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Medical Research Council Reproductive Biology Unit (G.M., A.S.M., R.M.S., P.T.K.S.), Edinburgh, EH3 9EW, United Kingdom; and the School of Biological and Molecular Sciences (L.R.E., N.P.G.), Oxford Brookes University, Headington, Oxford, OX3 0BP, United Kingdom
Address all correspondence and requests for reprints to: Dr. Philippa T. K. Saunders, Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom. E-mail: p.saunders{at}ed.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsReferences |
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In the rat, positive immunostaining for the 
-subunit of inhibin
() was first detectable on day 14.5 post coitum (p.c.), the first
day on which the testis could be morphologically distinguished from the
ovary. During fetal life, the -immunostaining was most prominent in
the fetal Leydig cells. In Sertoli cells, -immunostaining was
slightly stronger on days 14.5 and 15.5 p.c. compared with
16.5–20.5. After birth, -immunostaining remained intense in fetal
Leydig cells but declined following their replacement with their
adult-type counterparts; in contrast, -subunit increased in Sertoli
cells immediately after birth. Immunostaining with antibodies specific
to ßB-subunit showed a similar pattern to that of the -subunit,
except that positive immunostaining was first detectable on day
16.5 p.c., 2 days later than immunostaining for the -subunit.
The pattern of ßB-immunostaining in postnatal samples paralleled that
of the -subunit. Immunostaining using antibodies against the
ßA-subunit did not produce any significant reaction product in any
sample. Follistatin was undetectable in the fetal rat testis but
appeared in the Leydig cells immediately after birth and its expression
remained intense throughout postnatal development and in adult testis.
No evidence was obtained for expression of either the inhibin/activin
subunits or follistatin in the germ cells, peritubular myoid cells, or
other interstitial cells in any of the sections examined. In the human
fetal testis, both - and ßB-subunits were immunodetectable at 16,
18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger
immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months,
immunoexpression of the ßB-subunit was no longer detectable, whereas
the -immunostaining became weaker but was still present in both
Sertoli and Leydig cells. No positive immunostaining for ßA-subunit
or follistatin was detectable at any time point studied.
In conclusion, we have shown that, in the rat testis, the majority of
inhibin -subunit and inhibin/activin ßB-subunit is immunolocalized
to the fetal-type Leydig cells during fetal/neonatal life but,
following birth, immunoexpression in the Sertoli cells of both subunits
increases markedly while follistatin is immunodetectable only
postnatally.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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-subunit and one of the two similar, but
distinct ß-subunits (ßA and ßB). Activins are dimers of two
ß-subunits and the three possible activin dimers have been
designated, activin A (ßA/ßA), activin B (ßA/ßB), and activin
AB (ßA/ßB) (2). Apart from their action on FSH secretion, the
inhibins and activins have been shown to exert paracrine/autocrine
effects within the gonads (4, 5) and other tissues (6) and have been
proposed to have important paracrine function(s) during fetal
development (7). The proteins and messenger RNAs (mRNAs) of the three
subunits of inhibin/activin have been localized in the immature and
adult testis but little is known about the pattern of expression and
function of these peptides in the fetal and neonatal gonads (8, 9, 10)
.
In the adult testis, inhibin is mainly synthesized by Sertoli cells
with small amounts produced also in the Leydig cells (9, 10, 11). Previous
reports have described a similar pattern of expression in the fetal and
neonatal testis; however, in the limited data presented the
immunostaining in Leydig cells appeared to be more prominent in
immature animals than in adults (9, 10). A similar pattern of
expression has also been reported for the primate (rhesus monkey and
human) testis (8). In the rat, studies have indicated that
immunodetectable levels of the inhibin/activin subunits as well as
their mRNA levels in the testis vary greatly with age (9, 10). Specific
measurement of the levels of inhibin in circulation have usually been
based on assays that appear not to distinguish between dimeric (active)
inhibin and free -subunit (12), a problem which has now been
resolved by the development of highly specific monoclonal antibodies
that have been selected to recognize only the bioactive inhibin dimers
(13). Results using these assays have indicated that the
physiologically important form of inhibin in the male is inhibin B
(14). Immunocytochemical data generated using polyclonal antisera to
inhibin/activin peptides now merits revaluation.
Follistatins are a group of proteins reported to have the ability to suppress FSH secretion, but which are structurally unrelated to inhibins. So far, nine different-sized follistatin proteins have been described, all derived from a single gene by alternative splicing of its mRNA or posttranslational modifications (15). Studies by Kogawa and Nakamura and their colleagues (16, 17) have shown that follistatins bind to the activins and to lesser extent to inhibins (18). The role of the follistatins are not as yet completely elucidated, but the available data suggest that they may function primarily as regulators of activin bioavailability rather than as simple carrier molecules (19, 20). Follistatins are reported to be expressed both in the pituitary gland (16) and in the adult testis (21) but not in the fetal testis (22).
In the present study, we have used fixed tissue sections to undertake immunolocalization with new highly specific monoclonal antibodies raised against each of the inhibin/activin subunits and follistatin to study the ontogeny and cellular localization of the inhibin/activin subunits and follistatin in fetal testis from rat and human and to compare this to the postnatal rat testis. The results obtained have demonstrated that, in the rat, the majority of inhibin/activin is immunolocalized to the Leydig cells during fetal life, but, after birth, immunoexpression in the Sertoli cells becomes more significant. We believe this is the first detailed study describing the pattern of expression of immunodetectable inhibin/activin subunits and follistatin during fetal and neonatal life.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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Human fetal and postnatal testes were obtained from an archive collection at the Human Genetics Unit, Western General Hospital, Edinburgh, UK, and were derived from routine autopsies performed between 1990 and 1993. Testes from fetuses of 16, 18, and 24 (n = 2) weeks gestation and testes from 4-month-old neonates (n = 2) had been fixed in neutral buffered formaldehyde and were processed as above.
Immunocytochemistry
Sections (5 microns) were mounted on slides coated with
3-aminopropyl triethoxy-silane (TESPA; Sigma Chemical Co., St. Louis,
MO) and dried overnight at 50 C. Before incubation with primary
antibody, sections were dewaxed, rehydrated, in graded ethanols, washed
in water and TBS (0.05 M Tris-HCl pH 7.4, 0.85% NaCl)
following by blocking endogenous peroxidase by incubating the section
for 30 min in 1% H2O2 in TBS. Sections were
subjected to antigen retrieval (25) by microwaving in 0.01
M citrate buffer (pH 6.0) on full power for 20 min, and
thereafter left standing for 20 min without disturbance. Sections were
then washed for 5 min in TBS and blocked using normal rabbit serum
(Dako, High Wycombe, Buckinghamshire, UK) diluted 1:5 in TBS. Purified
monoclonal antibodies directed against -subunit [(26), code
173/9K] were used at a concentration of 2 µg/ml and antibodies
directed against the ßB-subunit, which had a 1% cross-reaction with
ßA-subunit, [(27), code 12/13] at a concentration of 0.12 µg/ml.
Monoclonal antibodies specific for the ßA-subunit [(28), code E4]
were used at a range of concentrations between 0.74 and 37 µg/ml.
Antibodies directed against recombinant FS-288 follistatin (code 17/2;
Evans and Groome, unpublished) were used at a concentration of 70
µg/ml; this antibody will recognize follistatin even when bound to
activin (L. R. Evans, unpublished). The immunostaining procedure was
similar with all antibodies that were diluted in TBS containing normal
rabbit serum (5:1, vol/vol) before incubation on sections under plastic
coverslips overnight at 4 C. The following day coverslips were removed,
sections washed twice in TBS (5 min each wash), incubated for 30 min
with biotinylated rabbit antimouse immunoglobulins (Dako) diluted 1:500
in TBS and then washed again in TBS (2 x 5 min). For detection of
bound antibodies, sections were first incubated with avidin-biotin
complex conjugated with horseradish peroxidase for 30 min and washed
twice in TBS (5 min each). Colour reaction product was developed by
incubating sections in a mixture of 0.05% (wt/vol)
3,3'-diaminobenzidine tetra-hydrochloride (DAB, Sigma) in 0.05
M Tris-HCl, pH 7.4, and 0.01% hydrogen peroxide. After
5–15 min, sections were washed in distilled water, counterstained with
hematoxylin, dehydrated in graded ethanols, cleared in xylene and
coverslipped using Pertex mounting medium (CellPath plc, Hemel
Hempstead, UK). Specificity of the antibodies was controlled by using
normal mouse serum instead of primary antibodies and by preabsorbing
the antibodies with the corresponding peptide.
Coimmunolocalization of inhibin- and 3ß-HSD
To detect inhibin -subunit and the steroidogenic enzyme
3ß-hydroxysteroid dehydrogenase (3ß-HSD) simultaneously in one
section, double fluorescent immunostaining was performed. Antibodies
raised in rabbits, against human 3ß-HSD (29), were kindly donated by
Professor Ian Mason (University of Edinburgh, Edinburgh, UK). Tissue
sections were blocked with normal goat serum and incubated with the
mixture of both primary antibodies (anti 3ß-HSD was used at a
dilution of 1:1000 and anti -inhibin at a dilution of 1:500)
overnight at 4 C. Incubation with primary antibodies was followed by
two washes in TBS and incubation with a mixture of fluorescent
secondary antibodies (goat antimouse FITC conjugated and goat
antirabbit TRITC conjugated, both from Sigma) at a dilution of 1:20 for
1 h at room temperature. Sections were washed again three times in
TBS and coverslipped using glycerol gelatin and examined under UV
light.
Analysis and photography
Images were captured into a Macintosh PowerPC computer using an
Olympus Provis Image analysis system (Olympus Optical Co., London)
equipped with a Kodak DCS420 camera (Eastman Kodak, Rochester, NY). For
comparative assessment, the intensity of immunopositive staining was
given a score as follows: - negative, ± barely detectable + faint, ++
clear positive, +++ intense positive.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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-subunit-subunit was first
detectable in the fetal rat testis on day 14.5 (Fig. 1a), the earliest age at which the testis could be
morphologically distinguished from the ovary. At this age, the
immunostaining appeared to be equally intense (+) in Sertoli and
interstitial cells. However, on day 15.5 (Fig. 1b), the immunostaining
was more intense in the interstitial cells (++) than in Sertoli cells
(+, arrowheads). Thereafter, the immunostaining remained
very intense in the fetal Leydig cells (+++) but was barely detectable
(±) in the fetal Sertoli cells (Fig. 1d). Double immunofluoresence
staining with anti--subunit and anti-3ß-HSD antibodies clearly
showed colocalization of 3ß-HSD and -subunit (Fig. 2) and therefore demonstrated that steroidogenically
active Leydig cells are the cells expressing inhibin -subunit in the
interstitium during fetal life. No positive immunostaining was detected
in the fetal ovary at any age examined (not shown).
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-subunit immunostaining in the interstitium
remained intense (+++) for several days after birth in the clusters of
fetal-type Leydig cells (Fig. 3a, arrows).
With progressive age and replacement of the fetal Leydig cells by their
adult-type counterparts, immunostaining reduced (+/++, Fig. 3b). In
contrast, to fetal life immunoexpression of the -subunit was
increased in the Sertoli cells after birth and differences between the
amount of immunostaining in individual tubule cross sections
(i.e. stage-dependent expression) from + to ++ could already
be distinguished on day 3 of life (Fig. 3a). Immunostaining of Sertoli
cells was clearly stage specific around day 27 (Fig. 3c);. in the adult
immunostaining of Sertoli cells in some tubules was very intense (+++),
whereas others were barely positive (±) (Fig. 3d).
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) but not on
days 14.5 or 15.5 p.c. Immunostaining in the Leydig cells remained
intense throughout fetal life (+++, Fig. 4, b and c). Like that for the
-subunit, immunoexpression of the ßB-subunit remained intense
postnatally in the fetal type-Leydig cells but became much weaker once
these cells were replaced by the adult generation of Leydig cells (Fig. 5, compare c + with a +++). Immunostaining for the
ßB-subunit was also observed within the tubules in the cytoplasm of
Sertoli cells, however, this staining was very weak (±) throughout
fetal development; no positive immunostaining was observed in fetal
ovaries (not shown). Postnatally, the immunostaining in the Sertoli
cells became more prominent soon after birth and remained prominent
throughout postnatal development (Fig. 5). Like the -subunit,
immunostaining for ßB-subunit in the adult testis was stage dependent
in Sertoli cells (+ to +++), with some immunostaining also observed in
interstitial cells (+).
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, a and c), the immunodetectable -and ßB-inhibin
subunits were present in both interstitial and Sertoli cells with
similar intensity (++). By 24 weeks (Fig. 6, b and d), most Sertoli
cells appeared to contain more immunoreactive protein (++/+++) than did
the Leydig cells (+/++), and this was also the case for
immunoexpression of -subunit in the two postnatal samples examined
(Fig. 6e). These results are consistent with the different time scales
of development and maturation of the rat and human gonad. In the human,
gonadal differentiation starts during the first trimester (30). FSH
production and secretion in the human fetus starts at around week 12 of
pregnancy and increased FSH secretion may be the trigger for the
expression of inhibin/activin subunits in Sertoli cells. As the
youngest testis studied was from a 16-week-old fetus, it is not known
if, like in the rat, the primary source of inhibin/activin subunits at
the very earliest stages of gonadal differentiation before onset of FSH
secretion are Leydig cells. The immunoexpression of - and
ßB-subunits in the human testis appeared to decline after birth,
possibly as a consequence of reduced FSH secretion and production
(31).
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Follistatin
Immunostaining for follistatin in the fetal rat testis failed to
detect any positive signal at any age. During neonatal life, positive
immunostaining (+++) was detected in clusters of fetal-type Leydig
cells (Fig. 7) and in their adult-type counterparts
(+++) at all ages examined. No positive signal for follistatin was
detected in the limited number of sections available from the fetal or
neonatal humans.
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-subunit immunoexpression
in both Sertoli and interstitial cells in 12-day-old rats with the
stronger staining being in the Sertoli cells. In these animals,
immunostaining with antibodies directed against the ßB-subunit
resulted in a similar intensity of staining in the interstitial and
Sertoli cells but did not detect any signal in clusters of Leydig
cells. However, Shaha et al. (10) did not detect any
significant immunoexpression of either - or ßB-subunits in
interstitial cells of fetal (day 18 p.c.) and neonatal testes
(days 1 and 7). In contrast, Roberts and co-workers (22, 33) observed
expression of the mRNA for the -subunit in both interstitial and
intratubular cells from day 14.5 p.c. onwards but were able to
detect ßB-subunit mRNA only in the intratubular compartment from day
14.5 p.c. onwards and found ßA-subunit mRNA in the interstitial
compartment just before birth in the rat. In the human and rhesus
monkey, Rabinovici et al. (8) reported strong expression of
both - and ßB-subunits proteins in the interstitial cells during
fetal life with the -subunit also being expressed in the Sertoli
cells, whereas the expression of ßB-subunit appeared to be confined
to the Leydig cells. The results obtained in the present study where
highly specific monoclonal antibodies were used, demonstrate intense
immunostaining for both - and ßB-subunits in the interstitial
cells within the fetal rat testis. mRNA for -inhibin was also
localized to the interstitial compartment (unpublished observations).
These results are in accordance with the findings of Rabinovici
et al. (8) for primate testis and with the results from
Roberts and Barth (22) on mRNA expression in the rat testis. However,
in the paper by Shaha et al. (10) in which the results for
the fetal testis were confined to frozen sections of testes from day
18 p.c. fetuses, immunostaining was most apparent around the base
of the seminiferous cords and interstitial staining was not prominent
in fetal Leydig cells. This difference may be explained by the poor
preservation of the tissue seen in the frozen sections. In the present
study in which fixed tissues were used, double immunoflourescent
staining using an anti-3ß-HSD antibody clearly showed exclusive
colocalization of -subunit and 3ß-HSD in the same cells on day
17.5 p.c., confirming that steroidogenically active Leydig cells
were the only cells expressing inhibin subunits in the interstitial
compartment at this age.
Immunoexpression of -subunit was evident at a very early stage of
testicular development, closely following the formation of the
testicular cords. Immunoexpression of the ßB-subunit was only
detectable 2 days after that of the -subunit and ßA-subunit was
undetectable in all the testes examined. It is possible that the early
onset of the -subunit synthesis is a part of a protective mechanism
ensuring the formation of inhibin rather than activin once ß-subunit
expression commences. The detection of immunostaining for the
ßB-subunit in the fetal rat testis on day 16.5 p.c. would be
consistent with the formation of inhibin B within the fetal gonad.
However, on this day the expression of both subunits was confined to
the fetal Leydig cells and occurred at a time when FSH expression is
not yet detectable (34, 35). Several studies have demonstrated the
potential effects of inhibins/activins on Sertoli cell proliferation
(36, 37) and on the function of the adult type Leydig cell (4, 38).
However, studies using mice in which the -subunit of inhibin has
been knocked out suggest that inhibin ßB is not essential for
testicular development during fetal life as these mice have an
apparently normal testis at the time of birth and are initially fertile
although they subsequently develop gonadal tumors (39) .
In the present study, we found that, in the rat, the patterns of
expression of both the - and ßB-subunits changed dramatically
after birth. While the level of immunostaining in the clusters of fetal
type Leydig cells remained high, expression of the -subunit in
Sertoli cells increased dramatically and was clearly increased on day 3
of life. The cause of this increase is unknown, but it could well be
connected changes in the levels of circulating FSH as this is known to
stimulate inhibin/activin production in the testis (40). In the rat
fetus, FSH is first detectable around day 19.5 p.c. (34, 35) rises
just before birth, and continues to increase during neonatal life.
Follistatins were originally identified as proteins, like inhibins, with the ability to suppress FSH secretion (41). However, additional studies have suggested that their primary role is to bind activins (16, 17) to prevent their biological effects (19, 20). Follistatin proteins are expressed in the adult testis (21) and pituitary gland (16) as detected by immunocytochemistry, but studies by Roberts and Barth (22) have failed to detect mRNA expression in the fetal rat testis. The immunocytochemical study presented in this paper confirmed their findings and have failed to detect follistatin in the fetal rat or human testis at any age using specific immunocytochemistry. We conclude that activin is either required in the fetal testis for normal development or alternatively that no bioactive activin is produced at this time and therefore there is no need for synthesis of follistatins to prevent its action. Postnatally, the expression of follistatin was detectable in the Leydig cells immediately after birth. This finding was rather surprising because, at that age, the Leydig cells are still of the fetal type, found prenatally. What triggers the presence of follistatin protein in these cells is not known. It is unlikely to be changes in LH, which regulates the steroidogenic function of Leydig cells during late fetal life (42), because LH is already present in the fetus on day 17.5 of gestation (34). It has been reported that follistatin mRNA is present in Sertoli and germ cells but not Leydig cells in adult rat testis (43); the question of the cellular site of synthesis of the immunodetectable follistatin, and the control of its production, therefore, requires further investigation. Studies by Boitani and co-workers (36) have suggested that Sertoli cell proliferation is stimulated by activin in the presence of FSH. Therefore, we speculate that the expression of follistatin in the testis postnatally could be involved in regulation of the effects of activin on Sertoli cells. No follistatin was detected in any of the human tissue sections examined. The antibodies used were raised against human follistatin, making it unlikely that they did not detect the protein, suggesting rather that follistatin is not produced by the fetal and neonatal human testis, and that either activin may be important for fetal testicular development in the human or alternatively, that there is no bioactive activin produced in the human fetal testis and therefore there was no need for synthesis of follistatin to prevent its actions.
The fetal-type Leydig cells differ from their adult counterparts in
several respects (42). It is believed that, postnatally, the fetal
Leydig cells do not transform into their adult type counterparts but
are replaced by new adult-type of Leydig cells that differentiate
from interstitial cells and populate the developing testis (42). The
data reported in this paper suggest yet another difference between
fetal-type and adult-type Leydig cells, namely in the expression of the
inhibin subunits. Both the - and ßB-subunits showed a similar
pattern of expression in the Leydig cells during neonatal life with
immunostaining for both subunits remaining prominent immediately after
birth in clusters of Leydig cells. In the rat, by around days 10–15 of
life, very few Leydig cells are evident within the testis, and when the
new adult generation of Leydig cells first appear they are scattered
between the tubules and are not in clusters as occurs for the fetal
generation of Leydig cells. Immunoexpression of - and ßB-subunits
in adult generation of Leydig cells was weaker than that seen in the
clustered fetal type Leydig cells during early neonatal life.
In conclusion, the data presented in this paper demonstrate for the
first time immunoexpression of - and ßB-inhibin/activin-subunits
in the Leydig cells of the fetal rat testis, suggesting that these
protein(s) play an important autocrine or paracrine roles at this stage
of development. In contrast, inhibin/activin subunits in the human
fetal testis have been detected in both Sertoli and interstitial cells,
a finding that can be explained by the different timing of gonadal
development in rodents and primates.
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Acknowledgments |
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Footnotes |
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Received October 15, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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5–4 isomerase from placenta:
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dehydrogenation/isomerization of C21 and C19 steroids. Endocrinology 126:2493–2498[Abstract]
-Inhibin is a tumour suppressor gene with gonadal specificity
in mice. Nature 360:313–319[CrossRef][Medline]
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S. J. McPherson, S. L. Mellor, H. Wang, L. W. Evans, N. P. Groome, and G. P. Risbridger Expression of Activin A and Follistatin Core Proteins by Human Prostate Tumor Cell Lines Endocrinology, November 1, 1999; 140(11): 5303 - 5309. [Abstract] [Full Text]
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I. Ketola, N. Rahman, J. Toppari, M. Bielinska, S. B. Porter-Tinge, J. S. Tapanainen, I. T. Huhtaniemi, D. B. Wilson, and M. Heikinheimo Expression and Regulation of Transcription Factors GATA-4 and GATA-6 in Developing Mouse Testis Endocrinology, March 1, 1999; 140(3): 1470 - 1480. [Abstract] [Full Text]
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R. A. Jarred, B. Cancilla, M. Richards, N. P. Groome, K. P. McNatty, and G. P. Risbridger Differential Localization of Inhibin Subunit Proteins in the Ovine Testis during Fetal Gonadal Development Endocrinology, February 1, 1999; 140(2): 979 - 986. [Abstract] [Full Text]
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A.-M. Andersson, J. Müller, and N. E. Skakkebæk Different Roles of Prepubertal and Postpubertal Germ Cells and Sertoli Cells in the Regulation of Serum Inhibin B Levels J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4451 - 4458. [Abstract] [Full Text]
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 F. Delfino and W. H. Walker Stage-Specific Nuclear Expression of NF-{kappa}B in Mammalian Testis Mol. Endocrinol., November 1, 1998; 12(11): 1696 - 1707. [Abstract] [Full Text]
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