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Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
Available online at www,sciencedirect.com Agricultural Sciences in China
2006, 5 ( 9 ) : 718-724
September 2006
Localization of Inhibin in Some Rat Tssues and Exploration of Its Transport Mechanism ZHANG Zhong-xia,XU Lu, DI He-shuang, YAN Yu-qin and WANG Gen-lin College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China
Abstract The aims of the study were to determine the distribution of inhibin and its a subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex (SABC) and to assess the transport mechanism of inhibin by investigating the localization of inhibin and a subunits in the central nervous system (mainly in hypothalamus and pituitary) of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its a subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin a subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its a subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and a subunit could be transported through the BBB by the method of “separation and reconstruction”.
Key words: inhibin, inhibin a subunit, blood-brain barrier (BBB), immunohistochemical localization
INTRODUCTION Inhibin is a dimer that consists of one alpha-chain (18 Ka) and one beta-chain (14 Ka) linked by disulfide bridges, which plays an important role in regulating the synthesis and secretion of the FSH in pituitary, in modulating the development of ovary and sperm, also as a sensitive marker of mucinous ovarian cancers, and as an inhibiting factor. Although the inhibin’s origin, biological properties, assay method, gene regulation and passive immunity, a,PA,PB mRNA structure, expression, signal transduction (Bernard et al. 2001), roles, and its receptor are known, the mechanism of action of inhibin still remains disputable. There are three hypotheses regarding the mechanisms of action
of inhibin: inhibin acting on hypothalamus, pituitary, or ovary. Investigation has revealed the blood-brain barrier (BBB) bioactivity. BBB is a specialized construct that is formed by brain capillary endothelial cell; it acts as an important selective barricade during the exchange of brain and blood matter. However, circulating inhibin must be transported through BBB to act on nerve center. Research showed that besides micromolecules, partial macromolecules such as insulin and other hormones could also be transported through BBB. Zhang et al. (2004) indicated that follicular inhibin could be transported across BBB in sows. Yan et al. (2004) found that glycoprotein-transporting carrier was present in brain tunica intima. Other studies have demonstrated that a (Wang et al. 2005) and P subunits (He et al. 1999) could be transported through
Received 16 May, 2006 Accepted 7 July, 2006 ZHANG Zhong-xia, MSc. E-mail: zhongxial02Qyahoo.com.cn: Correspondence WANG Gen-lin, Tel: +86-2544395045, Fax: +86-25-84395314, E-mail: glwang9njau. edu.cn
02M)6.CAAS
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Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
BBB, but to date, there is no evidence that inhibin molecule could be transported through BBB. Also, the mechanisms have not been explored. So the question is how inhibin is being transported through BBB when it acts in brain. Recent progress has demonstrated the expression of inhibin in various tissues and has implicated potential involvement in diverse physiological and pathophysiological processes (Meunier et aE. 1988; Chen and Johnson 1996; Peeters et al. 1997). But most studies were focused on a-subunit expression and research at mRNA level. Currently, there is no assay for ‘total’ inhibin using monoclonal antibodies. Therefore, the aims of the present study were 1) to determine the distribution of inhibin and its a subunits in extragonadal tissues in the rat by immunohistochemical method with specific monoclonal antibodies; 2) to assess the transport mechanism of inhibin through the blood-brain barrier.
MATERIALS AND METHODS Animals Fifty mature female Sprague-Dawley rats were bred for 7 days, and then they were divided into five groups: the intact group, ovariectomized group, and exogenous injection groups, which were intraperitoneally injected with inhibin crude extract (2 pg), inhibin a fragment (2 pg), or normal saline 7 days after ovariectomization. Six hours after injection, five groups were immediately dissected, and hypothalamus, pituitary, heart, kidney, spleen, adrenal gland, pancreatic gland, liver, lung, and submaxillary gland of all the rats were collected. The tissues were fixed in 10%neutral formalin for 14 h at room temperature.
Tissue preparation The fixed organs were thereafter dehydrated through increasing concentrations of ethanol, cleared and embedded in paraffin wax (m.p. 54-56°C). The paraffinembedded organs were serially sectioned at 6-pm thickness and placed on APES (3-aminopropyl-trithoxysi1ane)coated slides. After drying at 37°C for one night, slides were stored at 4°C for preemergency.
lmmunohistochemistry Primary monoclonal antibody was mouse monoclonal anti-inhibin-a antibody (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd, Code 12777) at a dilution of 1:500 and mouse monoclonal anti-inhibin antibody (Antibodyshop,Code HYB 224-01) at a dilution of 1:200. Inhibin extract was obtained from porcine ovary and purified in the laboratory, and porcine inhibin a-subunit (1 32) fragment was obtained from Sigma (Code 18641). Immunocytochemical staining was carried out according to the method proposed by Wang et al. (2005). In brief, sections were deparaffinized in xylene, rehydrated through graded ethanol, and washed in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for two times, 10 min each. All incubations were carried out at room temperature, and PBS was used for washing after each step. The sections were then incubated in 3% H,O, in methanol at room temperature for 10 min to quench nonspecific staining, followed by the addition of 0.01 mol L-’ citric acid-buffered saline (pH 6.0) at 92-98°C for 20 min to repair endogenous enzyme. Afterward, the sections were incubated for 20 min at room temperature with normal goat serum confining liquid and then incubated with primary monoclonal antibody against inhibin or a subunit at a dilution of 1:200 for 1 h at 37°C. After incubation, the sections were treated with 0.5% biotinylated goat antimouse secondary antibody (SABC kit) for 20 min at 37°C and subsequently incubated with 2% streptavidin-biotin complex (SABC kit) for 20 min at 37°C. The antibody bound to the sections was visualized by treating the sections with 0.05% 3.3’-diaminobenzidine tetrachloride (Wuhan Boster Biotechnology Co., Ltd, CodeARlM2) in 10mM Tris-buffered saline containing 0.01% H,O, for 3 min. Specificity of the antibody against inhibin or a subunit was examined using normal mouse serum instead of primary antibody. To identify the cell types in each organ, a serial section of each sample was also stained with hematoxylin. Then the sections were washed in tap water, dehydrated in alcohol, cleared in xylene, and mounted with neutro-resin.
RESULTS lmmunohistochemical localization of inhibin in
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some extragonadal tissues of the rat The results of the immunostaining of inhibin in intact extragonadal tissues of rats showed that inhibin-positive cells were observed in hypothalamus, pituitary, heart, pancreas, kidney, lung, liver, spleen, submaxillary gland, and adrenal cortex. In ovariectomized rats tissues, similar positive expressions were observed in heart, kidney, spleen, and pancreas, whereas no positive sites were seen in hypothalamus, pituitary, lung, liver, submaxillary gland, and adrenal gland (Table). Inhibin-positive cells were observed mainly in sarcolemma of the heart, circum-intralobular ductin of pancreas, the renal corpuscle kidney, and red pulp area of the spleen. In contrast, with normal mouse serum as the primary antibodies to detect inhibin and inhibin a, all the cells showed negative expression. It suggested that inhibin and a subunits were not only restricted to the gonads but also were present in a wide variety of nonreproductive organs and tissues, including the heart, pancreatic gland, kidney, and spleen. The result also demonstrated that the inhibin that was distributed in hypothalamus and pituitary of intact rats was from the ovary.
lmmunohistochemical localization of inhibin in hypothalamusand pituitary of ovariectomized rats The localization of inhibin and a subunit was investigated in the hypothalamus and pituitary of ovariectomized rats, which had been injected with inhibin a subunit fragment (2 pg) or inhibin crude extract (2 pg). The result showed that when inhibin-a monoclonal antibody or inhibin antibody was used along with inhibin a-subunit fragment ( 2 pg), both hypothalamus (Fig.- 1, -2) and pituitary (Fig.-3, -4)showed inhibin and asubunit positive cells; when inhibin antibody was used along with inhibin extract (2 pg), both again showed inhibin and a subunit positive cells (Fig.-5-8). In contrast, the normal saline treatment showed negative
cells. The result suggested that a-subunit and inhibin dimer could be transported through the RBI3 to reach the hypothalamus and pituitary.
DISCUSSION Inhibin as a member of the transformation growth factor P (TGF-P) gene superfamily, exhibiting paracrine and/or autocrine mechanisms of actions, plays roles in the regulation of cell proliferation and differentiation in a variety of tissues. Even though single a-subunit has no inhibin bioactivity, there might exist a distinct paracrine and/or autocrine function. Reports have showed that a-subunit can inhibit the binding of FSH with its receptor (Robertson et al. 1997). Excess inhibin a-subunit damages oocyte maturation (Blauer et al. 1992). Similarly, loss of a gene homozygote may cause gonadoma, etc. This study on the extragonadal distribution of inhibin and a-subunit confirmed previously described results of Meunier’s research on ewes (Meunier et al. 1988) and also showed some similarity with data produced in hen (Chen and Johnson 1996) using Northern blot analysis. However, there are still differences. For example, in Meunier’s report, heart, spleen, and pancreas were not mentioned, whereas Chen’s report on hen showed that a-subunit mRNA was less expressed in heart and adrenal gland, and PA-subunit mRNA was less expressed in heart, adrenal gland, liver, lung, and spleen. The diverse results reflect that inhibin is conservative in phylogeny, and interspecific difference also exists. This study suggested that inhibin and its a subunit might affect the function of the tissues by autocrine/paracrine mechanisms. Blauer et al. (1992) stated that in chicken embryo, a-subunit was localized in smooth muscle myoblasts as well as in developing cardiac muscle cells. Although Purkinje fibers were strongly immunoreactive, undifferentiated mesenchymal cell and terminal differentiated mature cardiac muscle cell had no positive
Table Distribution of inhibin in rat tissues Tiqrue ___ Intact rats Ovanectomized rats ~
“+”
~Hypothalamus
+
Pituitary ~ gland _
Heart _
_Pancreas ~
Kidney
Lung
Liver
Submaxrll~ygldnd
Spleen
+
+
+
+ +
+
+
+
+ +
represents inhibin immunostaining reaction positive;
+ ”-”
+
Adrenal gland
+
represents inhibin immunostaining reaction negative.
82006,CAAS All nghts resewed Publishedby Elsevier Lid
Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
72 1
Fig. Immunohistochemical localization of inhibin and a-subunit in hypothalamus and pituitary of the sexually matured ovariectomized rat (x400 DAB stained, arrows indicate positive area). 1,2, in hypothalamusand and 3.4, in pituitary 6 h after exogenous injection of inhibin a fragment; 5,6, in hypothalamus and 7, 8, in pituitary 6 h after exogenous injection of inhibin extract.
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722
expression. This suggests that a-subunit could be correlated with muscle differentiation and nerve conduction. Ohnishi et aZ. (2003) proved that FS could act in the treatment of pancreas fibrosis, which indicates that inhibin may play a role in inhibiting pancreatic fibrosis. Research has shown that inhibin a was invariably negative in both normal and fibrotic rat liver, and p immunoreactivity of p subunit was weak in parenchymal cells (PC). In fibrotic livers, PA showed strong immunoreaction (de Bleser et aZ. 1997). Activin A binding to its receptor can lead to increase in Ca2+concentration in endochylema. Moreover, increases in insulin secretion promote glucose production of rat liver cells. It is assumed that exogenous inhibin crude extracthnhibin a fragment may exert actions antagonistic to activin A to play a role in recovery with regard to liver ailment. To date, the precise role of inhibin in alimentary system remains unknown. Because inhibin can specifically act on the pituitary gland to selectively inhibit the release of follicle-stimulatinghormone (FSH) induced by GnRH, rat submaxillary glands, which can produce GnRH and hormone receptor (GnRHR) (Yao el al. 2003), might regulate GnRH function through paracrine mechanisms. As already known, spleen is the most important lymph organ. The localization of positively stained cells of inhibin and a subunits in spleen suggests that inhibin might be an important messenger between neuroendocrine system and immune system. The expression of inhibin and a subunits in adrenal gland of normal rats suggested that they might specifically regulate androgen secretion in the adrenal gland. In addition, it may be related to stress. On the basis of the report that circulating inhibin declined to almost undetectable levels after castration, it could be inferred that heart, pancreatic gland, kidney, and spleen may not contribute to the concentration of circulating inhibin. In the first study, it was demonstrated whether hypothalamus and pituitary could secrete inhibin. The result showed that in control rats, both hypothalamus and pituitary showed a considerable amount of immunoreactive inhibin. But after ovariectomy, both showed negative result. It is concluded that both hypothalamus and pituitary cannot secrete inhibin by themselves, and the inhibin in these areas mainly comes from the ovary.
ZHANG Zhong-xia el al.
As already known, circulating inhibin must penetrate BBB to act on nerve center. The question is as to how inhibin is transported through BBB, in the form of dimer or subunits. The ovariectomized rats were injected with inhibin extract in this study. With inhibin-specific antibody used in streptavidin-biotin complex (SABC), the positive reaction sites were observed in both hypothalamus and pituitary, which suggested that inhibin could be transported through BBB. However, normally BBB only allows small lipophilic moleculesto pass through. Inhibin is a nonlipophilic peptide with a molecular weight of 32 Ka, and no inhibin transcytosed protein has been found so far. It has been reported that peptide could be transported through BBB in injected pig by unsaturated transmembrane diffusion (Xu and Zheng 1998). In brain capillary wall, many kinds of hydrolytic enzymes are present, which may result in the instability of the giant molecule and their subsequent hydrolysis may break it into a micromolecule that could penetrate BBB. In this study, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats previously injected with inhibin crude extract by SABC with inhibin a-subunit-specific antibody, which suggested that inhibin was cleaved into a and p subunits by zymohydrolysis. Moreover, positive reaction sites were also observed in the hypothalamus and pituitary of ovariectomized rats previously injected with inhibin a subunit by SABC with inhibin specific antibody. These results suggested that inhibin dimer was formed in hypothalamus and pituitary. Recent progresses demonstrated that inhibin a subunit contains seven Cys, located at 30,59,63,95,96, 131, and 133, respectively. At least one of the seven Cys would be linked to inhibin p subunit by disulfide bridges (Sugino et al. 1992; Pangas and Woodruff 2002). Based on the evidence that p subunit is expressed in the nerve center, it is proposed that it was the injected a fragment that reformed inhibin dimer with p subunit. Taking everything into account, it is concluded that inhibin could be transported through BBB, and there may be a transport model of inhibin “separation and reconstruction”: Inhibin dimer would separate into a and P subunits in the event of transporting BBB, and thereafter subunits could be recombined into bioactive inhibin in the brain.
82006, CAAS. All mhtsmseNBd. Published by Elsevier Ltd.
Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
Moreover, the results concerning the cellular localization of inhibins are often conflicting and sometimes incomplete. Once inhibin a subunit has been demonstrated in the liver cells, and there was no inhibin expression after endogenous biotin was inhibited (Munro et a2. 1999). It has also been reported that inhibin level in the liver of ovariectomized ewes decreased sharply, whereas inhibin a subunit could still be detected (Peeters et al. 1997). Two explanations were suggested. One is the sensitivity of assay used in detection, another is the detection level. For example, a sensitive competitive RT-PCR system can detect inhibidalpha at mRNA level, whereas immunohistochemistry detects inhibid inhibin a subunit at protein level. The present findings illustrated that differences might be due to the procession of mRNA translating into ultimate inhibin protein. Also, it has been demonstrated that the expression level of inhibin is greatly mutable during the estrous cycle (Bernard and Woodruff 2001; Tilbrook et al. 1999). In addition, the transport of BBB is a complicated process and many factors could influence it. Zhang et al. (2004) have demonstrated that follicular inhibin could penetrate the blood-brain barrier in sows; transport of inhibin through vesicles was influenced by Na+ content, protein concentration of vesicle, reaction time, ion channel, osmosis, etc.; and transport abilities were different for different species. In summary, we postulated a novel concept of inhibin transport through BBB, and the presence of inhibin and a subunit in various tissues provided evidence that they might potentially have inter- and intracellular functions in multiple systems. To date, inhibin’s predictive value in reproductive medicine is known, but little is known about the relationship between inhibin and gut diseases; our result may support further progress.
Acknowledgements This work was supported by the Doctor Fund from the Ministry of Education, China (20010307007).
References Bernard D J, Chapman S C, Woodruff T K. 2001. An emerging role for co-receptors in inhibin signal transduction. Molecular and Cellular Endocrinology, 180,5562. Bernard D J, Woodruff T K. 2001. Inhibin binding protein in rats: alternative transcripts and regulation in the pituitary
123
across the estrous cycle. Molecular Endocrinology, 15,654667. Blauer M, Kohonen J, Leivonen 1, Vilja P, Tuohimaa P. 1992. Inhibin alpha- and beta A-subunit immunoreactivity in the chicken embryo during morphogenesis. Development, 115, 1079-1086. Chen C C, Johnson P A. 1996. Molecular cloning of inhibird activin beta A-subunit complementary deoxyribonucleic acid and expression of inhibidactivin alpha- and beta A-subunits in the domestic hen. Biology ofReproduction, 54,429-435. de Bleser P J, Niki T, Xu G, Rogiers V, Geerts A. 1997. Localization and cellular sources of activins in normal and fibrotic rat liver. Hepatology, 26,905-912. He Z Y, Liu H C, Mele C A, Barmat L, Veeck L L, Davis 0, Rosenwaks Z. 1999. Expression of inhibidactivin subunits and their receptors and binding proteins in human preimplantation embryos. Journal of Assisted Reproduction and Genetics, 16,73-80. Meunier H, Rivier C, Evans R M, Vale W. 1988. Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions. Proceedings of the National Academy of Sciences of the USA, 85, 247-251. Munro L M, Kennedy A, McNicol A M. 1999. The expression of inhibidactivin subunits in the human adrenal cortex and its turnours. The Journal of Endocrinology, 161,341-347. Ohnishi N, Miyata T, Ohnishi H, Yasuda H, Tamada K, Ueda N, Mashima H, Sugano K. 2003. Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis. Gut, 52,1487-1493. Pangas S A, Woodruff T K. 2002. Production and purification of recombinant human inhibin and activin. The Journal of Endocrinology, 172, 199-210. Peeters R, Vanmontfort D, van Isterdael J, Verhoeven G, Rombauts L, Decuypere E. 1997. Evidence for the presence of immunoreactive inhibin in extragonadal tissues of ovariectomized ewes. Animal Reproduction Science, 48,251-
268. Robertson D M, Cahir N, Findlay J K, Burger H G, Groome N. 1997. The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma. The Journal of Clinical Endocrinology & Metabolism, 82, 889-896. Sugino K, Nakamura T, Takio K. 1992. Purification and characterization of high molecular weight forms of inhibin from bovine follicular fluid. Endocrinology, 130,789-796. Tilbrook A J, de Kretser D M, Clarke I J. 1999. Seasonal changes in the negative feedback regulation of the secretion of the gonadotrophins by testosterone and inhibin in rams. The
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of GnRH and its receptor in rat submaxillary glands by
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immunohistochemical, in situ hybridimtion and RT-PCR. L$e Science, 72, 2895-2904.
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Yan B, Zhu H Q, Huang P T. 2004. Soluble expression and target
Sinica, 38, 171 -175. (in Chinese) X u J M, Zheng H M. 1998. Progress in the study of peptide
transfer across blood-brain barrier. Chinese Pharmacological Bullelin, 14,34-37.
Yao B, Huang W, Huang Y, Chui Y, Wang Y, Li H, Pu R, Wan L, Zhang R. 2003. A study on the localization and distribution
study to brain of anti- TfR ScFv. Chinere Journal
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Biotechnology, 20,342-347. (in Chinese) Zhang H L, Yang L G, Wang C, Mao D G. 2004. Study on kinetics of follicular inhibin transported by the blood-brain barrier of sows. Scientia Agricultuni Sinicu , 37, I21 2- I21 6. (in Chinese) (Edited by WANG Lu-han)
02006.CAAS. All rights resewed. Publishedby ElsevierLtd
2006, 5 ( 9 ) : 718-724
September 2006
Localization of Inhibin in Some Rat Tssues and Exploration of Its Transport Mechanism ZHANG Zhong-xia,XU Lu, DI He-shuang, YAN Yu-qin and WANG Gen-lin College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China
Abstract The aims of the study were to determine the distribution of inhibin and its a subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex (SABC) and to assess the transport mechanism of inhibin by investigating the localization of inhibin and a subunits in the central nervous system (mainly in hypothalamus and pituitary) of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its a subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin a subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its a subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and a subunit could be transported through the BBB by the method of “separation and reconstruction”.
Key words: inhibin, inhibin a subunit, blood-brain barrier (BBB), immunohistochemical localization
INTRODUCTION Inhibin is a dimer that consists of one alpha-chain (18 Ka) and one beta-chain (14 Ka) linked by disulfide bridges, which plays an important role in regulating the synthesis and secretion of the FSH in pituitary, in modulating the development of ovary and sperm, also as a sensitive marker of mucinous ovarian cancers, and as an inhibiting factor. Although the inhibin’s origin, biological properties, assay method, gene regulation and passive immunity, a,PA,PB mRNA structure, expression, signal transduction (Bernard et al. 2001), roles, and its receptor are known, the mechanism of action of inhibin still remains disputable. There are three hypotheses regarding the mechanisms of action
of inhibin: inhibin acting on hypothalamus, pituitary, or ovary. Investigation has revealed the blood-brain barrier (BBB) bioactivity. BBB is a specialized construct that is formed by brain capillary endothelial cell; it acts as an important selective barricade during the exchange of brain and blood matter. However, circulating inhibin must be transported through BBB to act on nerve center. Research showed that besides micromolecules, partial macromolecules such as insulin and other hormones could also be transported through BBB. Zhang et al. (2004) indicated that follicular inhibin could be transported across BBB in sows. Yan et al. (2004) found that glycoprotein-transporting carrier was present in brain tunica intima. Other studies have demonstrated that a (Wang et al. 2005) and P subunits (He et al. 1999) could be transported through
Received 16 May, 2006 Accepted 7 July, 2006 ZHANG Zhong-xia, MSc. E-mail: zhongxial02Qyahoo.com.cn: Correspondence WANG Gen-lin, Tel: +86-2544395045, Fax: +86-25-84395314, E-mail: glwang9njau. edu.cn
02M)6.CAAS
All rghts reservd.Publishedby Elsevier Ltd
719
Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
BBB, but to date, there is no evidence that inhibin molecule could be transported through BBB. Also, the mechanisms have not been explored. So the question is how inhibin is being transported through BBB when it acts in brain. Recent progress has demonstrated the expression of inhibin in various tissues and has implicated potential involvement in diverse physiological and pathophysiological processes (Meunier et aE. 1988; Chen and Johnson 1996; Peeters et al. 1997). But most studies were focused on a-subunit expression and research at mRNA level. Currently, there is no assay for ‘total’ inhibin using monoclonal antibodies. Therefore, the aims of the present study were 1) to determine the distribution of inhibin and its a subunits in extragonadal tissues in the rat by immunohistochemical method with specific monoclonal antibodies; 2) to assess the transport mechanism of inhibin through the blood-brain barrier.
MATERIALS AND METHODS Animals Fifty mature female Sprague-Dawley rats were bred for 7 days, and then they were divided into five groups: the intact group, ovariectomized group, and exogenous injection groups, which were intraperitoneally injected with inhibin crude extract (2 pg), inhibin a fragment (2 pg), or normal saline 7 days after ovariectomization. Six hours after injection, five groups were immediately dissected, and hypothalamus, pituitary, heart, kidney, spleen, adrenal gland, pancreatic gland, liver, lung, and submaxillary gland of all the rats were collected. The tissues were fixed in 10%neutral formalin for 14 h at room temperature.
Tissue preparation The fixed organs were thereafter dehydrated through increasing concentrations of ethanol, cleared and embedded in paraffin wax (m.p. 54-56°C). The paraffinembedded organs were serially sectioned at 6-pm thickness and placed on APES (3-aminopropyl-trithoxysi1ane)coated slides. After drying at 37°C for one night, slides were stored at 4°C for preemergency.
lmmunohistochemistry Primary monoclonal antibody was mouse monoclonal anti-inhibin-a antibody (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd, Code 12777) at a dilution of 1:500 and mouse monoclonal anti-inhibin antibody (Antibodyshop,Code HYB 224-01) at a dilution of 1:200. Inhibin extract was obtained from porcine ovary and purified in the laboratory, and porcine inhibin a-subunit (1 32) fragment was obtained from Sigma (Code 18641). Immunocytochemical staining was carried out according to the method proposed by Wang et al. (2005). In brief, sections were deparaffinized in xylene, rehydrated through graded ethanol, and washed in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for two times, 10 min each. All incubations were carried out at room temperature, and PBS was used for washing after each step. The sections were then incubated in 3% H,O, in methanol at room temperature for 10 min to quench nonspecific staining, followed by the addition of 0.01 mol L-’ citric acid-buffered saline (pH 6.0) at 92-98°C for 20 min to repair endogenous enzyme. Afterward, the sections were incubated for 20 min at room temperature with normal goat serum confining liquid and then incubated with primary monoclonal antibody against inhibin or a subunit at a dilution of 1:200 for 1 h at 37°C. After incubation, the sections were treated with 0.5% biotinylated goat antimouse secondary antibody (SABC kit) for 20 min at 37°C and subsequently incubated with 2% streptavidin-biotin complex (SABC kit) for 20 min at 37°C. The antibody bound to the sections was visualized by treating the sections with 0.05% 3.3’-diaminobenzidine tetrachloride (Wuhan Boster Biotechnology Co., Ltd, CodeARlM2) in 10mM Tris-buffered saline containing 0.01% H,O, for 3 min. Specificity of the antibody against inhibin or a subunit was examined using normal mouse serum instead of primary antibody. To identify the cell types in each organ, a serial section of each sample was also stained with hematoxylin. Then the sections were washed in tap water, dehydrated in alcohol, cleared in xylene, and mounted with neutro-resin.
RESULTS lmmunohistochemical localization of inhibin in
02M)6,CAAS.All rlghts R)sBNBd. PuMished by ElsevierLtd.
some extragonadal tissues of the rat The results of the immunostaining of inhibin in intact extragonadal tissues of rats showed that inhibin-positive cells were observed in hypothalamus, pituitary, heart, pancreas, kidney, lung, liver, spleen, submaxillary gland, and adrenal cortex. In ovariectomized rats tissues, similar positive expressions were observed in heart, kidney, spleen, and pancreas, whereas no positive sites were seen in hypothalamus, pituitary, lung, liver, submaxillary gland, and adrenal gland (Table). Inhibin-positive cells were observed mainly in sarcolemma of the heart, circum-intralobular ductin of pancreas, the renal corpuscle kidney, and red pulp area of the spleen. In contrast, with normal mouse serum as the primary antibodies to detect inhibin and inhibin a, all the cells showed negative expression. It suggested that inhibin and a subunits were not only restricted to the gonads but also were present in a wide variety of nonreproductive organs and tissues, including the heart, pancreatic gland, kidney, and spleen. The result also demonstrated that the inhibin that was distributed in hypothalamus and pituitary of intact rats was from the ovary.
lmmunohistochemical localization of inhibin in hypothalamusand pituitary of ovariectomized rats The localization of inhibin and a subunit was investigated in the hypothalamus and pituitary of ovariectomized rats, which had been injected with inhibin a subunit fragment (2 pg) or inhibin crude extract (2 pg). The result showed that when inhibin-a monoclonal antibody or inhibin antibody was used along with inhibin a-subunit fragment ( 2 pg), both hypothalamus (Fig.- 1, -2) and pituitary (Fig.-3, -4)showed inhibin and asubunit positive cells; when inhibin antibody was used along with inhibin extract (2 pg), both again showed inhibin and a subunit positive cells (Fig.-5-8). In contrast, the normal saline treatment showed negative
cells. The result suggested that a-subunit and inhibin dimer could be transported through the RBI3 to reach the hypothalamus and pituitary.
DISCUSSION Inhibin as a member of the transformation growth factor P (TGF-P) gene superfamily, exhibiting paracrine and/or autocrine mechanisms of actions, plays roles in the regulation of cell proliferation and differentiation in a variety of tissues. Even though single a-subunit has no inhibin bioactivity, there might exist a distinct paracrine and/or autocrine function. Reports have showed that a-subunit can inhibit the binding of FSH with its receptor (Robertson et al. 1997). Excess inhibin a-subunit damages oocyte maturation (Blauer et al. 1992). Similarly, loss of a gene homozygote may cause gonadoma, etc. This study on the extragonadal distribution of inhibin and a-subunit confirmed previously described results of Meunier’s research on ewes (Meunier et al. 1988) and also showed some similarity with data produced in hen (Chen and Johnson 1996) using Northern blot analysis. However, there are still differences. For example, in Meunier’s report, heart, spleen, and pancreas were not mentioned, whereas Chen’s report on hen showed that a-subunit mRNA was less expressed in heart and adrenal gland, and PA-subunit mRNA was less expressed in heart, adrenal gland, liver, lung, and spleen. The diverse results reflect that inhibin is conservative in phylogeny, and interspecific difference also exists. This study suggested that inhibin and its a subunit might affect the function of the tissues by autocrine/paracrine mechanisms. Blauer et al. (1992) stated that in chicken embryo, a-subunit was localized in smooth muscle myoblasts as well as in developing cardiac muscle cells. Although Purkinje fibers were strongly immunoreactive, undifferentiated mesenchymal cell and terminal differentiated mature cardiac muscle cell had no positive
Table Distribution of inhibin in rat tissues Tiqrue ___ Intact rats Ovanectomized rats ~
“+”
~Hypothalamus
+
Pituitary ~ gland _
Heart _
_Pancreas ~
Kidney
Lung
Liver
Submaxrll~ygldnd
Spleen
+
+
+
+ +
+
+
+
+ +
represents inhibin immunostaining reaction positive;
+ ”-”
+
Adrenal gland
+
represents inhibin immunostaining reaction negative.
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Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
72 1
Fig. Immunohistochemical localization of inhibin and a-subunit in hypothalamus and pituitary of the sexually matured ovariectomized rat (x400 DAB stained, arrows indicate positive area). 1,2, in hypothalamusand and 3.4, in pituitary 6 h after exogenous injection of inhibin a fragment; 5,6, in hypothalamus and 7, 8, in pituitary 6 h after exogenous injection of inhibin extract.
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722
expression. This suggests that a-subunit could be correlated with muscle differentiation and nerve conduction. Ohnishi et aZ. (2003) proved that FS could act in the treatment of pancreas fibrosis, which indicates that inhibin may play a role in inhibiting pancreatic fibrosis. Research has shown that inhibin a was invariably negative in both normal and fibrotic rat liver, and p immunoreactivity of p subunit was weak in parenchymal cells (PC). In fibrotic livers, PA showed strong immunoreaction (de Bleser et aZ. 1997). Activin A binding to its receptor can lead to increase in Ca2+concentration in endochylema. Moreover, increases in insulin secretion promote glucose production of rat liver cells. It is assumed that exogenous inhibin crude extracthnhibin a fragment may exert actions antagonistic to activin A to play a role in recovery with regard to liver ailment. To date, the precise role of inhibin in alimentary system remains unknown. Because inhibin can specifically act on the pituitary gland to selectively inhibit the release of follicle-stimulatinghormone (FSH) induced by GnRH, rat submaxillary glands, which can produce GnRH and hormone receptor (GnRHR) (Yao el al. 2003), might regulate GnRH function through paracrine mechanisms. As already known, spleen is the most important lymph organ. The localization of positively stained cells of inhibin and a subunits in spleen suggests that inhibin might be an important messenger between neuroendocrine system and immune system. The expression of inhibin and a subunits in adrenal gland of normal rats suggested that they might specifically regulate androgen secretion in the adrenal gland. In addition, it may be related to stress. On the basis of the report that circulating inhibin declined to almost undetectable levels after castration, it could be inferred that heart, pancreatic gland, kidney, and spleen may not contribute to the concentration of circulating inhibin. In the first study, it was demonstrated whether hypothalamus and pituitary could secrete inhibin. The result showed that in control rats, both hypothalamus and pituitary showed a considerable amount of immunoreactive inhibin. But after ovariectomy, both showed negative result. It is concluded that both hypothalamus and pituitary cannot secrete inhibin by themselves, and the inhibin in these areas mainly comes from the ovary.
ZHANG Zhong-xia el al.
As already known, circulating inhibin must penetrate BBB to act on nerve center. The question is as to how inhibin is transported through BBB, in the form of dimer or subunits. The ovariectomized rats were injected with inhibin extract in this study. With inhibin-specific antibody used in streptavidin-biotin complex (SABC), the positive reaction sites were observed in both hypothalamus and pituitary, which suggested that inhibin could be transported through BBB. However, normally BBB only allows small lipophilic moleculesto pass through. Inhibin is a nonlipophilic peptide with a molecular weight of 32 Ka, and no inhibin transcytosed protein has been found so far. It has been reported that peptide could be transported through BBB in injected pig by unsaturated transmembrane diffusion (Xu and Zheng 1998). In brain capillary wall, many kinds of hydrolytic enzymes are present, which may result in the instability of the giant molecule and their subsequent hydrolysis may break it into a micromolecule that could penetrate BBB. In this study, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats previously injected with inhibin crude extract by SABC with inhibin a-subunit-specific antibody, which suggested that inhibin was cleaved into a and p subunits by zymohydrolysis. Moreover, positive reaction sites were also observed in the hypothalamus and pituitary of ovariectomized rats previously injected with inhibin a subunit by SABC with inhibin specific antibody. These results suggested that inhibin dimer was formed in hypothalamus and pituitary. Recent progresses demonstrated that inhibin a subunit contains seven Cys, located at 30,59,63,95,96, 131, and 133, respectively. At least one of the seven Cys would be linked to inhibin p subunit by disulfide bridges (Sugino et al. 1992; Pangas and Woodruff 2002). Based on the evidence that p subunit is expressed in the nerve center, it is proposed that it was the injected a fragment that reformed inhibin dimer with p subunit. Taking everything into account, it is concluded that inhibin could be transported through BBB, and there may be a transport model of inhibin “separation and reconstruction”: Inhibin dimer would separate into a and P subunits in the event of transporting BBB, and thereafter subunits could be recombined into bioactive inhibin in the brain.
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Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism
Moreover, the results concerning the cellular localization of inhibins are often conflicting and sometimes incomplete. Once inhibin a subunit has been demonstrated in the liver cells, and there was no inhibin expression after endogenous biotin was inhibited (Munro et a2. 1999). It has also been reported that inhibin level in the liver of ovariectomized ewes decreased sharply, whereas inhibin a subunit could still be detected (Peeters et al. 1997). Two explanations were suggested. One is the sensitivity of assay used in detection, another is the detection level. For example, a sensitive competitive RT-PCR system can detect inhibidalpha at mRNA level, whereas immunohistochemistry detects inhibid inhibin a subunit at protein level. The present findings illustrated that differences might be due to the procession of mRNA translating into ultimate inhibin protein. Also, it has been demonstrated that the expression level of inhibin is greatly mutable during the estrous cycle (Bernard and Woodruff 2001; Tilbrook et al. 1999). In addition, the transport of BBB is a complicated process and many factors could influence it. Zhang et al. (2004) have demonstrated that follicular inhibin could penetrate the blood-brain barrier in sows; transport of inhibin through vesicles was influenced by Na+ content, protein concentration of vesicle, reaction time, ion channel, osmosis, etc.; and transport abilities were different for different species. In summary, we postulated a novel concept of inhibin transport through BBB, and the presence of inhibin and a subunit in various tissues provided evidence that they might potentially have inter- and intracellular functions in multiple systems. To date, inhibin’s predictive value in reproductive medicine is known, but little is known about the relationship between inhibin and gut diseases; our result may support further progress.
Acknowledgements This work was supported by the Doctor Fund from the Ministry of Education, China (20010307007).
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