GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

77,

Salmonid I. Distinct MASUMI

Cellular

NOZAKI,* YASUMITSU

NOBUKO

(1990)

Pituitary Gonadotrophs

Distributions

NAKAI,t

348-357

of Two Gonadotropins,

NArTo,t PENNY SWANSON,@ YOSHIHIKO OOTA,~ KUNIMASA HIROSHI KAWAUCHI~

GTH I and GTH II KATSUYUKI SUZUKI.$

MIYATA$ AND

*Primate Research Institute, K?;oto University. lnuyumu, Aichi 484. Jupun: fDepartmrnt of Anutomy, School of Medicine, Showu University, Shinagauw. Tokyo. Jupan; $School of Fisheries, Kitusato University, Sunriku, Iwate Japan; und #Department of Biology. Fucultv of Science, Shizuoka University. Oo?u. Shizuoku, Jupun

Accepted

May

9. 1989

Using antisera specific for the 0 subunits of two distinct coho salmon gonadotropins, GTH I and GTH II. an immunocytochemical study of rainbow trout and Atlantic salmon pituitaries was done. Cells which immunostained with anti-GTH I p were distributed in the periphery of the glandular cords of the proximal pars distalis (PPD), in close association with somatotrophs. On the other hand, cells immunostained with anti-GTH 11 p were located in the central parts of the glandular cords of the PPD. Neither the GTH I-producing nor the GTH II-producing cells stained with antisera against chum salmon growth hormone or the p subunit of human thyroid-stimulating hormone. Moreover, GTH I and GTH II were localized in distinctly different cells. In no case was colocalization of these GTHs in the same cell observed. Finally. it was concluded that classification of GTH cells as globular and vesicular forms does not reflect the type of hormone produced by the cell, but may reflect ‘c IYW k&mlc PE\S. IX differences in the physiological conditions of the cells.

Although many immunocytochemical and ultrastructural studies have been made of teleost pituitary gonadotrophs (for review, Van Oordt and Peute, 1983), the existence of one or two gonadotropin (GTH) cell types has been controversial. Since such morphological characteristics of the cells as the presence of granular or globular cytoplasmic inclusions have been used to classify gonadotrophs, the controversy has centered on whether two gonadotroph cell types are cells at different stages of synthetic activity (e.g., Ekengren et al., 1978; Peute et al., 1978) or cells which have distinctly different hormonal products (e.g., Olivereau, 1976; Ueda and Hirashima, 1979). This controversy has been unresolved since it has not been clear whether I TO whom reprint requests address: School of Fisheries, ton, Seattle, WA 98195.

the teleost pituitary produces one or more chemically distinct GTH molecules. Most investigators have isolated only a single GTH designated maturational GTH (Burzawa-Gerard, 1982) which is homologous to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) of tetrapods. Conversely, a second GTH. vitellogenie GTH. which does not appear to show chemical homology to LH or FSH has been isolated from several species of teleosts (see review, Idler and Ng, 1983). More recently. the definitive identification of two chemically distinct GTHs, designated GTH I and GTH II, has been provided by Kawauchi and associates (chum salmon: Kawauchi et al., 1987; Suzuki et (11.. 1988a, b, c, d; Itoh et al., 1988; and coho salmon: Swanson et al., 1987). Although both GTH I and GTH II exhibit similar steroidogenic activities, plasma and pituitary

should be sent. Present University of Washing-

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TWO

GTH

CELLS

levels of these two GTHs vary markedly during reproductive development (Suzuki ef al., 1988c, d). Furthermore, sequence studies have shown that the p subunit of chum salmon GTH II has only 31% sequence identity to GTH I (Kawauchi et al., 1987; Itoh et al., 1988) and that it appears to be almost identical to the sequence of chinook salmon maturational GTH p subunit reported by Trinh et al. (1986). Neither GTH I nor GTH II bear chemical similarity to the vitellogenic GTH isolated from several species of teleosts by Idler and associates (see Idler and Ng, 1983). In the present study, antibodies directed against the p subunits of coho salmon (Oncorhychus kisutch) GTH I and GTH II were used to determine whether these two chemically distinct GTHs are produced in the same or different pituitary cell types. A longitudinal study of the pituitary gonadotrophs during reproductive development in rainbow trout (Salmo gairdneri) is reported in the accompanying paper (Nozaki et al., 1990). MATERIALS

AND METHODS

Forty rainbow trout, Sdmo gclirdneri irideus, (body weights ranging from 85 g to 3.0 kg) of both sexes were obtained from a hatchery near Inuyama, Aichi, Japan, during the period from April 1986 to August 1987. Pituitaries from four sexually mature male Atlantic salmon (S. sahr) maintained in salt water were kindly collected by Dr. E. M. Plisetskaya, University of Washington, Seattle, Washington. during October 1986. The pituitary gland, with or without the attached brain, was removed immediately after decapitation and fixed in Bouin-Hollande sublimate for about 12 hr. Pituitaries were dehydrated through a series of increasing concentrations of ethanol. After 90% ethanol, the glands were washed in a solution containing iodine-potassium iodide in 90% ethanol for 24 hr to remove deposits of mercuric chloride. Pituitaries were embedded in Paraplast, and serial sections (4 to 6 pm) were mounted on glass slides. The rabbit anti-coho salmon GTH I p subunit (Lot No. 8622) and anti-coho salmon GTH 11 p subunit (Lot No, 8624) were raised by P. Swanson (Swanson et al.. 1987). Anti-human thyroid-stimulating hormone

IN

349

SALMONIDS

(hTSH) p subunit (Lot No. AFP-62323473) was obtained from Dr. S. Raiti, National Pituitary Agency, Baltimore, Maryland, and anti-chum salmon growth hormone (GH) was obtained from Dr. H. Kawauchi (Bolton er al., 1986). lmmunocytochemical staining was generally performed with a Vectastain ABC (avidin-biotin peroxidase complex) kit, but some sections were stained with a Vectastain ABC-AP (avidin-biotin alkaline phosphatase) kit. The staining procedures have been described elsewhere (Nozaki et al., 1988). To confirm the specificity of the immunostaining the following control stains were done: (1) replacement of primary antibody with normal rabbit serum (NRS), (2) omission of primary antibody, and (3) absorption of primary antibodies with purified coho GTH I p or GTH II p or purified u subunits of GTH I and II which were isolated by P. Swanson (Swanson et al., 1987).

RESULTS

Occurrence and Specificity of the Immunoreaction Typical examples of the immunoreactions which were obtained with respective antibodies in the rainbow trout pituitary are shown in Figs. la-ld. Four distinct cell types containing GTH I, GTH II, GH, or TSH could be demonstrated in the proximal pars distalis (PPD). Anti-GTH I p and antiGTH II p immunostained clearly distinct cells of the PPD in both the rainbow trout and Atlantic salmon pituitary (cf. Figs. la1b). The preabsorption experiments, however, failed to show the difference of the tissue affinities between anti-GTH I p and anti-GTH II p: preabsorption of each antiserum with either GTH I p or GTH II p equally abolished positive staining (Table I). In order to examine possible contamination of GTH I p in GTH 11 p, or vice versa, the minimum amount of protein needed for the complete elimination of positive staining was tested for each antiserum. However, no clear difference was observed in the neutral points between the preabsorption with GTH I p and that with GTH II p for either antiserum (Table 1). In contrast, preabsorption of the antisera with (Ysubunit

NOZAKI

ET

AL

FIG. 1. Four successive sagittal sections (a-d) through the dorsal proximal pars distalis (PPD) of a spermatogenie rainbow trout stained with (a) anti-salmon GTH I 6: (b) anti-salmon GTH II p; (c) anti-human TSH p; and (d) anti-salmon GH. Note that each antiserum stains distinctly different cell-types in the PPD. (a-d) x330.

preparations had a minimal effect on the immunoreaction (Table 1). Positive staining with anti-hTSH p was not eliminated by preabsorption with either GTH I or II p (Table 1).

Rainbow

Trout

The following descriptions were based on the results obtained with the vitellogenic and spermatogenic trout (300-600 g body

TWO

GTH

CELLS

IN

TABLE EFFECTS

OF PREABSOIWTION

OF ANTIBODIES

Proteins used for preabsorption

Antibodies Anti-GTH

I B

Anti-GTH

II B

Anti-hTSH

B

GTHIB GTH II B GTH a GTH I B GTH II B GTH (Y GTH I B GTH II B GTH a

WITH

351

SALMONIDS

1

CORRESPONDING

ANTIGENS

AND

RELATED

PROTEINS

Stain intensities after preabsorption (kg protein/O. 1 ml antisera at working dilution) 25

5

1

0.2

0.04

0.008

0.0016

0" 0 2 0 0 1 3 4 4

0 0 4 0 0 2 4

0 0 4 0 0 4

2 1

4 2

4 4

4 4

0 0

0 0

2 0

4 2

U 0, completely eliminated immunoreaction; I, largely reduced immunoreaction; munoreaction; 3, slightly reduced immunoreaction; 4, no reduced immunoreaction.

wt), but comparable results were also obtained in both juvenile and fully matured fish. (I) GTH I cells. Cells designated GTH I cells were stained specifically with antiGTH I l3 antiserum. These cells showed no immunoreaction to antisera against GTH II p, TSH l3, or GH (Fig. 1). GTH I cells were distributed mainly in the periphery of the glandular cords of the PPD in close association with the neurohypophysis (Figs. la and 2a). This topographic orientation of GTH I cells was typically observed in the dorsal PPD rather than the ventral PPD. GTH I cells were round in shape, similar to GH cells in size, and appeared to be more numerous in the dorsal PPD (Fig. 1). GTH I cells had a similar distribution to GH cells in the dorsal PPD and often encircled the GH cells (Fig. 3a). Some GTH I cells were found to invade the rostra1 pars distalis (RPD). However, GTH I cells were unstained with periodic acid-Shiff s reagent (PAS) or aldehyde fuchsin (AF). They were generally granulated in appearance. Vacuolated GTH I cells with vacant cytoplasmic inclusions were rarely observed in pituitaries from fish in prespawning stages or from recently spawned females (Fig. 2a). (2) GTH ZZ cells. Cells which were stained specifically with anti-GTH II l3 an-

2, moderately reduced im-

tiserum were designated GTH II cells. These cells were not stained with anti-GH, anti-GTH I l3, or anti-hTSH l3 antisera (Fig. 1). GTH II cells were generally elongated in shape and were located mainly in the central parts of the glandular cords of the PPD (Figs. lb and 2b). Cytoplasmic processes of GTH II cells protruded to the basal lamina, and the endings of these processes were often enlarged (Figs. lb and 2b). The GTH II cells appeared to be more numerous in the ventral PPD, although some cells invaded the RPD. GTH II cells were stained intensely with PAS and AF. In all pituitaries collected throughout the reproductive cycle, GTH II cells appeared granulated. (3) TSH cells. Cells designated TSH cells were stained intensely with anti-hTSH p, but showed no immunoreactivity to antiGTH I p, anti-GTH II l3, or anti-GH (see Fig. 1). TSH cells were distributed in the most rostra1 parts of the PPD close to the RPD (Fig. lc). They were round in shape and often encircled the blood sinus. TSH cells were stained intensely with both PAS and AF. (4) GH cells. GH cells were immunostained with anti-salmon GH antisera and were distributed in the periphery of glandular cords of the PPD in close association with the neurohypophysis (Fig. Id). They

352

NOZAKI

ET AL.

FIG. 2. Two successive sagittal sections (a and b) through the dorsal proximal pars distalis (PPD) vitellogenic rainbow trout stained with (a) anti-salmon GTH 1 p and (b) anti-salmon GTH II p. In (a). vacuolated cells (arrow) together with granulated cells (:arrowhead), whereas in (b), note granulated appeal of the GTH II cells. Also note the difference between G TH 1 and GTH II cells in their topographic location shape. (a,b) x330. FIG. 3. Two successive sagittal sections (a and b) through the dorsal proximal pars distalis (PPD) oi a spermatogenic rainbow trout stained with (a) anti-salmu In GTH I p and (b) anti-salmon GH. Note several CiTH I cells (arrows in a), each of which encircles GH cells ( arrows in b). (a,b) x470.

were ovoid in shape and were stained intensely with Orange G. None of the antibodies against salmon GTHs or hTSH 6 showed positive reaction to GH cells (see Fig. 1). GH cells and GTH I cells shared the same peripheral regions of the PPD and

were intimately (Fig. 3b).

associated with each o ther

Atlantic. Salmon

The results obtained

with the Atla .ntic

TWO

GTH

CELLS

salmon pituitary were similar to those of the rainbow trout with respect to tissue affinities for the antisera used in this study (Figs. 4a-4g). However, some differences in topographical location and appearance of the cells are described below. (I) GTH I cells. GTH I cells were distributed mainly in the dorsomedial part of the PPD close to the neurohypophysial stalk (Fig. 4a). GTH I cells were generally round or ovoid in shape and larger than any other cell types in the PPD (Fig. 4d). In contrast to the rainbow trout, GTH I cells had a basophilic element which was faintly stained with PAS, AF, and aniline blue. Additionally, unlike the trout, most of the Atlantic salmon GTH I cells appeared vacuolated (Fig. 4d). (2) GTH II crlls. GTH II cells were distributed mainly in the periphery of the PPD, and some cells invaded the RPD and pars intermedia (Fig. 4b). They were more numerous in the rostra1 region of the PPD. In the Atlantic salmon, GTH I and GTH II cells had different regional distributions in the PPD, but both types of cells intermingled near their regional boundaries (Figs. 4a and 4b). GTH II cells were generally midsized, but their shape varied from ovoid to slender depending on the location (Figs. 4e and 4f). GTH II cells were comparable to GTH I cells in number. Most GTH II cells which were located in the caudal region of the PPD were granulated, while those in the rostra1 PPD exhibited degranulated features (Figs. 4e and 4f). PAS, AF, and aniline blue intensely stained ail GTH II cells. (3) T,SH cells. TSH cells were distributed along the boundaries between the RPD and PPD (Fig. 4~). Most TSH cells were found in the most caudal parts of the RPD, but some were in the most rostra1 parts of the PPD. The topographic distribution of TSH cells was clearly different from the two gonadotroph cell types (Fig. 4~). TSH cells were generally ovoid in shape and similar to GTH II cells in size. They were relatively

IN

353

SALMONIDS

few in number and were stained intensely with PAS, AF, and aniline blue. DISCUSSION

The present study clearly shows that GTH I and GTH II are produced in distinctly different cells of the pituitary in both rainbow trout and Atlantic salmon. Although the preabsorption tests failed to show a difference in the tissue affinities between anti-GTH I p and anti-GTH II p, the precise reason for this is not known. Perhaps the three-dimensional structures of the two GTH ps show similarity despite only 31% amino acid sequence identity (Itoh et al., 1988). At the very least, it does not seem to be due to the contamination of GTH 1 p in GTH II p, or vice versa, since there was no clear difference between the two GTH preparations in the neutral points of the preabsorption tests. Moreover, the purity of these preparations was verified by reverse-phase high-performance liquid chromatography and each protein had a different single N-terminal amino acid residue (Swanson et al., 1987). GTH II cells, which are demonstrated by specific staining with anti-GTH II p, are typical basophils. No doubt, the GTH II cells correspond to the gonadotrophs of many previous studies (see Van Oordt and Peute, 1983; Olivereau and Nagahama, 1983; Farbridge and Leatherland, 1986). The other basophilic cells, which are located in the rostra1 region of the PPD, are highly immunoreactive to anti-hTSH p. Again, these cells clearly correspond to the TSH cells which have been reported in a number of teleostean species, including salmonids (Margolis-Kazan and Schreibman, 1981; Fridberg et al., 1981; Farbridge and Leatherland, 1986). In contrast to the GTH II and TSH cells, GTH I cells are most likely to have been described in several different ways, depending on the investigators approach and/or reproductive status of the fish.

FIG. 4. Three successive sagittal sections (a-c) through the pituitary of a reproductively mature male Atlantic salmon stained with (a) anti-salmon GTH I p; (b) anti-salmon GTH II p; and (c) anti-human TSH p. The areas in (a) and (b) outlined by rectangles are enlarged and shown in (d), (e), and (I). A serial section of area (e) which was stained with anti-chum salmon GH is shown in (g). Note four distinct cell types, each containing different hormones in the Atlantic salmon PPD (GTH I cells: a and d; GTH II cells: b, e, and f; TSH cells: c; GH cells: g). (a-c) x23; (d, e, and g) x200; (0 x380.

TWO

GTH

CELLS

Under light microscopy. Olivereau described two different types of putative GTH cells in several teleost species, including salmonids (Olivereau, 1976, 1977, 1978; Olivereau and Nagahama, 1983). In salmonids, the first type of putative GTH cell is typically basophilic and immunoreactive to GTH antisera, whereas the second type of putative GTH cell is largely chromophobic and stained dubiously with GTH antisera. Olivereau (1976) further reported that the first type of gonadotroph in the Atlantic salmon showed a gradual degranulation during gonadal maturation, while the second type showed a progressive vacuolization. The largely chromophobic cells described by Olivereau were also observed by Boddingius (1975), Ekengren et al. (1978), and Leatherland and Sonstegard (1980), but these authors did not recognize the possible gonadotropic function of these cells. The description of the first and second types of putative gonadotrophs by Olivereau generally conforms to that of the GTH II and GTH I cells, respectively, in mature salmonids. However, in reproductively immature rainbow trout, GTH I cells appear to correspond to the first type of GTH cell described by Olivereau (see below). Using electron microscopy, investigators have described two different gonadotrophs which have frequently been termed globular and vesicular (cisternal) forms (see Van Oordt and Peute, 1983). Some investigators claim that these two cell forms represent two different GTH cell types (e.g., Ueda and Hirashima, 1979; Ueda, 1980), whereas most other authors have considered that there is only one type of GTH cell whose ultrastructure is globular or vesicular, depending on the reproductive phase (Ekengren et al., 1978; Peute et al., 1978, 1980; van Putten et al., 1981). Since an ultrastructural study of GTH I and GTH II cells was not done, it is not possible to strictly classify these gonadotrophs as globular or vesicular. An electron microscopic study of

IN

SALMONIDS

355

GTH 1 and GTH II cells is now in progress (Naito et al., unpublished observations). In the accompanying paper, we demonstrate that in the rainbow trout, GTH I cells are present and appear to be active prior to the onset of spermatogenesis and vitellogenesis, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis (Nozaki et al., 1990). Olivereau (1976) and Ueda (1980) reported that in juvenile fish with immature gonads only globular cells were detectable. Therefore, it is likely that the globular cells in the immature fish described by these authors are GTH I cells. In contrast, the globular gonadotrophs in mature fish resemble GTH II cells. Thus, it is apparent that classitication of GTH cells into so-called globular and vesicular cells does not seem to reflect the difference between GTH I and GTH II cells at all stages of reproduction. The presence of GTH I and GTH II in clearly distinct cells within the pituitary of salmonids was surprising in view of the reports of coexistence of LH and FSH in the same cells in several species of tetrapod vertebrates (human: Phifer et al., 1973; monkey: Herbert, 1976; rat: Dada et al., 1983; Japanese quail: Mikami, 1986; frog: Gracia-Navarro and Licht, 1987). In no instance did we observe localization of GTH I and GTH II in the same cell in the salmonid pituitary. Nevertheless, a more detailed study of GTH I and GTH II cells at the electron microscopic level is necessary to confirm these results. Apart from the recent isolation of salmon GTH I and GTH II by Kawauchi and associates (see Kawauchi et al., 1987; Suzuki et al., 1988a, b; Itoh et al., 1988; Swanson et al., 1987), only Idler and associates (see Idler and Ng, 1983) have been successful in isolating two different fractions with different gonadotropic potencies. These were designated vitellogenic (Con AI) GTH and maturational (Con AII) GTH. However, neither salmon GTH I nor GTH II bear

356

NOZAKI

chemical similarity to vitellogenic GTH (Suziki et al., 1988a, b; Itoh et al., 1988). It is difficult to reconcile our immunocytochemical study with that reported by Burton et al. (1981) who used antibodies to Con AI GTH and Con AI1 GTH, as well as antibodies to Con AI TSH and Con AI1 TSH. They reported the general topographic distributions of immunoreactive cells to respective antibodies in the winter flounder pituitary, but did not study whether both Con AI GTH and Con AI1 GTH or Con AI TSH and Con AI1 TSH were present in the same or distinct cells. In conclusion, the present study clearly shows that there are two distinctly different types of GTH cells each producing different GTHs. Furthermore, our study shows that classification of GTH cells into globular and vesicular forms does not reflect the type of hormone produced by the cells, but may reflect the differences in physiological condition of each cell. The results of this study provide additional evidence for duality of teleost GTHs. Although the biological significance of these two GTHs is not clearly understood, a study of GTH I and GTH II cells during various stages of reproductive development sheds some light on this problem (see accompanying paper. Nozaki et al., 1990). ACKNOWLEDGMENTS Support for this study was provided by a fellowship to P.S. from the Japanese Society for Promotion of Science for research in Japan and by a grant-in-aid to H.K. from the Japanese Ministry of Education, Science, and Culture. We express our sincere gratitude to Dr. W. W. Dickhoff for his critical reading and comments of this manuscript and to Dr. E. Plisetskya for collection of Atlantic salmon pituitaries.

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ET AL,. J., and Hirano, T. (1986). Development and validation of a salmon growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 62, 230-238. Burton, M. P., Idler, D. R., and Ng, T. B. (1981). The immunofluorescent localization of teleost gonadotropins and thyrotropins in flounder pituitary. Gen. Comp. Endocrinol. 43, 13.5-147. Burzawa-Gerard, E. (1982). Chemical data on pituitary gonadotropins and their implication to evolution. Cunud. J. Fish. Aquat. Sci. 39, 80-91, Dada, M. 0.. Campbell. G. T.. and Blake, C. 4. (1983). A quantitative immunocytochemical study of the luteinizing hormone and follicle-stimulating hormone cells in the adenohypophysis of adult male rats and adult female rats throughout the estrous cycle. Endocrinology 113, 970-984. Ekengren. B., Peute, J., and Fridberg, G. (1978). Gonadotropic cells in the Atlantic salmon. Srrlmo srr/cru: An experimental, immunocytological. and electron microscopical study. Cell Ti.r.sur Rc7.t. 191, 187-203. Farbridge, K. J., and Leatherland, .I. F. (1986). A comparative immunohistochemical study of the pars distalis in six species of teleost fishes. Fish Physiol. Biochem. 1, 63-74. Fridberg. CT.. Lindahl, K., and Ekengren, B. (I981 I. The thyrotropic cells in the Atlantic salmon. Salmo s&r. Actu Zool. 62, 43-5 I. Gracia-Navarro. F.. and Licht, P. (1987). Subcellular localization of gonadotropic hormones LH and FSH in frog adenohypophysis using doublestaining immunocytochemistry. J. Hisroc-hem. Cytochem.

35, 763-769.

Herbert. D. C. (1976). Immunocytochemical evidence that luteinizing hormone (LH) and folliclestimulating hormone (FSH) are present in the same cell type in the Rhesus monkey pituitary gland. Endocrinology 98, 1554-1557. Idler. D. R.. and Ng, T. B. (1983). Teleost gonadotropins: Isolation, biochemistry and function. In “Fish Physiology” (W. S. Hoar. D. J. Randall, and E. M. Donaldson. Eds.). Vol. 9A. pp. 1X7-221. Academic Press, New York. ltoh. H.. Suzuki. K., and Kawauchi. H. (1988). The complete amino acid sequences of beta subunits of two distinct chum salmon gonadotropins Gen. Camp.

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71, 44384.51.

Kawauchi, H., Suzuki, K., Itoh, H.. Swanson. P.. and Nagahama, Y. (1987). Duality of salmon pituitary gonadotropins. In “Proc. 1st Congr. Asia and Oceania Sot. Comp. Endocrinol.. Nagoya, November 4-7, 1987” (E. Ohnishi. Y. Nagahama, and H. Ishizaki, Eds), pp. 15-18. Leatherland, J. F,, and Sonstegard, R. A. (1980). Seasonal changes in thyroid hyperplasia, serum thyroid hormone and lipid concentrations, and pitu-

“‘;_- I

TWO GTH CELLS IN SALMONJDS itary gland structure in Lake Ontario coho salmon, Oncorhynchus kisutch Walbaum and a comparison with coho slamon from Lake Michigan and Erie. J. Fish. Biol. 16, 539-562. Margolis-Kazan, H., and Schreibman, M. P. (1981). Cross-reactivity between human and fish pituitary hormones as demonstrated by immunocytochemistry. Cell Tissue Res. 221, 257-267. Mikami, S. (1986). Immunocytochemistry of the avian hypothalamus and adenohypophysis. Inr. Rev. CytoJ.

103, 189-248.

Nozaki, M., Miyata, K., Oota, Y., Gorbman, A., and Plisetskaya, E. P. (1988). Different cellular distributions of two somatostatins in brain and pancreas of salmonids, and their separate associations with insulin and glucagon secreting cells. Gen.

Comp.

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69, 267-280.

Nozaki, M., Naito, N., Swanson, P., Dickhoff, W. W., Nakai, Y., Suzuki, K., and Kawauchi, H. (1989). Salmonid pituitary gonadotroph cells. II. Ontogeny of GTH I and GTH 11 cells in the rainbow trout (Salmo gairdneri irideus). Gen. Comp. Endocrinol.

11, 358-367.

Olivereau, M. (1976). Les cellules gonadotropes hypophysaires du saumon de I’Atlantique: Unicite our dualite? Gen. Comp. Endocrinol. 28, 82-95. Olivereau, M. (1977). Gonadotropic cells in teleost fish. Invest. Pesq. 41, 67-68. Olivereau, M. (1978). Les cellules gonadotropes ches les salmonides. Ann. Biol. Anim. Bioch. Biophys. 18, 793-798.

Olivereau, M.. and Nagahama, Y. (1983). Immunocytochemistry of gonadotropic cells in the pituitary of some telesot species. Gen. Comp. Endocrinol. 50, 252-260.

Peute, J., Goos, H. J. Th., de Bruyn, M. B. A., and Van Oordt. P. G. W. J. (1978). Gonadotropic cells of the rainbow trout pituitary during the annual cycle: Ultrastructure and hormone content. Ann.

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Phifer, R. F., Midgley, A. R., and Spicer, S. S. (1973). Immunohistologic and histologic evidence that follicle-stimulating hormone and luteinizing hormone are present in the same cell type in the

human pars distalis. J. C/in. Endocrinol. Metub. 17-5-141. Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988a). Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands. Cert. Comp. Endocrinol. 71, 292-301. Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988b). isolation and characterization of subunits of two distinct gonadotropins from chum salmon pituitary glands. Gen. Comp. Endocrinol. 71, 30236,

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Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988~). Steroidogenic activities of two distinct salmon gonadotropins. Gen. Comp. Endocrinol. 71, 45p467.

Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988d). Development of salmon GTH I and GTH II radioimmunoassays Cert. Comp. Endocrinol. 71, 450-458.

Swanson, P., Suzuki, K., and Kawauchi, H. (1987). Isolation and biochemical characterization of two distinct pituitary gonadotropins from coho salmon. Oncorhynchus kisutch. Amer. Zoo/. 27, 79A. Trinh, K.-Y., Wang, N. C., Hew, C. L., and Crim. L. W. (1986). Molecular cloning and sequencing of salmon gonadotropin beta subunit. Eur. J. Biothem.

159, 619-624.

Ueda, H. (1980). Changes of two types of pituitary gonadotrophs in white-spotted charr, Salvelinus Ieucomaenis, during gonadal development. Bull. Fat.

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31, l-15.

Ueda, H., and Hirashima, T. (1979). On two different types of putative gonadotropins in the pituitary gland of the masu salmon, Oncorhynchus masou. Annot.

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52, 114-124.

Van Oordt, P. G. W. J., and Peute, J. (1983). The cellular origin of pituitary gonadotropins in teleosts. In “Fish Physiology” (W. S. Hoar, D. J. Randall, and E. M. Donaldson, Eds.), Vol. 9A, pp. I37186. Academic Press, New York. Van Putten, L. J. A., Peute, J., Van Oordt, P. G. W.J., Goes, H. J. Th., and Breton, B. (1981). Glycoprotein gonadotropin in the plasma and its cellular origin in the adenohypophysis of shamoperated and ovariectomized rainbow trout. Salmo

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218, 439448.