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Original Studies |
Nutrition and Development, The Rowett Research Institute (T.J.M., R.G.L.), Bucksburn, Aberdeen, Scotland AB21 9SB; and Departments of Obstetrics and Gynecology (T.J.M., P.A.F., D.R.A., R.G.L.) and Medical Genetics (N.H.), University of Aberdeen, Foresterhill, Aberdeen, Scotland AB25 2ZD
Address all correspondence and requests for reprints to: Dr. Richard G. Lea, Division of Maternal-Fetal Biology, The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland AB21 9SB. E-mail: rgl{at}rri.sari.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/C17–20-lyase (P450c17; messenger
ribonucleic acid and protein) were confined to the Leydig cells. The
number of immunopositive cells increased between 13 and 19 weeks
(P < 0.001). P450c17 mRNA (in situ
hybridization) and protein were localized to the same population of
interstitial Leydig cells. Androgen receptor and Bcl-2 protein
(anti-apoptotic) were gradually restricted to the peritubular myoid
cells as gestation progressed. Conversely, Bax protein (pro-apoptotic)
was predominantly localized to the tubule Sertoli cells, whereas the
germ cells were Bax immunonegative. In conclusion, human fetal Leydig cell hyperplasia is characterized by increasing numbers of proliferating cells and increased expression of steroidogenic enzymes. The Bcl-2-positive, Bax-negative status of the peritubular myoid cells may be a strategy for cell survival.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, the male gonad begins differentiation in the sixth week of gestation with the gradual development of testicular cords and interstitium from gonadal blastomas (22). Testicular cords are formed by the aggregation of Sertoli cells, and the germ cells are enclosed within these structures. The interstitial fetal Leydig cells begin differentiation during the eighth week of gestation (23) and undergo rapid proliferation until week 18, where upon a plateau is reached, and Leydig cell number remains at this level until the third trimester (24, 25). The period of Leydig cell hyperplasia is also coincident with a peak in fetal testosterone synthesis between 14 and 18 weeks gestation (23). Consequently, this period of Leydig cell hyperplasia is thought to be important in the development of the normal testis.
The balance among tissue growth, differentiation, and apoptosis is
crucial for normal fetal gonad development (26, 27). To
investigate the proliferative vs. apoptotic pathways in the
human XY gonad between 13 and 19 weeks gestation, we used
immunohistochemistry to localize expression of proliferating cell
nuclear antigen (PCNA), c-Myc, Bcl-2, and Bax proteins. We have
coordinated these expression profiles with the sequential
differentiation of Leydig and peritubular myoid cells using markers
for 3ß-hydroxysteroid dehydrogenase (3ß12HSD), cytochrome P450
17-hydroxylase/C17–20-lyase (P450c17), and
the androgen receptor (AR).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ethical committee approval for the study was obtained from the Grampian Health Board and University of Aberdeen joint ethical committee (Project 2470), and tissue collection abided by the Polkinghorne committee recommendations for the use of human fetal material in research. Informed consent was obtained from women undergoing surgical termination of normally progressing second trimester pregnancies, and fetuses were collected between 13 and 19 weeks gestation. Testes were grouped according to gestational age as follows: 1) 13–14 weeks (n = 4), 2) 15–16 weeks (n = 4), 3) 17 weeks (n = 4), and 4) 18–19 weeks (n = 4). Testes were removed and immersion-fixed in Bouin’s solution for 5.5 h at 4 C, rinsed, and stored in 70% ethanol until routinely embedded in wax.
Immunohistochemistry
Sections (5 µm) were cut onto 3-aminopropyltriethoxysilane (Sigma, Poole, UK)-coated slides for the immunohistochemical localization of 1) PCNA; 2) protooncogene products c-myc, bcl-2, and bax; 3) steroidogenic enzymes P450c17 and 3ßHSD; and 4) AR. Commercially available antibodies were used for the detection of PCNA and AR (Novacastra Laboratories Ltd., Newcastle, UK), c-Myc and Bax (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), and Bcl-2 (DAKO Corp., High Wycombe, UK). Antibodies for the detection of P450c17 and 3ßHSD were supplied by Prof. Ian Mason (University of Edinburgh, Edinburgh, UK).
Tissue sections were dewaxed in Histoclear (National Diagnostics, Hessel Hull, UK), rehydrated through a graded ethanol series, and washed in 0.1 mol/L Tris-buffered saline (pH 7.6) twice for 5 min each time. Pretreatment of the sections was necessary for localization of Bcl-2, Bax, 3ßHSD, P450c17, and AR (see below). Treatment with 3% hydrogen peroxide in water for 5 min at room temperature quenched endogenous peroxidase activity. All tissue sections were subjected to an endogenous biotin block using an avidin/biotin blocking kit (Vector Laboratories, Inc., Peterborough, UK) in accordance with the manufacturer’s protocol. This was followed by a nonimmune block using normal horse serum (Vector Laboratories, Inc.) for 20 min at room temperature. Tissues were then incubated with the appropriate primary antibody as follows: 1) immunolocalization of PCNA: monoclonal mouse anti-rat PCNA diluted 1:100 in normal horse serum for 60 min at room temperature; 2) immunolocalization of c-Myc: monoclonal mouse anti-human c-Myc at a dilution of 1:50 overnight at 4 C; 3) immunolocalization of Bcl-2: monoclonal mouse anti-human Bcl-2 at a dilution of 1:40 overnight at 4 C; 4) immunolocalization of Bax: polyclonal rabbit anti-human Bax at a dilution of 1:50 for 60 min at room temperature; and 5) immunolocalization of 3ßHSD and P450c17: rabbit anti-human 3ßHSD (type 1) and anti-pig P450c17 were both used after diluting 1:500, and tissue sections were incubated overnight at 4 C.
All tissue sections were labeled using an avidin-biotin-peroxidase detection system (Vector Laboratories, Inc.) followed by incubation with 3,3'-diaminobenzidine (DAB; Vector Laboratories, Inc.) for 5–15 min for brown color development. Finally, sections were counterstained with hematoxylin, dehydrated in graded ethanols, cleared in xylene, and coverslipped using Pertex mounting medium (Cellpath, Hemel Hempstead, UK). Negative controls were performed by replacing the primary antibody with normal mouse IgG at the same concentration.
Pretreatment of sections for Bcl-2, 3ßHSD, P450c17, and AR
To expose these epitopes, tissue sections were subjected to antigen retrieval by microwaving in 0.01 mol/L citrate buffer (pH 6.0) on full power twice for 5 min each time (Bax), three times for 5 min each time (Bcl-2), or four times for 5 min each time (3ßHSD, P450c17, and AR) and were allowed to stand for an additional 20 min.
Double immunostaining for AR and 3ßHSD, and PCNA and 3ßHSD
AR and 3ßHSD epitopes were exposed by microwaving three times for 5 min each time on full power and then were allowed to stand for an additional 20 min. Rabbit polyclonal anti-human AR was used after diluting 1:10, and tissue sections were incubated overnight at 4 C followed by biotinylated anti-rabbit IgG. The avidin-biotin-peroxidase labeling system was used and developed with DAB to produce a brown color on positive cells. After washing in Tris-buffered saline-Tween, the sections were incubated with the rabbit anti-3ßHSD antibody followed by biotinylated anti-rabbit IgG. The same labeling system was employed; however, Very Intense Purple (Vector Laboratories, Inc.) was used to develop the reaction, thereby generating a pink color. The following controls were carried out to ensure specific staining was observed 1) anti-AR was replaced by rabbit IgG before incubating with anti-3ßHSD; 2) anti-AR was followed by rabbit IgG in place of anti-3ßHSD; and 3) both primary antibodies were replaced with rabbit IgG. To ensure that staining could not be attributed to antibody cross-reactivity, the staining procedure was repeated with the primary antibodies switched.
Double immunostaining for PCNA and 3ßHSD was carried out using a similar protocol. As PCNA immunoreactivity is destroyed by microwaving, no antigen retrieval procedure was employed. Cells immunopositive for PCNA were detected using DAB as substrate, whereas 3ßHSD was visualized using Very Intense Purple. Specific staining was verified after running the controls described above, and antibody cross-reactivity was considered negligible after repeating the staining procedure with the primary antibodies switched over.
In situ hybridization for P450c17
Riboprobes for human P450c17. Human P450c17-pUC18 plasmid was provided by Prof. Stephen Hillier (University of Edinburgh, Edinburgh, UK). The plasmid was linearized with AccIII or BamHI for transcription with SP6 or T7 ribonucleic acid (RNA) polymerase to generate antisense and sense probes, respectively. Probes were radioactively labeled with 35S using a commercially available kit (Promega Corp. UK Ltd., Southampton, UK). Briefly, probes were incubated for 60 min at 37 C with 40 mmol/L Tris (pH 7.9), 6 mmol/L MgCl2, 2 mmol/L spermidine, 10 mmol/L NaCl, 10 mmol/L dithiothreitol (DTT), 40 U ribonuclease inhibitor, 1.85 MBq [35S]UTP (NEN Life Science Products, Boston, MA), and the appropriate RNA polymerase. After the addition of deoxyribonuclease, the probes were incubated at 37 C for an additional 30 min, after which purification was performed using RNA spin columns (Sigma).
In situ hybridization. In situ hybridization was carried out as previously described by Hoggard et al. (28) with a few minor modifications. Briefly, 5 µm sections cut over diethylpyrocarbonate (Sigma)-treated water and taken onto 3-aminopropyltriethoxysilane-coated slides were dewaxed with xylene and rehydrated through a decreasing ethanol series. Slides were immersed in 0.2 mol/L HCl for 20 min and then placed in 2 x SSC (1 x SSC contains 0.15 mol/L NaCl and 15 mmol/L sodium citrate, pH 7.0) for 30 min. Tissue sections were then treated with 2 µg/mL proteinase K (Sigma) in 0.2 mol/L Tris-HCl and 0.05 mol/L ethylenediamine tetraacetate (pH 7.6) for 20 min at 37 C, postfixed in 0.4% paraformaldehyde (Sigma) in phosphate-buffered saline for 20 min at 4 C, and washed in phosphate-buffered saline. Slides were acetylated with 0.25% acetic anhydride (Sigma) in 0.1 mol/L triethanolamine for 10 min before immersion in 2 x SSC and finally dehydration through graded ethanols, then air-dried.
Sections were hybridized with 106 cpm [35S]RNA in 60 µL hybridization buffer at 59 C overnight. Next day, sections were washed in 4 x SSC for 20 min, incubated in ribonuclease A (Sigma) solution (0.02 mg/mL in 0.01 mol/L Tris-HCl, 0.5 mol/L NaCl, and 0.1 mol/L ethylenediamine tetraacetate, pH 7.6) for 30 min at 37 C, and washed in 2 x SSC-DTT (SSC with 1 mmol/L DTT; Sigma). Slides were immersed in 1 x SSC-DDT for 10 min and in 0.5 x SSC-DTT for 10 min, then washed at 60 C with 0.1 x SSC-DTT before cooling in 0.1 x SSC. Sections were dehydrated in graded ethanols, air-dried, dipped in K5 nuclear emulsion (Ilford Ltd., Mobberley, UK), and exposed for 2 weeks at 4 C.
Slides were immersed in Phenisol developer (Ilford Ltd.) for 5 min, washed briefly, and fixed in Hypam fixing solution (Ilford Ltd.) for an additional 5 min. Slides were counterstained with Harris hematoxylin (Shandon International, Runcorn, UK) and examined by light microscopy.
Quantification and image analysis
For each marker, sections were visually scored on an arbitrary intensity scale by the same operator (T.M.). PCNA, c-Myc, 3ßHSD, and P450c17 were then selected to be quantified by computer-aided image analysis. The system was composed of an Olympus Corp. microscope (x20 objective; New Hyde Park, NY) and Hamamatsu digital camera (Hamamatsu, Bridgewater, NJ) connected to a computer running Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The tubular regions visible on captured images were digitally removed, and quantification was performed on the remaining interstitial tissue. The total area of positively stained cells (brown color) was measured and expressed as a percentage of the interstitial tissue area. In addition, PCNA staining within the tubules was quantified separately. Quantification was carried out over six randomly selected fields of view, after which the mean and SE had stabilized. Computer-aided image analysis was not performed for Bax, as no obvious changes were noted by visual scoring. Bcl-2 and AR showed an increase in staining intensity, but not area; therefore, quantification using digitized images was not possible.
Statistical analysis
Image analysis data were subjected to one-way ANOVA, and the Bonferroni/Dunn post-hoc test was used to compare values for PCNA, c-Myc, 3ßHSD, and P450c17 at different stages of gestation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the human fetal testis, PCNA-immunopositive cells were detected
in the tubules at all gestational ages examined (Fig. 1
, a–c),
possibly including the Sertoli population. PCNA immunostaining was also
detected in the Leydig cellcontaining interstitial area between 15
and 17 weeks gestation (Fig. 1b); however, only low level
immunostaining was observed at earlier (Fig. 1a) and later (Fig. 1c)
gestational stages. Image analysis confirmed that the number of
PCNA-immunopositive interstitial cells exhibited a significant increase
from 13–14 weeks to 15–16 weeks gestation (by ANOVA,
P < 0.05), followed by a significant decrease at
18–19 weeks (by ANOVA, P < 0.05; Fig. 2a). PCNA-positive cells within the
tubules were consistently observed at a high level (Fig. 2a). c-Myc was
exclusively localized to the cells in the interstitial area (Fig. 1m),
and image analysis showed that expression levels did not change
significantly between 13 and 19 weeks gestation (range expressed as the
mean percent positive cells ± SEM,
36.88 ± 8.35 at 13–14 weeks, 43.07 ± 7.24 at 15–16 weeks;
P > 0.05).
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Bax was predominantly localized to the Sertoli cells within the
tubules, although the germ cells were Bax immunonegative (Fig. 1
, d–f). Light immunostaining was consistently observed in the
interstitial area. Immunostaining intensity for Bax was independent of
gestational age. Testicular cells immunopositive for Bcl-2 were
detected in the interstitium and in the flattened peritubular myoid
cells (PMCs) surrounding the tubules (Fig. 1, g–i). As gestation
progressed from 13 to 19 weeks, the Bcl-2 immunostaining intensity
markedly increased in PMCs, but decreased in the interstitial area.
PMCs were notably Bax immunonegative while preserving high levels of
Bcl-2.
Immunolocalization of 3ßHSD and P450c17
Immunostaining for 3ßHSD (Fig. 1, j–l) and P450c17 (Fig. 1n) allowed identification of the steroidogenically active Leydig
cell population in the interstitial area. Subsequent image analysis
showed the number of P450c17- and 3ßHSD-immunopositive cells in the
interstitium significantly increased (by ANOVA, P <
0.001) over the period of investigation (Fig. 2b).
Double immunolocalization of AR and 3ßHSD: comparison with Bcl-2 immunostaining
AR was predominately expressed by the PMCs, with low level
immunostaining in the interstitial area (Fig. 1, j–l). Conversely,
3ßHSD was specifically localized to the Leydig cell-containing
interstitium. The expression profiles of 3ßHSD and AR are therefore
markedly different. Examination of Bcl-2-immunostained serial sections
revealed that AR and Bcl-2 were colocalized to the PMC population (Fig. 1, g—i, Bcl-2; Fig. 1, j—l, AR and 3ßHSD).
Double immunolocalization of PCNA and 3ßHSD
PCNA and 3ßHSD were immunolocalized to two distinct populations
of interstitial cells. No double labeling was observed (Fig. 1o).
Expression of P450c17 messenger RNA (mRNA)
In situ hybridization using a
35S-labeled P450c17 complementary RNA probe
resulted in hybridization in the interstitial area of the 13-week
gestation testis (Fig. 3a). The silver
grain clusters indicative of hybridization were more numerous in testis
collected from later gestational stages (Fig. 3b). In all cases sense
controls showed no signal above background levels (Fig. 3c). P450c17
mRNA and protein were therefore colocalized (Fig. 3, mRNA; Fig. 1n, protein).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the Leydig population increased in number between 13 and 19 weeks, the proliferating interstitial cells were not steroidogenic. Furthermore, our data suggest that at the mRNA and protein levels, the increased steroid-secreting capacity of the fetal testis reflects increased numbers of Leydig cells rather than increased synthesis. This suggests that precursors of Leydig cells exist in the interstitial region before differentiation and that after undergoing a phase of rapid division they are recruited into the nonproliferative steroid-secreting population. This emphasizes the importance of proliferation of Leydig cell precursors to meet the steroidogenic requirements of the human fetal testis. In support of this, mature fetal Leydig cells have been shown to have no mitotic activity in the rat (29). In the human, however, proliferation of other interstitial cell types cannot be discounted.
Although proliferation was assessed through localization of PCNA and c-Myc, these markers had different temporal and spatial expression patterns during the gestational window examined. PCNA was detected in tubules where levels remained constant and consistently higher than those observed in the interstitial area from 13–19 weeks gestation. This supports previous studies that reported Sertoli cell proliferation during human and animal fetal life (30). The persistent interstitial expression of c-Myc may reflect its central role in the regulation of cellular growth and differentiation during this phase of rapid growth of the Leydig cells.
The balance between cell proliferation and cell death by apoptosis is critical during morphogenesis, and this is controlled in part through the expression of a large family of related apoptotic regulatory genes. The prototype apoptosis inhibitor is the protein product of the protooncogene bcl-2. The apoptosis-inhibiting effect of Bcl-2 occurs through homodimerization or heterodimerization with apoptosis- inducing factors such as Bax. Overexpression of Bax negates the protective mechanism of Bcl-2 and promotes apoptotic cell death (31). It follows, therefore, that the Bcl-2/Bax ratio is an important determinant of cell survival. The localization of Bax to Sertoli cells and the up-regulation of Bcl-2 in the interstitium along with consistent expression of c-Myc suggest that the competing pathways regulating the balance between proliferation and apoptosis favor expansion of the interstitium during this developmental period.
A critical observation in the current study was the colocalization of Bcl-2 and AR to the PMCs. These data indicate that the PMCs may play an important role during the development of the human fetal testis. Androgens are known to be important in the regulation of testicular apoptosis in the adult (32, 33) and may play a similar role in the fetus.
In adults the Sertoli cells are understood to be the main site for the action of androgens (34); however, adult human and rat PMCs and Leydig cells have also been reported to contain AR (35, 36). In contrast, in the fetal rat AR was specifically localized to the PMCs (37). In support of these studies, our data in the human fetal testis show that AR is also localized to PMCs, with no expression in Sertoli cells.
In contrast to that of PMCs, animal studies indicate that apoptotic cell death of fetal germ cells is a normal feature of testis development late in gestation (27, 38). Germ cell apoptosis also occurs in adult rats and mice, and this is thought to account for the 25–75% loss of the expected sperm yield during normal spermatogenesis (27). In the human fetal testis Bax was localized to the tubules, although the germ cells were notably Bax immunonegative. As the germ cell Bcl-2/Bax ratio is not in favor of apoptosis, germ cell death may not occur from 13–19 weeks gestation in the human. However, other apoptotic regulatory genes may be involved, and confirmatory studies are necessary to localize cells with fragmented DNA in human fetal testis.
In conclusion, the period of Leydig cell hyperplasia in human fetal testis has been characterized by 1) a transient, but significant, increase in the number of proliferating cells and 2) a significant increase in the number of interstitial cells expressing the steroidogenic enzymes 3ßHSD and P450c17. The proliferating interstitial cells were not immunopositive for 3ßHSD and are therefore probably Leydig cell precursors. AR and Bcl-2 proteins were gradually restricted to the peritubular myoid cells as gestation progressed from 13 to 19 weeks, and the predominance of Bcl-2 may be a strategy for survival of this cell population. In contrast, Bax was mainly localized to the tubules, although the germ cells were negative. These studies suggest that the regulation of apoptosis and cell proliferation in the developing human fetal testis is critical for steroidogenesis, steroid action, and normal male development.
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Acknowledgments |
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Footnotes |
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Received December 16, 1999.
Revised May 22, 2000.
Revised August 29, 2000.
Accepted September 2, 2000.
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References |
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-hydroxylase/C17–20
lyase in the fetal rat testis is reduced by maternal exposure to
exogenous estrogens. Endocrinology. 137:1063–1070.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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), PCNA-tubules (
), 3ßHSD (
), and
P450c17 (•) in human fetal testis between 13 and 19 weeks
gestation. Where common superscripts are shown above
symbols, the values are significantly different (by ANOVA,
P < 0.05) for each variable, not between
variables. Values are expressed as the mean ± SEM
(n = 4).