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Original Studies |
Department of Growth and Reproduction, University of Copenhagen, DK-2100 Copenhagen, Denmark
Address all correspondence and requests for reprints to: Anna-Maria Andersson, M.Sc, Ph.D., Department of Growth and Reproduction, section GR 5064, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail: anna{at}rh.dk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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and ßB) were examined in adult testicular tissue with
normal spermatogenesis, spermatogenic arrest, or Sertoli cell only
tubules (SCO) as well as in normal testicular tissue from an infant and
a prepubertal boy. Adult men with testicular biopsy showing normal
spermatogenesis (n = 8) or spermatogenic arrest (n = 5) had
median inhibin B levels of 148 pg/mL (range, 37–463 pg/mL) and 68
pg/mL (range, 29–186 pg/mL), respectively, corresponding to normal or
near-normal levels of our reference population (165 and 31–443 pg/mL;
n = 358). Men with SCO (n = 9) had undetectable or barely
detectable (n = 1) serum levels of inhibin B. In contrast to
adults, prepubertal boys with SCO (n = 12) all had measurable
serum inhibin B levels that corresponded to our previously determined
normal range in healthy prepubertal boys (n = 114). However, in
postpubertal samples from the same SCO boys, inhibin B levels were
undetectable as in the adult SCO men. Intense inhibin -subunit
immunostaining was evident in Sertoli cells in both prepubertal and
adult testes. In the prepubertal testis, positive immunostaining for
the ßB-subunit was observed in Sertoli cells. In the adult testis,
intense immunostaining for the ßB-subunit was evident in germ cells
from the pachytene spermatocyte to early spermatid stages and to a
lesser degree in Leydig cells, but not in Sertoli cells or other stages
of germ cells. Thus, surprisingly, in adult men the two subunits
constituting inhibin B were expressed by different cell types. We
speculate that during puberty Sertoli cell maturation induces a change
in inhibin subunit expression. Thus, immature Sertoli cells express
both and ßB inhibin subunits, whereas fully differentiated
Sertoli cells only express the -subunit. The correlation in adult
men between serum inhibin B levels and spermatogenesis may be due to
the fact that inhibin B in adult men is possibly a joint product of
Sertoli cells and germ cells, including the stages from pachytene
spermatocytes to early spermatids. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit and a ßA-subunit, and the
inhibin B dimer, consisting of an -subunit and a ßB-subunit (1).
The subunits are covalently linked by a disulfide bond. By the
implementation of a new immunoassay format that made it possible to
measure these two bioactive inhibin forms specifically, it has been
shown that inhibin B is the principal circulating bioactive inhibin
form in men (2). Activins are structurally related dimers that are
composed of two ß-subunits and that can stimulate FSH release.
Inhibin B is presumed to be produced primarily by the Sertoli cells of
the testis based on in vitro studies (3, 4, 5, 6) and the
localization of inhibin subunit peptide and messenger ribonucleic acid
in testicular tissue from rats (7, 8, 9), primates (10), and humans (9) by
immunohistochemistry and in situ hybridization. However, the
regulation of inhibin B secretion is still largely unknown. Studies of
the role of gonadotropins or androgens in inhibin regulation are
equivocal. Several studies indicate that the presence of specific germ
cell types may stimulate the production and secretion of inhibin as
shown in the rat (11, 12, 13, 14) and indicated in men with impaired
spermatogenesis, who generally have very low or undetectable serum
inhibin B levels (15). On the other hand, we have recently shown that
prepubertal boys, who do not have spermatogenesis and therefore lack
the relevant germ cell types, secrete inhibin B in readily
measurable levels (16, 17). To further elucidate the role of germinal
cells in the regulation of inhibin B secretion we measured serum
inhibin B levels in men with either normal ongoing spermatogenesis,
spermatogenic arrest, or Sertoli cell only syndrome (SCO) based on a
testicular biopsy. In addition, we measured serum inhibin B levels in
boys and adolescents previously treated for acute lymphoblastic
leukemia (ALL), some of whom became SCO as a result of the treatment.
The expression and cellular localization of inhibin - and
ßB-subunits were investigated using immunohistochemistry in
testicular biopsies from adult men with normal or abnormal
spermatogenesis and from prepubertal boys. Based on our observations, we hypothesize that inhibin B in adult men may be produced by Sertoli cells and spermatocytes in collaboration.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Prepubertal boys. Simultaneous testicular biopsies and blood samples were available from 34 boys previously treated for ALL, who had testicular biopsy performed for screening for lymphoblastic infiltration. Of these boys, 12 acquired a SCO pattern as a consequence of their treatment, 13 boys had testes with normal numbers of germinal cells, and 9 boys had testes with reduced numbers of germinal cells after termination of treatment for ALL. From 17 boys, longitudinal blood samples and testicular biopsies were available, and from 6 of these boys postpubertal blood samples were also available. For immunocytochemistry, testicular tissue was obtained at autopsy from 2 boys, aged 4.5 months and 7 yr, who had died in sudden accidents.
Adult men. Concurrent testicular biopsies and blood samples were obtained from men referred to our clinic for infertility and from testicular cancer patients who had biopsy performed as screening for carcinoma in situ in the contralateral testis. Based on the testicular biopsy and semen parameters, men were grouped according to the presence of normal spermatogenesis (n = 8), spermatogenic arrest (n = 5), or SCO syndrome (n = 9). The men with normal spermatogenesis included five men with obstruction of the efferent ducts, two testicular cancer patients with one remaining normal testis after unilateral orchidectomy, and one patient with normal spermatogenesis in one testis and spermatogenic arrest of the other. Men with spermatogenic arrest were defined as having bilateral spermatogenic arrest and azoospermia. The SCO men were defined as having SCO and azoospermia and included four men with idiopathic bilateral SCO and five unilaterally orchidectomized men with SCO in the remaining testis due to irradiation for carcinoma in situ.
Serum samples and testicular biopsies
Serum was separated after clotting and was stored at -20 C
until hormone measurements. Adult serum samples were measured within a
few weeks from the time of sampling, whereas the time of storage of
serum samples from ALL treated boys varied from 15 yr to a few months.
Serum inhibin B was measured in an enzyme immunometric assay,
previously described (18). This assay is specific for the bioactive
inhibin B dimer (-ßB). The sensitivity of the inhibin B assay was
18 pg/mL, and the intra- and interassay coefficients of variation were
less than 12% and less than 17%, respectively.
Testicular biopsies of approximately 2 x 2 mm (boys) or 3 x 3 mm (adult men) were obtained by open surgery and fixed in Stieve’s fixative. For evaluation of spermatogenic status, slides were stained with hematoxylin-eosin before evaluation by light microscopy.
Testicular biopsy and blood sampling in boys treated for ALL as screening for testicular relapse was approved by the local ethical board. In the adult subjects, testicular biopsies and blood samples were performed as part of a clinical routine evaluation. The patients had given informed consent to the procedures.
Immunohistochemistry
Sections (4 µm) were mounted on SuperFrost Plus slides
(Menzel, Braunschweig, Germany) and dried overnight at 50 C. Before
incubation with primary antibody, sections were dewaxed, rehydrated in
graded ethanol, and washed in water and 0.05 mol/L Tris-HCl (pH 7.4)
and 0.15 mol/L NaCl (TBS). Sections were subjected to antigen retrieval
by microwaving in 0.01 mol/L citrate buffer (pH 5.5) on full power (750
watts) for 2 min and on 40% power for an additional 18 min. Sections
were allowed to cool at room temperature for 20 min before being washed
for 5 min in TBS and subsequently incubated in 1%
H2O2 in TBS for 30 min to block endogenous
peroxidase. Purified monoclonal antibodies directed against -subunit
(19) (code R1) were used at a concentration of 2 µg/mL, and two
different antibodies against the ßB-subunit (18) (code C5 and 12/13)
were used at concentrations of 25 and 5 µg/mL, respectively. The C5
and 12/13 antibodies directed against the ßB-subunit had 0.5–1%
cross-reactivity with the ßA-subunit (18). Primary antibodies were
diluted in TBS and incubated on sections in a humid chamber overnight
at 4 C. The following day sections were washed three times in TBS (3
min/wash), incubated with biotinylated goat antimouse Igs (Zymed
histostain kit, Zymed Laboratories, Inc., San Francisco,
CA) for 30 min, and then washed again three times in TBS. For detection
of bound antibodies, sections were subsequently incubated with
peroxidase-conjugated streptavidin-biotin complex (Zymed Histostain)
for 30 min, followed by washing three times in TBS. Color was developed
in an aminoethyl carbazole substrate solution (Zymed Histostain kit).
After 6–10 min of color development, sections were washed in tap
water, counterstained with hematoxylin, and mounted in glycerol vinyl
alcohol (Zymed Laboratories, Inc.). On sections
from prepubertal testes an alternative immunostaining procedure was
also used. In the alternative procedure the second layer of antibody
was exchanged with unlabeled rabbit antimouse antibody (Dako Corp., Glostrup, Denmark) diluted 1:50 in TBS, and the third
layer was exchanged with alkaline phosphatase-antialkaline phosphatase
complex (Dako Corp.) diluted 1:25 in TBS. After a final
wash in 0.05 mol/L Tris-HCl (pH 8.7), color was developed in a
new fuchsin substrate solution (Sigma, St. Louis,
MO). The specificity of the antibodies was controlled by using normal
mouse serum instead of primary antibodies and by preabsorbing the
antibodies with the corresponding peptide.
Statistics
Differences in inhibin B levels among men with normal spermatogenesis, spermatogenic arrest, and SCO were tested by the Mann-Whitney U test. The influence of storage on serum inhibin B levels was tested by linear regression on data that were square root transformed to obtain a good approximation to the normal distribution.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The median and range of serum inhibin B levels within the
different groups are summarized in Table 1
. For comparison, serum inhibin B levels
in a normal population of 114 prepubertal boys and 358 adult men, which
have been described previously (17), are also included in the
table.
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. Sera
from boys treated for ALL had been stored at -20 C from a few weeks up
to 15.3 yr (median, 4.8 yr) before being analyzed for inhibin B.
Because of the large variation in length of storage, the long term
storage stability of inhibin B had to be considered. There was no time
trend in prepubertal inhibin B levels when plotted according to storage
time (r2 = 0.005; P = 0.638), indicating
that inhibin B was relatively stable during long term storage.
Furthermore, serum inhibin B levels in prepubertal boys treated for ALL
with normal testis pattern were all within the normal range for
prepubertal boys, as were the levels in prepubertal boys with SCO. In
all postpubertal samples from boys who acquired a SCO pattern, serum
inhibin B levels were undetectable (Fig. 1).
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Expression of inhibin B subunits in human prepubertal and adult testes
In prepubertal testes, intense positive immunostaining for inhibin
-subunit was observed that was localized to Sertoli and interstitial
cells (Fig. 2A). Positive immunostaining
for inhibin ßB-subunit was also observed in Sertoli cells of
prepubertal testes (Fig. 2B). In adult testes with ongoing
spermatogenesis, positive immunostaining for inhibin -subunit was
observed in both Sertoli cells and Leydig cells (Fig. 3, A and B). A similar -subunit
immunostaining pattern was observed in testes with spermatogenic arrest
(Fig. 4A) and in testes with SCO (Fig. 4B). In adult testes with normal spermatogenesis and with spermatogenic
arrest, intense immunostaining for the inhibin ßB-subunit was located
in germ cells from pachytene spermatocytes to round spermatids (Figs. 3, C and D, and 4C). An identical staining pattern was observed using
two different monoclonal anti-ßB antibodies. In contrast, no staining
for the inhibin ßB-subunit was observed in Sertoli cells;
spermatogonia; preleptotene, leptotene, or zygotene spermatocytes; or
late spermatids (from types Sb–Sd). However, Leydig cells were also
positive for ßB-subunit immunostaining. In adult testes with SCO, no
intratubular immunostaining for the ßB-subunit was observed (Fig. 4D). Occasionally, faint ßB-subunit immunostaining was observed in a
few Leydig cells, but, in general, Leydig cells were also negative for
ßB-subunit staining in SCO testes.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit assay, was highest in the
presence of late spermatids. Human adult serum inhibin B levels, however, were not dependent on complete spermatogenesis, as serum inhibin B levels were measurable in all five men with bilateral spermatogenic arrest at the spermatocyte level, although the inhibin B levels tended to be lower than those observed in a normal male population, including men with documented complete spermatogenesis. Furthermore, levels of serum inhibin B in men with only one normal functioning testis were well within the normal range, indicating that the remaining testis after unilateral orchidectomy may be able to compensate for the missing testis with respect to inhibin B secretion.
The regulation of inhibin B secretion in prepubertal boys is not clear.
The localization of both - and ßB-subunits to interstitial and
Sertoli cells in the prepubertal human testis observed in this study is
in agreement with previous findings in the rat (8, 9) and human (9). We
recently showed that serum inhibin B levels are temporarily highly
elevated postnatally, presumably as a consequence of the temporary
activation of the pituitary shortly after birth (17), as also shown
previously (20), indicating that inhibin B secretion in the newborn is
under the influence of gonadotropins. Later in childhood, gonadotropin
levels are low, but perhaps sufficient to sustain the readily
measurable serum levels of inhibin B observed throughout childhood
(16). Thus, serum inhibin B levels in hypogonadotropic boys are in the
low range of healthy boys (21), although this may be due to a reduced
number of Sertoli cells rather than to decreased inhibin B production
caused by low FSH levels. Compared to the staining intensity observed
for the ßB-subunit in the adult testis, the staining in prepubertal
testes was relatively weak. This was also true in the testis from a boy
aged 4.5 months, although this is an age when serum inhibin B levels is
known to be increased above adult levels (17).
Several studies showing localization of the inhibin -subunit in
human testis exist that, in general, agree on the localization of this
subunit to the Sertoli cells and interstitial/Leydig cells in adult
testes (22, 23, 24, 25). Inhibin ßB-subunit expression has previously been
localized to Sertoli cells in the adult rat (8, 9) and monkey (10).
Recently, a study on the immunolocalization of the inhibin/activin
ßB-subunit in the adult human testis using the same antibodies as in
our present study was published that showed colocalization of the -
and ßB-subunits in the Sertoli cells (26). In contrast to these
findings, we found that the ßB-subunit was localized to pachytene
spermatocytes and round spermatids as well as to Leydig cells, whereas
the -subunit was localized to Sertoli cells and Leydig cells in the
adult human testis. We do not know the reason for this discrepancy, but
it may be due to differences in the fixation and processing of the
tissue. The facts that we were able to eliminate our ßB-subunit
staining pattern by preabsorbing the antibody with a ßB peptide, that
the staining pattern was highly reproducible in several different
tissue section from different men, and that our ßB-subunit staining
was restricted to selected germ cell types with no or negligible
background staining of other cell types strongly indicate that our
ßB-subunit staining was specific. However, as both subunits are
needed to produce inhibin B, our findings raises the question of where
circulating inhibin B is produced in the adult human testis. As both
subunits are present in Leydig cells, it cannot be excluded that these
cells contribute to the circulating levels of inhibin B. However,
studies in rats have shown that the major route via which inhibin
reaches the bloodstream is through secretion into the seminiferous
tubular fluid, thus supporting the idea of an intratubular production
of inhibin (27). Alternatively, it may be speculated that the Sertoli
cells express the inhibin ßB-subunit at levels below the sensitivity
of our immunohisto-chemistry.
The site of dimerization of inhibin subunits is not known. By electron
microscopy, evidence of transfer of inhibin -subunit from Sertoli
cells to spermatocytes has been reported in the human testis (24).
However, although inhibin subunit dimerization may take place
intracellularly in the spermatocytes, it could also theoretically take
place extracellularly. Creation of a disulfide bond from the sulfhydryl
groups of two cystein residues is a passive reaction that does not
require energy and that takes place in a nonreducing environment, such
as in the endoplasmic reticulum, Golgi, coated vesicles, or even
extracellular fluid. Manson et al. have shown that the
propeptide sequences present in the ß-subunit precursors are
necessary for dimerization, presumably by providing the proper
configuration for the involved cystein residues to be exposed (28, 29).
However, several studies have shown that a high proportion of the
inhibin forms in plasma consist of unprocessed or partly processed
precursor dimers (30, 31), indicating that the structural elements
necessary for dimerization of subunits are still present after
secretion. Further processing of inhibin/activin precursors into the
bioactive hormone by cleavage of the proprotein region can also take
place extracellularly (32).
We speculate that in the adult human testis, inhibin B may be a joint product of Sertoli cells and spermatocytes, and hence the change in localization of the ßB subunit during maturation may explain why serum inhibin B levels are germ cell dependent in the mature testis and not in the immature testis in man. Furthermore, our hypothesis that inhibin subunit dimerization and processing may take place postsecretionally may explain the abundance of non- or partly processed as well as monomeric forms of inhibin in serum. The five men with spermatogenic arrest who were included in this study all had spermatogenic progress that included the level of pachytene spermatocytes and all had measurable serum inhibin B levels. The somewhat lower serum inhibin B levels in these men could be explained by the fact that, in general, they have fewer spermatocytes beyond the pachy-tene stage and no spermatids. If, as we believe, ßB-subunit expression by pachytene spermatocytes and round spermatids correlates with serum inhibin B levels, we would expect men with spermatogenic arrest at a level before the pachytene stage to have very low or unmeasurable serum inhibin B levels.
ßB-subunits may also bind together as homodimers to make activin B. Thus, the expression of the ßB-subunit by certain stages of spermatocytes may not have anything to do with inhibin B, but, rather, may imply expression of activin B by these types of spermatocytes. As no activin B dimer-specific antibody is available to our knowledge, it is not possible by immunohistochemistry or in situ hybridization to distinguish whether the observed ßB peptide expression in spermatocytes is present as free ßB-subunits or activin B dimers. To our knowledge no specific activin B immunoassays are presently available, but development of such an assay would facilitate the specific quantitative measurement of activin B in enriched spermatocyte preparations and thus aid in solving this question.
In conclusion, during pubertal maturation of the human male, testicular inhibin B production becomes germ cell dependent. This change in inhibin B regulation may be explained by a maturational change in the localization of expression of the inhibin ßB-subunit from the Sertoli cells to specific germ cell stages in the spermatogenic process, namely to cells from the pachytene spermatocyte to round spermatid stages. Our suggestions that inhibin B is a joint product, and that the subunits of which may originate from different cell types propose to our knowledge a new concept in the regulation of peptide hormone and growth factor expression. This maturational change in the production and regulation of inhibin B levels thus provides a close link between serum inhibin B levels (and thus FSH feedback regulation) and spermatogenesis.
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Acknowledgments |
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Footnotes |
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Received June 23, 1998.
Revised August 27, 1998.
Accepted September 17, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and ßB subunits during the
cycle of the rat seminiferous epithelium. Endocrinology. 124:987–991.[Abstract]
-subunit mRNA levels by specific germ cell types. Mol Cell
Endocrinol. 72:13–22.[CrossRef][Medline]
-subunit in the human testis. A light-
and electron-microscopy study. Cell Tissue Res. 269:221–227.[CrossRef][Medline]
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A.-M. Andersson, J. H. Petersen, N. Jorgensen, T. K. Jensen, and N. E. Skakkebaek Serum Inhibin B and Follicle-Stimulating Hormone Levels as Tools in the Evaluation of Infertile Men: Significance of Adequate Reference Values from Proven Fertile Men J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2873 - 2879. [Abstract] [Full Text] [PDF]
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 K. Almstrup, J. E. Nielsen, M. A. Hansen, M. Tanaka, N. E. Skakkebaek, and H. Leffers Analysis of Cell-Type-Specific Gene Expression During Mouse Spermatogenesis Biol Reprod, June 1, 2004; 70(6): 1751 - 1761. [Abstract] [Full Text] [PDF]
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A. M. Wikstrom, T. Raivio, F. Hadziselimovic, S. Wikstrom, T. Tuuri, and L. Dunkel Klinefelter Syndrome in Adolescence: Onset of Puberty Is Associated with Accelerated Germ Cell Depletion J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2263 - 2270. [Abstract] [Full Text] [PDF]
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N. Lahlou, I. Fennoy, J.-C. Carel, and M. Roger Inhibin B and Anti-Mullerian Hormone, But Not Testosterone Levels, Are Normal in Infants with Nonmosaic Klinefelter Syndrome J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1864 - 1868. [Abstract] [Full Text] [PDF]
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P.-M. G. Bouloux, E. Nieschlag, H. G. Burger, N. E. Skakkebaek, F. C.W. Wu, D. J. Handelsman, G. H.W. Baker, R. Ochsenkuehn, A. Syska, R. I. Mclachlan, et al. Induction of Spermatogenesis by Recombinant Follicle-Stimulating Hormone (Puregon) in Hypogonadotropic Azoospermic Men Who Failed to Respond to Human Chorionic Gonadotropin Alone J Androl, July 1, 2003; 24(4): 604 - 611. [Abstract] [Full Text] [PDF]
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