| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
INSERM-INRA U-418 and Université Paris 7, Tour 33/43 (V.R.F., L.L., C.G., R.H.), 75251 Paris Cedex 05; and INSERM-INRA U 418 and IFR d’Endocrinologie, Hôpital Debrousse (J.M.S.), 69222 Lyon, France
Address all correspondence and requests for reprints to: Prof. 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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Northern blot analysis revealed one major IGF-I transcript of 7–7.5 kb and two minor transcripts of 3.8 and 1.8 kb in 20.5 day fetal testis. IGF-I was detected by RIA in 16.5 fetal day testes, and the amounts of IGF-I secreted by 16.5 and 20.5 fetal day testes in vitro were much greater than the amounts contained in the testes, indicating active synthesis in culture. The secretion of IGF-I by the fetal testis in vitro was increased with testicular age and time in culture. It was not modified by gonadotropins or (Bu)2cAMP.
Testosterone secretion by fetal testes explanted 13.5, 16.5, 18.5, and 20.5 days after conception and cultured in the presence or absence of 100 ng/ml LH for 3 days was not affected by the addition of 50 ng/ml IGF-I to the medium. In contrast, the addition of IGF-I to dispersed fetal testicular cells cultured for 3 days in the presence or absence of LH increased the number of Leydig cells identified by a positive cytochemical reaction for 3ß-hydroxysteroid dehydrogenase (3ßHSD). This was more pronounced with cells from 16.5- day-old fetuses (stage when the fetal Leydig cells are differentiating in vivo) than with 20.5-day-old fetuses cells (stage when the number and the function of fetal Leydig cells are stable or decreasing). It results from both an increased differentiation of mesenchymal cells in fetal Leydig cells and an increase in the mitotic index of the fetal Leydig cells, as inferred from the small increase in the percentage of bromodeoxyuridine/3ßHSD-positive cells. Both LH and IGF-I increased significantly testosterone production by day 16.5 cells. In the presence of LH, a high amount of testosterone was produced per 3ßHSD-positive cell; IGF-I further increased this production. This effect was not observed with day 20.5 cells. The amounts of testosterone produced per 3ßHSD-positive cell cultured in the presence of both LH and IGF-I were more than additive. Like IGF-I, insulin (50 ng/ml) increased testosterone secretion per 3ßHSD-positive cells in cultures of day 16.5 cells, but not in those of day 20.5, cells. Lastly, IGF-I also increased the steroidogenic activity of each Leydig cell in cultures containing (Bu)2cAMP, but its effects were weaker than those observed in the presence of LH. This suggests that IGF-I has sites of action both upstream and downstream cAMP generation.
These results suggest that IGF-I acts as paracrine/autocrine factor in the differentiation and activity of fetal Leydig cells.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Type I receptors for IGF-I have been found on human, pig, and rat Leydig cells (14, 15, 16), and IGF-I enhances the differentiated functions of Leydig cells (5, 6, 17). IGF-I stimulates the hCG-supported production of cAMP and testosterone by cultures of rat (18) and pig (19) Leydig cells. The response to cAMP analogs is also enhanced (16, 19), suggesting that IGF-I potentiates the action of LH/hCG at sites both proximal and distal to cAMP generation. IGF-I increases the number of LH/hCG receptors (16, 20) and the amount of LH/hCG receptor mRNA (21) as well as the activities of several steroidogenic enzymes and the amounts of mRNAs encoding them (21, 22, 23).
Further evidence for the importance of IGF-I for Leydig cell maturation and steroidogenic capacity was obtained by in vivo studies showing that treatment of GH-deficient Snell dwarf mice with hGH or IGF-I for 7 days produced a marked increase in the number of hCG receptors and in the steroidogenic responsiveness to this hormone (24). Lastly, the crucial role of IGF-I in the development and function of Leydig cells was obtained in studies of IGF-I gene knock-out mice (25). The testes of these animals were reduced in size and had fewer and smaller Leydig cells than normal, and the plasma testosterone levels were markedly reduced. Basal and LH-stimulated testosterone productions by testicular slices in vitro were also impaired.
All of these studies were performed on adult animals, and there are no data available on the role of IGF-I in the fetal testis. To determine whether IGF-I can be an intratesticular modulator of fetal Leydig cell function and differentiation, we first investigated the expression and secretion of IGF-I by the fetal testis, and second we studied its effect on testosterone production by organotypic cultures and dispersed fetal Leydig cells in vitro. The effects of IGF-I were also compared with those of insulin.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chemicals and solutions
The explants were placed in medium 199 containing 4.18
mM sodium bicarbonate (Flow Laboratories, Rockville, MD),
0.35% glutamine (Flow Laboratories), 100 IU/ml Specilline G (Specia,
France), 40 µg/ml gentamicin (Schering-Plough, Levallois-Perret,
France), and 5 µg/ml transferrin (Sigma Chemical Co., St. Louis, MO).
Dispersed Leydig cells were cultured in Ham’s F-12-DMEM (1:1; Life
Technologies, Grand Island, NY) containing 5 µg/ml transferrin
(Sigma) and 40 µg/ml gentamicin (Gentalline, Schering-Plough). Ovine
(o) LH (NIH LH S19; 1.01 NIH LH.S1 units/mg) was a gift from Dr. Parlow
(NIDDK, Bethesda, MD). Recombinant human (h) FSH (12000 IU/mg) was a
gift from Dr. B. Mannaerts (Organon International, Oss, The
Netherlands) (27). (Bu)2cAMP, soybean trypsin inhibitor,
deoxyribonuclease I, nitro blue tetrazolium (NBT), NAD, and human
insulin were purchased from Sigma. Collagenase was obtained from Serva
(Heidelberg, Germany). Recombinant IGF-I was obtained from Kabi-Vitrum
(Stockholm, Sweden).
Hybridization probe
A 2.6-kb fragment of IGF-I complementary DNA (a gift from M.
Jansen, Utrecht, The Netherlands) (28), was labeled to a specific
activity of 109 dpm/µg DNA using
[
-32P]deoxy-CTP and the Amersham Megaprime DNA
labeling kit (Amersham France, Les Ulis, France).
Organ cultures
Testes removed on day 13.5 were cultured on stainless steel
grids placed in organ culture dishes containing 0.8 ml culture medium
(Falcon, Grenoble, France) (26). Testes explanted at later ages were
cultured on Millipore culture filters (Millipore, Bedford, MA) as
previously described (29). Briefly, 16.5- and 18.5-day-old fetal testes
were cut into two pieces, and 20.5-day-old fetal testes into eight
pieces. All pieces from the same testis were placed on a Millipore
filter (pore size, 0.45 µm). The filter bearing the pieces of testis
was floated on 1.5 ml culture medium in 35-mm diameter tissue culture
dishes and maintained at 37 C. To study IGF-I secretion, eight testes
from day 20.5 fetuses and 32 testes from day 16.5 fetuses were pooled
on the same Millipore filter to obtain detectable secretion of IGF-I.
Testes were cultured for 3 days at 37 C in a humidified atmosphere
containing 95% air-5% CO2. The medium was changed every
24 h.
Culture of dispersed fetal testicular cells
The technique used was essentially that described by Habert and
Brignaschi (29) with minor modifications (30). Briefly, at least 40
testes from 20.5-day-old fetuses or 60 from 16.5-day-old fetuses were
carefully minced and incubated for 30 min at 32 C in 5 ml calcium- and
magnesium-free Dulbecco’s PBS (Life Technologies) containing 0.2 mg/ml
collagenase, 0.05 mg/ml soybean trypsin inhibitor, 0.01 mg/ml
deoxyribonuclease, and 40 µg/ml gentamicin, with gentle shaking. The
testes were also disrupted mechanically at 15 and 25 min by repeated
pipetting. The digested testes were diluted with 30 ml Dulbecco’s PBS,
and the cells were collected by centrifugation for 20 min at 100
x g. The pellets were resuspended in 30 ml Dulbecco’s PBS
and allowed to sediment under gravity for 12 min. The undigested
tubules settled first, and the interstitial cells, containing the
Leydig cells, were collected in the supernatant. The supernatant was
centrifuged at 100 x g for 20 min, and the resulting
pellet was resuspended in culture medium. The cells were counted in an
hemocytometer, and less than 10% of the cells were associated in
clumps of more than 5 cells. Cell viability was tested by trypan blue
(0.04%) exclusion, and more than 90% of the cells were viable. The
cells were placed in 96-well culture dishes (diameter, 6.4 mm) at
0.7 x 105–105 cells/100 µl/well and
precultured in a humidified atmosphere of 5% CO2 in air in
DMEM-Ham’s F-12 (1:1) plus 0.4% FCS for the first day (D0) to allow
the cells to attach. On the following days (D1 to D3), the cells were
cultured without FCS in the presence or absence of IGF-I (50 ng/ml),
insulin (50 ng/ml), LH (100 ng/ml) or (Bu)2cAMP (1
mM), and the medium was changed every 24 h.
Bromodeoxyuridine (BrdU) was added to some cultures for the last 3
h.
At the end of D3, the cells were harvested by trypsinization (trypsin-EDTA solution, Life Technologies) and counted in a hemocytometer. In other wells, the Leydig cells were identified by cytochemical detection of 3ß-hydroxysteroid dehydrogenase activity (3ßHSD) and counted. Briefly, multiwell dishes containing attached cells and culture medium were frozen and stored at -80 C. They were thawed immediately before use, and the reaction mixture was added to the culture medium to final concentrations of 0.25 mg/ml NBT, 0.28 mg/ml nicotinamide, 0.60 mg/ml NAD, and 0.05 mg/ml dehydroepiandrosterone (previously diluted in dimethylformamide). The dishes were incubated for 4 h at 37 C in 95% air-5% CO2. The dark blue positive cells (3ßHSD positive) in each well were counted under an inverted microscope. The percentage of 3ßHSD-positive cells was 1.31 ± 0.11% for day 16.5 fetal cultures and 1.01 ± 0.06% for day 20.5 fetal cultures (three to five experiments with two to six determinations for each experiment).
Colocalization of BrdU with 3ßHSD staining
Dispersed testicular cells, taken from fetal testes on day 16.5,
were cultured in a glass Lab-Tek chamber (Lab-Tek, Naperville, IL).
They were labeled by incubation with BrdU and 5-fluoro-2'-deoxyuridine
(labeling reagent diluted 1:100 according to the manufacturer’s
instructions, Amersham, Aylesbury, UK) on D3 for the last 3 h of
culture. Then, the medium was replaced by control medium, and the glass
Lab-Tek chamber containing attached cells and culture medium was frozen
and stored at -20 C. Immediately before use, the Lab-Tek chamber was
thawed, and the reaction mixture was added to the culture medium to
give final concentrations of 0.25 mg/ml NBT, 0.28 mg/ml nicotinamide,
0.60 mg/ml NAD, and 0.05 mg/ml dehydroepiandrosterone (from a stock
solution in dimethylformamide). The dishes were incubated for 4 h
at 37 C. BrdU incorporation into proliferating cells was detected by
immunocytochemistry according to the manufacturer’s recommendations.
Briefly, cells were washed with culture medium and fixed in Bouin’s
fluid. The cells were washed with PBS and incubated with 0.3%
H2O2 in methanol for 30 min at 20 C to
inactivate endogenous peroxidases. Cells were then washed several times
with PBS and incubated with a mouse anti-BrdU monoclonal antibody (cell
proliferation kit, Amersham) for 1 h at 20 C. The antibody bound
to the nuclei was detected by a peroxidase-linked antimouse IgG. Cells
were then stained with 3,3'-diaminobenzidine (Sigma) and counterstained
by brief immersion in hematoxylin. The percentage of dark blue positive
cells (3ßHSD positive) with a positive immunoreaction to BrdU was
counted (Fig 1
).
|
-androstan-3-one,
which is the only steroid that cross-reacts (64%) significantly with
testosterone, is secreted in minute amounts by the fetal rat testis
(31). When testosterone production was too low (day 13.5 testis and
testicular cells from fetuses cultured in the absence of LH), the
medium was first concentrated by extraction with ethyl acetate as
previously described (32).
IGF-I RIA
IGF-I was separated from its binding proteins (IGFBP) by acid-
ethanol extraction (12, 33). Briefly, fetal testes (eight testes
from day 20.5 fetuses and 32 testes from day 16.5 fetuses) were
homogenized in acetic acid (50 mM) in a Kontes
microhomogenizer (Kontes Co., Vineland, NJ). The homogenates (for IGF-I
content) and culture media (for secreted IGF-I) were mixed with acetic
acid (1 M, final concentration). Then, ethanol was added
(1:4). After centrifugation, the supernatants were lyophilized, and
IGF-I was measured in the dry extracts by RIA using recombinant IGF-I
as a standard (34). Briefly, samples were incubated with a specific
polyclonal anti-IGF-I rabbit antibody for 1 h at 4 C and then
labeled IGF-I was added overnight. Antibody-bound and free IGF-I were
separated using a goat antirabbit IgG antibody and polyethylene glycol
(6%). The limit of sensitivity of the assay was 250 pg/ml, and the
interassay coefficient of variation was 8.2% for a sample containing 5
ng/ml IGF-I.
The accuracy of the IGF-I determination in the culture medium was assessed by adding three amounts of IGF-I (1, 5, and 20 ng/ml) to control and conditioned media from the fifth day of testicular culture, as conditioned media may contain IGFBP, which could interfere with RIA. The amounts of IGF-I measured in the control and conditioned media were very similar to those expected (regression coefficient, r = 1.000 and r = 0.997; slope of regression line = 0.963 and 1.005; y-intercept of the regression line = 0.130 and 0.066 ng/ml for control and conditioned medium, respectively; n = 3–6).
Northern blot analysis of IGF-I transcripts
Twenty testes from 20.5-day-old rat fetuses and one testis from
a 19-day-old immature rat were harvested in 4 M guanidine
thiocyanate. Total RNA was extracted (35) and denatured by heating (65
C for 15 min), and aliquots (30 µg) were electrophoresed through a
1% agarose gel containing 10% formaldehyde. The RNAs were transferred
by capillarity to Hybond-N nylon membranes (Amersham) and cross-linked
to the membrane by irradiation with UV light for 2 min and baking at 80
C for 2 h. The membranes were prehybridized for at least 2 h
at 50 C in 50% formamide; 0.9 M NaCl, 50 mM
sodium phosphate (pH 7.5), and 5 mM EDTA; 5 x
Denhardt’s solution (0.1% Ficoll 400, 0.1% polyvinylpyrolidone, and
0.1% BSA); 0.1% SDS; 10% dextran sulfate; and 100 µg/ml boiled
salmon sperm DNA. The hybridization buffer contained 106
dpm/ml 32P-labeled probe, and hybridization was performed
overnight at 50 C. The resulting membranes were washed twice in 2
x NaCl/Cit (NaCl/Cit = 150 mM NaCl and 15
mM sodium citrate, pH 7.4)-0.1% SDS at room temperature
for 15 min each, then twice in NaCl/Cit-0.1% SDS at 50 C for 10 min
and autoradiographed by exposure to Kodak X-Omat-AR films (Eastman
Kodak, Rochester, NY) for 4–8 days at -70 C.
Statistical analysis
All values are the mean ± SEM. The
significance of the differences between the mean values from each
testis of the same fetus was evaluated by Student’s paired
t test. Other means were compared using one-way ANOVA
(Fisher’s test).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
|
Fetal testes in organ culture secreted immunoreactive IGF-I, which
became detectable on the second day in culture (Fig. 3). This production was age dependent and
increased with time in culture. The IGF-I content of day 16.5 fetal
testes after 5 days in culture was 18.7 ± 2.5, and that of 20.5
day-old fetuses was 78.8 ± 5 pg/testis (n = 6). These values
were similar to those measured at the beginning of the culture (Fig. 3). Consequently, IGF-I seems to be synthesized by the fetal testis in
organ culture.
|
).
|
) or presence (Fig. 5B) of 100 ng/ml
LH, with or without 50 ng/ml IGF-I. IGF-I did not alter the basal or
LH-stimulated testosterone secretions regardless of the age of the
testis.
|
). This increase was greater when
insulin and/or IGF-I were added to the medium. The effect of IGF-I was
significantly greater than that of insulin. There was no additive
effect of insulin and IGF-I. The amount of testosterone secreted by
fetal day 20.5 cells cultured with LH alone also increased during the
first and second days in culture, but not thereafter. Adding IGF-I
and/or insulin further increased testosterone production, but this
effect was less pronounced than for day 16.5 cells. There was, again,
no additive effect of insulin and IGF-I.
|
, A and B).
|
For day 20.5 cells cultured in the presence of LH, the total number of testicular cells did not significantly change during culture and was unaffected by the addition of IGF-I, The number of 3ßHSD-positive cells decreased during culture, and IGF-I prevented this decrease.
To evaluate the mitotic activity of the fetal Leydig cells, BrdU was
added for 3 h at the end of D3 in culture of day 16.5 cells, and
the percentage of 3ßHSD-positive cells in which BrdU was detected was
determined (Fig. 7C
). In the absence of LH and IGF-I, this
percentage was very low, but it was significantly increased in response
to LH or IGF-I. Furthermore, the effects of LH and IGF-I were
additive.
Effects of LH, IGF-I, and insulin on testosterone production by
cultured fetal Leydig cells
To evaluate the steroidogenic activity of fetal Leydig cells, the
amount of testosterone secreted into the medium on D3 was divided by
the number of 3ßHSD-positive cells counted at the end of this day
(Fig. 8). In the absence of LH, day 16.5
fetal Leydig cells secreted minute amounts of testosterone. Both IGF-I
and insulin caused an increase in testosterone secretion. For cells
cultured in the presence of LH, large amounts of testosterone were
produced per 3ßHSD-positive cells on D3 (Fig. 8). IGF-I or insulin
further increased this production for day 16.5 cells, but not for day
20.5 cells. For cultures in the presence of (Bu)2cAMP, the
positive effect of IGF-I was less than that in the presence of LH.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IGF-I was detected in the fetal testis as early as fetal day 16.5. Until now, the youngest stage studied showing IGF-I protein in the testis was postnatal day 3 (9). Testes from day 16.5 and 20.5 fetuses contained very little IGF-I before or after culture compared with the amounts secreted into the medium, indicating a high de novo synthesis in vitro. This production increased with age at explantation and with time in culture. This increasing production of IGF-I with time also occurred in cultures of Leydig and Sertoli cells from immature rats and piglets (12, 13), but the reason for it is unknown.
Gonadotropins and (Bu)2cAMP did not alter the secretion of IGF-I by the fetal testis in vitro. This is in contrast to the situation in immature and adult testes. LH and FSH increase IGF-I mRNA in the testes of immature hypophysectomized rats (37). IGF-I secretion by immature Leydig and Sertoli cells in culture is also stimulated by LH (Leydig) and FSH (Sertoli) in the rat (12) and the pig (13, 38). In contrast, hCG decreases the amount of IGF-I mRNA in purified Leydig cells from adult rats (39). These results, therefore, suggest that the regulation of IGF-I production by gonadotropins is age dependent and is not yet established at fetal stages.
The second part of our study examined the effect of IGF-I on fetal Leydig cell function using organotypic and dispersed fetal Leydig cell cultures. IGF-I did not alter testosterone secretion by organotypic cultures at any of the stages studied. On the contrary, IGF-I increased the number of Leydig cells in cultures of day 16.5 and 20.5 fetal cells and had a positive effect on steroidogenesis only by day 16.5 cells. There are at least three possible explanations for the difference between our two in vitro systems. First, the organ cultures were performed on Millipore filters, and IGF-I can be absorbed by the filter. However, this possibility must be discarded, because IGF-I had no effect on testosterone produced by testes cultured on stainless steel grids (data not shown). Second, in several studies, IGFBPs have been shown to inhibit the positive effects of IGF-I on Leydig cell functions (40, 41). Thus, if the cultured testes contained a concentration of endogenously produced IGFBPs it could inhibit the effect of IGF-I. However, no IGFBP was detected in our culture medium by Western blotting (data not shown), suggesting that IGFBPs are not involved to any major degree. Third, the local IGF-I concentration in organ cultures could be very high, so that the biological effect is maximal; therefore, exogenous IGF-I may not stimulate testosterone secretion. This is supported by data from immature rats showing that IGF-I has no action in cocultures of Leydig and Sertoli cells pretreated with LH plus FSH (42). However, addition of anti-IGF-I antiserum to this coculture decreased steroid output, suggesting a maximal para/autocrine effect of endogenous secreted IGF-I. In contrast, the concentration of secreted IGF-I is low in our dispersed fetal testicular cells cultures and is probably insufficient to cause a maximal biological effect. Indeed, partial removal of tubular cells during the preparation of dispersed cells may contribute to a lower IGF-I secretion because tubular cells are the main source of IGF-I in neonatal testis (9). Other studies in our laboratory have shown the same difference in the effect of transforming growth factor-ß1 as a function of the culture system. This factor inhibits testosterone production by dispersed fetal Leydig cells in culture, but has no effect in organotypic culture (30).
Using a dispersed fetal Leydig cell culture system validated previously (30, 43), this study reports for the first time a positive effect of IGF-I on fetal Leydig cell proliferation, differentiation, and function. We also used this model to study the effect of LH during the onset of steroidogenesis (day 16.5), although in vivo fetal Leydig cell differentiation, proliferation, and function are LH independent until fetal day 19.5 (32, 44). For fetal day 16.5 cells, LH and IGF-I increased the number of 3ßHSD-positive cells present in the wells. These positive effects may be due to either an increase in the mitotic activity of fetal Leydig cells and/or an increase in the differentiation of mesenchymal cells. Although it has been reported that fetal-type Leydig cells have no mitotic activity (45), our results demonstrate that both IGF-I and LH caused an increase in the number of 3ßHSD-positive cells labeled with BrdU. However, as only 2–3% of 3ßHSD-positive cells were BrdU positive, whereas the number of 3ßHSD-positive cells increased 2- to 2.5-fold, the present results indicate that IGF-I and LH act mainly by increasing the differentiation of mesenchymal precursors into Leydig cells. IGF-I also increases the proliferation of Leydig cell precursors and immature Leydig cells from young rats (46, 47) and immature pigs (19), but not that of mature rat Leydig cells (16). Recent studies of IGF-I gene knock-out mice have shown that in these animals the number of adult Leydig cells was markedly reduced (25), confirming the effects of IGF-I on cell multiplication and maturation of precursors of adult-type Leydig cells. IGF-I may also act by decreasing apoptosis of Leydig cells. However, although it has been shown that IGF-I may protect cells from apoptosis in some cell types (48) no antiapoptotic effect on Leydig cells has been reported until now.
The steroidogenic functions of day 16.5 fetal Leydig cells in culture rapidly and strongly decreased in the absence of LH, revealing an intense dedifferentiation in vitro. IGF-I partially reduced this dedifferentiation. When cultures were performed in the presence of LH, high amounts of testosterone were produced per 3ßHSD-positive cells. IGF-I further increased this production. Thus, our results clearly demonstrated that IGF-I potentiates the steroidogenic effect of LH on fetal Leydig cells when both factors are added together. In the same way, it has been demonstrated that IGF-I alone has long term trophic effects on the differentiated functions of immmature and adult Leydig cells (reviews in Refs. 5, 6, 49, and 50).
In the present study, the trophic effect of IGF-I on the steroidogenic activity was higher in the presence of LH than in the presence of (Bu)2cAMP, suggesting that IGF-I acts on both upstream and downstream steps of the cAMP pathway. IGF-I also acts at sites both proximal and distal to cAMP generation in adult type Leydig cells. It increases the number of LH/hCG receptors (16, 20) and the amounts of LH/hCG receptor mRNA (21) as well as the activities of several steroidogenic enzymes and their mRNA concentrations (6, 15, 21, 22, 23).
Interestingly, the positive effects of IGF-I on the steroidogenic activity of fetal day 16.5 Leydig cells do not occur in fetal day 20.5 cells. Similarly, the ability of IGF-I to cause an increase in the number of 3ßHSD-positive cells in vitro was largely reduced from day 16.5 to day 20.5. These developmental changes correlate well with the in vivo development of fetal Leydig cell number and function. During fetal development, Leydig cell number and function are strongly increasing on fetal day 16.5, while the number of Leydig cells per testis stops increasing around day 20.5 (51), and the testosterone production per Leydig cell becomes stable or even declines (31, 52). Hence, IGF-I could be an important regulator of the development of steroidogenesis in the fetal testis.
There is considerable evidence that insulin plays a role in the regulation of testosterone production by adult-type Leydig cells, and there are insulin receptors on purified rat Leydig cells (16, 53). Insulin also stimulates testosterone production by purified rat and pig Leydig cells (16, 19). We find that insulin has a positive effect on the steroidogenic activity of fetal day 16.5 cells. As the insulin concentrations used were low, it is unlikely that it acted via the IGF-I receptor. This is the first evidence that insulin receptors are present on rat Leydig cells during fetal life. The positive effect of insulin on steroidogenesis by fetal day 16.5 Leydig cells was not found with fetal day 20.5 cells as it was for IGF-I. As the concentration of insulin in the fetal plasma on day 16.5 is undetectable, and it is very high on day 20.5 (54), the physiological role of insulin in the control of fetal testicular steroidogenesis remains to be established.
In conclusion, we have demonstrated that IGF-I is present in the fetal testis, has a mitogenic effect on Leydig cells from 16.5 day-old fetuses, and stimulates their differentiation from mesenchymal cells. These effects were weaker or absent in cells from fetal day 20.5 testes. These results can be linked to the development of Leydig cells in vivo and thus provide evidence that IGF-I has a paracrine/autocrine action on Leydig cell function and differentiation during early development.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received December 17, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Culty, R. Thuillier, W. Li, Y. Wang, D. B. Martinez-Arguelles, C. G. Benjamin, K. M. Triantafilou, B. R. Zirkin, and V. Papadopoulos In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat Biol Reprod, June 1, 2008; 78(6): 1018 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lin, R.-S. Ge, G.-R. Chen, G.-X. Hu, L. Dong, Q.-Q. Lian, D. O. Hardy, C. M. Sottas, X.-K. Li, and M. P. Hardy Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero PNAS, May 20, 2008; 105(20): 7218 - 7222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Villalpando, E. Lira, G. Medina, E. Garcia-Garcia, and O. Echeverria Insulin-Like Growth Factor 1 Is Expressed in Mouse Developing Testis and Regulates Somatic Cell Proliferation Experimental Biology and Medicine, April 1, 2008; 233(4): 419 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Delbes, C. Duquenne, J. Szenker, J. Taccoen, R. Habert, and C. Levacher Developmental Changes in Testicular Sensitivity to Estrogens throughout Fetal and Neonatal Life Toxicol. Sci., September 1, 2007; 99(1): 234 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L F C Brito, A D Barth, N C Rawlings, R E Wilde, D H Crews Jr, Y R Boisclair, R A Ehrhardt, and J P Kastelic Effect of feed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, testosterone, and on sexual development in bulls Reproduction, July 1, 2007; 134(1): 171 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Colon, F. Zaman, M. Axelson, O. Larsson, C. Carlsson-Skwirut, K. V. Svechnikov, and O. Soder Insulin-Like Growth Factor-I Is an Important Antiapoptotic Factor for Rat Leydig Cells during Postnatal Development Endocrinology, January 1, 2007; 148(1): 128 - 139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R. Manna, S. P. Chandrala, S. R. King, Y. Jo, R. Counis, I. T. Huhtaniemi, and D. M. Stocco Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells Mol. Endocrinol., February 1, 2006; 20(2): 362 - 378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ivanova, K. M. Dobrzycka, S. Jiang, K. Michaelis, R. Meyer, K. Kang, B. Adkins, O. A. Barski, S. Zubairy, J. Divisova, et al. Scaffold Attachment Factor B1 Functions in Development, Growth, and Reproduction Mol. Cell. Biol., April 15, 2005; 25(8): 2995 - 3006. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. H. Ing, A. M. Laughlin, D. D. Varner, T. H. Welsh Jr., D. W. Forrest, T. L. Blanchard, and L. Johnson Gene Expression in the Spermatogenically Inactive "Dark" and Maturing "Light" Testicular Tissues of the Prepubertal Colt J Androl, July 1, 2004; 25(4): 535 - 544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Livera, C. Pairault, R. Lambrot, M. Lelievre-Pegorier, J.M. Saez, R. Habert, and V. Rouiller-Fabre Retinoid-Sensitive Steps in Steroidogenesis in Fetal and Neonatal Rat Testes: In Vitro and In Vivo Studies Biol Reprod, June 1, 2004; 70(6): 1814 - 1821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. K. Jordan, J. H.-C. Shen, R. Olaso, H. A. Ingraham, and E. Vilain Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/{beta}-catenin synergy PNAS, September 16, 2003; 100(19): 10866 - 10871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Matsui and T. Takahashi Mouse Testicular Leydig Cells Express Klk21, a Tissue Kallikrein That Cleaves Fibronectin and IGF-Binding Protein-3 Endocrinology, November 1, 2001; 142(11): 4918 - 4929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Perrot, E. B. Moiseeva, Y. Gozes, S. J. Chan, and B. Funkenstein Insulin-Like Growth Factor Receptors and Their Ligands in Gonads of a Hermaphroditic Species, the Gilthead Seabream (Sparus aurata): Expression and Cellular Localization Biol Reprod, July 1, 2000; 63(1): 229 - 241. [Abstract] [Full Text]
|
|
|
|
|
|
B. Boulogne, C. Levacher, P. Durand, and R. Habert Retinoic Acid Receptors and Retinoid X Receptors in the Rat Testis During Fetal and Postnatal Development: Immunolocalization and Implication in the Control of the Number of Gonocytes Biol Reprod, December 1, 1999; 61(6): 1548 - 1557. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
