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Home , Activin and inhibin

Endocrine Journal 1996, 43(4), 375-385

immunoh istochemical Localiza tion of Activin A and Foil istatin in Human Tissues

MIcHIKO WADA, YASUMI SHINTANI, MASAAKI KOSAKA, TosHIAKI SANO*, KAzuo HIZAWA*, AND SHIRO SAITO

First Department of Internal Medicine and *First Department of Pathology, School of Medicine, The University of Tokushima, Tokushima 770, Japan

Abstract. We immunohistochemically investigated the localization of activin A and in various human tissues with specific to recombinant human (rh-) activin A and rh-follistatin. Specific immunostaining of activin A was detected in Leydig and Sertoli cells of the testis. In the , granulosa cells of mature follicle and luteal cells of the stained for activin A. Immunoreactive activin A was present in somatotrophs of the and -positive B cells of the pancreatic islets. Immunoreactivity for activin A was also found in follicular cells, adrenocortical cells, neuronal cells of the cerebrum and monocytoid cells in the bone marrow. Follistatin, an activin-binding , was immunostained in the same tissues as activin A. These findings indicated that activin A and follistatin are widely distributed in human tissues, suggesting that activin plays important roles as a common regulator in various tissues under the control of co-existing follistatin.

Key words: Activin , Follistatin, Inhibin, Immunohistochemistry (Endocrine Journal 43: 375-385,1996)

ACTIVIN and inhibin are both dimeric logical activities. It regulates erythropoiesis [6], originally isolated from porcine follicular fluid and promotes nerve cell survival [7], inhibits neural able to influence FSH release from the pituitary differentiation [8], induces embryonic mesoderm [1-3]. There are three forms of activin, activin A, formation [9], and stimulates insulin [10]. activin AB and activin B, each of which consists of Activin subunits and follistatin mRNAs have been two subunits (flA or f3B) [1-3]. Inhibin is com- detected in gonadal and various extragonadal tis- posed of one a-subunit and one fJ-subunit (/3A or sues [11, 12], implying that they have diverse fB) [2, 3]. All of these subunits are structually functions. similar, and activins and inhibin belong to the It has been difficult to obtain a specific transforming growth factor-f3 (TGF-f3) superfamily to activin because its structure is highly homolo- [3, 4]. Follistatin, an activin-binding protein, is a gous in various species and it shares a common glycosylated single-chain protein also isolated from /3-subunit with inhibin. Since we succeeded in pre- ovarian follicular fluid and has the potency to sup- paring for specific polyclonal antibodies to press FSH release by binding to activin [5]. recombinant human (rh-) activin A and rh-follista- Activin has been reported to exhibit various bio- tin [13, 14], we immunohistochemically examined the localization of activin A and follistatin in vari- ous human tissues to elucidate the distribution and Received: December 25, 1995 Accepted: March 7, 1996 possible roles of activin and follistatin in vivo. We Correspondence to: Dr. Michiko WADA, First Department also examined the distribution of the inhibin u- of Internal Medicine, School of Medicine, The University subunit to compare it with those of activin A and of Tokushima, Kuramoto-cho 3, Tokushima 770, Japan follistatin. 376 WADA et a!.

Materials and Methods Immunohistochemistry

Tissue preparation Three ,um thick paraffin-embedded sections were stained by the streptoavidin-biotin peroxidase com- Human normal tissues obtained at autopsy or plex method (Histofine SAB-PO kit; Nichirei Co., surgery were fixed in 10% buffered formalin and Tokyo, Japan). The deparaffinized sections were embedded in paraffin. The ages of 10 patients at immersed in 0.3% H2O2 in 100% methanol for 30 autopsy ranged from 18 to 69 years (mean, 56 years, min at room temperature to block endogenous per- 8 men and 2 women). None of the patients had oxidase activity. Possible background staining was received therapy. Each type of organ also blocked by applying normal goat serum for was obtained from at least 3 patients. were 20 min at room temperature. Anti-activin A anti- obtained from 12 patients (between 25 and 45 years body was applied to the sections at 1:1000 dilution of age; mean 36 years) at the time of oophorecto- in phosphate-buffered saline, and followed by in- my for ovarian cyst or uterine myoma. Testes cubation at 4 °C in a moist chamber for 12 h. They biopsied for sterility in 3 patients of the 4th de- were then treated with biotinylated goat anti-rab- cade, and removed by castration from 3 patients bit IgG conjugated to streptoavidin-biotinylated of the 7th decade were used. Bone marrow speci- peroxidase and developed with DAB hydrogen mens were obtained from 3 healthy men aged peroxide substrate (45 mg 3,3' DAB in 150 ml Tris- twenty. Informed consent was obtained from all buffered saline solution containing 3 p/ H202). The these patients. sections were counterstained with methyl green or hematoxylin. Control sections were treated in the Antibody preparation same way, except that the antibody was preab- sorbed with 500 jig rh-activin A with affinity Anti-activin A polyclonal antibody was generat- chromatography units (Nalgene Co., USA). This ed with rh-activin A supplied by Ajinomoto Central preabsorbed antibody did not show any signifi- Research Laboratories, Kawasaki, Japan as de- cant reaction with rh-activin A on evaluation by scribed [13]. This antibody cross-reacted only with ELISA. Commercial preparations of antibodies bovine inhibin (3.2%) and porcine activin AB were also used in studies of the co-localization of (10.0%), but not with porcine activin B (<0.5%) [13]. 13-FSH(1:100 dilution, Immunotech, France), J3-LH The addition of excessive follistatin minimally af- (1:100 dilution, Immunotech), GH (1:800 dilution, fected ir-activin A recovery in the DAKO, Denmark), PRL (1:100, dilution, Immuno- radioimmunoassay (unpublished data), and the tech), fl-TSH (1:100 dilution, Immunotech) and presence of activin-follistatin complex was suggest- ACTH (1:1000 dilution, UCB-bioproducts, USA) in ed on gel chromatographic analysis of serum and adjacent sections of the pituitary, and of insulin follicular fluid [13, 14]. Taken together with these (1:400 dilution, DAKO) and (1:1000 dilu- findings, this polyclonal antibody was supposed tion, DAKO) in adjacent sections of pancreatic to detect both follistatin-bound and -unbound ac- islets. The localizations of follistatin and inhibin tivin A. A polyclonal antibody to rh-follistatin were examined by the same procedure with anti- (Ajinomoto Central Research Laboratories) was also follistatin antibody (1:200 dilution) and anti-inhibin prepared with the same procedure as that for ac- a-subunit antibody (1:200 dilution). tivin A [15]. By the immunoradiometric assay this antibody did not cross-react with bovine inhibin and rh-activin A (<0.5%). A mouse monoclonal Results antibody to bovine inhibin a-subunit was a gift from Prof. Y. Hasegawa (Kitasato University School The localizations of immunoreactive (ir-) activin of Veterinary Medicine and Animal Sciences, Tow- A, follistatin and inhibin a-subunit in human tis- ada, Japan) [16] and was used for the comparison. sues are summarized in Table 1. Activin A and follistatin were almost concurrently localized in various tissues such as the reproductive system, ACTIVIN AND FOLLISTATIN IN HUMAN TISSUES 377

Table 1. Localization of activin A , follistatin and the inhibin a-subunit in human tissues

endocrine organs, brain and bone marrow. In the pituitary, adenohypophysial cells were In the testis, intense immunostaining for activin immunostained with anti-activin A antibody. Im- A was observed diffusely in the cytoplasm of Ley- munohistochemical studies with adjacent sections dig cells, most of which were grouped in clusters, revealed that ir-activin A (Fig. 3A) was co-local- and partly in the cytoplasm of Sertoli cells adja- ized in GH-positive cells (Fig. 3B). The other cent to the basement membrane of seminiferous including FSH (Fig. tubules (Fig. 1A). No variation in the localization 3C), LH, PRL, TSH and ACTH were not co-local- of ir-activin A was observed with age. This strong ized with activin A. In the , activin A immunostaining was noticeably reduced by pre- was strongly immunostained in islets of Langer- absorption of the antibody with excessive rh-activin hans. It was demonstrated that ir-activin A (Fig. A (Fig. 1B). In the ovary, ir-activin A was detected 4A) was co-localized with insulin in B cells (Fig. in the granulosa cells of mature follicle (Fig. 2A), 4B), but not with glucagon in A cells (Fig. 4C) by but not in those of immature follicles such as pri- immunostaining on adjacent sections. In the thy- mary, secondary and small Graafian follicles. roid, follicular cells, but not parafollicular cells, Ir-activin A was also detected in granulosa lutein were immunostained with anti-activin A antibody cells of the corpus luteum (Fig. 2D). However, no (Fig. 5A). The adrenal cortex was diffusely immu- significant immunostaining for activin A was ob- nostained for activin A, but the adrenal medulla served in thecal cells at any stage of follicular was not immunostained (Fig. 5C). Besides these development. endocrine tissues, ir-activin A was also detected in 378 WADA et al.

Fig. 1. Localization of activin A, follistatin and inhibin a-subunit in human testis. Immunoreactivity for activin A is strongly positive in interstitial Leydig cells and in Sertoli cells adjacent to the basement membrane of the tubules in human testis (A). The specific immunostaining of activin A is noticeably reduced when antibody preabsorbed with excess rh-activin A is used (B). Immunoreactive follistatin is detectable in Leydig cells and Sertoli cells (C). Immunoreactive inhibin a-subunit is detectable in Leydig cells (D). Magnification: A, C and D, x 400; B, x 300.

the neurons of the cerebrum (Fig. 6A) and in mono- cytoid cells of the bone marrow (Fig. 6C). Discussion Follistatin showed similar distribution to activin A (Table 1), testicular Leydig and Sertoli cells (Fig. In the present study, we investigated the immu- 1C), granulosa cells of mature follicle (Fig. 2B) and nohistochemical distribution of activin A and luteal cells of the corpus luteum in the ovary (Fig. follistatin in human tissues. The immunohis- 2E), pituitary somatotrophs (Fig. 3D), insulin-posi- tochemical detection of activin and f ollistatin does tive B cells of pancreatic islets, thyroid follicular not always mean their local production, but it is cells (Fig. 5B), adrenocortical cells (Fig. 5D), cere- certain that their distribution is closely related to bral neuronal cells (Fig. 6B) and monocytoid cells their functions in vivo. Since the anti-activin A in the bone marrow (Fig. 6D). antibody used here slightly cross-reacted with bo- Immunoreactivity of the inhibin a-subunit was vine inhibin (3.2%) [13], this antibody may bind to detected in Leydig cells of the testis (Fig. 1D), gran- inhibin through the common 13-subunit at low af- ulosa cells of mature follicle (Fig. 2C) and luteal finity. The localization of the inhibin a-subunit cells in the ovary (Fig. 2F) and pituitary somatotro- was therefore simultaneously examined by using phs, but not in the pancreas, thyroid, , anti-inhibin a-subunit antibody. cerebrum or bone marrow (Table 1). Ir-activin A was predominantly present in Ser- toli cells of the testis, insulin-positive B cells of the ACTIVIN AND FOLLISTATIN IN HUMAN TISSUES 379

Fig. 2. Localization of activin A, follistatin and inhibin a-subunit in human ovary . Immunoreactive activin A (A), f ollistatin (B) and inhibin a-subunit (C) are detected in granulosa cells of the mature follicle . Immunoreactive activin A (D), follistatin (E) and inhibin a-subunit (F) are detected in luteal cells of the corpus luteum . A, B and C, x 600; D, E and F, x 400.

pancreatic islets, thyroid follicular cells, adreno- immunostaining for the inhibin a-subunit protein cortical cells, neuronal cells of the cerebrum and was negative in this study. This discrepancy may monocytoid cells of the bone marrow , because these have resulted from either the low sensitivity of im- tissue specimens were stained with anti-activin A munohistochemistry with this monoclonal antibody antibody, but not with anti-inhibin a-subunit anti- or a very small amount of inhibin produced in body. These results indicate that activin A is more these tissues. Of interest is that all of the activin widely distributed than inhibin as an important A-positive tissues were co-stained with anti-follista- local regulator in these tissues. Activin A and in- tin antibody, suggesting that activin-follistatin hibin A are supposed to be present in Leydig cells complex is present in these tissues. This is in ac- of the testis, granulosa cells of mature ovarian fol- cordance with the reports demonstrating that licle and luteal cells of corpus luteum , and pituitary follistatin mRNA is co-expressed generally with somatotrophs, judged by positive immunostain- those of activin subunits [11, 12]. As follistatin is ing of both activin A and inhibin a-subunit . known to antagonize the activin action in various Although inhibin a-subunit mRNA was demon- tissues such as granulosa and pituitary cells [17 , strated in rat brain and adrenal gland [11] , 18], it is accordingly estimated that follistatin is im- 380 WADA et al.

Fig. 3. Localization of activin A and follistatin in human pituitary. In adjacent sections, immunoreactive activin A (A, arrow) is co-localized with the most of the immunoreactive GH (B, arrow), but not with immunoreactive FSH (C). Immunoreactive follistatin (D, arrow) is also co-localized with immunoreactive GH (B). x 500.

Fig. 4. Localization of activin A in human pancreas. In adjacent sections of the pancreatic islet, activin A-positive cells (A) are co-localized with insulin-positive cells (B) but not with glucagon-positive cells (C). x 400. ACTIVIN AND FOLLISTATIN IN HUMAN TISSUES 381

Fig. 5. Localization of activin A and follistatin in human thyroid and adrenal gland. The thyroid follicular cells are immunostained with anti-activin A antibody (A) and anti-follistatin antibody (B). The adrenocortical cells (upper part of each photograph), but not the medulla (bottom of photographs), are stained for activin A (C) and follistatin (D). A and B, x 600, C and D, x 400.

portant in regulating the local function of activin According to Roberts et al., inhibin a- and fB- in each tissue. The precise mechanism of this reg- subunits are intensely immunostained and the ulation is not clear at present, but it was recently f3A-subunit is weakly stained in Sertoli cells in adult shown that activin stimulates the expression of fol- rat testis [23]. In human fetal testis, the immu- listatin mRNA in rat pituitary and granulosa cells noreactivity of a-subunit was demonstrated in [19-21]. In addition, it has been demonstrated that Sertoli cells and the a-, f3A- and fiB-subunits were follistatin binds to detected in Leydig cells [24]. In this study, we on the cell surface [22], implying that follistatin have detected ir-activin A, follistatin and inhibin may be involved in the reservation of activin for a-subunit in Leydig cells, supporting the view that cell use or protecting its degradation by capturing activin inhibits the LH-stimulated of the activin molecule on the cell surface. Consider- from Leydig cells [25]. The presence of ing that activin has extremely diverse functions, it ir-activin A and follistatin in Sertoli cells also sup- is also possible that locally-produced follistatin pre- ports the hypothesis that activin A stimulates vents the bioactive activin molecule from spreading spermatogonial proliferation [26] and acts as an to other tissues. Further studies are required to autocrine regulator of function [27], and elucidate the interaction between activin and fol- is consistent with the fact that and listatin, at least in the tissues where we detected follistatin mRNAs were detected in rat Sertoli cells positive immunostaining. [28], but the reason why the immunoreactivity of 382 WADA et al.

Fig. 6. Localization of activin A and follistatin in human cerebrum and bone marrow The cytoplasm of neurons in the cerebrum is stained for activin A (A) and follistatin (B). Activin A (C) and follistatin (D) are detected in monocytoid cells (arrows) in the bone marrow. A and B, x 400, C and D, x 1000. a-subunit was not detected in Sertoli cells remains This discrepancy leads us to consider that activin to be determined. and follistatin produced by the human ovary are were originally purified from so small in amount that the serum levels of activin ovarian fluids and supposed to play important and follistatin do not necessarily reflect the ovari- roles in the development of ovarian follicles. In an release of these proteins. this study, ir-activin A, inhibin a-subunit and fol- Activin A has various functions in the pituitary listatin were all detected in granulosa cells of other than FSH releasing activity. Activin A in- mature follicles and luteal cells. Yamoto et al. [29] hibits basal and GHRH-stimulated GH release, and Roberts et al. [30] demonstrated that three sub- somatotroph growth and TRH-stimulated PRL re- units of inhibin are present in human granulosa lease [34-36]. On serial sections, we have cells of small antral follicles as well as preovulato- demonstrated that ir-activin A, follistatin and in- ry follicles. Our observation of ir-activin A, inhibin hibin a-subunit are co-localized in human and follistatin in granulosa cells of only mature somatotrophs, but not in gonadotrophs, inconsis- follicles and luteal cells is in accordance with the tent with the report by Roberts et al. showing the previous report that the human serum inhibin lev- presence of both inhibin a- and /3B-subunits only el peaks at and remains high in the luteal in gonadotrophs [37]. The reason for this is not phase [31], but inconsistent with the observation yet clear, but there have been several additional that serum levels of activin and follistatin did not reports concerning this matter. A surface compo- vary during the normal menstrul cycle [32, 33]. nent of GH3 cells, which express GH but not FSH, ACTIVIN AND FOLLISTATIN IN HUMAN TISSUES 383 has been found to recognize activin and inhibin plasm of neurons in the cerebrum, supporting the [38], and the pituitary cells, in which cytosolic free view that activin is important in the survival and calcium concentration was increased in response differentiation of nerve cells [7, 8]. We also dem- to activin A, were normal rat pituitary somatotro- onstrated ir-activin A and follistatin in monocytoid phs, but not gonadotrophs [39], indicating that the cells in the bone marrow, which is in accordance activin A-induced biological events are closely re- with the report that activin A is produced by mono- lated to pituitary somatotrophs. In addition, cytes/ in the bone marrow and is mRNA of the inhibin f3A-subunit has been demon- known to affect the differentiation of erythroid pro- strated in the human pituitary [40], suggesting that genitors [47-49]. It is interesting that ir-activin A activin A as well as activin B is important as a and follistatin were detectable in the thyroid folli- local factor in modifying pituitary function. cular cells. As we recently demonstrated that Activin A stimulates insulin secretion from rat activin A stimulates thyroid growth irrespective of pancreatic islets [10]. There have been several dif- cAMP-accumulation and inhibits thyroid function ferent reports on the localization of ir-activin A in [50], the activin/follistatin system may also play rat pancreatic islets [41, 42]. We demonstrated here some roles in the thyroid. that ir-activin A and f ollistatin are both present in In conclusion, we demonstrated that activin A the majority of insulin-producing B cells. We have and follistatin are widely distributed in various also noticed that activin A-positive islets cells dis- human tissues such as those of the endocrine, he- appear together with insulin-positive B cells and mopoietic and nervous systems. These results the remaining glucagon-positive A cells are not suggest that activin plays important roles in the stained with anti-activin A antibody in the Long autocrine/paracrine regulation of many tissues Evans Tokushima Lean (LETL) rat [43], which is under the regulation of co-existing follistatin. an animal model of spontaneously occurring insu- lin-dependent diabetes mellitus [44]. These findings strongly suggest that activin A is an auto- Acknowledgements crine regulator of insulin secretion. Ir-activin A and follistatin were also found in The authors thank Dr. Y. Eto, Ajinomoto Central adrenocortical cells. Although ir-inhibin a-subunit Research Laboratories (Kawasaki, Japan) for sup- was detected in the human adult adrenal gland plying rh-activin A/erythroid differentiation factor [45], immunostaining of the inhibin a-subunit was (EDF) and rh-follistatin, Prof. Y. Hasegawa, Kita- equivocal in this study. Considering this together sato University School of Veterinary Medicine and with the reports that activin A inhibits the prolif- Animal Sciences (Towada, Japan) for providing eration of human fetal adrenal cells [46] and anti-inhibin antibody, and Prof. H. Sugino, Insti- enhances the ACTH-stimulated synthesis, tute for Enzyme Research, University of Tokushima activin and follistatin may be locally involved in (Tokushima, Japan) for supplying porcine activin the steroidogenesis of the adrenal cortex. Activin AB and porcine activin B. A and follistatin were also detected in the cyto-

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