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INSERM-INRA U 418 (R.O., C.P., B.B., R.H.), Université Paris 7, Tour 33/43, 75251 Paris Cedex 05, France; INSERM-INRA U 418 (P.D.), Hôpital Debrousse, 69222 Lyon, France
Address all correspondence and requests for reprints to: Professor R. Habert, INSERM U 418, Université Paris 7, Tour 33/43, 2 Place Jussieu, 75251 Paris Cedex 05, France. E-mail: habert{at}paris7.jussieu.fr
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Introduction |
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The way in which this pattern of development is regulated is poorly understood. Gonocytes proliferate when genital ridges and their associated mesonephroi are cultured in the absence of hormones or growth factors (12). Similarly, the gonocytes spontaneously reenter mitosis in cultured neonatal testes (9). This suggests that these events are controlled by local factors. There is now evidence that some factors are involved in the intratesticular control of gonocytes development because they are present in the developing testis and/or they affect gonocytes proliferation and/or survival in vitro. These factors are the anti-Müllerian hormone (13), the fibroblast growth factor 2 (14), the leukemia inhibitory factor, the ciliary neurotropic factor (15), the oncostatin M (16), the platelet-derived growth factor and estradiol (17).
Another family of growth factors, the transforming growth factor ß family (TGFßs), is believed to act as a local regulator of adult testicular activities (18, 19). This family consists of three isoforms, TGFß1, TGFß2, and TGFß3, in mammals (20, 21, 22, 23). TGFßs signal by binding to two specific receptors termed type I (TßRI) and type II (TßRII), which are both transmembrane serine/threonine kinases (24, 25).
Our recent immunohistochemical studies showed that both TGFß1 and TGFß2 are present in the developing fetal and neonatal rat testis (26, 27). Immunoreactive material was found in the Sertoli cells as early as fetal day 13.5 for TGFß2 and 14.5 for TGFß1 and both became faint or undetectable from fetal day 18.5 onward. Positive reactions for both isoforms appeared in the fetal-type Leydig cells on fetal day 16.5 and became very intense from day 18.5 onward. The gonocytes had no detectable TGFß1 immunoreactivity, whereas a positive reaction for TGFß2 appeared on fetal day 20.5, was maximal on postnatal day 4, and decreased thereafter. Furthermore, the testes from 20.5-day-old fetuses contain TGFß1 messenger RNA transcripts as revealed by Northern blot analysis and the fetal testes explanted at the same age secreted bioactive TGFß1-like material in culture (28). The effect of TGFßs on the establishment of the germ cell lineage has only been studied at primordial germ cells stage. It was shown that TGFß1 decreases the number of primordial germ cells in cultures taken from 8.5-day-old mouse embryo (29) but has no effect on primordial germ cells taken from 10.5-day-old mouse embryo (5). These data led us to investigate whether TGFßs are involved in the control of gonocytes in the rat testis during fetal and neonatal life. We first examined the effect of TGFßs on the number, proliferation, and survival of gonocytes in organ culture, in which the testicular architecture and intercellular communications are preserved. Then, we investigated the immunolocalization of the transducing TGFß receptors in the fetal and neonatal testis throughout development to obtain a clearer picture of the way TGFßs act.
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Materials and Methods |
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The TGFß receptor antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were raised by immunizing rabbits against synthetic peptides that correspond to amino acids 158 to 179 of human TßRI and 246 to 266 of human TßRII. These sequences are the conserved kinase domains in rodents, humans, and other species (30) and are the same in rats and humans. These receptor antibodies do not cross-react with each other. They have recently been used to study the distribution of TßRI and TßRII in the pig testis throughout development (19).
Animals
Rats of the Wistar strain (Iffa Credo, l’Arbresle, France) were
housed and bred as previously described (31). Briefly, females were
caged with the males for the night, and the day following an overnight
mating was counted as day 0.5 post conception (0.5 dpc). Natural birth
occurred between day 21.5 at 1400 h and day 22.5 at 1800 h.
Because precise timing of the postnatal development was desired, only
pups born between 1900 h on 21.5 dpc and 0800 h on 22.5 dpc
(i.e. 60% of the neonates) were kept. Each litter contained
eight pups.
Pregnant rats from gestational days 13.5 to 20.5 were anesthetized between 1400 h and 1600 h by an ip injection of 4 mg/100 g sodium pentobarbital (Sanofi, Libourne, France), and the fetuses were rapidly removed from the uterus. Fetuses were dissected under a binocular microscope. On fetal day 13.5, the sex of the gonad is not yet morphologically recognizable, and the sex of the fetuses was determined with the sex chromatin test performed on the amniotic membrane as described by Jost (32). At this stage, the two gonads and their associated mesonephroi were collected together because they are closely attached to each other and because there are germ cells in the mesonephric tissue (33). From day 14.5 of fetal life, the sex was determined by the morphology of the gonads, and the testes could be dissected free of other structures. Neonates on day 3 post partum (3 dpp) were killed by cervical dislocation between 1400 h and 1600 h and their testes immediately removed.
Culture procedure
All tissues were cultured as previously described (31). Briefly,
for the oldest stages, the testes were cut into small pieces (4 pieces
on 17.5 dpc and 18 pieces on 3 dpp). The whole testes and their
associated mesonephroi (13.5 dpc), or all the pieces from the same
testis (17.5 dpc and 3 dpp) were placed on a Millipore (Saint Quentin
Yvelines, France) filter (pore size: 0.45 µm) floating on the medium.
The explants were kept at 37 C in a humidified chamber gassed with 95%
air and 5% CO2 for 2 days without changing the medium. To
measure testicular responsiveness to TGFßs, one testis cultured in
the control medium was compared with the contralateral testis cultured
in medium containing TGFßs for stages 17.5 dpc and 3 dpp. For fetal
day 13.5, the comparisons were performed between two fetuses from the
same mother.
Gonocytes counting
The whole explant was fixed in Bouin’s fluid for 2 h,
embedded in paraffin, and sectioned at 5 µm thickness. All the serial
sections from one explant were mounted on slides, deparaffinized, and
rehydrated as previously described (26). After staining with
hematoxylin-eosin, the gonocytes were identified by their round, large,
spherical, lightly stained nucleus containing fine chromatin granules
and two or more globular nucleoli and by a clearly visible cytoplasmic
membrane (1). All the gonocytes in every three sections (13.5 dpc
testis) and in every twenty sections (17.5 dpc and 3 dpp testes) were
counted, and the total number of gonocytes counted per testis was
designed the total count (TC). This number was multiplied by 3 (13.5
dpc testis) or by 20 (17.5 dpc and 3 dpp testes) to obtain the crude
count (CC) of gonocytes per testis. To correct for any double counting
resulting from the appearance of a single cell in two successive
sections, the Abercombrie formula was used: TC = CC x S/(S +
D) where TC is the true count, S is the section thickness (5 µm), and
D is the mean diameter of the gonocyte nucleus (34). D is the true mean
diameter of the gonocytes nuclei. It is equal to the average of the
nuclear diameters measured on the section (DM) divided by 
/4 to
correct for the overrepresentation of smaller profiles in sections
through spherical particles. DM was measured on each testis studied, by
at least 100 random determinations using a micrometer inserted within
the ocular lens and calibrated with an object micrometer on the
microscope plate. All these countings and measurements were done
blind.
Measurement of bromodeoxyuridine incorporation index
Cultured 13.5 dpc testes (and associated mesonephroi) were
labeled with 5-bromo-2'-deoxyuridine (BrdU) and
5-fluoro-2'-deoxyuridine (labeling reagent diluted 1:100 according to
the instructions of the cell proliferation kit, Amersham,
Buckinghamshire, UK) during the last hour of culture. BrdU
incorporation into proliferating cells was detected by
immunocytochemistry according to the manufacturer’s recommendations.
Briefly, some sections randomly chosen from the control and
paired-treated Bouin-fixed explants were mounted on the same slides,
washed with PBS and incubated with 0.3% H2O2
in methanol at 20 C for 30 min to inactivate endogenous peroxidases.
Sections were then washed several times with PBS and incubated with a
mouse anti-BrdU monoclonal antibody (cell proliferation kit, Amersham)
at 20 C for 1 h. The antibody bound to the nuclei was detected by
a peroxidase-linked antimouse IgG. Finally, slides were stained with
DAB (3,3'-diaminobenzidine, Sigma Chemical Co., St. Louis, MO),
conterstained by brief immersion in hematoxylin and eosin, and
dehydrated by standard procedures (27). The BrdU incorporation index
(% cells showing a clear positive immunoreaction to BrdU) was obtained
from a blind counting of all the gonocytes or the Sertoli cell nuclei
presents on the sections (i.e. at least 700 nuclei). In
preliminary experiments, triplicate counts of the same slides were
performed in blind and the SEM within the triplicates in
the percentage of labeled cells was 0.24 ± 0.05 (n = 4).
Measurement of DNA fragmentation index
Apoptotic cells were detected in situ using a
modified version of the terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end labeling (TUNEL) method (35). Cultured
13.5 dpc testes (and associated mesonephroi) were fixed in buffered 4%
formaldehyde for 15 min at 4 C. Tissue sections (5 µm), randomly
chosen from the control and paired-treated Bouin-fixed explants, were
mounted on the same slides, washed twice in PBS, and incubated in
permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate)
for 2 min at 4 C. They were given four washes in PBS and incubated in
TdT buffer containing TdT (0.6 U/µl, Boehringer Mannheim, Mannheim,
Germany) and fluorescein-deoxy-UTP (dUTP) (Boehringer Mannheim) for
1 h at 37 C in a humidified chamber. The sections were washed in
PBS (x4) and incubated with antifluorescein antibody conjugated to
peroxidase for 30 min at 37 C in a humidified chamber. The slides were
washed in PBS (x4), stained with DAB, and conterstained as above.
Positive controls were incubated with DNAse I (100 µg/ml) for 10 min
at 20 C to induce DNA strand breaks. Negative controls were incubated
without TdT. The DNA fragmentation index (% cells with a clear
positive TUNEL staining) was obtained from a blind counting of all the
gonocytes nuclei presents on the sections (i.e. at least 700
nuclei). In preliminary experiments, triplicate counts of the same
slides were performed in blind and the SEM within the
triplicates in the percentage of labeled cells was 0.18 ± 0.09
(n = 4).
Immunohistochemical staining
Immunostaining was performed with the Vectastain Elite ABC kit
(Vector Laboratories, Burlingame, CA) (36). Briefly, deparaffinized
sections were rehydrated. Those to be used for TßRII immunostaining
were placed in citrate buffer and heated in a microwave oven for 3
x 5 min at 750 W. Endogenous peroxidases and nonspecific protein
binding were blocked by incubations in hydrogen peroxide followed by
normal goat serum. The sections were subsequently incubated overnight
in the anti-TßRI antibody (1 µg/ml) or with the anti-TßRII
antibody (0.5 µg/ml) in a humidified chamber at 4 C. The
distributions of these primary antibodies were revealed with a
biotinylated goat antirabbit secondary antibody and an
avidin-biotin-peroxidase complex. Peroxidase was visualized with
3,3'-diaminobenzidine. Sections were rinsed in PBS between each
step.
The specificity of TßRI and II staining was checked using three negative controls: by antibody presaturated with 1 µg/ml synthetic peptides used for immunization, by antibody diluted to the extinction limit of staining (dilution 1/5000), and by replacing the antibody with nonimmune IgG. Staining for TßRs was considered to be specific when the three negative controls gave no staining. For each of the studied ages, three to four tissue blocks were made and three to six sections from each block were analyzed for immunostaining.
Statistics
All values are means ± SEM. One-way ANOVA was
used to compare between data from more than two groups. Furthermore,
the statistical significance of the difference between the mean values
for the treated testes and the corresponding untreated controls were
evaluated using Student’s paired t test.
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Results |
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). The number of gonocytes
in the different groups were significantly different (P
< 0.001) in the ANOVA. In the control medium, the number of gonocytes
increased 2.5-fold after 1 day in culture and 5.1-fold after 2 days.
TGFß1 (10 ng/ml) had no effect during the first day of culture but
greatly reduced the increase in the number of gonocytes during the
second day in culture, so that the number of gonocytes in the
TGFß1-treated testis was about 50% that of the control testis. The
diameter of the gonocytes nucleus was not affected by TGFßs
(9.12 ± 0.14 µm for TGFß1-treated cells and 9.13 ± 0.08
µm for control cells, n = 4).
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2) Effect of TGFß1 on the number of gonocytes in the testis in
culture as a function of the age at explantation
The same method was used to assess the effect of TGFß1 on the
number of gonocytes as a function of the age at explantation (Fig. 2). Testes explanted on 17.5 dpp
(beginning of the quiescent period in vivo) showed no change
in the number of gonocytes during the two days of culture in control
medium. TGFß1 (10 ng/ml) had no effect. With testes explanted on
postnatal day 3 (when mitosis resumes), ANOVA revealed differences
between the different groups (P < 0.05). The number of
gonocytes slightly increased after 2 days of culture in control
medium. TGFß1 (10 ng/ml) produced a slight but statistically
significant decrease in the number of gonocytes after 2 days in culture
compared with the paired-control culture. TGFß1 had no effect on the
diameter of the nucleus (9.86 ± 0.11 µm (TGFß1) and 9.70
± 0.10 µm (control) for 17.5 dpc testis; and 11.86 ± 0.24 µm
(TGFß1) and 11.68 ± 0.05 µm (control) for 3 dpp testis,
n = 3).
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and 4). This percentage was remarkably high
(50%) and was not altered by TGFß1 or TGFß2. TGFßs also had no
effect on the BrdU incorporation index in the Sertoli cells.
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and 6).
Fragmentation of DNA was frequent in the gonocytes but was never
detected in other cell types in control or in TGFß-treated testes.
The percentage of TUNEL positive gonocytes in TGFß1 or
TGFß2-treated testes was 25% higher than in the control samples and
comparison of the different groups by ANOVA revealed statistical
differences (P < 0.01).
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).
Gonocytes were more strongly stained for TßRII than for TßRI, and
staining for both receptors increased from fetal day 18.5 onward.
TGFß receptors were already present in the migrating primordial germ
cells on fetal day 13.5 and they were found in a portion of the
cytoplasm on fetal day 18.5; staining had extended to the whole
cytoplasm and the plasma membrane at the other stages. The Sertoli
cells showed no immunoreactivity for either receptor at any time. In
the Leydig cells, positive specific stainings for both receptors
appeared on fetal day 16.5 and became intense from fetal day 18.5
onward. Leydig cells always stained stronger for TßRI than for
TßRII. Finally, sections incubated with antibodies presaturated with
their respective antigens (Fig. 7, I and J) or with nonimmune serum
(data no shown), gave no detectable signal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To our knowledge, apoptosis has not been studied in the developing fetal and neonatal testis, and we began by using the in situ TUNEL method to quantify this phenomenon and find out which cell types were undergoing apoptosis. This method detects DNA fragmentation, a biochemical event that is thought to herald the irrevocable commitment of cells to apoptosis, although necrosis can also generate a fair number of stainable DNA ends (41). However, necrotic cells cannot be confused with apoptotic cells in cultured fetal testis. There were very few necrotic cells in our organ cultures even after 5 days of culture (42). Necrosis also appeared as area formed by many cells and not as individual cells among living cells. Therefore, the in situ TUNEL-positive reaction, which is a good marker for apoptosis in adult germ cells (43), also appears to be relevant in the fetus. TGFßs clearly induce apoptosis in the gonocytes and the TUNEL-positive reaction was found only in gonocytes, and the other testicular cell types were not labeled. This suggests that apoptosis is confined to the germ cell lineage in the developmental testis and does not affect somatic cells. Similarly, Billig et al. (43) recently reported a specific TUNEL-positive reaction in germ cells in prepuberal rat testis.
The gonocytes were immunostained for both TßRI and TßRII from fetal day 13.5 onward. This strongly suggests that TGFßs act directly on gonocytes. They could also act via the Sertoli cells because the number of germ cells depends on the number of Sertoli cells (44). But this indirect action of TGFßs seems unlikely in our model because TßRs were not detected in Sertoli cells, and the TGFßs did not affect the percentage of BrdU-positive and did not induce TUNEL-positive Sertoli cells.
The inhibitory effect of TGFßs on cultured testis was clearly age dependent. It disappeared with 17.5 dpc testis and rose again with 3 dpp testis. This change is not due to any variation in the ability of our culture system to support the survival or mitosis of gonocytes. The total number of gonocytes was maintained when testes were explanted at a quiescent stage (17.5 dpc) and slightly increased when they were explanted at a mitosis-resuming stage (3 dpp), and explants at this later stage contained 26 ± 4% (n = 4) BrdU-positive germ cells after 2 days of culture in the control medium, whereas they contained only 4 ± 1% (n = 4) at the time of explantation (data not shown).
This age-related change in the response to TGFßs parallels the pattern of apoptotic activity in the gonocytes in vivo. Recent unpublished experiments showed a sharp decrease in the percentage of TUNEL-positive gonocytes between 15.5 dpc and 18.5 dpc, followed by undetectable numbers until 3 dpp and a new surge thereafter (Boulogne, B., R. Olaso, C. Levacher, and R. Habert, unpublished data). The change in the intracellular distribution of the receptors may also account for the age-related change in the ability of TGFßs to cause gonocytes apoptosis. The TßRI and TßRII receptors lie in an area of cytoplasm forming a cap near the nucleus in gonocytes from 17.5 dpc to 20.5 dpc. This may be due to the concentration of the organelles near the nucleus at this stage of development (45). Whether TßRs are confined in these organelles or distributed on the plasma membrane during this cap phase remains to be investigated.
If TGFß1 and/or TGFß2 physiologically act to increase apoptosis in gonocytes, it can be hypothesized that these factors originate from the Sertoli cells during the fetal surge of apoptosis because they are both found only in this cell type before fetal day 16.5. The second surge of apoptosis that occurs after postnatal day 3 may depend on the isoform TGFß2 acting via an autocrine mechanism because immunostaining for this isoform in gonocytes is maximum at this period (27). It could also result from a paracrine control by TGFß1 or TGFß2 from the Leydig cells because these cells contain high amounts of both isoforms (26, 27), and the blood testis barrier has not yet differentiated at this developmental stage (3).
Lastly, there was intense staining for both TßRs in the Leydig cells. This suggests that these cells express many of these receptors during fetal and neonatal life because TßRs cannot originate from another cell type. Unlike gonocytes, Leydig cells staining for TßRI was higher than that for TßRII. The large number of TßRs in the fetal Leydig cells agrees with our recent results showing that TGFßs have a potent negative effect on steroidogenesis in these cells (37). Because TGFß1 and TGFß2 appear in large amounts in the Leydig cells during late fetal life (26, 27), these factors may be involved in the autocrine control of the decrease of the testosterone production that occurs in vivo during this developmental period (46, 47). Our recent observations indicate that the Leydig cells in adult rat are much less well stained for TßRs and exhibit stronger staining for TßRII than for TßRI (Olaso et al., unpublished data). These differences in the TßRs profiles of adult and fetal Leydig cells offer new support for the concept of two Leydig cell types during testis development: the fetal-type Leydig cell generation, which is maintained during the 2 first weeks of neonatal life, and the adult-type generation, which emerges thereafter.
In conclusion, this study shows, for the first time, that identified intratesticular factors, the TGFßs, can be involved in the paracrine/autocrine regulation of the number of gonocytes during the early testicular development.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from Ministère de la Recherche et
de la Technologie.
Received July 28, 1997.
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