Cell and Tissue Research
Cell Tissue Res. 202, 505-508 (1979)
9 by Springer-Verlag 1979
Short Communication Immunohistochemical Localization of Thyrotropin-Releasing Hormone (TRH) in Skin of Rana pipiens Janice L. Bolaffi and Ivor M.D. Jackson Endocrine Division, New England Medical Center Hospital, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.
Summary. Using immunofluorescent techniques thyrotropin releasing hormone ( T R H ) is demonstrated in skin o f Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer. Key words: Thyrotropin releasing hormone - Frog skin - Immunofluorescence - APUD.
T R H has been identified in significant amounts not only in the hypothalamus, but also in extrahypothalamic nervous tissue of all vertebrate species and more recently, outside the nervous system altogether in rodent gastro-intestinal tract and anuran skin (reviewed by Jackson and Reichlin, 1979). Such tissue distribution also occurs with other biologically active peptides (Polak and Pearse, 1970; Erpsamer and Melchiorri, 1973; Dockray and Hopkins, 1975; H6kfelt et al., 1975; Van Noorden et al., 1977; Brown et al., 1977; Rivier et al., 1978), and is consistent with the A P U D (amine precursor uptake decarboxylation) hypothesis which postulates that these peptides arise in tissue derived from neuroectoderm (Pearse, 1976). It became important to determine the exact anatomical location of T R H within frog skin, since the cutaneous glands, thought to originate embryologically from neuroectoderm related tissue, have been proposed as the location of amphibian skin peptides (Polak and Pearse, 1970; Erpsamer and Melchiorri, 1973; Dockray and Hopkins, 1975). Send offprint requests to: Dr. Janice L. Bolaffi, Endocrine Division, New England Medical Center
Hospital, 171 Harrison Avenue, Boston, Massachusetts 02111, USA We wish to thank Dr. Ronald DeLellisand Ms. Mary Blount for their expert adviceand guidance in the immunohistochemical techniques. This investigation was supported by NIH National Research ServiceAward ~ 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.
0302-766X/79/0202/0505/$01.00
506
J.L. Bolaffi and I.M.D. Jackson
Materials and Methods
Frozen sections(6-8 I~m)of dorsal or ventral skin of adult Ranapipiens or R. catesbeianawereprocessed according to the indirect immunofluorescence technique of Coons (1958) to demonstrate TRH. Sections were exposedto normal goat serum 1 : 20 (Cappel) for 30minutes, TRH antibody [preparedas previously described Oackson and Reichlin, 1974)] at 1:20 to 1:100 for 30-60minutes and fluorescein-isothyocyanate (FITC)-conjugated anti-rabbit gamma globulin (Cappel) at 1:10 for 30 minutes. Adjacent sections served as controls and were treated with normal rabbit serum or TRHantibody adsorbed with synthetic TRH at 250 lag/ml instead of primary antibody.
Results
As can be seen in Fig. 1 a, such treatment of dorsal skin of the frog produces a remarkable pattern of fluorescent cell bodies throughout all the cell layers of the epidermis as well as the epithelium lining the large cutaneous glands, but not in dermal tissue. The fluorescence, striking at TRH-antibody concentrations from I : 20-1 : 100, diminishes with decreasing antibody concentration, and is eliminated by preadsorption of antiserum with synthetic T R H or substitution of primary antibody with normal rabbit serum (Fig. 1 b). Ventral skin preparations, and dorsal and ventral skin from R. catesbeiana show similar but less intense distribution of immunofluorescent-TRH (these observations not shown).
Discussion
The widespread distribution and extraordinary degree of TRH-linked fluorescence in dorsal skin preparations are in good agreement with the very high amounts of T R H in dorsal skin measured by radioimmunoassay (Jackson and Reichlin, 1977). The antibody has been well characterized in this laboratory and does not cross react significantly with arginine vasopressin, norepinephrine, corticotropin, 1-proline, 1-histidine, 1-glutamine or various combinations of these amino acids. Furthermore the fluorescence decreases as the antibody is diluted, and is completely eliminated by preadsorbing the antibody with synthetic TRH. Although such data are not absolutely conclusive, they are good indices that the fluorescence seen is specific for T R H in frog skin. Immunofluorescent-TRH is observed in epithelium lining the cutaneous glands but is not observed within the glands themselves although any T R H stored within the glands might have been washed out during the histochemical processing. In most favorable preparations, Rana skin shows a gradient, within the epidermis, of TRH-immunofluorescence which increases in the cell layers towards the basement membrane with m a x i m u m immunofluorescence in the stratum germinativum; this distribution is similar to that proposed for N a § K § -ATPase sites in the frog skin (Mills etal., 1977), and is consistent with the proposed osmoregulatory, role for T R H (Grimm-Jorgensen and Vofite, 1979) and other neurohypophysial hormones (Bentley, 1974) in anuran skin.
Thyrotropin-Releasing Hormone in Frog Skin
507
Fig. 1. a Dorsal skin of R. pipiens processed for TRH-immunofluorescence as described in the text, was exposed to TRH-antibody (1 : 25) for 45 minutes. Magnification x 280 (upper). b Control sections of dorsal skin, adjacent to those sections seen in Fig. 1 and processed identically except normal rabbit serum (1 : 25) was substituted for the TRH antibody. Magnification x 280 (lower)
508
J.L. Bolaffi and I.M.D. Jackson
In addition to TRH, amphibian skin is rich in other biologically active peptides which have also been identified in the brain and gastrointestinal tract of mammals, birds, amphibia and fish (Erpsamer and Melchiorri, 1973; Polak and Pearse, 1970; Dockray and Hopkins, 1975, VanNoorden et al., 1977). The localization of TRH in Rana skin to epidermis as well as cutaneous glands is consistent with the view that these tissues are of specialized neuroectodermal derivation and are part of the APUD system, and may suggest a possible role in skin function for this peptide as a neuromodulator.
References Bentley, P.J.: Actions of neurohypophyseal peptides in amphibians, reptiles and birds. In: Handbook of Physiology, Vol. IV. The pituitary gland and its neuroendocrine control (E. Knobil and W.H. Sawyer, eds.) pp. 545-563 (1974) Brown, M., Rivier, J., Vale, W.: Bombesin: Potent effects on thermoregulation in the rat. Science 196, 998-1000 (1977) Coons, A.H.: In general cytochemical methods (Y.F. Danielli, ed.), pp. 339-422. New York: Academic Press (1958) Dockray, G.J., Hopkins, C.R.: Caerulein secretion by dermal glands in Xenopus laevis. J. Cell. Biol. 64, 724-733 (1975) Erpsamer, V., Melchiorri, R.I.: Active polypeptides of the amphibian skin and their synthetic analogues. Pure and Appl. Chem. 35, 463 (1973) Grimm-Jorgensen, Y., Vofite, C.L.: A possible role of thyrotropin-releasing hormone in the seasonal adaptation of salt transport in the frog. Gen. Comp. Endocrinol. 37, 482-486 (1979) H6kfelt, T., Fuxe, K., Johansson, D., Jeffcoate, S., White, N.: Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry. Europ. J. Pharmacol. 34, 389-392 (1975) Jackson, I.M.D., Reichlin, S.: Thyrotropin-releasing hormone TRH: Distribution in hypothalamic and extrahypothalamic brain tissue. Endocrinology 93, 854-862 (1974) Jackson, I.M.D., Reichlin, S.: Thyrotropin-releasing hormone: abundance in the skin of the frog, Rana pipiens. Science 198, 414-415 (1977) Jackson, I.M.D., Reichlin, S.: Distribution and biosynthesis of TRH in the nervous system. In: Central nervous system effects of hypothalamic hormones and other pepides (Collu etal., eds.), pp. 3-54. New York: Raven Press (1979) Mills, J.W., Ernst, S.A., DiBona, D.R.: Localization ofNa § pump sites in frog skin. J. Cell. Biol. 73, 88110 (1977) Pearse, A.G.E.: Peptides in brain and intestine. Nature 262, 92-94 (1976) Polak, J.M., Pearse, A.G.E.: Anti-gastrin immunofluorescence in the skin of Hyla crepitans and the cytochemistry of the cells involved. Experientia 26, 288-289 (1970) Rivier, C., Rivier, J., Vale, W.: The effect of bombesin and related peptides on prolactin and growth hormone secretion in the rat. Endocrinology 102, 519-522 (1978) Van Noorden, S., Polak, J.M., Negri, L., Pearse, A.G.E.: Common peptides in brain, intestine and skin: embryology, evolution and significance. J. Endocrinol. 75, 33-34 (1977) Accepted July2, 1979
Cell Tissue Res. 202, 505-508 (1979)
9 by Springer-Verlag 1979
Short Communication Immunohistochemical Localization of Thyrotropin-Releasing Hormone (TRH) in Skin of Rana pipiens Janice L. Bolaffi and Ivor M.D. Jackson Endocrine Division, New England Medical Center Hospital, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.
Summary. Using immunofluorescent techniques thyrotropin releasing hormone ( T R H ) is demonstrated in skin o f Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer. Key words: Thyrotropin releasing hormone - Frog skin - Immunofluorescence - APUD.
T R H has been identified in significant amounts not only in the hypothalamus, but also in extrahypothalamic nervous tissue of all vertebrate species and more recently, outside the nervous system altogether in rodent gastro-intestinal tract and anuran skin (reviewed by Jackson and Reichlin, 1979). Such tissue distribution also occurs with other biologically active peptides (Polak and Pearse, 1970; Erpsamer and Melchiorri, 1973; Dockray and Hopkins, 1975; H6kfelt et al., 1975; Van Noorden et al., 1977; Brown et al., 1977; Rivier et al., 1978), and is consistent with the A P U D (amine precursor uptake decarboxylation) hypothesis which postulates that these peptides arise in tissue derived from neuroectoderm (Pearse, 1976). It became important to determine the exact anatomical location of T R H within frog skin, since the cutaneous glands, thought to originate embryologically from neuroectoderm related tissue, have been proposed as the location of amphibian skin peptides (Polak and Pearse, 1970; Erpsamer and Melchiorri, 1973; Dockray and Hopkins, 1975). Send offprint requests to: Dr. Janice L. Bolaffi, Endocrine Division, New England Medical Center
Hospital, 171 Harrison Avenue, Boston, Massachusetts 02111, USA We wish to thank Dr. Ronald DeLellisand Ms. Mary Blount for their expert adviceand guidance in the immunohistochemical techniques. This investigation was supported by NIH National Research ServiceAward ~ 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.
0302-766X/79/0202/0505/$01.00
506
J.L. Bolaffi and I.M.D. Jackson
Materials and Methods
Frozen sections(6-8 I~m)of dorsal or ventral skin of adult Ranapipiens or R. catesbeianawereprocessed according to the indirect immunofluorescence technique of Coons (1958) to demonstrate TRH. Sections were exposedto normal goat serum 1 : 20 (Cappel) for 30minutes, TRH antibody [preparedas previously described Oackson and Reichlin, 1974)] at 1:20 to 1:100 for 30-60minutes and fluorescein-isothyocyanate (FITC)-conjugated anti-rabbit gamma globulin (Cappel) at 1:10 for 30 minutes. Adjacent sections served as controls and were treated with normal rabbit serum or TRHantibody adsorbed with synthetic TRH at 250 lag/ml instead of primary antibody.
Results
As can be seen in Fig. 1 a, such treatment of dorsal skin of the frog produces a remarkable pattern of fluorescent cell bodies throughout all the cell layers of the epidermis as well as the epithelium lining the large cutaneous glands, but not in dermal tissue. The fluorescence, striking at TRH-antibody concentrations from I : 20-1 : 100, diminishes with decreasing antibody concentration, and is eliminated by preadsorption of antiserum with synthetic T R H or substitution of primary antibody with normal rabbit serum (Fig. 1 b). Ventral skin preparations, and dorsal and ventral skin from R. catesbeiana show similar but less intense distribution of immunofluorescent-TRH (these observations not shown).
Discussion
The widespread distribution and extraordinary degree of TRH-linked fluorescence in dorsal skin preparations are in good agreement with the very high amounts of T R H in dorsal skin measured by radioimmunoassay (Jackson and Reichlin, 1977). The antibody has been well characterized in this laboratory and does not cross react significantly with arginine vasopressin, norepinephrine, corticotropin, 1-proline, 1-histidine, 1-glutamine or various combinations of these amino acids. Furthermore the fluorescence decreases as the antibody is diluted, and is completely eliminated by preadsorbing the antibody with synthetic TRH. Although such data are not absolutely conclusive, they are good indices that the fluorescence seen is specific for T R H in frog skin. Immunofluorescent-TRH is observed in epithelium lining the cutaneous glands but is not observed within the glands themselves although any T R H stored within the glands might have been washed out during the histochemical processing. In most favorable preparations, Rana skin shows a gradient, within the epidermis, of TRH-immunofluorescence which increases in the cell layers towards the basement membrane with m a x i m u m immunofluorescence in the stratum germinativum; this distribution is similar to that proposed for N a § K § -ATPase sites in the frog skin (Mills etal., 1977), and is consistent with the proposed osmoregulatory, role for T R H (Grimm-Jorgensen and Vofite, 1979) and other neurohypophysial hormones (Bentley, 1974) in anuran skin.
Thyrotropin-Releasing Hormone in Frog Skin
507
Fig. 1. a Dorsal skin of R. pipiens processed for TRH-immunofluorescence as described in the text, was exposed to TRH-antibody (1 : 25) for 45 minutes. Magnification x 280 (upper). b Control sections of dorsal skin, adjacent to those sections seen in Fig. 1 and processed identically except normal rabbit serum (1 : 25) was substituted for the TRH antibody. Magnification x 280 (lower)
508
J.L. Bolaffi and I.M.D. Jackson
In addition to TRH, amphibian skin is rich in other biologically active peptides which have also been identified in the brain and gastrointestinal tract of mammals, birds, amphibia and fish (Erpsamer and Melchiorri, 1973; Polak and Pearse, 1970; Dockray and Hopkins, 1975, VanNoorden et al., 1977). The localization of TRH in Rana skin to epidermis as well as cutaneous glands is consistent with the view that these tissues are of specialized neuroectodermal derivation and are part of the APUD system, and may suggest a possible role in skin function for this peptide as a neuromodulator.
References Bentley, P.J.: Actions of neurohypophyseal peptides in amphibians, reptiles and birds. In: Handbook of Physiology, Vol. IV. The pituitary gland and its neuroendocrine control (E. Knobil and W.H. Sawyer, eds.) pp. 545-563 (1974) Brown, M., Rivier, J., Vale, W.: Bombesin: Potent effects on thermoregulation in the rat. Science 196, 998-1000 (1977) Coons, A.H.: In general cytochemical methods (Y.F. Danielli, ed.), pp. 339-422. New York: Academic Press (1958) Dockray, G.J., Hopkins, C.R.: Caerulein secretion by dermal glands in Xenopus laevis. J. Cell. Biol. 64, 724-733 (1975) Erpsamer, V., Melchiorri, R.I.: Active polypeptides of the amphibian skin and their synthetic analogues. Pure and Appl. Chem. 35, 463 (1973) Grimm-Jorgensen, Y., Vofite, C.L.: A possible role of thyrotropin-releasing hormone in the seasonal adaptation of salt transport in the frog. Gen. Comp. Endocrinol. 37, 482-486 (1979) H6kfelt, T., Fuxe, K., Johansson, D., Jeffcoate, S., White, N.: Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry. Europ. J. Pharmacol. 34, 389-392 (1975) Jackson, I.M.D., Reichlin, S.: Thyrotropin-releasing hormone TRH: Distribution in hypothalamic and extrahypothalamic brain tissue. Endocrinology 93, 854-862 (1974) Jackson, I.M.D., Reichlin, S.: Thyrotropin-releasing hormone: abundance in the skin of the frog, Rana pipiens. Science 198, 414-415 (1977) Jackson, I.M.D., Reichlin, S.: Distribution and biosynthesis of TRH in the nervous system. In: Central nervous system effects of hypothalamic hormones and other pepides (Collu etal., eds.), pp. 3-54. New York: Raven Press (1979) Mills, J.W., Ernst, S.A., DiBona, D.R.: Localization ofNa § pump sites in frog skin. J. Cell. Biol. 73, 88110 (1977) Pearse, A.G.E.: Peptides in brain and intestine. Nature 262, 92-94 (1976) Polak, J.M., Pearse, A.G.E.: Anti-gastrin immunofluorescence in the skin of Hyla crepitans and the cytochemistry of the cells involved. Experientia 26, 288-289 (1970) Rivier, C., Rivier, J., Vale, W.: The effect of bombesin and related peptides on prolactin and growth hormone secretion in the rat. Endocrinology 102, 519-522 (1978) Van Noorden, S., Polak, J.M., Negri, L., Pearse, A.G.E.: Common peptides in brain, intestine and skin: embryology, evolution and significance. J. Endocrinol. 75, 33-34 (1977) Accepted July2, 1979
