GE.NERAL
AND
COMPARATIVE
ENDOCRINOLOGY
37, 343-349
(1979)
Chum Salmon Pituitary Fractions: Somatotropic Activity and Cytoimmunofluorescence Studies’ M. P. KOMOURDJIAN~
Accepted
AND D. R. IDLER
December
20, 1978
Highly purified chum salmon pituitary fractions were bioassayed for their ability to stimulate elongation in hypophysectomized rainbow trout, Salnzo gcrirdnwi. One fraction, previously identified as the Na-retaining principle, prolactin, was not active. Of the active fractions, only the most potent one elicited a dose response. An antiserum against this fraction permitted the specific labeling of somatotropic cells in pituitary sections by an indirect immunocytofluorescence procedure. An antiserum against the Na-retaining principle led to the specific fluorescence of the follicular “prolactin cells.” However, neither antiserum appeared to discriminate between acidophile type in pituitary sections from frogs or rats.
A role for somatotropin in growth promotion in Salmo species is well established (Swift, 1954; Komourdjian, 1976a,b). Hypophysectomy in salmonids interrupts normal growth which is reestablished by somatotropin therapy. This suggests the presence of a somatotropic hormone in salmonid pituitaries and constitutes the basis for a sensitive and reliable assay for somatotropic activity in purified pituitary material (Komourdjian ef al., 1978). While. hitherto, most studies were conducted using mammalian hormones, advances have now been described in the isolation of teleostean pituitary hormones (Farmer et rrl., 1975, 1976). Recently, Idler c’t cl/. (1978) reported the is,olation of highly purified pituitary fractions from chum salmon, O~~C~&V~C~~~IS dicta. One of these fractions possessed potent Na-retaining activity, a characteristic property of teleost prolactin-like principles. ' M.S.R.L.
Contribution No. 328. z Present address: Department of Mathematics Natural Sciences, College of Cape Breton. P.O. 760. Sydney. Nova Scotia. BlP 63 I. Canada.
and Box
The present presence of fractions. fluorescence of the main ing fractions.
study seeks to determine the somatotropic activity in these Additionally, immunocytois used to assess the specificity somatotropic and Na-retain-
MATERlALS
AND
METHODS
Homrone frurtions. The purification and some properties of these preparations have been described in detail elsewhere (Idler PI al., 1978). Briefly, the non-glycoprotein fraction Con A- I (Idler et al., 1975) was obtained from whole frozen chum salmon, 0. krta, pituitary homogenate by affinity chromatography. This was further fractionated by gel filtration and ion exchange. Fraction I was the unadsorbed fraction from DEAE eluted in 0.005 M ammonium bicarbonate at pH 9.0. Fractions 2.3. and 4, the adsorbed fractions, were eluted at ammonium bicarbonate tpH 9.0) concentrations of 0.016 to 0.024, 0.024 to 0.04, and 0.04 to 0.084 M respectively. Anribodies. Antisera were raised to the fractions with highest sodium-retaining and somatotropic activities, i.e., 1 and 2 respectively. Male NZW rabbits, approximately 2.2 kg in weight. received Freund’s complete adjuvant. emulsified with I25 kg of antigen, intradermally. and intramuscular boosters of the suspension containing 238 pg of antigen 2 months thereafter. Each time, 0.1 ml of pertussis vaccine was in-
343 0016~6480/79/020343-07$01.00/O Copyright Q 1979 by Academic Press. Inc. All right!, of reproduction in any form recerved.
344
KOMOURDJIAN
jetted subcutaneously. Animals were bled 1 month after the final injection. Fluorescein labeled goat anti-rabbit y globulin was purchased from Antibodies Incorporated (Davis, Calif.). Immrfnocvrnfoyy. Pituitaries from 22-cm male rainbow trout, Sulmo guirdneri. were fixed in ice-cold Bouin’s formol-picric-acetic acid fixative. After 24 hr. tissues were dehydrated, embedded, and sectioned at 7-8 pm. Sections were rapidly hydrated and transferred to phosphate buffer. pH 7.4, for 10 min. O-rings were then placed over the sections and filled with antiserum. After 90 mitt, slides were washed in several changes of buffer. The labeled monospecific antibody, diluted I: IO with buffer, was then applied to the sections for I hr. Slides were washed in buffer and mounted using a 3:1 glycerol:phosphate buffer mixture. Sections of pituitary tissue from 350-g male Sprague-Dawley and 50-g frogs, Ronu pipirns. were processed similarly. In control sections normal rabbit serum replaced antiserum to fractions I and 2, or alternatively, the labeled antibody was omitted. Additionally, the specificity of each antiserum was studied by adsorbing it with the corresponding antigen prior to immunofluorescent labeling. In all cases serial sections were stained with periodic acid-Schiff ‘s-hematoxylin-orange Cl (Komourdjian et ai., 1976b) or using the Cleveland-Wolfe technique. Biouusuy for somutrotropic uctir,iry. Rainbow trout, S. guirdneri. were obtained from Goossens trout farm, Otterville, Ontario. Animals, I5 to I9 cm in length, were hypexed according to the procedure of Komourdjian and Idler (1977) and held under the conditions described in that study. Briefly, two tanks were subdivided to accommodate four groups. Dilute seawater (I I oioo salinity) was recirculated and cooled to 11°C. An 8 hr photophase was maintained throughout the study. Fish were fed to satiation three times daily with Ewos 5P trout pellets. Two months after hypophysectomy biweekly morphometric determinations and injections were commenced. Each time, all fish were weighed to the nearest decigram, their fork lengths measured to the nearest millimeter. and injected intraperitoneally with 0.2 ml of IO mM CAPS (cyclohexylaminopropanesulfonic acid) buffer. adjusted to pH 9.5 with NaOH. After 2 weeks each group received a different DEAE hormone test fraction. Each fish was injected with 25 pg of the particular preparation dissolved in 0.2 ml CAPS buffer; this treatment was continued for 3 weeks. The dose was then doubled for the groups which responded positively. in order to investigate the existence of a dose-effect response. Sufficient fraction 4 material was not available. The pertinent group and that receiving fraction I were injected with porcine somatotropin to assess whether the fish were capable of showing higher growth rates.
AND
IDLER
Instantaneous linear growth rates were computed according to the equation 100 (log,Y, - log,Y,)/(T-t) where Y, and Y, are mean lengths at times T and t (Brown, 1957). Results were examined by analyses of variance and Duncan’s multiple range test.
RESULTS
trout All groups of hypophysectomized showed specific linear growth rates of 0.07% or less per day during the ICday control period when they received injections of CAPS buffer. Injections of 25pg doses of the purified peptide preparations stimulated growth in three of the four groups (Fig. 1). Fraction 2 produced the largest stimulation followed by fractions 3 and 4 respectively: fraction I (prolactin) did not stimulate growth. Doubling the dose to 50 pg produced a significant (p < 0.001) increase in growth with fraction 2 but not with fraction 3. Injections of porcine STH in groups II and IV elicited a significantly higher growth rate than the basal (CAPS) rate. The growth rate of all groups gradually approached initial levels when the fish were again injected with CAPS. Fluorescence in trout pituitary sections was localized in the erythrosinophilic cells of the rostra1 pars distalis (prolactin cells) using antiserum to fraction 1 (Fig. 2) and in the orangeophiles (somatotrops) of the proximal pars distalis with antiserum to fraction 2 (Fig. 3). The fluorescence in the follicular cells tended to be concentrated in the apical region but in the proximal pars orangeophiles it was more evenly distributed. Similar results were obtained with pituitary sections from Atlantic salmon. However, either antiserum appeared to elicit fluorescence in both types of rat pituitary acidophiles. Similarly, no discrimination was evident in the labeling of the amphibian tissues. None of the control sections revealed any fluorescence except for the native fluorescence found in the teleostean saccus vasculosus.
SOMATOTROPIN
1N
SALMON
345
PITUITARY
0.45
z 0.40 8 & Go.35 L m
I
go30 IL
0
I (7)
GROUP GROUPII
gj
(6)
GROUP
ILK
(6)
GROUP
I!Z
(5)
F
3
o-25
22 (3
0 rTT
3 020 z
i
2
O-15
8 $
O-IO
5 k-J *o-o5
III1 DAYS
l-14
lcJ “1 I
14-35
35-46
46-67
-
67-92
FIG. I. Instantaneous linear growth rates (mean 2 SEM) of hypophysectomized trout injected intraperitoneally with chromatographic fractions from chum salmon pituitaries (F) or porcine somatotropin (pSTH). Identical superscripts denote significant differences (p < 0.01); asterisk indicates no significant difference compared to initial (basal) rate during CAPS treatment.
DISCUSSION
Our results confirm the presence of a potent growth promoting principle, or somatotropin, in pituitaries of chum salmon, and verify earlier identifications of the cells of origin of the principle (see Introduction). The principle appeared to be concentrated in the fraction eluted at ammonium bicarbonate concentrations of 0.024 to 0.04 M. A study on the somatotropic activity of Tilapia pituitary extracts in sockeye salnerka. (Clarke et al., mon, Oncorhynchus 1977) suggests that the chum salmon principle in the present study had higher activity than the former; nevertheless, the differences between the two studies defy a strict comparison. However, the salmonid prepa-
ration is likely of the order of two magnitudes more active than the porcine somatotropin used in our studies (also Komourdjian and Idler, 1978). The determination of reliable dose-response curves must now await the isolation of larger amounts of hormone. The significance of the activity found in two of the other fractions is not clear although the presence of contamination in the case of fraction 3 and alternate hormonal forms (fraction 4) might be subjects of speculation. Current studies have shown some immunoreactive material in fractions 3 and 4, but none in fraction 1, by radioimmunological methods using an antiserum to the more active fraction 2 (Komourdjian and Idler, unpublished).
346
KOMOURDJIAN
FIG. 2. lar prolactin
Sagittal cells.
section 160x.
of S. ~aird~c~i
pituitary
AND
IDLER
gland
showing
fluorescent
staining
of the follicu-
SOMATOTROPIN
IN
SALMON
PITUITARY
347
348
KOMOURDJlAN
The specific localization of fluorescence in our study, by an approach considered to be highly sensitive (Hayashida, 1973), suggests that the antigenic portions of the two native salmon hormones, somatotropin and prolactin, are probably structurally distinct. This is an encouraging finding towards the development of a specific radioimmunoassay for these principles. This specificity is of special interest following reports of structural similarities between prolactin and somatotropin from Tilapia (Farmer rf (II., 1977; Clarke et al., 1977). Some overlap may exist in the biological activities of the salmonid hormones. At a very high dose, chum salmon somatotropin mimicked Na-retaining activity (Idler et al., 1978). Yet, the current study did not reveal any somatotropic activity in the chum pituitary fraction identifiable as proiactin. This suggests that teleostean prolactin and somatotropin are distinguishable as had earlier been suggested by Ball (1965). This is the first report of immunofluorescent labeling of rainbow trout pituitary cells with antisera to salmonid hormones. Our work is in agreement with earlier studies which had characterized the somatotropic cells based on their tinctorial characteristics and on histophysiological evidence, mainly a correlation between cellular hypertrophy and an increased growth rate of the animal (Olivereau, 1954; Olivereau and Ridgway, 1962; Komourdjian et ml., 1976b). Additional evidence had been provided by immunohistochemical studies antigens using heterologous (ovine) (McKeown and VanOverbeeke, 1971), although the reliability of such approaches has been questioned (Ingleton and Stribley, 1977). The evidence for the cellular origin of prolactin in salmonids derives from similar histophysiological (Olivereau, 1969) and immunohistochemical studies (Aler, 197 1; McKeown and VanOverbeeke, 1971). Studies on eel, Anguilla anguilla pituitaries using conspecific antisera (Ingleton and
AND
IDLER
Stribley, 1977) have also been valuable since these glands and those of salmonids share fundamental morphological features. Our study extends these findings to positively relate the Na-retaining principle of salmonids (Idler ef al., 1978) to their cells of origin. The absence of fluorescence within the lumen of each follicle of the rostra1 pars distalis supports the idea that the intraluminar material is not identifiable with prolactin. The origin of this material is not yet understood. REFERENCES Aler, G. (1971). The study of prolactin in the pituitary gland of the Atlantic eel (Anguilla anguilla) and the Atlantic salmon (Salmo s&r) by immunofluorescent technique. Actu Zool. (Stockholm)
52, 145-156.
Ball, .I. N. (1965). Partial hypophysectomy in the teleost Poecilia: Separate identities of teleostean growth hormone and teleostean prolactin-like hormone. Gen. Camp. Endocrinol. 5, 654-661. Brown, M. E. (1957). Experimental studies on growth in “Physiology of Fishes” (M. E. Brown, ed.), Vol. I, pp. 361-400. Academic Press, New York. Clarke. W. C., Farmer, S. W., and Hartwell, K. M. ( 1977). Effect of teleost pituitary growth hormone on growth of Tilupia mossambicu and on growth and seawater adaptation of sockeye salmon (Uncorhynchus
nerku).
Gen.
Cump.
Endocrinol.
33.
174-178. Farmer, S. W., Clarke, W. C., Papkoff, H., Nishioka, R. S., Bern, H. A.. and Li, C. H. (1975). Studies on the purification and properties of teleost prolactin. L$e Sci. 16, 149-159. Farmer, S. W., Papkoff, H., Hayashida, T., Bewley, T. A.. Bern, H. A., and Li, C. H. (1976). Purification and properties of teleost growth hormone. Gen. Camp. Endocrinol. 30, 91-100. Farmer, S.-W., Papkoff, H., Bewley, T.-A., Hayashida. T.. Nishioka, R. S., Bern, H. A., and Li, C. H. (1977). Isolation and properties of teleost prolactin. Gen. Camp. Endocrinol. 31, 60-71. Hayashida, T. (1973). Immunology of prolactin. In “Peptide Hormones,” Vol. 2A: “Methods in Investigative and Diagnostic Endocrinology” (S. A. Berson and R. S. Yalow, eds.), pp. 577-579. American Elsevier, New York. Idler, D. R., Bazar, L. S., and Hwang, S. J. (1975). Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography. Endocrine Res. Commun.
2, 215-235.
SOMATOTROPIN Idler,
IN
D. R., Shamsuzzaman. K., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Gen. Comp. Endocrinol. 35, 409-418. Ingleton, P.-M., and Stribley, M.-F. (1977). Immunofluorescent identification of the cells of origin of eel (Anguillu unguillu) pituitary hormones separated by polyacrylamide gel electrophoresis. Gen. Comp. Endocrinol. 31. 37-44. Komourdjian, M. P., Saunders, R. L., and Fenwick, J. C. (1976a). The effect of porcine somatotropin on growth, and survival in seawater of Atlantic salmon. (Saln~o scrlur) Parr. Cunud. J. Zoo/. 54, 531-535. Komourdjian, M. P., Saunders. R. L.. and Fenwick. J. C. (1976b). Evidence for the role of growth hormone as a part of a ‘light-pituitary axis’ in growth and smoltification of Atlantic salmon (Salvo suIur). Cunud. 3. Zool. 54, 544-551. Komourdjian, M.-P., and Idler, D.-R. (1977). of rainbow trout, Sulmo Hypophysectomy ,guirdneri. and its effect on plasmatic sodium regulation. Gcn. Comp. Endocrinol. 32, 536-542. Komourdjian. M. P., Burton. M. P., and Idler, D. R.
SALMON
PITUITARY
349
( 1978). Growth of rainbow trout, Sulmo yuirdnrri, after hypophysectomy and somatotropin therapy. Gen. Comp. Endocrinol. 34, 158- 162. McKeown, B. A., and VanOverbeeke, A. P. (1971). Immunohistochemical identification of pituitary hormone producing cells in the sockeye salmon (Oncorhynchus nrrku, Walbaum). Z. Zellfbrsch. 112. 350-362. Olivereau, M. (1954). Hypophyse et glande thyrdide chez les poissons. &de histophysiologique de quelques corr&lations endocriniennes en particulier chez Sulmo sulur L. Ann. Inst. OcPunogr. Puris 29, 95-296. Olivereau, M. (1969). Functional cytology of prolactin-secreting cells. Gen. Camp. Endocrinol. Suppl. 2, 32-41. Olivereau, M., and Ridgway. G. J. (1962). Cytologie hypophysaire et antigen sirique en relation avec la maturation sexuelle chez Oncorhynchus species. Compt. Rend. H. 254, 753-755. Swift, D. R. (1954). Influence of mammalian growth hormone on rate of growth of fish. Nuture (London) 173, 1096.
AND
COMPARATIVE
ENDOCRINOLOGY
37, 343-349
(1979)
Chum Salmon Pituitary Fractions: Somatotropic Activity and Cytoimmunofluorescence Studies’ M. P. KOMOURDJIAN~
Accepted
AND D. R. IDLER
December
20, 1978
Highly purified chum salmon pituitary fractions were bioassayed for their ability to stimulate elongation in hypophysectomized rainbow trout, Salnzo gcrirdnwi. One fraction, previously identified as the Na-retaining principle, prolactin, was not active. Of the active fractions, only the most potent one elicited a dose response. An antiserum against this fraction permitted the specific labeling of somatotropic cells in pituitary sections by an indirect immunocytofluorescence procedure. An antiserum against the Na-retaining principle led to the specific fluorescence of the follicular “prolactin cells.” However, neither antiserum appeared to discriminate between acidophile type in pituitary sections from frogs or rats.
A role for somatotropin in growth promotion in Salmo species is well established (Swift, 1954; Komourdjian, 1976a,b). Hypophysectomy in salmonids interrupts normal growth which is reestablished by somatotropin therapy. This suggests the presence of a somatotropic hormone in salmonid pituitaries and constitutes the basis for a sensitive and reliable assay for somatotropic activity in purified pituitary material (Komourdjian ef al., 1978). While. hitherto, most studies were conducted using mammalian hormones, advances have now been described in the isolation of teleostean pituitary hormones (Farmer et rrl., 1975, 1976). Recently, Idler c’t cl/. (1978) reported the is,olation of highly purified pituitary fractions from chum salmon, O~~C~&V~C~~~IS dicta. One of these fractions possessed potent Na-retaining activity, a characteristic property of teleost prolactin-like principles. ' M.S.R.L.
Contribution No. 328. z Present address: Department of Mathematics Natural Sciences, College of Cape Breton. P.O. 760. Sydney. Nova Scotia. BlP 63 I. Canada.
and Box
The present presence of fractions. fluorescence of the main ing fractions.
study seeks to determine the somatotropic activity in these Additionally, immunocytois used to assess the specificity somatotropic and Na-retain-
MATERlALS
AND
METHODS
Homrone frurtions. The purification and some properties of these preparations have been described in detail elsewhere (Idler PI al., 1978). Briefly, the non-glycoprotein fraction Con A- I (Idler et al., 1975) was obtained from whole frozen chum salmon, 0. krta, pituitary homogenate by affinity chromatography. This was further fractionated by gel filtration and ion exchange. Fraction I was the unadsorbed fraction from DEAE eluted in 0.005 M ammonium bicarbonate at pH 9.0. Fractions 2.3. and 4, the adsorbed fractions, were eluted at ammonium bicarbonate tpH 9.0) concentrations of 0.016 to 0.024, 0.024 to 0.04, and 0.04 to 0.084 M respectively. Anribodies. Antisera were raised to the fractions with highest sodium-retaining and somatotropic activities, i.e., 1 and 2 respectively. Male NZW rabbits, approximately 2.2 kg in weight. received Freund’s complete adjuvant. emulsified with I25 kg of antigen, intradermally. and intramuscular boosters of the suspension containing 238 pg of antigen 2 months thereafter. Each time, 0.1 ml of pertussis vaccine was in-
343 0016~6480/79/020343-07$01.00/O Copyright Q 1979 by Academic Press. Inc. All right!, of reproduction in any form recerved.
344
KOMOURDJIAN
jetted subcutaneously. Animals were bled 1 month after the final injection. Fluorescein labeled goat anti-rabbit y globulin was purchased from Antibodies Incorporated (Davis, Calif.). Immrfnocvrnfoyy. Pituitaries from 22-cm male rainbow trout, Sulmo guirdneri. were fixed in ice-cold Bouin’s formol-picric-acetic acid fixative. After 24 hr. tissues were dehydrated, embedded, and sectioned at 7-8 pm. Sections were rapidly hydrated and transferred to phosphate buffer. pH 7.4, for 10 min. O-rings were then placed over the sections and filled with antiserum. After 90 mitt, slides were washed in several changes of buffer. The labeled monospecific antibody, diluted I: IO with buffer, was then applied to the sections for I hr. Slides were washed in buffer and mounted using a 3:1 glycerol:phosphate buffer mixture. Sections of pituitary tissue from 350-g male Sprague-Dawley and 50-g frogs, Ronu pipirns. were processed similarly. In control sections normal rabbit serum replaced antiserum to fractions I and 2, or alternatively, the labeled antibody was omitted. Additionally, the specificity of each antiserum was studied by adsorbing it with the corresponding antigen prior to immunofluorescent labeling. In all cases serial sections were stained with periodic acid-Schiff ‘s-hematoxylin-orange Cl (Komourdjian et ai., 1976b) or using the Cleveland-Wolfe technique. Biouusuy for somutrotropic uctir,iry. Rainbow trout, S. guirdneri. were obtained from Goossens trout farm, Otterville, Ontario. Animals, I5 to I9 cm in length, were hypexed according to the procedure of Komourdjian and Idler (1977) and held under the conditions described in that study. Briefly, two tanks were subdivided to accommodate four groups. Dilute seawater (I I oioo salinity) was recirculated and cooled to 11°C. An 8 hr photophase was maintained throughout the study. Fish were fed to satiation three times daily with Ewos 5P trout pellets. Two months after hypophysectomy biweekly morphometric determinations and injections were commenced. Each time, all fish were weighed to the nearest decigram, their fork lengths measured to the nearest millimeter. and injected intraperitoneally with 0.2 ml of IO mM CAPS (cyclohexylaminopropanesulfonic acid) buffer. adjusted to pH 9.5 with NaOH. After 2 weeks each group received a different DEAE hormone test fraction. Each fish was injected with 25 pg of the particular preparation dissolved in 0.2 ml CAPS buffer; this treatment was continued for 3 weeks. The dose was then doubled for the groups which responded positively. in order to investigate the existence of a dose-effect response. Sufficient fraction 4 material was not available. The pertinent group and that receiving fraction I were injected with porcine somatotropin to assess whether the fish were capable of showing higher growth rates.
AND
IDLER
Instantaneous linear growth rates were computed according to the equation 100 (log,Y, - log,Y,)/(T-t) where Y, and Y, are mean lengths at times T and t (Brown, 1957). Results were examined by analyses of variance and Duncan’s multiple range test.
RESULTS
trout All groups of hypophysectomized showed specific linear growth rates of 0.07% or less per day during the ICday control period when they received injections of CAPS buffer. Injections of 25pg doses of the purified peptide preparations stimulated growth in three of the four groups (Fig. 1). Fraction 2 produced the largest stimulation followed by fractions 3 and 4 respectively: fraction I (prolactin) did not stimulate growth. Doubling the dose to 50 pg produced a significant (p < 0.001) increase in growth with fraction 2 but not with fraction 3. Injections of porcine STH in groups II and IV elicited a significantly higher growth rate than the basal (CAPS) rate. The growth rate of all groups gradually approached initial levels when the fish were again injected with CAPS. Fluorescence in trout pituitary sections was localized in the erythrosinophilic cells of the rostra1 pars distalis (prolactin cells) using antiserum to fraction 1 (Fig. 2) and in the orangeophiles (somatotrops) of the proximal pars distalis with antiserum to fraction 2 (Fig. 3). The fluorescence in the follicular cells tended to be concentrated in the apical region but in the proximal pars orangeophiles it was more evenly distributed. Similar results were obtained with pituitary sections from Atlantic salmon. However, either antiserum appeared to elicit fluorescence in both types of rat pituitary acidophiles. Similarly, no discrimination was evident in the labeling of the amphibian tissues. None of the control sections revealed any fluorescence except for the native fluorescence found in the teleostean saccus vasculosus.
SOMATOTROPIN
1N
SALMON
345
PITUITARY
0.45
z 0.40 8 & Go.35 L m
I
go30 IL
0
I (7)
GROUP GROUPII
gj
(6)
GROUP
ILK
(6)
GROUP
I!Z
(5)
F
3
o-25
22 (3
0 rTT
3 020 z
i
2
O-15
8 $
O-IO
5 k-J *o-o5
III1 DAYS
l-14
lcJ “1 I
14-35
35-46
46-67
-
67-92
FIG. I. Instantaneous linear growth rates (mean 2 SEM) of hypophysectomized trout injected intraperitoneally with chromatographic fractions from chum salmon pituitaries (F) or porcine somatotropin (pSTH). Identical superscripts denote significant differences (p < 0.01); asterisk indicates no significant difference compared to initial (basal) rate during CAPS treatment.
DISCUSSION
Our results confirm the presence of a potent growth promoting principle, or somatotropin, in pituitaries of chum salmon, and verify earlier identifications of the cells of origin of the principle (see Introduction). The principle appeared to be concentrated in the fraction eluted at ammonium bicarbonate concentrations of 0.024 to 0.04 M. A study on the somatotropic activity of Tilapia pituitary extracts in sockeye salnerka. (Clarke et al., mon, Oncorhynchus 1977) suggests that the chum salmon principle in the present study had higher activity than the former; nevertheless, the differences between the two studies defy a strict comparison. However, the salmonid prepa-
ration is likely of the order of two magnitudes more active than the porcine somatotropin used in our studies (also Komourdjian and Idler, 1978). The determination of reliable dose-response curves must now await the isolation of larger amounts of hormone. The significance of the activity found in two of the other fractions is not clear although the presence of contamination in the case of fraction 3 and alternate hormonal forms (fraction 4) might be subjects of speculation. Current studies have shown some immunoreactive material in fractions 3 and 4, but none in fraction 1, by radioimmunological methods using an antiserum to the more active fraction 2 (Komourdjian and Idler, unpublished).
346
KOMOURDJIAN
FIG. 2. lar prolactin
Sagittal cells.
section 160x.
of S. ~aird~c~i
pituitary
AND
IDLER
gland
showing
fluorescent
staining
of the follicu-
SOMATOTROPIN
IN
SALMON
PITUITARY
347
348
KOMOURDJlAN
The specific localization of fluorescence in our study, by an approach considered to be highly sensitive (Hayashida, 1973), suggests that the antigenic portions of the two native salmon hormones, somatotropin and prolactin, are probably structurally distinct. This is an encouraging finding towards the development of a specific radioimmunoassay for these principles. This specificity is of special interest following reports of structural similarities between prolactin and somatotropin from Tilapia (Farmer rf (II., 1977; Clarke et al., 1977). Some overlap may exist in the biological activities of the salmonid hormones. At a very high dose, chum salmon somatotropin mimicked Na-retaining activity (Idler et al., 1978). Yet, the current study did not reveal any somatotropic activity in the chum pituitary fraction identifiable as proiactin. This suggests that teleostean prolactin and somatotropin are distinguishable as had earlier been suggested by Ball (1965). This is the first report of immunofluorescent labeling of rainbow trout pituitary cells with antisera to salmonid hormones. Our work is in agreement with earlier studies which had characterized the somatotropic cells based on their tinctorial characteristics and on histophysiological evidence, mainly a correlation between cellular hypertrophy and an increased growth rate of the animal (Olivereau, 1954; Olivereau and Ridgway, 1962; Komourdjian et ml., 1976b). Additional evidence had been provided by immunohistochemical studies antigens using heterologous (ovine) (McKeown and VanOverbeeke, 1971), although the reliability of such approaches has been questioned (Ingleton and Stribley, 1977). The evidence for the cellular origin of prolactin in salmonids derives from similar histophysiological (Olivereau, 1969) and immunohistochemical studies (Aler, 197 1; McKeown and VanOverbeeke, 1971). Studies on eel, Anguilla anguilla pituitaries using conspecific antisera (Ingleton and
AND
IDLER
Stribley, 1977) have also been valuable since these glands and those of salmonids share fundamental morphological features. Our study extends these findings to positively relate the Na-retaining principle of salmonids (Idler ef al., 1978) to their cells of origin. The absence of fluorescence within the lumen of each follicle of the rostra1 pars distalis supports the idea that the intraluminar material is not identifiable with prolactin. The origin of this material is not yet understood. REFERENCES Aler, G. (1971). The study of prolactin in the pituitary gland of the Atlantic eel (Anguilla anguilla) and the Atlantic salmon (Salmo s&r) by immunofluorescent technique. Actu Zool. (Stockholm)
52, 145-156.
Ball, .I. N. (1965). Partial hypophysectomy in the teleost Poecilia: Separate identities of teleostean growth hormone and teleostean prolactin-like hormone. Gen. Camp. Endocrinol. 5, 654-661. Brown, M. E. (1957). Experimental studies on growth in “Physiology of Fishes” (M. E. Brown, ed.), Vol. I, pp. 361-400. Academic Press, New York. Clarke. W. C., Farmer, S. W., and Hartwell, K. M. ( 1977). Effect of teleost pituitary growth hormone on growth of Tilupia mossambicu and on growth and seawater adaptation of sockeye salmon (Uncorhynchus
nerku).
Gen.
Cump.
Endocrinol.
33.
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