European Journal of Endocrinology (1997) 137 523–529
ISSN 0804-4643
Follistatin-like immunoreactivity in the cytoplasm and
nucleus of spermatogenic cells in the rat
Kenji Ogawa, Osamu Hashimoto1, Masamichi Kurohmaru1, Takeo Mizutani, Hiromu Sugino2
and Yoshihiro Hayashi1
Laboratory Animal Research Center, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-01 Japan,
1
Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113 Japan and
2
Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, 770 Japan
(Correspondence should be addressed to K Ogawa)
Abstract
Immunohistochemistry using an antiserum raised against the synthetic follistatin peptide (residues
123–134) was used, in the present study, to detect the stage-specific appearance of immunoreactive
follistatin in the rat testis. Follistatin immunoreactivity was not found in Sertoli and Leydig cells, while
it was clearly detected in spermatogenic cells. Follistatin-like immunoreactivity was detected in the
cytoplasm and nucleus of late pachytene spermatocytes. Although the reaction in the cytoplasm
disappeared after meiosis, it continued to be intense in the nucleus from pachytene spermatocytes to
round spermatids. This finding indicated that follistatin or its closely related peptide produced in late
pachytene spermatocytes migrates from the cytoplasm to the nucleus. We subjected rat testis
homogenate to affinity chromatography on a sulfate-cellulofine and anti-follistatin Cys (123–134)–
Affi-Gel Hz column followed by reverse-phase HPLC and analyzed the resulting fractions by Western
blotting using follistatin antiserum. Three major bands at 57, 45 and 39 kDa or four bands at 52, 44,
39 and 34 kDa were detected in crude preparations from rat testis homogenate, under reducing or
non-reducing SDS-PAGE respectively. The protein from rat testis, which was recognized by antifollistatin (123–134) antiserum, exhibited a characteristic pattern for follistatin on SDS-PAGE, i.e.
slower migration under reducing conditions than under non-reducing conditions, suggesting that it
was follistatin or its closely related protein. Follistatin or its closely related protein may be a stagespecific modulator of spermatogenesis. Since follistatin-like immunoreactivity was not found in oocytes
in any stage of development from embryonic to adult rats, it may act in an event specific to
spermatogenesis, such as nuclear condensation.
European Journal of Endocrinology 137 523–529
Introduction
Activin, a member of the transforming growth factor
(TGF)-b family, was initially isolated from gonads as a
stimulator of follicle-stimulating hormone (FSH) secretion
from the pituitary gland. It was found to be a dimer of the
b subunit of inhibin which was an inhibitor of FSH
secretion (1–4). Follistatin, originally obtained from
porcine follicular fluid during the isolation of inhibin
and activin, is structurally different from inhibin/activin
in spite of its inhibin-like activity (5, 6). Activin and
follistatin produced in the gonads were thought to serve
as modulators of FSH release in a long loop feedback
manner to the pituitary. However, their broad distribution
throughout the body, including the pituitary gland (7, 8),
suggests that they act in a paracrine mechanism.
Like other members of the TGF-b family, activin exhibits
potent activities in diverse biological processes, including
promotion of erythroid differentiation (9), induction of
mesoderm tissue formation (10), stimulation of early
embryogenesis (11), promotion of folliculogenesis (12),
q 1997 Society of the European Journal of Endocrinology
modulation of pituitary and pancreatic hormone release
(13–15), and bone formation (16).
Nakamura et al. (17) found that follistatin is an
activin-binding protein. Follistatin binds to both inhibin
and activin through the common b subunit (18).
Neutralization of activin’s activity by follistatin is
proven in various systems, such as the stimulation of
FSH secretion in cultured pituitary cells (8), induction of
mesoderm tissue formation in Xenopus oocytes (19), and
differentiation of rat granulosa cells (20). These results
indicated that follistatin is a functional regulator of
activin’s activity.
Several investigations have been carried out to clarify
the role of inhibin and activin in spermatogenesis (21–
23). Inhibin reduces spermatogonial number (21), and
activin stimulates spermatogonial proliferation in vitro
(22). While the studies have shown the significance of
inhibin/activin, the function of follistatin in spermatogenesis is still unclear.
To detect the localization of follistatin in the rat testis, the
present study was undertaken by immunohistochemistry
524
K Ogawa and others
using an antiserum raised against the synthetic peptide of
follistatin.
Materials and methods
Materials
Recombinant human follistatin (rhFS) was kindly
provided by Dr M Muramatsu (Department of Biochemistry, Faculty of Medicine, Saitama Medical School,
Japan) and Dr Y Eto (Ajinomoto Co. Inc., Central
Research Laboratories, Kawasaki, Japan). Avidin–
biotin–peroxidase (ABC) reagent was obtained from
Vector Laboratories Inc. (Burlingame, CA, USA). The
peptide fragment of residues 123–134 of human
follistatin and diphtheria toxoid-conjugated Cys (123–
134) of human follistatin were obtained from Chiron
Mimotopes Peptide Systems (Victoria, Australia). CNBractivated Sepharose 4B was obtained from Pharmacia
LKB (Uppsala, Sweden). The Affi-Gel Hz and Econo-Pac
10DG desalting column were from BioRad (Richmond,
CA, USA). Sulfate-cellulofine was from Seikagaku Kogyo
(Tokyo, Japan). The HPLC 5TMS-300 column (Cosmosil
Packed Column, 4.6 × 250 mm) was obtained from
Nacalai Teque (Kyoto, Japan). ECL Western blotting
detection reagents were obtained from Amersham
International plc (Amersham, Bucks, UK).
Animals
Wistar Imamichi strain rats were obtained from the
Imamichi Institute for Animal Reproduction (Ibaraki,
Japan). All animals were maintained in air-conditioned
quarters and food and water were available ad libitum.
Preparation of tissue sections
For immunohistochemical examinations, the testes
were excised from 4-, 6-, 8-, 12- and 18-day plus 6-,
8- and 10-week-old rats (four animals were used at each
age). Under ether anesthesia, the testes were excised
from 4-, 6-, 8- and 12-day-old rats and were fixed in
Bouin’s fixative for 4 to 6 h. Older groups (18-day, 6-, 8and 10-week-old rats) were similarly anesthetized and
perfused transcardially with saline followed by Bouin’s
fixative. Testes excised from each animal were sliced into
slabs and immersed in the same fixative for 4 to 6 h.
After fixation, tissues were dehydrated in a graded
ethanol series, cleared in xylene and embedded in a
paraffin block. Sections were cut on a sliding microtome
and then subjected to immunohistochemical procedure
using follistatin antiserum.
Immunohistochemical procedure
A rabbit polyclonal antiserum against synthetic follistatin peptide (residues 123–134) (24–26) was used for
immunohistochemistry. The amino acid sequence of
EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
residues 123–134 is identical in human (6), rat (27),
mouse (28), ovine (29), bovine (30) and porcine (31)
follistatin. The antiserum was purified by affinity
chromatography on protein A–Sepharose before performing immunohistochemistry.
The ABC procedure was performed as described
previously (32). After deparaffinizing, the sections
were incubated in H2O2 in methanol for 30 min to
eliminate endogenous peroxidase. They were placed in a
moist chamber, covered first with 5% normal goat
serum for 30 min to minimize non-specific staining,
followed by rabbit antiserum diluted 1:1200 with 0.3%
BSA/0.01 mol/l PBS for overnight incubation at room
temperature. After washing with 0.01 mol/l PBS, they
were incubated with biotinylated goat anti-rabbit IgG
for 30 min and reacted with ABC reagent for 45 min. A
mixture of 0.05% 3,30 -diaminobenzidine tetrahydrochloride in 0.01 mol/l PBS–0.01% H2O2 was added for
7 to 10 min to develop the peroxidase reaction.
To confirm the method and antiserum specificity,
control sections were incubated with non-immune
rabbit serum from the same rabbit from which the
antiserum was obtained or primary antiserum absorbed
with excess (400 ng) rhFS. Additional controls were
stained without secondary antiserum or ABC reagent.
Preparation of affinity chromatography
column
The peptide fragment residues Cys (123–134) of
human follistatin coupled with diphtheria toxoid was
given to adult rabbit as an antigen. The rabbit was
injected with 500 mg of the conjugate emulsified in
Freund’s complete adjuvant every 2 weeks. Serum was
collected by heart puncture 5 weeks after first immunization. The serum was purified by affinity chromatography on follistatin peptide (123–134)-coupled
Sepharose 4B column. The antiserum recognized both
follistatin from porcine follicular fluid (pFS) and rhFS in
SDS-PAGE and immunoblotting analysis (data not
shown). IgG from serum was partially purified by
ammonium sulfate precipitation, and then coupled to
Affi-Gel Hz.
Preparation of testicular extract
The testes excised from 20 adult rats (8 to 10 weeks old)
were homogenized in buffer I (20 mmol/l Tris–HCl buffer,
pH 7.3, containing 2 mmol/l EDTA, and 5 mmol/l benzamidine–HCl) containing 0.15 mol/l NaCl, 1 mmol/l
di-isopropylfluorophosphate, 1 mmol/l N-ethylmaleimide, and 1 mmol/l phenylmethylsulphonyl fluoride.
The homogenates were centrifuged at 1600 g for
15 min. NaCl and Triton X-100 were added to the
supernatant to make the final concentrations of
0.35 mol/l and 1% respectively. The resulting solution
was stirred for 30 min and centrifuged at 9900 g for
30 min. Sulfate-cellulofine (10 ml) was added to 20 ml
Follistatin-like immunoreactivity in spermatogenic cells
EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
525
Table 1 The immunoreaction of testicular cells with anti-follistatin (123–134) at various ages.
Cells
Spermatogenic cells
Spermatogonia
Spermatocyte
Round spermatid
Elongated spermatid
Somatic cells
Sertoli cells
Leydig cells
4 days
6 days
8 days
12 days
18 days
6 weeks
8 weeks
10 weeks
¹
/
/
/
¹
/
/
/
¹
/
/
/
¹
/
/
/
¹
¹
/
/
¹
þ
þ
/
¹
þþþ
þþþ
¹
¹
þþþ
þþþ
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
¹
The intensity of reaction is given on a scale of ¹ to þ þ þ: (¹) negative, (þ) moderately positive, (þ þ þ) intensely positive. The symbol /
means the absence of corresponding cells.
of each supernatant and stirred gently for 12 h. The gels
were washed with buffer I containing 0.35 mol/l NaCl
and 0.1% Triton X-100, resuspended in the same buffer,
and packed into the column (1.6 × 20 cm). The column
was then eluted with buffer I containing 1.5 mol/l NaCl
and 0.1% Triton X-100. The elute was dialyzed against
10 mmol/l Tris, pH 7.5, overnight, and then applied to the
anti-follistatin–Affi-Gel Hz column. The column was
Figure 1 Histochemical micrographs showing localization of follistatin-like immunoreactivity in the rat testis (bar ¼ 50 mm). (A) Moderate
immunoreaction with anti-follistatin (123–134) serum was found in the cytoplasm and nucleus of the spermatocyte and an intense
reaction was found in the nucleus of the round spermatids, whereas it was not detected in the spermatogonia in the 6-week-old rat.
(B) Although follistatin-like immunoreactivity was not detected in pachytene spermatocytes, it was intense in the nucleus but not in the
cytoplasm of round spermatids in stages I to VI of seminiferous epithelia in the 8-week-old rat. (C) In stages VII to VIII, follistatin-like
immunoreaction was found in both the cytoplasm and nucleus of pachytene spermatocytes. Intense immunoreaction was also found in the
nucleus of round spermatids in stage VII to VIII (8-week-old rat). (D) In stages XI to XII, the reaction was found to be intense in the nucleus
and moderate in the cytoplasm of pachytene spermatocytes (8-week-old rat). When the antiserum was absorbed with an excess amount
(400 ng) of rhFS, the reaction was not found in any type of cell in the testis.
526
K Ogawa and others
EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
Figure 2 Schematic representation of follistatin-like immunoreactivity in spermatogenic
cells at different stages of the seminiferous
cycle.
washed with 10 mmol/l Tris–HCl, pH 7.5, followed
by 10 mmol/l Tris–HCl, pH 7.5/0.5 mol/l NaCl, and
then eluted by 100 mmol/l glycine, pH 2.5. The eluate
was desalted by using an Econo-Pac 10DG column
(4.6 × 250 mm), equilibrated with 0.1% trifluoroacetic
acid (TFA). The desalting sample was injected into a
5TMS-300 column (4.6 × 250 mm), equilibrated with
0.1% TFA, and eluted with a linear gradient of 16–40%
(v/v) acetonitrile in 0.1% TFA at a flow rate of 0.5 ml/min.
The eluted fractions were examined by SDS-PAGE and
immunoblotting analysis using anti-follistatin (123–
134).
SDS-PAGE and immunoblotting
pFS was purified from follicular fluid as described
previously (8). The pFS, rhFS and aliquots of eluted
fractions were dissolved in SDS-PAGE sample buffer
(10 mmol/l Tris, 1% SDS, 2% b-mercaptoethanol and
0.04% bromophenol blue) and heated at 100 8C for
5 min. Electrophoresis was carried out in 12.5% gels as
described by Laemmli (33), and the protein was
transferred to Immobilon membranes (Millipore). The
membrane was blocked with Block Ace (Dainihonseiyaku Inc., Osaka, Japan) and incubated with antifollistatin (123–134) serum diluted 1:3000 with 0.3%
BSA/0.3% Tween-20/0.01 mol/l PBS overnight at room
temperature. After washing in 0.01 mol/l PBS/0.3%
Tween-20, the membranes were incubated for 2 h with
horse-radish peroxidase-labeled goat anti-rabbit IgG
(Vector Laboratories Inc, Burlingame, CA, USA). Immunoreactive protein bands were detected using the
enhanced chemiluminescence detection method (ECL
Western blotting detection reagent; Amersham International plc). To determine non-specific blots, the
antiserum prepared with excess rhFS (1–2 mg) was
used in a parallel experiment.
Results
Localization of follistatin-like
immunoreactivity in rat spermatogenic cells
The immunoreactions of testicular cells with antifollistatin (123–134) are summarized in Table 1. In
this study, follistatin-like immunoreactivity was not
detected in the testis from 4- to 18-day-old rats. In 6week-old rats, although matured spermatids were not
observed, spermatocytes and round spermatids were
found in some testicular tubules. A moderate immunoreaction with anti-follistatin (123–134) was found in
the cytoplasm and the nucleus of spermatocytes and in
the nucleus of round spermatids, whereas it was not
detected in spermatogonia (Fig. 1A). Follistatin immunoreactivity was not detected in Sertoli and Leydig cells in
6-week-old rats.
In mature rats (8 and 10 weeks old), the reaction
with anti-follistatin (123–134) serum was detected in
both nucleus and cytoplasm of late pachytene spermatocytes (Fig. 1C and D). The immunoreactivity was also
detected in the nucleus of round spermatids but was not
in their cytoplasm (Fig. 1B, C and D). When the
antiserum was absorbed with an excess amount
(400 ng) of rhFS, the reaction was abolished. Immunoreactivity of follistatin was not detected in either Sertoli
or Leydig cells.
Figure 2 summarizes our results obtained from
histochemical analyses with anti-follistatin (123–134)
serum in testicular sections from mature rats. Spermatogonia were not stained at any stage examined in this
EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
Follistatin-like immunoreactivity in spermatogenic cells
527
(lane 2) respectively. In the preparation from rat testis
homogenate, three major bands at 57, 45 and 39 kDa
were detected with anti-follistatin (123–134) (lane 3).
Under non-reducing conditions, four bands were
detected at 35, 32, 30 and 28 kDa for pFS (lane 4) and
at 37, 35, 34 and 32 kDa for rhFS (lane 5). Four bands at
52, 44, 39 and 34 kDa were detected in the preparation
from rat testis homogenate (lane 6). When the antiserum
was absorbed by the excess of rhFS (1–2 mg), the
immunoreaction was markedly reduced.
Discussion
Figure 3 Western blotting analysis of follistatin from porcine
follicular fluid, rhFS and crude preparations from rat testis
homogenates. pFS (50 ng; lanes 1 and 4), rhFS (50 ng; lanes
2 and 5) and aliquots of eluted fractions from reverse-phase
HPLC (lanes 3 and 6) were subjected to reducing (lanes 1–3)
or non-reducing (lanes 4–6) SDS-PAGE, electroblotted onto
Immobilon membranes, and incubated with anti-follistatin
(123–134) antiserum. The numbers on the left represent the
molecular size of the protein molecular mass standard (BioRad).
study. No reaction was found in preleptotene to zygotene
spermatocytes. Pachytene spermatocytes were not
stained in stages I to V but did stain in stages VI to
XII. The reaction in spermatocyte cytoplasm disappeared in the diplotene phase (stage XIII). Immunoreaction in the pachytene spermatocyte nucleus was
slight in stage VI, moderate in stages VII to IX, and
intense in stages X to XII. Although the reaction in the
cytoplasm disappeared after meiosis, it continued to be
intense in the nucleus from pachytene spermatocytes to
round spermatids. The reaction in nuclei appeared to be
weaker in step 8 spermatids and disappeared in step 10.
The reaction was not found in spermatids after step 10
or in spermatozoa.
Western blotting analysis of follistatin-like
immunoreactivity in the rat testis
We subjected rat testis homogenate to affinity chromatography on sulfate-cellulofine and anti-follistatin Cys
(123–134) column followed by reverse-phase HPLC and
analyzed the resulting fraction by Western blotting using
anti-follistatin (123–134) serum. Follistatin immunoreactivity with the crude preparation from the homogenate was detected in fraction 11 to 14 (data not shown).
The results obtained in Western blotting analysis are
shown in Fig. 3. When the anti-follistatin (123–134)
was used for immunoblotting under reducing conditions, pFS and rhFS exhibited three major bands at
41, 39 and 37 kDa (lane 1), and at 42, 40 and 39 kDa
The present study demonstrated that follistatin-like
immunoreactivity is located in rat spermatogenic cells
by immunohistochemistry. During the development of
spermatogenic cells, follistatin-like immunoreactivity is
first detected in the cytoplasm and nucleus of late
pachytene spermatocytes. Although the reaction in the
cytoplasm disappeared after meiosis, it continued to be
in the nucleus from pachytene spermatocytes to round
spermatids. This finding suggests that follistatin or its
closely related protein produced in late pachytene
spermatocytes migrates from the cytoplasm to the
nucleus. A previous study has demonstrated that the
level of follistatin mRNA signal is high at stages IX to XI
of the cycle (34). This is in agreement with our results
which show a high level of follistatin-like immunoreactivity in cytoplasm of spermatocytes at stages X to
XII. However, Kaipia et al. (34) showed follistatin mRNA
expression in Sertoli cells. The possibility still remains
that follistatin is produced in Sertoli cells and then
rapidly taken up by spermatocytes. In previous studies,
the immunoreactive bA subunit of inhibin/activin has
been detected in the cytoplasm (35) and the nucleus of
spermatogenic cells (36). It is, therefore, conceivable
that follistatin binds to activin in the cytoplasm and
nucleus of spermatogenic cells.
Follistatin-like immunoreactivity was detected in
spermatogenic cells from meiotic prophase to the
period just before initiation of nuclear condensation.
Because inhibin and activin act in oocyte meiotic
resumption (37, 38), follistatin may also act in meiotic
regulation during spermatogenesis. In females, however, follistatin-like immunoreactivity was not found in
oocytes in any stage of development from embryonic
to adult rats (K Ogawa, unpublished observations),
whereas it was detected in granulosa cells in preovulatory follicle by the same antiserum (25). It is, therefore,
conceivable that follistatin may act in an event specific
to spermatogenesis, such as nuclear condensation.
The results of Western blot analyses indicated the
existence of follistatin or its closely related protein in rat
testis. A protein extracted from rat testis, which was
recognized by anti-follistatin (123–134) antiserum,
exhibited a characteristic pattern for follistatin on SDSPAGE, i.e. slower migration under reducing conditions
than under non-reducing conditions (17).
528
K Ogawa and others
In addition to activin-binding activity, follistatin has
been reported to have affinity for nucleic acid-like
substances, such as heparin (7), dextran sulfate (8) and
heparan sulfate (39). Taken together with the finding
that follistatin-like immunoreactivity is located in the
nucleus, follistatin may be a DNA-binding protein of
spermatogenic cells. Postmeiotic male germ cell differentiation into mature spermatozoa involves remarkable
structural and biochemical changes, including nuclear
DNA compaction and acrosome formation. Expression
of various genes, such as protamine (40), calspermin
(41) and transition protein-1 (40, 42), is thought to be
required for this process. In this study, follistatin-like
immunoreactivity was highly accumulated in the
nucleus in postmeiotic germ cells, suggesting that it
may be associated with a transcriptional activator, such
as the cyclic AMP-responsive element modulator (40–
42), particularly during the haploid phase. It may be
responsible for the activation of several haploid cellspecific genes. Further observations will be needed to
clarify this point.
In conclusion, follistatin-like immunoreactivity was
detected in both the nucleus and cytoplasm in
spermatogenic cells, and the immunoreactivity varied
in intensity depending on the stage of the cycle.
Follistatin or its closely related protein may be a stagespecific modulator of spermatogenesis.
Acknowledgements
The authors wish to thank Dr M Muramatsu and Dr Y
Eto for providing rhFS.
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Received 17 February 1997
Accepted 7 April 1997