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Department of Veterinary Preclinical Studies (P.J.O.’S., P.J.B.), University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom; Biocenter Oulu and Department of Biochemistry (M.H., S.V.), University of Oulu, FIN-90571 Oulu, Finland; Department of Molecular and Cellular Biology (A.P.M.), The BioLabs, Harvard University, Cambridge, Massachusetts 02138
Address all correspondence and requests for reprints to: P. J. O’Shaughnessy, Department of Veterinary Preclinical Studies, University of Glasgow Veterinary School, Bearden Road, Glasgow G61 1QH, UK. E-mail: P.J.OShaughnessy{at}vet.gla.ac.uk
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Abstract |
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-androstanediol and/or testosterone were the major
androgens secreted from day 30 onwards. These results show that fetal
Leydig cells do not express significant levels of a reductive isoform
of 17ßHSD/17KSR and that androstenedione is the major androgen
secreted by these cells. Production of testosterone up until puberty is
dependent upon 17ßHSD/17KSR activity in the seminiferous tubules—a
"two cell" requirement for testosterone synthesis. Expression of
the 17ßHSD/17KSR type 3 isoform (the main reductive isoform in the
testis) declines in the seminiferous tubules before puberty but then
reappears in the developing adult Leydig cell population. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (P450c17) and 17ß-hydroxysteroid
dehydrogenase/17-ketosteroid reductase (17ßHSD/17KSR). Each of these
enzymes is expressed in the fetal testis (1, 2, 3) and localization
studies in fetal/neonatal animals have shown that P450scc, 3ßHSD, and
P450c17 are expressed specifically within the Leydig cell compartment
(2, 4, 5). The final step in the biosynthesis of active androgens is
17-keto reduction of androstenedione (a weak androgen) to testosterone
catalyzed by 17ßHSD/17KSR. To date, eight different isoforms of
17ßHSD/17KSR have been isolated in the human, mouse, and rat (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
and the nomenclature used in this report is that used by Peltoketo
et al. (20). Isoform types 1, 3, 5, and 7 show
17-ketosteroid reductase activity in the presence of 17ß-
hydroxysteroids although only isoform types 1, 3, and 5 can utilize
androstenedione as a substrate (8, 12, 16, 18). In the testis it is
clear that 17ßHSD/17KSR type 3 is the major isoform involved in
testosterone biosynthesis because loss of this isoform leads to a
failure of normal masculinization during development and a rise in
circulating androstenedione with a reduction in circulating
testosterone in the adult (8, 21). We have shown previously that in the
adult mouse testis the type 3 isoform is expressed only in the Leydig
cells (22) but more recent work from our group has raised the
possibility that in the fetal/neonatal testis this isoform may be
expressed in the seminiferous tubules (23). This would be important to
our understanding of testis development since it would suggest that the
fetal Leydig cells do not produce significant levels of active androgen
but are dependent on the tubules for a "two cell" synthesis of
androgen. Alternatively, it is possible that fetal Leydig cells express
one of the other reductive isoforms of 17ßHSD/17KSR and we have
already shown that the type 1 isoform is expressed in the
fetal/neonatal mouse testis (2). In this study, we have examined the
localization of 17ßHSD/17KSR isoform expression and 17-ketosteroid
reductase enzyme activity in the testis during development and have
characterized the major androgens produced by the testicular
interstitial compartment. Results show that fetal/neonatal Leydig cells
lack significant levels of 17ßHSD/17KSR expression and 17-ketosteriod
reductase activity and that androstenedione is the major androgen
produced until shortly before puberty. |
Materials and Methods |
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Isolation of interstitial tissue and seminiferous tubules
For studies on localization of enzyme activity, isoform
expression, and androgen production tubular and interstitial
compartments of the testis were mechanically separated. Testes were
decapsulated and placed in a culture dish containing culture medium
(DMEM/Ham’s F12 (1/1) [Life Technologies, Inc., Paisley,
UK) (DMEM/F12) containing NaHCO3 (2.4 g/liter)
and BSA (0.1%)]. Under a dissecting microscope tubules were teased
from the testis using fine forceps. In animals aged 10 days and above,
this is a relatively straightforward procedure and the webs of
interstitial tissue obtained can clearly be seen to be free of tubular
components. The procedure can also be used in animals aged 1 or 5 days
but is more difficult because the tubules are more fragile. With care
it is possible to be confident that the interstitial webs contain no
whole tubules although small pieces of tubule or tubular contents may
remain. It was not possible to use this procedure on fetal testes
because the tubules were too fragile and a few animals on the day of
birth could not be used for the same reason. The seminiferous tubules
from all ages of animals prepared using this procedure contained some
interstitial contamination.
Tissue incubations
Whole testes, webs of interstitial tissue or isolated
seminiferous tubules were incubated in culture medium (DMEM/F12) under
5% CO2 in air. To measure androgen production
interstitial webs from animals aged 1 to 20 days were incubated in 2.5
ml of medium at 37 C for up to 5 h while tissue from animals aged
30 to 90 days were incubated in 3.5 ml at 34 C. Tissue from one testis
of each animal was incubated under basal conditions while the
contralateral testis was incubated with human CG (hCG)
(Sigma-Aldrich Corp. Ltd., Poole, UK) at a concentration
of 200 mIU/ml to stimulate androgen synthesis. At the end of the
incubation period medium was stored frozen until assayed for androgen
content by RIA. To measure reduction of androstenedione to testosterone
tissue was incubated in DMEM/F12 and
[3H]androstenedione (0.5 µCi, 1
µM final concentration) (Amersham Pharmacia Biotech Ltd., Poole, UK) was added in 20 µl DMSO. Aliquots of
medium (100 µl) were removed at intervals during the incubation and
20 µl NaOH (1 N) added. At the end of the incubation
period 50 µg of the nonradioactive carrier steroids androstenedione,
testosterone, dihydrotestosterone and androstanediol were added to each
sample along with [14C]testosterone (2000
dpm/sample) to measure recovery.
Extraction and chromatography
Samples from the incubation of tissue with
[3H]androstenedione were extracted twice with
5ml toluene and steroids were separated by TLC using polyester-backed
silica gel plates (Whatman Ltd., Maidstone, UK) in
chloroform/ether (7/1) (24). Steroids were visualized under UV light or
by staining with phosphomolybdic acid (10% in ethanol) and the areas
representing each steroid were cut out and counted in a scintillation
counter.
RT-PCR
Total RNA was extracted from tissues using RNAzol
(Biogenesis Ltd., Little Chalfont, UK). RNA was
reverse transcribed using random hexamers and Moloney murine leukemia
virus reverse transcriptase (Superscript, Life Technologies, Ltd.) as described previously (25, 26). The PCR reactions were
carried out in Tris/HCl buffer (75 mM, pH 9.0 at 25 C)
containing
(NH4)2SO4
(20 mM), Tween 20 (0.01%), MgCl2 (2
mM), dNTPs (0.2 mM each), Taq
polymerase (2 U per 100 µl), primers (200 nM
each) and template (0.1–2 µl) in a total reaction volume of 30
µl.
The primers used were based on previously published sequences for mouse 17ßHSD/17KSR isoform types 1, 3, 5, and 7 (10, 12, 15, 16)
type 1 5'-ACT GTG CCA GCA AGT TTG CG-3' (bases 498–517)
5'-AAG CGG TTC GTG GAG AAG TAG-3' (bases 807–787)
product size 310 bp
type 3 5'-ATT TTA CCA GAG AAG ACA TCT-3' (bases 365–385)
5'-GGG GTC AGC ACC TGA ATA ATG-3' (bases 731–711)
product size 367 bp
type 5 5'-CCA TCC GAA GCA AGA TAG CAG (bases 191–211)
5'-GCT GCC TGC GGT TAA AGT TGG (bases 517–497)
product size 327 bp
type 7 5'-TGC AGA GGA AGT CAA GCA AAA-3' (bases 282–301)
5'-CTT CTT TGC ATT GCG AGA GGA-3' (bases 591–571)
product size 310 bp
Additional primers used to test cross-contamination of testicular compartments were:
3ßHSD type I:
5'-ACT GCA GGA GGT CAG AGC T-3' (bases 214–232)
5'-GCC AGT AAC ACA CAG AAT ACC-3' (bases 778–758) product size 565 bp (27)
Androgen-binding protein (ABP):
5'-ACC CAC GCA GAA TTC AGT CTC-3' (bases 700–720)
5'-CAG GCA GAA GGA AGC AGA AGA-3' (bases 1098–078)
product size 399 bp (28).
Primers for ß-actin have been described previously (29). Products from PCR reactions were separated on 1% agarose gels and visualized with ethidium bromide.
In situ hybridization
Testes from mice on E18-5 and postnatal day 90 were fixed either
in 4% paraformaldehyde or in Bouins and prepared for in
situ hybridization as described previously (3). Sense and
antisense cRNA probes for mouse 17ßHSD/17KSR type 3, 3ßHSD type 1
and AMH were prepared from complementary DNA (cDNA) clones as described
previously (22, 23, 30) using T3 and T7 polymerase in the presence of
[32P]dUTP. Tissue sections were incubated
overnight at 55 C with probe (100,000 cpm/µl) in hybridization buffer
[50% deionized formamide, 10% dextran sulfate, 50
mM dithiothreitol, 500 µg/ml DNA from calf
thymus, 1x Denhardt’s reagent, 20 mM Tris-HCl
(pH 8.0), 5 mM EDTA, 10 mM
sodium phosphate (pH 6.8)]. Following a number of post hybridization
washes and dehydration, the slides were allowed to air dry.
Autoradiography was carried out using Ilford K5 emulsion and the slides
were stained with Mayer’s Hematoxylin and Eosin.
RIA
Tissue incubation medium was assayed for androstenedione,
testosterone, and androstanediol using RIAs specific for each steroid
as previously described (31). The limits of detection of the assays
were 125, 100 and 250 fmol/ml respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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show that types 1, 3, and 7 are
expressed on day 1 while types 3 and 7 are expressed in the adult
testis. The type 5 isoform was not expressed in the mouse testis,
although there was clear expression in the liver, which acted as a
positive control (Fig. 1). To determine whether 17ßHSD/17KSR isoforms
1, 3, and 7 are expressed in interstitial tissue or seminiferous
tubules the testicular compartments were separated in neonatal and
adult animals and isoform expression examined by RT-PCR (Fig. 2). As a measure of the possible
cross-contamination between compartments expression of 3ßHSD type I
and ABP was also examined (Fig. 2). Results show that in the neonatal
testes 17ßHSD/17KSR types 1, 3, and 7 are all expressed predominantly
in the seminiferous tubule compartment with only low levels of
expression apparent in the interstitial compartment. In the adult,
there was no detectable expression of 17ßHSD/17KSR type 1 (the 800-bp
product in the seminiferous tubule lane is derived from genomic DNA),
whereas the type 3 and type 7 isoforms were expressed largely in the
interstitial tissue (Fig. 2). As expected, 3ßHSD type 1 expression
was predominantly in the interstitial tissue, whereas ABP was
predominantly in the tubular compartment, although there was clearly
some low level cross contamination between compartments (Fig. 2).
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show that up to 10 days enzyme
expression is largely or completely within the seminiferous tubules. On
day 20, enzyme expression was undetectable in either testicular
compartment, although there was clear amplification of the control
actin bands. Enzyme expression returned on day 30 but was confined to
the interstitial compartment, and this pattern persisted until
adulthood.
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show that the type 3 isoform was expressed only in the seminiferous
tubules in fetal testes and in the interstitial tissue in adult testes.
Expression of 3ßHSD type 1 (a Leydig cell marker) was confined to the
interstitial tissue in fetal testes while expression of AMH (a Sertoli
cell marker) was confined to the tubules.
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). Seminiferous
tubules from both neonatal and adult testes expressed high levels of
enzyme activity producing mainly testosterone from added
[3H]androstenedione (Fig. 5).
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). From day 15, production of
5-androstanediol by interstitial tissue began to increase although
androstenedione was still the major C19 steroid
produced until after day 20 (Fig. 6). Between days 20 and 30, there was
a 20- to 30-fold increase in total androgen production with
androstenedione levels less than 15% of the combined
17ß-hydroxysteroid (androstanediol plus testosterone) levels. This
pattern continued up to adulthood with the only change that
androstanediol levels declined at 40 days and testosterone became the
major androgen produced.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the adult mouse testis, the type 3 17ßHSD/17KSR isoform is expressed solely in the Leydig cell compartment (22), and it might have been expected that a similar localization would be seen in the fetal/neonatal testis, especially because all other components of the androgen biosynthetic pathway are found only within the fetal Leydig cells (3, 4, 5). It is clear, however, from the several lines of evidence reported here, that Leydig cells in the fetal/neonatal testis lack significant levels of 17ßHSD/17KSR type 3 isoform and any other isoforms of the enzyme capable of reducing androstenedione to testosterone. It is likely that most of the conversion of androstenedione to testosterone by the interstitial tissue of prepubertal animals in our experiments is due to cross contamination by tubular components arising during the separation procedure. Androstenedione is a weak androgen and our results show that formation of the bioactive 17ß-hydroxy-C19 steroids in the fetal/neonatal testis is dependent on 17ßHSD/17KSR localized in the seminiferous tubules, probably within the Sertoli cells.
In mammalian species so far studied it is clear that two populations of Leydig cells arise during development. Soon after testicular differentiation a fetal population develops which is responsible for masculinization of the fetus while an adult population, responsible for development of secondary sex characteristics and fertility in older animals, begins to develop at a later stage (38, 39). In the mouse, evidence now suggests that the adult population of cells begins to arise around postnatal day 10 (3). Because 17ßHSD/17KSR activity and expression of the type 3 isoform both appear first in the interstitial tissue between days 20 and 30 it is highly likely that fetal Leydig cells lack 17ßHSD/17KSR and appearance of this enzyme in the interstitial tissue is due to development of the adult Leydig cell population.
The reason for the loss of 17ßHSD/17KSR type 3 expression in the Sertoli cells between days 10 and 20 is not clear, but it coincides with a number of changes that occur in the testis at this time including loss of Sertoli cell proliferation, formation of occlusive junctions between Sertoli cells, start of germ cell meiosis and development of the adult Leydig cell population (3, 40, 41, 42). In addition to loss of the type 3 isoform, Sertoli cells also lose expression of the type 1 and type 7 isoforms during postnatal development. We have shown previously that expression of both type 1 and type 3 isoforms is lost at the same time in the mouse testis as it approaches puberty suggesting that there may be a common mechanism (2). It has recently been shown that Leydig cells appear to inhibit some aspects of Sertoli cell function (43), and it is possible that the developing adult Leydig cell population inhibits expression of Sertoli cell 17ßHSD/17KSR isoforms. Despite the loss of expression of 17ßHSD/17KSR types 1, 3, and 7, the seminiferous tubules in the postpubertal animal continue to show high levels of 17-ketosteroid reductase activity. This suggests that there is a further, as yet unidentified, isoform expressed in the seminiferous tubules and supports previous studies showing that seminiferous tubules in the adult rat express 17-ketosteroid reductase activity, which is biochemically distinct from that expressed in the interstitial tissue (44). The presence of a novel isoform of 17ßHSD/17KSR in the tubules may offer an explanation for the observed virilization that occurs around puberty in individuals lacking the type 3 isoform of 17ßHSD/17KSR (21). This virilization is caused by a marked rise in plasma testosterone and it is generally considered that extragonadal conversion of androstenedione to testosterone is the most likely explanation of this phenomenon. The demonstration that adult seminiferous tubules probably express another isoform of the enzyme after puberty offers an alternative explanation suggesting that the testes themselves will be capable of limited testosterone synthesis by cooperation between tissue compartments, a phenomenon which occurs normally during fetal development.
These results may have some relevance to our understanding of Leydig cell evolution. From comparative studies of vertebrate testicular anatomy and cell biology, it has been proposed that Leydig cells arrived relatively late within the vertebrate testis and that they evolved originally as a source of circulating steroids (45). Before this time Sertoli cells are likely to have been the major steroidogenic cell within the testis, providing an appropriate steroid environment for germ cell development. It is proposed that in mammals Leydig cells came, eventually, to dominate steroid production and Sertoli cells lost most of their steroidogenic potential (46). This is supported by a number of studies, including this one, showing remnants of steroidogenic enzyme activity in the mammalian Sertoli cell (47, 48) and by the demonstration that in culture Sertoli cells can be induced to express StAR protein and P450scc (49, 50). Our observation that fetal Leydig cells lack 17ßHSD/17KSR type 3 while Sertoli cells in fetal/neonatal animals and adult Leydig cells express the enzyme may suggest that the fetal Leydig cell population is evolutionarily older than the adult population. Thus, fetal Leydig cells are dependent on the Sertoli cells for synthesis of active androgens, while the adult population may have arisen to be largely independent.
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Acknowledgments |
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Footnotes |
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The following trivial steroid names have been used: androstenedione
(4-androstene-3,17-dione), testosterone (4-androstene-17ß-ol,3-one),
dihydrotestosterone (5-androstane-17ß-ol,3-one) and
5-androstanediol (5-androstane-3,17ß-diol and
5-androstane-3ß,17ß-diol).
Received December 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biol Reprod 58:520–525-hydroxysteroid dehydrogenase-activity. J Biol
Chem 268:12964–12969-hydroxysteroid
dehydrogenases from rat and human prostate. J Biol Chem 272:15959–15966-hydroxylase protein and mRNA in the testis of the testicular
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