Molecular Human Reproduction, Vol. 9, No. 4, 219-225,
April 2003
© 2003 European Society of Human Reproduction and Embryology
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Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads
Submitted on July 19, 2002; resubmitted on December 16, 2002; . accepted on January 23, 2003
1 Cell Biology Department, Research Society, Bai Jerbai Wadia Hospital for Children, AD Marg, Parel, Mumbai 400 012 and 2 Department of Pathology, King Edward Memorial Hospital, Parel Mumbai 400 012, India
3 To whom correspondence should be addressed at: Primate Biology Division, National Institute for Research in Reproductive Health (NIRRH), J.M. Street, Parel Mumbai 400 012, India. e-mail: deepaknmodi{at}hotmail.com
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The purpose of the study was to examine the occurrence of programmed cell death (apoptosis) in normal and chromosomally aneuploid testis and ovaries during the second trimester of human development. Such information may be useful in understanding normal and abnormal germ cell development and disorders associated with infertility in adult life. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis in human fetal ovaries (n = 16) and testis (n = 14) between 9 and 23 weeks of development, in ovaries of four Turner’s syndrome fetuses (45X) and in the gonad of an XO/XY fetus. In normal fetal testis, a small proportion of germ cells, Sertoli cells and Leydig cells undergo apoptosis. In normal fetal ovaries, some developing oocytes and granulosa cells were detected as TUNEL positive. Semiquantitative analysis of fetal ovaries revealed that
3–7% of oocytes were apoptotic. In abnormal fetal testis (XO/XY genotype). TUNEL analysis revealed that only germ cells not enclosed in seminiferous tubules undergo apoptosis. TUNEL analysis of the Turner’s syndrome (45X) ovaries studied at 15 and 20 weeks of development revealed massive apoptosis of the oocytes. Nearly 50–70% of the oocytes were TUNEL positive in these ovaries. These results suggest that germ cell apoptosis is a common event occurring during development of human gonads. Chromosomal defects by some means accelerates apoptosis that probably leads to gonadal dysgenesis later in life. Key words: apoptosis/germ cells/human fetal testis/human fetal ovaries/Turner’s syndrome/45X/XO/XY
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Introduction |
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Among the various cell types present in the gonads, the germ cells are the cells that are important in determining the fertility potential of an individual, as they will form the sperm or the oocytes. A critical event involved in gamete formation is the initial recruitment and the successful maintenance of the germ cells in the gonads during development. Failure of the germ cells to survive during development may lead to defective or no gamete production and hence infertility.
It has been estimated that 700–1300 germ cells are present in the genital ridges of both the sexes at 5 weeks of human gestation. The number of germ cells reaches 6x105 by 8 weeks; by 16–20 weeks, the human fetal ovary contains 6x106 germ cells. This number falls to 1–2x106 by term (reviewed in Baker, 1963; Rabinovic and Jaffe, 1990; Forabosco et al., 1991). Thus there is a drastic loss of germ cells in the human ovary during development itself. Extensive studies in mice and some studies in humans have suggested that the prenatal loss of germ cells occurs by apoptosis (reviewed in Reynaud and Draincourt, 2000).
Little is known regarding the loss of germ cells from the normally developing testis. Experimental evidence in cryptorchidism (a birth defect affecting 1% of newborn males that is associated with perinatal germ cell loss) suggests apoptosis as a possible mechanism of germ cell loss (Yin et al., 2002); recently apoptosis of germ cells has also been reported in human fetal testis (Helal et al., 2002).
Most sex chromosome aneuploidies are associated with gonadal dysgenesis owing to absence of germ cells in the gonads. Turner’s syndrome (45X genotype), a frequently observed sex chromosome aneuploidy in humans, is associated with infertility as the ovaries of most patients are streak ovaries that lack germ cells. Developmental studies in 45X fetuses reveal that the germ cells in the early genital ridges are normal in number, indicating that the differentiation and migration of germ cells occur normally in these gonads. However, by mid-gestation, the numbers of germ cells are dimuted significantly, leaving only streak fibrous tissue at birth in most cases (Singh and Carr, 1966; Cunniff et al., 1991). How these germ cells are lost during fetal life itself is unclear. Since germ cells in the normal gonads are probably lost via apoptosis it is possible that germ cell loss in the chromosomally abnormal gonads may also occur by apoptosis.
The aim of the present study was to examine the occurrence of germ cell apoptosis in normal human fetal testis and ovaries and in the gonads of fetuses with sex chromosome anomalies.
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The study was approved by the local ethics committees of Bai Jerbai Wadia hospital for children and King Edward Memorial Hospital (KEMH).
Normal human fetal gonads (n = 30; 16 ovaries, 14 testes) were collected from legal abortions performed at the Obstetric and Gynecology Department of KEMH after obtaining consent from the patients. 45X gonads were available from four cases (two cases at 15 weeks, one case at 20 weeks and one case at 22 weeks) that were aborted due to presence of multiple malformations detected on ultrasonography. In one case the gonads were obtained from a spontaneously aborted fetus at 14 weeks that was received for diagnostic autopsy. Externally, the fetus was grossly normal but the external sex could not be well defined. In all the cases, the age of the fetus was estimated on the basis of the date of last menstrual period and/or the foot length (Robinson, 1995).
The gonadal tissue in all cases was fixed in 10% buffered formaldehyde for 24 h and processed for routine paraffin embedding and sectioning. At all stages care was taken to avoid nuclease contamination. Sections 5 µm thick were cut and mounted on aminosailine coated glass slides and dried at 37°C and stored at room temperature until use.
Fluorescent in-situ hybridization (FISH)
The chromosomal anomalies in the abnormal fetuses was detected by interphase FISH on the cells obtained from the gonadal tissue. In the case of the spontaneously aborted fetus, FISH for X and Y chromosomes was performed as the right gonad was a dysgenetic testis and the left was a streak detected histologically (described below).
For the purpose of FISH, three paraffin sections (each 5 µm thick) were collected and incubated in xylene at 60°C for 30 min to extract the paraffin. After three changes of xylene, the tissues were rehydrated in grades of ethanol and incubated in 0.01 N HCl of 30 min followed by a digestion in pepsin solution prepared in 0.01 N HCl at a concentration of 4 mg/ml at 37°C for 3 h. The digested sections were washed twice in water and refixed in freshly prepared chilled fixative (3:1 methanol:acetic acid) overnight. The cells were then spread on subbed slides, air-dried and dehydrated in grades of ethanol. The probes used for FISH (chromosome X, Y and 18) were specific for the centromere and were purchased from Vysis (UK). The probe for chromosome X was labelled in spectrum green, Y with spectrum red and 18 with spectrum aqua. Diluted probe cocktail (5 µl) was applied onto dehydrated cells, covered with a coverslip, sealed and incubated at 42°C for 10 min. The target DNA and the probes were simultaneously denatured at 88°C for 14 min and hybridized overnight at 42°C. The slides were then washed in 0.2xstandard saline citrate (SSC) at 70°C for 3 min and in 2xSSC for 2 min at room temperature. The slides were mounted in an antifade medium (3% DABCO in 90% glycerol) containing diaminophinylindole (DAPI) as a counterstain. The cells were viewed under an Olympus BX-60 fluorescence microscope (Japan) equipped with a triple band pass filter to view spectrum red, green and DAPI simultaneously. Spectrum aqua was separately visualized using a specific filter (Vysis, UK). At least 100 diploid cells (two signals for chromosome 18) were counted for the number of signals using the criteria described previously (Modi et al., 1999). The rate of false positive hybridization was determined by performing FISH on cells obtained from ovaries of four normal XX fetuses.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)
The paraffin sections were deparaffinized in xylene, hydrated and treated with 0.3% Triton X-100 for 15 min. The tissues were then digested with 0.01 mg/ml Proteinase K (Gibco BRL USA) for 15 min at 37°C. Apoptosis was detected in situ by TUNEL using a commercial kit (Roche, Germany). Briefly, the digested sections were extensively rinsed in distilled water and incubated for 10 min in the reaction buffer provided with the kit. The sections were then incubated in the reaction cocktail containing the buffer, CoCl2, digoxigenin-labelled UTP, dATP and 125 IU of terminal transferase enzyme. The incubation was carried out for 30 min at 37°C followed by stopping the reaction in 2 mmol/l EDTA for 10 min and in 0.1 mol/l Tris HCl buffer (pH 7.5) for 10 min. The sections were blocked in a blocking solution containing 0.3% Triton X-100 and 2% normal sheep serum prepared in 0.1 mol/l Tris–HCl buffer (pH 7.5) for 30 min. The sections were then incubated in alkaline phosphatase-conjugated anti-digoxigenin antibody (diluted 1:500 in the blocking solution; Roche) overnight at 4°C. After extensive washing in Tris–HCl buffer (pH 7.5) the slides were incubated in 0.1 mol/l Tris–HCl (pH 9.5) for 10 min. The colour was developed using Nitro Blue Tetrazolium (NBT)–5-bromo-4-chloroindolylphosphate (BCIP) (Roche) solution for 15 min at room temperature. The slides were washed in water and mounted in an aquamount. For negative controls the slides were treated identically as above except that terminal transferase was omitted from the TUNEL reaction mixture.
All samples were analysed under a light microscope on a blinded basis and in duplicates.
For semiquantification the number of TUNEL positive and negative oocytes were counted in randomly selected high power fields (x100 objective) and 
500 oocytes from each ovary were counted. In the case of the 20 week old fetal Turner’s syndrome ovary, as the number of oocytes was low only 200 oocytes per ovary could be counted. The apoptotic index was calculated as the number of TUNEL positive oocytes/total number of oocytes countedx100. The intra- and inter-observer correlation were high (r = 0.97 and r = 0.98 respectively; data not shown). All testicular samples were only evaluated qualitatively.
Immunohistochemical localization of steroid acute regulatory (StAR) protein
As the morphology of the germ cells detected in the stroma of the XO/XY gonad partially resembled Leydig cells (large spindle-shaped cells), immunostaining for StAR protein was performed as described below.
Paraffin sections of the XO/XY mosaic gonad and of an age-matched control testis were deparaffinized in xylene and hydrated in grades of ethanol and washed in 0.01 mol/l phosphate-buffered saline (PBS) for 10 min. The endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 for 30 min. The sections were subsequently incubated at 4°C overnight in rabbit anti-mouse StAR polyclonal antibody (gift from Dr D.B.Hales, University of Chicago, USA) diluted 1:50 in the blocking solution. The slides were then extensively washed in PBS and incubated in horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG for 2 h at room temperature. After extensive washings to remove the unbound antibody, the colour was developed using diaminobenzidene (Sigma, USA) as substrate. The sections were then dehydrated, cleared in xylene and mounted in DPX. Testis sections incubated without the antibody were used as negative control.
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FISH
The hybridization efficiency of the probe used was 96%. Our lower limit of detecting a separate cell line was 4%, which corresponded to the mean ±2 SD of the false positive as reported previously (Amiel et al., 1996; Modi et al., 1999). As the true false negative values cannot be estimated, the upper limit of detecting mosaicism was arbitrarily assumed as 95% (Modi et al., 1999). Using the above criteria, all the Turner’s syndrome fetuses were non-mosaic XO in the gonadal tissue. In the case of the spontaneously aborted fetus with poorly defined external sex, both the gonads differed morphologically. On the right side, the gonad had some seminiferous tubules enclosing germ cells and an epidydmis; the left gonad was a streak with no evidence of any tubules. Müllerian derivatives were detected histologically on this side of the gonad. To rule out any sex chromosome abnormality, FISH for the sex chromosome was performed on the cell obtained from this gonad, where 30% of cells had XY genotype, and nearly 70% lacked the Y chromosome, i.e. had the XO genotype.
Apoptosis in the normal testis
Testes from 14 fetuses (9–22 weeks gestation) were examined by TUNEL in the present study. Apoptosis was not detected in the testis at early stages of development. After 14 weeks gestation until 22 weeks, TUNEL positive germ cells were detected in the testis (Figure 1); a small proportion of Sertoli cells and Leydig cells were TUNEL positive in the second trimester fetal testis. However, the numbers were extremely low (data not shown).
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Apoptosis in the normal fetal ovaries
Sixteen second trimester (13–23 weeks) fetal ovaries were analysed by TUNEL in this study. Apoptotic cells were detected in fetal ovaries at all stages of development investigated. At 14 weeks of development when follicular structures were not evident, most TUNEL positive cells were identified as oogonia or oocytes. At all subsequent stages most apoptotic germ cells were identified as oocytes, these oocytes were not enclosed by the follicular cells. At
16 weeks, a small proportion of granulosa cells were also detected as TUNEL positive (Figure 2).
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Semiquantitative analysis of the ovarian sections revealed that
3–7% of oocytes were undergoing apoptosis in normal fetuses studied from 13 to 22 weeks of development (Figure 3A).
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Immunohistochemical localization of StAR in the normal and XO/XY fetal testis
As expected, strong StAR immunoreactivity was detected in Leydig cells and Sertoli cells of normal fetal testis (Figure 4). As for normal testis, StAR immunoreactivity was detected in Sertoli cells and Leydig cells in the stroma of testis of the XO/XY gonad. StAR immuno reactivity was not detected in germ cells that partially resembled Leydig cells in morphology.
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Apoptosis in chromosomally aneuploid gonads
Most of the germ cells found in the stroma of the XO/XY gonad (testis) were apoptotic; germ cells enclosed in the seminiferous tubules were negative for TUNEL (Figure 1). Apoptotic germ cells or somatic cells were not detected in the streak (left) gonad at this stage of development (data not shown).
At 15 and 20 weeks of development no follicular organization was evident in the 45X gonads; all TUNEL positive cells were oocytes. As compared with age-matched control ovaries, a massive increase in the TUNEL positive cells was detected in the 45X ovaries studied at 15 and 20 weeks of development (Figure 2). However, normal or TUNEL positive oocytes were not detected in the 45X ovary studied at 22 weeks of development. Semiquantitative analysis of the 45X ovaries reveals that at 15 weeks, nearly 50% of oocytes were apoptotic in the 45X ovaries; at 20 weeks nearly 70% of oocytes were TUNEL positive (Figure 3B).
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Discussion |
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The results of the present study demonstrate that apoptosis of germ cells occurs during both normal male and female development, and that the numbers of germ cells undergoing apoptosis are higher in the sex chromosomally aneuploid gonads.
Little is known regarding germ cell loss from human male fetal gonads. In the present study we detected TUNEL positive cells in the developing testis from 14 weeks of development until 20 weeks. These results are in partial agreement with a recent report (Helal et al., 2002) where apoptosis of germ cells was detected in the testis as early as at 12 weeks of development until term. However, in the present study we were unable to detect germ cell apoptosis in the testis before 14 weeks of development. At present the reason for this discrepancy is unclear, it is possible that we may have missed some apoptotic cells in the early developing testis as our analysis is based on TUNEL alone, whereas Helal et al. (2002) used a combination of TUNEL and morphology to detect apoptosis.
Along with the germ cells a small proportion of somatic (Sertoli and Leydig) cells was also found to be apoptotic during second trimester fetal development. However the functional significance of this finding at present is unclear, but as the proportion of somatic cells undergoing apoptosis is low (Helal et al., 2002; present study), it is possible that these cells may be dying as a result of physiological death and may not have a functional relevance.
In the case of the XO/XY fetus, the right gonad had some seminiferous tubules; most germ cells were seen scattered in the gonadal stroma. Morphologically, these cells had a large nucleus and abundant cytoplasm that did not typically resemble those of the germ cells of the testis. To rule out the possibility that these cells were Leydig cells, we performed immunohistochemical localization of StAR protein that can serve as a marker of Leydig cells (Lin et al., 1995). As expected, StAR was specifically localized to the Leydig cells and the Sertoli cells in the normal and XO/XY testis; expression was not detectable in the germ cells ruling out the possibility that the presumptive germ cells within the stroma may be Leydig cells.
TUNEL analysis on the sections of this gonad revealed that most germ cells found within the gonadal parenchyma were apoptotic whereas those within the tubules were normal (did not stain positive for TUNEL). These observations tempt us to suggest that spontaneous degeneration of germ cells is a rare phenomenon in the developing testis if they are enclosed within a correct milieu, and that loss of support from the somatic cells may cause germ cell loss via apoptosis. Indeed, germ cells at ectopic sites such as the adrenal are reported to degenerate spontaneously (Upadhyay and Zamboni, 1982).
The hallmark of ovarian differentiation is the entry of germ cells in meiotic prophase. In human fetuses by 11–12 weeks, although vigorous mitotic activity is continuing in the superficial layers of the ovarian cortex, a few germ cells in the deepest layers enter the leptotene stage of meiotic prophase (Motta et al., 1997). Progressively, an increasing number of oogonia enter the meiotic pool and become enclosed by a layer of somatic cells to form a primordial follicle. The future reproductive capacity of the female is thought to depend on the number of follicles recruited in this pool. The dynamics of follicular development have been extensively studied in human fetal ovaries using quantitative cytological and morphometric methods. Baker (1963) was the first to demonstrate that germ cells in the genital ridges, which are minimal initially, undergo rapid multiplication to reach 6x106 by mid-trimester after which they reduce to 2x106 by term. This indicates that the number of germ cells in the ovary at birth is <20% of their peak number. Similar observations have been made in the ovaries of other mammalian species (Ericson, 1966; Reynaud and Driancourt, 2000). This is evidence that the normal fate of female germ cells during oogenesis is death.
It was not known how germ cells were lost from fetal ovaries until recently when it was shown that these cells are lost via apoptosis. Three lines of evidence arising from experimental animals support this view. Flow cytometric analysis of germ cells and detection of DNA ladder from cells obtained from fetal mouse ovaries suggested that apoptosis was the mechanism of cell death in the ovary (Cocouvanis et al., 1993; Ratts et al., 1995). This suggestion was confirmed by in-situ detection of apoptosis (TUNEL) and electron microscopic studies of human and mouse fetal ovaries (DePol et al., 1997; 1998; Driancourt et al., 1997; Vaskivou et al., 2001).
Consistent with these reports, in the present study nearly 3% of cells were TUNEL positive at 14 weeks, and the number increased to 7% by 20 weeks. These results indicate that germ cells are lost from the ovaries even before the initiation of folliculogenesis; the numbers increase as follicle organization proceeds. However, the number of degenerating germ cells identified by TUNEL in human fetal ovaries differed in various studies. Using TUNEL technique, nearly 7–10% of germ cells were found to be apoptotic between 18 and 20 weeks of development (DePol et al., 1997); higher numbers have recently been reported (Vaskivou et al., 2001). According to Vaskivou et al. (2001), at 13 weeks of development nearly 10% of oocytes are TUNEL positive, and as many as 15–17% of oocytes are detected as TUNEL positive at 20 weeks of development. In the present study, however, a maximum of 8% cells were found to be TUNEL positive and we were unable to confirm the observations of Vaskivou et al. (2001) even after additional analysis with a third independent observer. The reason for the discrepancy observed in the number of apoptotic germ cells between the present study and that reported previously (Vaskivou et al., 2001) is unclear.
Despite these differences, the frequency of apoptotic germ cells estimated in the human and murine fetal ovaries is markedly lower than the magnitude of the germ cells that have been estimated to be lost from normal fetal ovaries. It has been suggested that since apoptotic cells disappear very rapidly (Gumienny et al., 1999), it is likely that the number of apoptotic oocytes detected in various studies is an underestimate (Reynaud and Driancourt, 2000). Alternatively, it is possible that some other mechanisms may also exist that may contribute to the loss of germ cells from fetal ovaries, i.e. oocytes may also leave the developing ovary via the surface epithelium, resulting in cell loss (Motta et al., 1997).
Currently, it is unclear why some oocytes in the normal fetal ovary die while others survive. Two major phases of cell death have been described in the mouse fetal ovary. First, a major risk of death occurs through the meiotic prophase; second, there is limited risk during primordial follicle formation (discussed in Reynaud and Driancourt, 2000). In human fetuses, apoptotic germ cells were detected in the ovaries from 13 weeks of development. The process was intensified as follicle formation was initiated (16 weeks onwards). Furthermore, apoptosis of germ cells is almost negligible in the ovaries of newborn infants (Vaskivou et al., 2001; D.N.Modi, unpublished observations). These observations suggest that, unlike the murine counterpart, the risk of germ cell degeneration in the human ovary is throughout the meiotic prophase and even during folliculogenesis. The risk is almost negligible upon completion of follicle formation.
Along with the germ cells, apoptosis of some granulosa/ pre-granulosa cells was also detected in the developing ovary after 16 weeks, but the exact number of apoptotic somatic cells could not be estimated with confidence. Interestingly, Vaskivou et al. (2001) also demonstrated apoptosis of some granulosa cells in second trimester human fetal ovaries but the functional significance of this phenomenon is currently obscure.
In the fetal XO ovaries, the numbers of apoptotic oocytes were higher as compared to age-matched controls. At 15 weeks nearly 50% of oocytes were TUNEL positive; 70% of germ cells were apoptotic in the ovary of 20 week old XO fetus. In comparison, only 3–5% of germ cells were apoptotic in age-matched normal XX ovaries. Although these results are based on a limited number of fetuses it is tempting to suggest that the massive germ cell loss that occurs in the ovaries of Turner’s syndrome fetuses (Singh and Carr, 1966) is by programmed cell death in early gestation itself; by late second trimester the ovary is devoid of oocytes, resulting in a streak gonad in infancy and adulthood (Rivelis et al., 1978; Cunniff et al., 1991). To the best of our knowledge this is the first report demonstrating apoptosis as a mechanism of germ cell loss in the XO gonads. However, it is of interest to note that not all oocytes in the XO ovaries studied at 15 and 20 weeks of development were apoptotic, suggesting that some of the normal oocytes may form primordial follicles at least in some cases. Indeed, follicles have been detected in a proportion of adolescent girls with Turner’s syndrome (Hreinsson et al., 2002).
At present, it is unclear why germ cells degenerate rapidly in the XO fetal ovaries; defective meiotic paring due to absence of the second sex chromosome has been postulated as a possible reason for this germ cell degeneration (Burgoyne and Baker, 1985). However, XO mice have normal ovaries and are even fertile, albeit they suffer from premature ovarian failure (McLaren, 1991). Furthermore, in human XO ovaries, the arrest in oogenesis occurs at the very early stages of meiotic prophase even before chromosome paring is established (Speed, 1986; Cunniff et al., 1991). Thus it is unlikely that abnormal paring during meiosis may cause germ cell death in fetal XO ovaries. Thus the precise mechanism(s) involved in accelerated rates of germ cell apoptosis in the XO gonads needs to be established.
During growth of mammalian oocytes, the coupling of granulosa cells amongst themselves and with the oocyte occur via gap junctions and tight junctions (Motta et al., 1997; reviewed in Kidder and Mhawi, 2002). Defective coupling leads to early germ cell loss from fetal ovaries (Juneja et al., 1999; Kidder and Mhawi, 2002). At present we do not know if gap and tight junctions in 45X ovaries are defective; but ultrastructural studies reveal a lack of tight junctions in the endometrium of 45XO adults (Rogers et al., 1992). Thus it is possible that in 45X ovaries absence of gap and/or tight junctions may lead to defective oocyte–granulosa cell coupling, impairing primordial follicle formation and hence germ cell apoptosis.
Not much is known regarding the molecular mechanisms regulating apoptosis in fetal gonads. Recent studies have shown that at the molecular level, prenatal oocyte apoptosis appears to be regulated by Bcl–Bax interactions, Fas, Fas ligand, stem cell factor, and transcription factors such as GATA-4 (Felici et al., 1999; Matsui et al., 2000; Rucker et al., 2000; Vaskivou et al., 2001). Several key apoptotic factors such as Bcl, Fas, FasL and caspase are thought to regulate apoptosis in the postnatal testis (Dolci et al., 1991; Lee et al., 1997; Kierszenbaum, 2001). Recently, it has also been demonstrated that germ cell apoptosis occurs by p53 dependent and independent mechanisms. p53 independent germ cell apoptosis is reported to be mediated by Fas dependent mechanisms at least in the cryptorchid mouse testis (Yin et al., 2002).
In summary, the present study demonstrates that depletion of germ cells occurs in human fetal ovaries and testis during mid-gestation by apoptosis. Chromosome aneuploidy by some means accelerates germ cell apoptosis in the gonads during mid-trimester, leading to gonadal dysgenesis in adulthood.
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Acknowledgements |
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This work is a part of the PhD thesis work of D.M. We are thankful to Dr Sudha Gangal (Ex director BJWHC) for her constant support and critical comments. We are extremely grateful to Dr L.Vasudevan and Dr L.Rajgopal (Anatomy Department KEMH) and Dr M.Bhattacharya and Dr V.Salvi (Obestetrics and Gyenecology Dept KEMH) for their permission and help in collection of normal fetal gonads. We thank Dr A.Maitra, Molecular Endocrinology Department, NIRRH for the StAR antibody.
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