing partner for the single X chromosome. However, like XY females, many oocytes survive in XO females. In fact, the XO female mouse is fertile. It has been proposed that survival of any individual germ cells in the XO ovary might depend on the ability of the univalent X chromosome to form a nonhomologous association with itself or with an autosome [28]. In the XY mouse ovary, however, the Y chromosome remains single throughout meiotic prophase [18]. The X chromosome is occasionally seen associated with an autosome pair, but its contribution to oocyte survival has not been established. In human XO females, most germ cells are lost during early meiotic prophase, and this oocyte loss cannot be attributed to pairing failure at the P stage [28]. It is possible that a single dosage of X-encoded genes may be responsible for the excessive loss of oocytes in both XY and XO females.
Comparison of Meiotic Progress In Vivo and In Vitro
It is generally known that in vitro development is inferior to in vivo development due to lack of proper nutrients or blood circulation. Therefore, it was our concern that our observations in culture may not represent the developmental events in vivo. Unfortunately, we have not yet established a method to follow the fate of oocytes by BrdU labeling in vivo because injection of BrdU resulted in a broader and uncertain duration of BrdU labeling and often resulted in loss of oocytes (unpublished data). Nonetheless, our morphological observations of ovarian development in vivo (Fig. 2) suggest that meiotic progress in culture is comparable to that in vivo. First, a significant number of oocytes at the P stage first appeared in the XX ovary at 17.7 dpc in vivo. This is consistent with our finding that the germ cells in the premeiotic S phase at 13.3 dpc reached the Z/P stage after 4.5 days in culture. Second, a significant number of oocytes at the D stage first appeared in the XX ovary at 19.7 dpc in vivo. Assuming that these oocytes represented the population that had entered meiosis early at 13.3–13.7 dpc, it took 6.0–6.5 days for an oocyte to reach the D stage in vivo. This is consistent with our finding that the oocytes at the premeiotic S phase at 13.7 dpc reached the D stage after 6.5 days in culture. Third, the deficit in the oocytes at the P and D stages in the XY ovary at 17.7 dpc and later is consistent with our observations in culture.
We were also concerned with a possible adverse effect of BrdU incorporation on meiotic progress. Although we confirmed the presence of BrdU-labeled oocytes at the D stage after a prolonged culture, their number was not as large as anticipated. It remains possible that BrdU-labeled oocytes were more frequently eliminated than unlabeled oocytes during the P stage. This problem must not invalidate the comparisons between XX and XY ovaries processed under the same conditions.
The Cause of Oocyte Loss
The cause of oocyte loss (or survival) in the normal ovary remains unknown. If trophic factors play a role, our results may suggest that the production of trophic factors is more limited in the central region than the peripheral region of a fetal ovary, since germ cells begin to enter meiosis in the central region. If the cause of oocyte loss is intrinsic, we speculate that the germ cells that prematurely enter meiosis may not have accumulated sufficient cytoplasmic factors, such as mitochondria and meiotic proteins, that are essential for completing the meiotic prophase. On the other hand, our results do not support the hypothesis that pairing failure is the major cause of oocyte loss. Clarifying the cause of the excessive loss of oocytes in both XY and XO females will promote our understanding of oocyte loss in normal fetal ovaries.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: T. Taketo, Urology Research Laboratory, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, Quebec, Canada H3A 1A1. FAX: 514 843 1457; teruko.taketo{at}much.mcgill.ca
Received: 26 March 2003.
First decision: 14 April 2003.
Accepted: 28 July 2003.
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40 red spots show clustering of centromeres. No BrdU incorporation is seen. b) Oocyte at the late Z stage from an XY ovary. Pairing between homologous chromosomes is almost complete except for a few regions. Heavy BrdU incorporation can be seen over the nucleus. c) Oocyte at the mid P stage from an XY ovary. Pairing between homologous chromosomes is complete. Partial BrdU incorporation can be seen, particularly at centromeres, where the overlapping of red and green fluorescence gives yellow color. d) Oocyte at the mid P stage from an XY ovary. The Y chromosome (arrow) is apart from the X chromosome (arrowheads). No BrdU incorporation is seen except for some centromeres. Original magnification x150