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 follistatin in various human tissues with specific antibodies 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 ovary, granulosa cells of mature follicle and luteal cells of the corpus luteum stained for activin A. Immunoreactive activin A was present in somatotrophs of the pituitary gland and insulin-positive B cells of the pancreatic islets. Immunoreactivity for activin A was also found in thyroid follicular cells, adrenocortical cells, neuronal cells of the cerebrum and monocytoid cells in the bone marrow. Follistatin, an activin-binding protein, 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 proteins 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 secretion [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 antibody 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 hormone therapy. Each type of organ also blocked by applying normal goat serum for was obtained from at least 3 patients. Ovaries 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 glucagon (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 anterior pituitary hormones 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 pancreas, 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).