Molecular Human Reproduction, Vol. 5, No. 8, 737-741,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Oncogenes and tumour suppressor genes in first trimester human fetal gonadal development
1 Division of Obstetrics and Gynaecology, City Hospital, Hucknall Road, Nottingham, NG5 1PB, 2 Department of Obstetrics and Gynaecology and 3 Department of Immunology, University of Liverpool, Liverpool L69 3BX, UK
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Abstract |
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Tumour suppressor genes and oncogenes that control proliferation and apoptosis are known to play an important role in embryogenesis, second trimester fetal oocyte loss, adult ovulation, and in adult male testicular degeneration. We have examined tumour suppressor genes, oncogenes and oestrogen receptors during first trimester human gonadal differentiation to investigate their role at this crucial phase in development. Immunohistochemistry was used to localize the gene products of Bcl-2, c-erB-2, c-myc, p53, nm23 and oestrogen receptor. As gonadal development occurred at 6–12 weeks gestation, a changing pattern of expression was observed that varied in different cell types. The oestrogen receptor was not present in oogonia, spermatogonia and supporting cells during the first trimester. This study highlights the importance of oncogenes and tumour suppressor genes in first trimester gonadal development.
cell cycle/development/gonadal/oestrogen receptors
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Introduction |
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Oncogenes and tumour suppressor genes that control cellular proliferation, differentiation and apoptosis are known to play an important role both in early mammalian development and gonadal function in adult life. Apoptosis and those genes that control this process have been found in preimplantation mouse and human embryos (Jurisicova et al., 1998
; Warner et al., 1998), mouse blastocyts (Hardy, 1997), during early placental invasion and differentiation (Uckan et al., 1997; Quenby et al., 1998) and second trimester human fetal oocyte loss (De Pol et al., 1997). In adults, ovulation is dependent on a complex balance between pro- and anti-apoptosis susceptibility genes (Tilly et al., 1997). Adult male testicular degeneration is also affected by apoptosis (Brinkworth et al., 1997).
During the first 3 months of gonadal life, extensive cellular proliferation and sexual differentiation are known to occur. Epidemiological evidence suggests that it is during the first trimester that fetal gonadal proliferation and differentiation are most susceptible to damage. This evidence from comes from studies of adults who were exposed to diethylstilboestrol (DES) as fetuses. DES was given to pregnant women during the first trimester for the prevention of miscarriage. Male children exposed to DES were found to have significantly lower sperm counts (Henderson et al., 1976) and adult males exposed to DES in utero have an increased risk of testicular cancer (Aria et al., 1983). In the female, DES exposure in the first trimester has been reported to cause an increase in clear cell adenocarcinoma of the cervix and vagina (Herbst et al., 1979), but no increase in carcinoma of the ovary.
Gonadal development starts with the migration of primordial germ cells (PGCs) from the yolk sac to the gonadal anlage in the sixth week of gestation. No sexual differences are thought to be apparent in the gonads up to 8 weeks gestation (6 weeks post-conception) (Drews, 1995). After this time, male gonads have formed primitive testicular cords that can be recognized using light microscopy. Both PGC and epithelial cells are essential for normal gonadal development (Drews, 1995). In the male gonad, outside the testicular cords, Leydig cells differentiate, proliferate and secrete testosterone that is important in testicular descent and inducing the masculine phenotype.
This study examined the presence of gene products that affect the probability that any cell will engage in an apoptotic as opposed to a proliferative programme (Evan et al., 1995). Bcl-2 inhibits programmed cell death (Bagg and Cossman, 1993). The c-erB-2 oncogene encodes a truncated form of the epidermal growth factor receptor. Cells expressing this oncogene behave as though they are constantly being signalled to proliferate by a growth factor (Alberts et al., 1994). c-myc is part of the post receptor intracellular signalling pathway for the stimulation of cell proliferation by a growth factor (Alberts et al., 1994). Cells in which c-myc expression is switched on independently of growth factors undergo apoptosis (Alberts et al., 1994). p53 has been shown to induce apoptosis (Yonish-Rouch et al., 1991). When a DNA fault is detected, p53 acts in the presence of large amounts of damage, to push the cell into apoptosis (Perry and Levine, 1993). nm23 (non-metastatic) is a tumour suppressor gene which seems to have a role in preventing tumour metastasis (Golden et al., 1993), but its molecular mechanism of action has yet to be fully understood (DeLa Rosa et al., 1994). However, in detailed work on mouse embryogenesis, nm23 expression has been found to be associated with increasing differentiation (Lakso et al., 1992).
For oestrogenic compounds to affect fetal gonadal development, oestrogen detecting receptors should be present as proliferation and differentiation occurs. Oestrogen receptors have been found to have important physiological effects on adult male reproduction in mammals (Hess et al., 1997).
We have examined human fetal gonads at 6–12 weeks gestation for gene products thought to be involved in proliferation, apoptosis and differentiation and, in neighbouring sections, oestrogen receptors.
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Material and methods |
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Permission was obtained from the Local Ethical Committee to collect embryonic tissue and all patients gave informed consent to the research. First trimester embryos of gestational age 6–12 weeks were obtained following elective termination of apparently normal pregnancies, from women who were not taking any oestrogenic drugs. Embryos were dated using a combination of pre-operative ultrasound, crown–rump length and post-abortion measurement of the foot length. Visualization of testicular cords indicated male sex and those without gonadal cords were assigned female sex. Two embryos of each sex were used for each gestational age studied. The specimens (n = 16) were washed in phosphate-buffered saline (PBS) within 5 min and examined under a binocular dissecting microscope. Fetal pelvises were selected and fixed in freshly prepared 4% paraformaldehyde (4 g in 100 ml of 100 mmol/l PBS, pH 7.2) at 4°C for 24 h. The fetal pelvises were then embedded in paraffin wax with the gonads as close to the surface of the block as possible. Sections (5 µm) were cut and mounted on poly-L-lysine coated slides. Every tenth slide was stained with haematoxylin and eosin and examined in order to identify the fetal gonads.
Immunohistochemistry
The slides containing fetal gonads were air-dried and heated for 20 min at 60°C before dewaxing with xylene and rehydration. Six different mouse monoclonal antibodies were used in this study (Table I). Two negative controls were used, the primary antibody was replaced either with Tris-buffered saline (TBS), containing 0.1% bovine serum albumin (BSA) or by mouse immunoglobulin G (IgG) at the same concentration as the primary antibody. Positive controls were tissues known to express the antigen being studied (Table I) and, in the case of oestrogen receptors, maternal decidua obtained from the same pregnancy. In order to maximize visualization of the gene product, each antibody was tested at a series of dilutions, with and without antigen retrieval (unmasking of epitopes after paraformaldehyde fixation and paraffin embedding) and in the presence and absence of enhancement steps. Antigen retrieval conditions (pH of the buffer, heating intensity and time) were altered in order to ascertain optimal antigen detection with minimal background staining for each antibody used (Shi et al., 1997). For Bcl-2, c-erB-2, p53, and oestrogen receptor, optimization of immunohistochemical staining was achieved by pressure cooking the sections in 10 mmol/l citrate buffer (pH 6.0), for 4 min at low pressure in an aluminium pressure cooker. Antigen retrieval did not enhance the staining for c-myc and nm23. The antibody dilutions found to give optimal staining are shown in Table I. After dilution of the antibody in TBS containing 0.1% BSA, sections were incubated at room temperature for 60 min. The second antibody, a rabbit anti-mouse Ig antiserum (Dako Ltd, Bucks, UK) was then applied at 1:50 dilution for 30 min at room temperature. This was followed by treatment with alkaline phosphatase anti-alkaline phosphatase complex (APAAP) (1:50 dilution for 30 min at room temperature, Dako Ltd). To enhance the intensity of the APAAP labelling reaction, the second and third incubation steps were repeated (each step for 30 min). For development of alkaline phosphatase, the new fuchsin method was employed, the substrate solution containing Tris–HCl, pH 8.8 (20 min, room temperature). 0.2 mmol/l levamisole was used to inhibit endogenous alkaline phosphatase. Finally the sections were counterstained with haematoxylin for 20 s and mounted in Aquamount (BDH Laboratories, Poole, UK).
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Results |
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The negative controls, performed by leaving out the primary antibody, showed no staining with APAAP and Fuchsin Red, indicating that endogenous alkaline phosphatase had been effectively destroyed. There was also no staining with the mouse IgG controls except at a dilution of 1:10 when a light background staining was observed.
All immunohistochemistry was repeated twice on different days to ensure the intensity and pattern of the staining was reproducible. Two embryos of each sex were studied at each gestational age. Each slide was examined by three observers; one not blinded (SQ), one blinded to the gestation (MRG) and one blinded to the antibody used and the gestation (CB). Each observer reported the presence of strong staining, equivalent to that seen in the positive controls, weak staining or absence of staining. The results are shown in Tables II and III, where each entry represents the most frequently reported pattern seen by the three observers on two embryos of the same gestation.
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We found that at 6 weeks gestation, some gonads showed larger cells identified as oogonia irregularly interspersed with smaller epithelial cells (Figure 1a
). Other gonads had cells that were lined up in columns of cells which were identified as primitive testicular cords (Figure 1b). Those gonads with early testicular cords were assigned male sex. However, at 6 weeks gestation, the gene expression observed in the gonads was the same in those gonads assigned female and male sex (Tables II and III
), which is consistent with their lack of differentiation at this gestation.
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Bcl-2 was seen in the supporting cells of both the fetal ovary (Figure 1a
) and testis (Figure 1b) at 6–8 weeks gestation, but only in ovarian epithelial cells at 10 and 12 weeks (Table III). A negative control for Bcl-2 in fetal ovary is shown in Figure 1c. During weeks 8, 10 and 12, Bcl-2 was observed in the Leydig cells of the testis (Figure 1d: negative control Figure 1e, Table III). c-erB-2 was seen in the proliferating gonadal supporting cells in both fetal testis and ovary (Tables II and III, Figure 1f). c-erB-2 was also present in the Leydig cells of the testis at all gestations examined (Table II). c-myc was present in spermatogonia and Sertoli cells from 8–12 weeks gestation, and at 12 weeks in the Leydig cells (Table II). In the ovary, c-myc was demonstrated in oogonia only at 10 and 12 weeks (Table III). The p53 gene product was optimally observed when a high concentration (1:10) of the primary antibody was used. When mouse IgG was used as a control at the same concentration, weak cytoplasmic (but not nuclear) staining was seen. Therefore, only nuclear staining was counted as positive in this case. p53 nuclear staining was present in ~half the spermatogonia at 10 and 12 weeks gestation, and half the oogonina at 8 weeks gestation but all oogonia were positively stained with p53 at 10 and 12 weeks gestation (Tables II and III). The nm23 gene product was found in the oogonia between 8 and 12 weeks gestation, but only in the spermatogonia at 10 and 12 weeks. nm23 was also demonstrated in differentiating Leydig cells. In decidual tissue, nuclear staining for the oestrogen receptor was observed in glandular epithelial cells as expected (Salmi et al., 1996). In the gonadal tissue, only positive cytoplasmic staining was seen and there was no nuclear staining: these tissues were, therefore, considered negative for the oestrogen receptor (Salmi et al., 1996). |
Discussion |
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First trimester germ cell differentiation and proliferation must be tightly regulated as uncontrolled stem cells are potentially malignant, but insufficient proliferation may later be a causative factor in adult infertility. Other gonadal cells that surround these germ cells play an important role in gonadal development and function. The inter-dependence of gonadal germ cells and their surrounding cells has been recently highlighted (Perez and Tilly, 1997
). Mouse oocyte apoptosis was found to be affected by the surrounding cumulus cells.
We have found that the genes (Bcl-2, c-erB-2, c-myc, p53, nm23) were present during the first trimester of human gonadal development, furthermore their pattern of expression changed as the first trimester progressed and different cell types expressed varying combinations of genes. Bcl-2 was not found in germ cells; thus they were not protected from apoptosis by this tumour suppressor gene (Tables II and III). Bcl-2 in ovarian epithelial cells could aid cell population expansion by preventing apoptosis. Rapid proliferation of ovarian epithelial cells may be necessary for enough cells to be produced to surround the oogonia. In the Sertoli cells, the expression of Bcl-2 dramatically changed from being present at 6–8 weeks then absent at 10–12 weeks gestation. This changing expression of Bcl-2 could be explained by the fact that Sertoli cells proliferate in waves (Andersen and Byskov, 1996). c-erB-2, a proliferation promoting oncogene, was present on all supporting cells but absent from the oogonia and only weakly detected on 10 and 12-week spermatogonia (Tables II and III). This suggests that stimulation of proliferation in germ cells may be different from that in supporting cells. The presence of c-myc in gonadal germ cells (Tables II and III) indicated that these cells could be susceptible to apoptosis in certain conditions. One mechanism for control of abnormal germ cell proliferation may be effected by p53, whose immunolocalization represents physiological up-regulation of the p53 gene (Milner, 1991; Marzusch et al., 1995). p53 was localized to spermatogonia and oogonia at the end of the first trimester. This early gestation is an ideal time to promote apoptosis in cells containing damaged DNA, as abnormal germ cell DNA that survives to adulthood from this gestation may lead to reproductive failure or malignancy. nm23 was seen on the most differentiated germ cells, the more mature spermatogonia and the medullary oogonia. This finding of increasing nm23 expression with differentiation is in accordance with a previous publication on nm23 in mouse embryogenesis (Lakso et al., 1992).
Although cytoplasmic staining was seen, there was no nuclear staining for oestrogen receptors in either the male or female first trimester gonads. This is in contrast to the epidemiological evidence, suggesting that any effects of oestrogen on first trimester gonads would have to be indirect.
This study has revealed a complex pattern of tumour suppressor gene and oncogene expression in human first trimester fetal gonads. Further research is necessary to elucidate the exact processes that influence this critical period of human development.
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Notes |
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4 To whom correspondence should be addressed
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Submitted on February 5, 1999; accepted on May 13, 1999.
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