- Email: [email protected]
Gonadotrophic activity in the buccal lobe (Rachendachhypophyse) of the pituitary gland of the rabbit fish Hydrolagus colliei (Chondrichthyes: Holocephali)
GENERAL
AND
COMPARATIVE
48, 174-180
ENDOCRINOLOGY
(1982)
Gonadotrophic Activity in the Buccal Lobe (Rachendachhypophyse) of the Pituitary Gland of the Rabbit Fish Hydrolagus colliei (Chondrichthyes: Holocephali) J. M. DODD, Department
of Zoology,
M. H. I. DODD,
University
College
of North
J. P. SUMPTER,’ Wales,
Bangor,
AND N. JENKINS~ Gwynedd
LL57
2WW,
United
Kingdom
Accepted October 28, 1981 The ventral lobe of the pituitary gland of the selachoid elasmobranch, Scyliorhinus has been shown in earlier work to be the main source of gonadotrophin in this fish and it has been suggested that the buccal lobe (Rachendachhypophysis) in holocephalan fishes, because of its several similarities to the ventral lobe, may be its physiological equivalent; in this paper we adduce evidence in favour of this view. Extracts of buccal lobe of the rabbit fish, Hydrolugus colliei, like those of ventral lobe of S. canicula, are powerfully steroidogenic in an in vitro assay based on quail testicular tissues and less active, though significantly so, in promoting the uptake of 32P by the day-old chick testis. It is suggested, albeit on the basis of heterologous bioassays, that the buccal lobe of the holocephalan Hydrolagus colliei is the main source of gonadotrophin in this fish. canicula,
The adenohypophysis of aquatic verte- blood supply; both are vascularised by the brates is morphologically and functionally internal carotid arteries and in neither case subdivided to an extent that is not found in is there any direct vascular connection with tetrapods (Dodd, 1963; Holmes and Ball, the rest of the pituitary (Meurling, 1967). The possibility that the two structures are 1974). This is particularly striking in the cartilaginous fishes (elasmobranchs and homologous, on the basis of embryological holocephalans) in which the intracranial criteria, has been examined by several pituitary consists of rostral, median, and workers (Sathyanesan, 1965; Honma, 1969; neurointermediate lobes and there is an- Meurling, 1967). The VL of elasmobranchs other, more or less completely segregated, arises from paired lateral lobes of Rathke’s lobe called the ventral lobe (VL) in elasmopouch which bend downwards during debranchs and the Rachendachhypophysis or velopment and fuse beneath the main part buccal lobe (BL) in holocephalans. In elas- of the gland (Baumgartner, 1915) whereas, mobranchs, the VL is connected by a according to Honma (1969) the BL of the strand of tissue of variable thickness to the holocephalan Hydrolagus colliei originates median lobe throughout life whereas in as an unpaired outgrowth from Rathke’s adult Holocephali separation of the BL pouch; thus, on the embryological evidence from the rest of the pituitary is complete the VL and BL do not appear to be homoland it lies isolated, embedded in lymphoid ogous. Furthermore, the strand of tissue tissue, in the roof of the buccal cavity (Figs. connecting the isolated lobe with the rest of 1 and 2). Like the VL, the BL lacks a portal the pituitary in elasmobranchs usually lies between it and the posterior region of the r Present address: Department of Applied Biology, median lobe (Fig. lA), whereas in the School of Biological Sciences, Brunei University, UxHolocephali it originates from the anterior bridge, Middlesex UB8 3PH, United Kingdom. ’ Department of Physiology and Biochemistry, The end of the rostra1 lobe (Fujita, 1963) and disappears in H. colliei by the time the larUniversity, Whiteknights, Reading RG6 2AJ, United Kingdom. val fish are free-swimming (Honma, 1969, 174 0016~6480/82/100174-07$01.00/0 Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
GONADOTROPHIC
ACTIVITY
IN Hydrofogus
coffiei
BUCCAL
LOBE
175
FIG. 1. Diagrammatic sagittal sections of an (A) elasmobranch and (B) holocephalan pituitary gland, to show the positions and relationships of the individual lobes. RL, rostral lobe; ML, median lobe; NIL, neurointermediate lobe; VL, ventral lobe; BL, buccal lobe; L, lymphoid tissue; C, cartilage; OC, optic chiasma; III: third ventricle.
and Fig. 1B). Norris (1941), however, states that in some elasmobranchs also, the VL occupies an anterior site and the interhypophysial stalk is attached to the rostral lobe of the pituitary. So far as the function of these lobes is concerned, the VL of elasmobranchs was at first thought to be vestigeal and functionless (Norris, 1941) but it is now known to be the main gonadotrophic and thyrotrophic region of the pituitary (Dodd et ul., 1960; Dent and Dodd, 1961; Jackson and Sage, 1973; Sumpter eC al., 1978b). No physiological studies have as yet been carried out on the BL of Holocephali though its large size and glandular appearance in mature adult H. colliei
suggest that it also has an important function (Sathyanesan, 1965). Because of this and other similarities between it and the elasmobranch VL it has frequently been suggested that the two lobes may have similar functions; evidence in support of this view is presented below. MATERIALS
AND METHODS
Source of Material Material for gonadotrophin assays came from several hundred adult specimens of both sexes of the rabbit fish, H. co&i, which were collected by trawling at 40-50 fathoms off Lopez Island near the Friday Harbor Laboratories of the University of Washington in July 1977. The fish were decapitated at the time of capture, the heads kept on ice and immediately on
176
DODD
ET
AL.
FIG. 2. Roof of the buccal cavity (lower jaw removed) of a young H. colliei (length 12 cm) with oral mucous membrane removed to show the pituitary complex in situ (anterior is at left side). BL, buccal lobe; L, lymphoid tissue; LI, lobi inferiores; ML, median lobe; NIL, neurointermediate lobe; RL, rostra1 lobe.
return to the laboratory the pituitary glands were removed and separated into buccal lobes, rostra1 and median lobes (RML), and neurointermediate lobes (NIL). These were freeze-dried and stored at -20” prior to bioassay for gonadotrophic activity in two well-established systems, some 6 months later. The biometric data presented in Fig. 3 were obtained from immature and mature fish of both sexes trawled in the same area between April and mid-July 1977. Each fish used was weighed to an accuracy of 1.0 g, its BL was then carefully removed and weighed to an accuracy of 0.1 mg. Gonads and reproductive tracts were also examined and weighed to assess stage of maturity.
Assay Techniques The freeze-dried pituitary lobes were weighed on a Cahn electrobahmce to the nearest microgram, and homogenised in phosphate-buffered saline. The homogenates were centrifuged at 3000g for 30 mitt, after which the supernatants were removed and stored frozen prior to assay. (a) Quail testicular cell assay (QTCA). This assay was performed according to the method of Maung and Follett (1977) as modified by Jenkins et al. (1978). It is based on the stimulation of androgen synthesis and release from isolated testicular ceils (about 30% of which are Leydig cells) in vitro. After incubation of the cells with extracts of single pituitary lobes (see Table l), androgens in the incubation medium were measured directly, as described by Maung and Follett (1977). The antiserum employed showed a 66% cross-
reaction with dihydrotestosterone compared with 100% for testosterone; thus the assay measures both androgens, although the data are expressed in terms of testosterone. (b) Chick 32P-assay. This assay, based on the fact that gonadotrophins stimulate the uptake of radioactive phosphorous (3*P) by the testes of day-old chicks, was performed according to the technique of Follett and Farner (1966). Extracts were prepared by homogenising 2 BL and 45 each of RML and NIL. All lobes were assayed at three different doses, using 6 chicks at each dose level. An ovine luteinizing hormone (NIH-LH-S19) was used as standard in both bioassays. Potency estimates were calculated according to Bliss (1952) using the log-linear regions of the dose-response curves. The mean index of precision (A) for the QTCA assay was 0.09 2 0.01 (SEM, n = 4), while the single V-chick bioassay had a A of 0.144.
RESULTS
Regressions of the data presented in Fig. 3 were carried out to determine the best equation which would predict the weight of the BL
from
the total
body
weight of the
fish, the two sexes being dealt with separately. For both males and females the BL weight was related to body weight with a power equation, and the curves are drawn
GONADOTROPHIC
ACTIVITY
IN
04 0
FIG. 3. The relationship between body weight and buccal lobe weight in male (0) and female (0) Hydrolagus colliei. Power curves fitted by log-log regression: males: BL, 0.00173W’.351(n = 45; S,,,2 = 0.132); females; BL, 0.00577W1.165 (n = 72; S,,z = 0.179).
in Fig. 3. In both sexes the power curve, fitted by log-log regression, was a significantly better fit than a straight line (F tests; P < 0.0001 in both sexes). The existence of a curved relationship was further demonstrated by showing that the fitted values of the powers were significantly greater than 1, the value which would apply to straight line (t tests; males; P < 0.01; females; P < 0.002). Females weighing less than 0.6 kg and males of less than 0.4 kg were found to be sexually immature.
Hydrolagus
Pituitary lobe
ACTIVITY
RML NIL a Mean -C SE of 3 determinations
177
LOBE
1
OF THE PITUITARY LOBES OF H. OUAIL TESTICULAR CELL BIOASSAY
Dry weight of each individual lobe bg)
BL
BUCCAL
Replicated assays of extracts of freeze dried individual pituitary lobes the results of which are summarised in Table 1, show clearly that the BL of the pituitary gland of H. colliei contains considerable amounts of a substance which is highly active in stimulating steroidogenesis in the QTCA. The other lobes of the pituitary contain a substance which is similarly active, but is present in much smaller quantities; more than 95% of the gonadotrophic activity present in the entire pituitary gland resides in the BL when the QTCA is used as the bioassay. The dose-response curve of each pituitary lobe, and the ovine LH standard, had similar slopes. When the thick-32P bioassay was used to assess gonadotrophic activity, very little activity could be detected in any of the pituitary lobes. The highest dose of the BL produced a significant response, giving a potency of 8.55 (7.24-10.09) pg NIH-LHS19 equivalents/lobe, but very high concentrations of either the RML or NIL (3 lobes/chick) gave no response, giving both these lobes a potency of less than 0.4 pug NIH-LH-S19 equivalents/lobe. An antiserum raised against a purified dogfish gonadotrophin, isolated from ventral lobes (anti-CM2; see Sumpter et al., 1978a) had no significant effect on the potency in the QTCA of an extract prepared from BL of H. colliei (BL + normal rabbit
TABLE GONADOTROPHIC
coNki
3.75 2.55 6.94 3.30 0.95 3.50 2.38 of an individual lobe.
colliei, MEASURED
NIH-LHS19 Per lobe ? 7.6” loo 140 277 2.91 2 0.94a 0.39 3.01 k 0.81” 1.17
IN THE
(pg equivalents) Per mg dry weight 26.7 54.9 39.9 0.88 0.41 0.86 0.49
178
DODD
serum: 123 + 6.6 pg; BL + anti-CM2: 108 4 11.3 pg, both expressed as I.tg equivalents NIH-LH-S19/lobe). The same antiserum completely neutralised an extract prepared from dogfish, S. canicula, ventral lobe (Fig. 4). DISCUSSION
The bioassay results reported above show that the BL of the pituitary gland of H. colliei contains a considerable amount of a substance that is highly active in stimulating steroidogenesis by quail testicular tissue in the QTCA bioassay system. This assay has previously been shown to respond by increased steroidogenesis to a wide range of vertebrate gonadotrophins including those of mammalian, avian and fish species (Jenkins et al., 1978). Extracts of the VL of elasmobranchs from five different families have all been found to possess marked gonadotrophic activity, ranging from an average of 100 pg NIH-LH-S19 equivalents/lobe in mature female dogfish, S. canicula, to more than 2 mg equivalents in the larger ventral lobe of a single mature male blue shark, Prionace gluucu (Sumpter et al., 1980). On a lobe-weight basis the
VL+anti-CM2
oJ
10
100 Dose
hg/ml) 0.0001
0.001 Dose
(lobe/tube)
FIG. 4. Effects of an extract of H. colliei buccal lobe (BL) or S. canicltfa ventral lobe (VL) on androgen release from quail testicular cells in vitro, and their neutralization by antibody. Five microlitres of either normal rabbit serum (NRS) or an antibody raised against puritied dogfish gonadotrophin (anti-CM2; see Sumpter et al., 1978a, b) was added 30 mm prior to the addition of testicular cells where indicated. Control tubes received no gonadotrophin. Each point represents the mean of duplicate determinations.
ET AL.
gonadotrophin extracted from VL of S. cuniculu was one of the most potent of all tested in the assay (Jenkins et al., 1978). BL of H. colliei collected in July, well before the peak of reproductive activity, and from both sexes, showed a higher potency per lobe than this. The distribution of gonadotrophic activity in the pituitary gland of H. colliei resembles very closely that found in S. cuniculu, with the vast majority of the activity being found in the VL of the elasmobranch pituitary and in the BL of the holocephalan pituitary. In both groups, however, small quantities of a steroidogenic gonadotrophin were present in all other lobes of the pituitary (Sumpter et al., 1978b). Thus it appears that in several phylogenetically ancient fishes the ventral lobe of the elasmobranch pituitary and the BL of the H. colliei pituitary contain a similar molecule that can act as a steroidogenic gonadotrophin in at least one class of vertebrates, the birds. The molecule, however, is not identical in the two main kinds of contemporary cartilaginous fishes since the antiserum raised against a purified dogfish gonadotrophin isolated from the ventral lobes of S. cuniculu (Sumpter et al., 1978b) is not capable of neutralizing the gonadotrophic activity present in the BL of H. colliei (Fig. 4), though it neutralized the biological activity of S. cuniculu VL extract, and that of five other species of elasmobranchs, including both sharks and rays (Sumpter et al., 1980). Although the BL extracts were extremely potent in the QTCA, they were much less active (5% relative to the same standard) in stimulating uptake of 32P by the day-old chick testis, even though both are based on avian tissue. Another fish gonadotrophin, purified from salmon pituitaries, has also been shown to have very different potencies in the two bioassays, though it was much more potent when measured in the chick 32P-bioassay than in the QTCA (Jenkins et al., 1978). This indicates one of the difficulties experienced when interpreting data based on the use of heterologous as-
GONADOTROPHIC
ACTIVITY
says, another being that, even though extracts of H. colliei BL show gonadotrophic activity in two heterologous bioassays, this does not necessarily prove that the BL produces a gonadotrophin active in Hydrolugus. It has been known for some time that the biological activities of a hormone in the species which produces it may be markedly different from those evoked by the same hormone in a different species. A good example of this has recently been provided by MacKenzie et al., (1978) who showed than an amphibian (bullfrog) luteinizing hormone (LH) which in the homologous species was gonadotrophic, not only functioned as a thyroid stimulating hormone (TSH) in a number of heterologous (reptilian) species but was much more potent in this respect than bullfrog TSH. Thus we cannot be certain that the molecule possessing gonadotrophic activity in our heterologous bioassays also functions as a gonadotrophin in H. colliei, though it would be very surprising if it was not either a gonadotrophin or TSH. However, support for the view that the BL is, in fact, producing a gonadotrophin functionally active in H. colliei, comes from the data presented in Fig. 3. These clearly demonstrate that the BL to body weight ratio is not constant throughout the life cycle, but increases in both male and female fish as, and after, they attain sexual maturity. To date, the reproductive physiology of the Holocephali is virtually unexplored, though Dean (1906) reports that H. colliei reproduces throughout the year, with a possible peak of activity in late summer and early autumn; our own preliminary studies support this view. This means that between April and mid-July, the period during which the data presented in Fig. 3 were collected, the adult fish were probably at the nadir of their reproductive activity, and least likely to exhibit sexually associated differences in BL weight. In S. canicula, which also has an extended breeding period, and shows a marked annual cycle,
IN
Hydrolagus
colliei
BUCCAL
LOBE
179
significant changes in VL size (Dodd et al., unpublished), and gonadotrophin content (Sumpter and Dodd, 1979), have been demonstrated, both being greatest in female fish in the middle of their breeding season, and least in immature fish (Sumpter, 1977). ACKNOWLEDGMENTS This research was supported by grants from the Science Research Council and from the Browne Fund of the Royal Society to J.M.D., and by postgraduate studentships from the Natural Environment Research Council and Science Research Council to J.P.S. and N.J., respectively. Thanks are also due to Dr. William Marshall for assistance in collecting Hydrolagus glands, the National Institutes of Health for the gifts of ovine LH, and Dr. T. J. Pitcher for the statistical analysis. The gland material on which this paper is based was collected at the Friday Harbor Marine Laboratories of the University of Washington and the Bamfield Marine Station, British Columbia, Canada, and we wish to acknowledge the generous cooperation of the Directors.
REFERENCES Baumgartner, E. A. (1915). The development of the hypophysis in Squalus acanthias. J. Morphol. 26, 391-446. Bliss, C. I. (1952). “Statistics of Bioassay.” Academic Press, New York. Dean, B. (1906). “Chimaeroid Fishes and Their Development.” Carnegie Inst. of Washington, Washington, D.C., Publ. 32. Dent, J. N., and Dodd, J. M. (1961). Some effects of mammalian thyroid stimulating hormone, elasmobranch pituitary extracts and temperature on thyroidal activity in newly hatched dogfish (Scyliorhinus canicula). J. Endocrinol. 22, 395 -402. Dodd, J. M. (1963). The pituitary complex. In “Techniques in Endocrine Research” (P. Eckstein, and F. Knowles, eds.), pp. 161-185. Academic Press, New York. Dodd, J. M., Evenett, P. J., and Goddard, C. K. (1960). Reproductive endocrinology in cyclostomes and elasmobranchs. Syrup. 2001. Sot. London 1, 77-103. Follett, B. K., and Farner, D. S. (1966). Pituitary gonadotrophin in the Japanese quail (Coturnix coturnix japonica) during photoperiodically induced gonadal growth. Gen. Conp. Endocrinol. 7, 125-131. Fujita, T. (1963). Uber das Zwischenhim-Hypophy-
180
DODD
sensystem von Chimaera monstrosa. Z. Zetl60, 147-162. Holmes, R. L., and Ball, J. N. (1974). “The Pituitary Gland.” Cambridge Univ. Press, London/New York. Honma, Y. (1969). Some evolutionary Aspects of the Morphology and Role of the adenohypophysis in Fishes. Gunma Symp. Endocrinol. 6, 19-37. Jackson, R. G., and Sage, M. (1973). A comparison of the effects of mammalian TSH on the thyroidal glands of the teleost Gateichthys felis and the elasmobranch Dasyatis sabina. Comp. Biochem. Physiol. 44A, 867-870. Jenkins, N., Sumpter, J. P., and Follett, B. K. (1978). The effects of vertebrate gonadotrophins on androgen release in vitro from testicular cells of Japanese quail, and a comparison with their radioimmunoassay activities. Gen. Camp. Endocrinol. 35, 309321. MacKenzie, D. S., Licht, P., and Papkoff, H. (1978). Thyrotrophin from amphibian (Rana catesbeiana) pituitaries and evidence for heterologous activity of bullfrog luteinizing hormone in reptiles. Gen. forsch.
Comp.
Endocrinol.
36, 566-574.
Maung, 2. W., and Follett, B. K. (1977). Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testes. Gen. Camp. Endocrinoi.
33, 242-253.
Meurling, P. (1967). The vascularization of the pituitary in elasmobranchs. Sarsia 28, l-104. Norris, H. W. (1941). “The Plagiostome Hypophysis,
ET
AL.
General Morphology and Types of Structure.” Frinnell, Iowa. Sathyanesan, A. G. (1965). The hypophysis and hypothalamohypophyseal system in the chimaeroid fish Hydrolagus colliei (Lay and Bennett) with a note on their vascularisation. J. Morphol. 116, 413-450. Sumpter, J. P. (1977). “Biochemical and Physiological Studies on the Reproductive Hormones of the Dogfish.” Ph.D. thesis, Univ. of Wales. Sumpter, J. P., and Dodd, .I. M. (1979). The annual reproductive cycle of the female lesser spotted dogfish, Scyliorhinus canicula L. and its endocrine control. .I. Fish Biol. 15, 687-695. Sumpter, J. P., Follett, B. K., Jenkins, N., and Dodd, J. M. (1978a). Studies on the purification and properties of gonadotrophin from ventral lobes of the pituitary gland of the dogfish (Scyliorhinus canicula L.). Gen. Comp. Endocrinol. 36, 264-274. Sumpter, J. P., Jenkins, N., and Dodd, J. M. (1978b). Gonadotrophic hormone in the pituitary gland of the dogfish (Scyliorhinus canicula L.): distribution and physiological significance. Gen. Camp. Endocrinol.
36, 275-285.
Sumpter, J. P., Jenkins, N., Duggan, R. T., and Dodd, J. M. (1980). The steroidogenic effects of pituitary extracts from a range of elasmobranch species on isolated testicular cells of quail and their neutralisation by a dogfish anti-gonadotrophin. Gen. Comp.
Endocrinol.
40, 331.
AND
COMPARATIVE
48, 174-180
ENDOCRINOLOGY
(1982)
Gonadotrophic Activity in the Buccal Lobe (Rachendachhypophyse) of the Pituitary Gland of the Rabbit Fish Hydrolagus colliei (Chondrichthyes: Holocephali) J. M. DODD, Department
of Zoology,
M. H. I. DODD,
University
College
of North
J. P. SUMPTER,’ Wales,
Bangor,
AND N. JENKINS~ Gwynedd
LL57
2WW,
United
Kingdom
Accepted October 28, 1981 The ventral lobe of the pituitary gland of the selachoid elasmobranch, Scyliorhinus has been shown in earlier work to be the main source of gonadotrophin in this fish and it has been suggested that the buccal lobe (Rachendachhypophysis) in holocephalan fishes, because of its several similarities to the ventral lobe, may be its physiological equivalent; in this paper we adduce evidence in favour of this view. Extracts of buccal lobe of the rabbit fish, Hydrolugus colliei, like those of ventral lobe of S. canicula, are powerfully steroidogenic in an in vitro assay based on quail testicular tissues and less active, though significantly so, in promoting the uptake of 32P by the day-old chick testis. It is suggested, albeit on the basis of heterologous bioassays, that the buccal lobe of the holocephalan Hydrolagus colliei is the main source of gonadotrophin in this fish. canicula,
The adenohypophysis of aquatic verte- blood supply; both are vascularised by the brates is morphologically and functionally internal carotid arteries and in neither case subdivided to an extent that is not found in is there any direct vascular connection with tetrapods (Dodd, 1963; Holmes and Ball, the rest of the pituitary (Meurling, 1967). The possibility that the two structures are 1974). This is particularly striking in the cartilaginous fishes (elasmobranchs and homologous, on the basis of embryological holocephalans) in which the intracranial criteria, has been examined by several pituitary consists of rostral, median, and workers (Sathyanesan, 1965; Honma, 1969; neurointermediate lobes and there is an- Meurling, 1967). The VL of elasmobranchs other, more or less completely segregated, arises from paired lateral lobes of Rathke’s lobe called the ventral lobe (VL) in elasmopouch which bend downwards during debranchs and the Rachendachhypophysis or velopment and fuse beneath the main part buccal lobe (BL) in holocephalans. In elas- of the gland (Baumgartner, 1915) whereas, mobranchs, the VL is connected by a according to Honma (1969) the BL of the strand of tissue of variable thickness to the holocephalan Hydrolagus colliei originates median lobe throughout life whereas in as an unpaired outgrowth from Rathke’s adult Holocephali separation of the BL pouch; thus, on the embryological evidence from the rest of the pituitary is complete the VL and BL do not appear to be homoland it lies isolated, embedded in lymphoid ogous. Furthermore, the strand of tissue tissue, in the roof of the buccal cavity (Figs. connecting the isolated lobe with the rest of 1 and 2). Like the VL, the BL lacks a portal the pituitary in elasmobranchs usually lies between it and the posterior region of the r Present address: Department of Applied Biology, median lobe (Fig. lA), whereas in the School of Biological Sciences, Brunei University, UxHolocephali it originates from the anterior bridge, Middlesex UB8 3PH, United Kingdom. ’ Department of Physiology and Biochemistry, The end of the rostra1 lobe (Fujita, 1963) and disappears in H. colliei by the time the larUniversity, Whiteknights, Reading RG6 2AJ, United Kingdom. val fish are free-swimming (Honma, 1969, 174 0016~6480/82/100174-07$01.00/0 Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
GONADOTROPHIC
ACTIVITY
IN Hydrofogus
coffiei
BUCCAL
LOBE
175
FIG. 1. Diagrammatic sagittal sections of an (A) elasmobranch and (B) holocephalan pituitary gland, to show the positions and relationships of the individual lobes. RL, rostral lobe; ML, median lobe; NIL, neurointermediate lobe; VL, ventral lobe; BL, buccal lobe; L, lymphoid tissue; C, cartilage; OC, optic chiasma; III: third ventricle.
and Fig. 1B). Norris (1941), however, states that in some elasmobranchs also, the VL occupies an anterior site and the interhypophysial stalk is attached to the rostral lobe of the pituitary. So far as the function of these lobes is concerned, the VL of elasmobranchs was at first thought to be vestigeal and functionless (Norris, 1941) but it is now known to be the main gonadotrophic and thyrotrophic region of the pituitary (Dodd et ul., 1960; Dent and Dodd, 1961; Jackson and Sage, 1973; Sumpter eC al., 1978b). No physiological studies have as yet been carried out on the BL of Holocephali though its large size and glandular appearance in mature adult H. colliei
suggest that it also has an important function (Sathyanesan, 1965). Because of this and other similarities between it and the elasmobranch VL it has frequently been suggested that the two lobes may have similar functions; evidence in support of this view is presented below. MATERIALS
AND METHODS
Source of Material Material for gonadotrophin assays came from several hundred adult specimens of both sexes of the rabbit fish, H. co&i, which were collected by trawling at 40-50 fathoms off Lopez Island near the Friday Harbor Laboratories of the University of Washington in July 1977. The fish were decapitated at the time of capture, the heads kept on ice and immediately on
176
DODD
ET
AL.
FIG. 2. Roof of the buccal cavity (lower jaw removed) of a young H. colliei (length 12 cm) with oral mucous membrane removed to show the pituitary complex in situ (anterior is at left side). BL, buccal lobe; L, lymphoid tissue; LI, lobi inferiores; ML, median lobe; NIL, neurointermediate lobe; RL, rostra1 lobe.
return to the laboratory the pituitary glands were removed and separated into buccal lobes, rostra1 and median lobes (RML), and neurointermediate lobes (NIL). These were freeze-dried and stored at -20” prior to bioassay for gonadotrophic activity in two well-established systems, some 6 months later. The biometric data presented in Fig. 3 were obtained from immature and mature fish of both sexes trawled in the same area between April and mid-July 1977. Each fish used was weighed to an accuracy of 1.0 g, its BL was then carefully removed and weighed to an accuracy of 0.1 mg. Gonads and reproductive tracts were also examined and weighed to assess stage of maturity.
Assay Techniques The freeze-dried pituitary lobes were weighed on a Cahn electrobahmce to the nearest microgram, and homogenised in phosphate-buffered saline. The homogenates were centrifuged at 3000g for 30 mitt, after which the supernatants were removed and stored frozen prior to assay. (a) Quail testicular cell assay (QTCA). This assay was performed according to the method of Maung and Follett (1977) as modified by Jenkins et al. (1978). It is based on the stimulation of androgen synthesis and release from isolated testicular ceils (about 30% of which are Leydig cells) in vitro. After incubation of the cells with extracts of single pituitary lobes (see Table l), androgens in the incubation medium were measured directly, as described by Maung and Follett (1977). The antiserum employed showed a 66% cross-
reaction with dihydrotestosterone compared with 100% for testosterone; thus the assay measures both androgens, although the data are expressed in terms of testosterone. (b) Chick 32P-assay. This assay, based on the fact that gonadotrophins stimulate the uptake of radioactive phosphorous (3*P) by the testes of day-old chicks, was performed according to the technique of Follett and Farner (1966). Extracts were prepared by homogenising 2 BL and 45 each of RML and NIL. All lobes were assayed at three different doses, using 6 chicks at each dose level. An ovine luteinizing hormone (NIH-LH-S19) was used as standard in both bioassays. Potency estimates were calculated according to Bliss (1952) using the log-linear regions of the dose-response curves. The mean index of precision (A) for the QTCA assay was 0.09 2 0.01 (SEM, n = 4), while the single V-chick bioassay had a A of 0.144.
RESULTS
Regressions of the data presented in Fig. 3 were carried out to determine the best equation which would predict the weight of the BL
from
the total
body
weight of the
fish, the two sexes being dealt with separately. For both males and females the BL weight was related to body weight with a power equation, and the curves are drawn
GONADOTROPHIC
ACTIVITY
IN
04 0
FIG. 3. The relationship between body weight and buccal lobe weight in male (0) and female (0) Hydrolagus colliei. Power curves fitted by log-log regression: males: BL, 0.00173W’.351(n = 45; S,,,2 = 0.132); females; BL, 0.00577W1.165 (n = 72; S,,z = 0.179).
in Fig. 3. In both sexes the power curve, fitted by log-log regression, was a significantly better fit than a straight line (F tests; P < 0.0001 in both sexes). The existence of a curved relationship was further demonstrated by showing that the fitted values of the powers were significantly greater than 1, the value which would apply to straight line (t tests; males; P < 0.01; females; P < 0.002). Females weighing less than 0.6 kg and males of less than 0.4 kg were found to be sexually immature.
Hydrolagus
Pituitary lobe
ACTIVITY
RML NIL a Mean -C SE of 3 determinations
177
LOBE
1
OF THE PITUITARY LOBES OF H. OUAIL TESTICULAR CELL BIOASSAY
Dry weight of each individual lobe bg)
BL
BUCCAL
Replicated assays of extracts of freeze dried individual pituitary lobes the results of which are summarised in Table 1, show clearly that the BL of the pituitary gland of H. colliei contains considerable amounts of a substance which is highly active in stimulating steroidogenesis in the QTCA. The other lobes of the pituitary contain a substance which is similarly active, but is present in much smaller quantities; more than 95% of the gonadotrophic activity present in the entire pituitary gland resides in the BL when the QTCA is used as the bioassay. The dose-response curve of each pituitary lobe, and the ovine LH standard, had similar slopes. When the thick-32P bioassay was used to assess gonadotrophic activity, very little activity could be detected in any of the pituitary lobes. The highest dose of the BL produced a significant response, giving a potency of 8.55 (7.24-10.09) pg NIH-LHS19 equivalents/lobe, but very high concentrations of either the RML or NIL (3 lobes/chick) gave no response, giving both these lobes a potency of less than 0.4 pug NIH-LH-S19 equivalents/lobe. An antiserum raised against a purified dogfish gonadotrophin, isolated from ventral lobes (anti-CM2; see Sumpter et al., 1978a) had no significant effect on the potency in the QTCA of an extract prepared from BL of H. colliei (BL + normal rabbit
TABLE GONADOTROPHIC
coNki
3.75 2.55 6.94 3.30 0.95 3.50 2.38 of an individual lobe.
colliei, MEASURED
NIH-LHS19 Per lobe ? 7.6” loo 140 277 2.91 2 0.94a 0.39 3.01 k 0.81” 1.17
IN THE
(pg equivalents) Per mg dry weight 26.7 54.9 39.9 0.88 0.41 0.86 0.49
178
DODD
serum: 123 + 6.6 pg; BL + anti-CM2: 108 4 11.3 pg, both expressed as I.tg equivalents NIH-LH-S19/lobe). The same antiserum completely neutralised an extract prepared from dogfish, S. canicula, ventral lobe (Fig. 4). DISCUSSION
The bioassay results reported above show that the BL of the pituitary gland of H. colliei contains a considerable amount of a substance that is highly active in stimulating steroidogenesis by quail testicular tissue in the QTCA bioassay system. This assay has previously been shown to respond by increased steroidogenesis to a wide range of vertebrate gonadotrophins including those of mammalian, avian and fish species (Jenkins et al., 1978). Extracts of the VL of elasmobranchs from five different families have all been found to possess marked gonadotrophic activity, ranging from an average of 100 pg NIH-LH-S19 equivalents/lobe in mature female dogfish, S. canicula, to more than 2 mg equivalents in the larger ventral lobe of a single mature male blue shark, Prionace gluucu (Sumpter et al., 1980). On a lobe-weight basis the
VL+anti-CM2
oJ
10
100 Dose
hg/ml) 0.0001
0.001 Dose
(lobe/tube)
FIG. 4. Effects of an extract of H. colliei buccal lobe (BL) or S. canicltfa ventral lobe (VL) on androgen release from quail testicular cells in vitro, and their neutralization by antibody. Five microlitres of either normal rabbit serum (NRS) or an antibody raised against puritied dogfish gonadotrophin (anti-CM2; see Sumpter et al., 1978a, b) was added 30 mm prior to the addition of testicular cells where indicated. Control tubes received no gonadotrophin. Each point represents the mean of duplicate determinations.
ET AL.
gonadotrophin extracted from VL of S. cuniculu was one of the most potent of all tested in the assay (Jenkins et al., 1978). BL of H. colliei collected in July, well before the peak of reproductive activity, and from both sexes, showed a higher potency per lobe than this. The distribution of gonadotrophic activity in the pituitary gland of H. colliei resembles very closely that found in S. cuniculu, with the vast majority of the activity being found in the VL of the elasmobranch pituitary and in the BL of the holocephalan pituitary. In both groups, however, small quantities of a steroidogenic gonadotrophin were present in all other lobes of the pituitary (Sumpter et al., 1978b). Thus it appears that in several phylogenetically ancient fishes the ventral lobe of the elasmobranch pituitary and the BL of the H. colliei pituitary contain a similar molecule that can act as a steroidogenic gonadotrophin in at least one class of vertebrates, the birds. The molecule, however, is not identical in the two main kinds of contemporary cartilaginous fishes since the antiserum raised against a purified dogfish gonadotrophin isolated from the ventral lobes of S. cuniculu (Sumpter et al., 1978b) is not capable of neutralizing the gonadotrophic activity present in the BL of H. colliei (Fig. 4), though it neutralized the biological activity of S. cuniculu VL extract, and that of five other species of elasmobranchs, including both sharks and rays (Sumpter et al., 1980). Although the BL extracts were extremely potent in the QTCA, they were much less active (5% relative to the same standard) in stimulating uptake of 32P by the day-old chick testis, even though both are based on avian tissue. Another fish gonadotrophin, purified from salmon pituitaries, has also been shown to have very different potencies in the two bioassays, though it was much more potent when measured in the chick 32P-bioassay than in the QTCA (Jenkins et al., 1978). This indicates one of the difficulties experienced when interpreting data based on the use of heterologous as-
GONADOTROPHIC
ACTIVITY
says, another being that, even though extracts of H. colliei BL show gonadotrophic activity in two heterologous bioassays, this does not necessarily prove that the BL produces a gonadotrophin active in Hydrolugus. It has been known for some time that the biological activities of a hormone in the species which produces it may be markedly different from those evoked by the same hormone in a different species. A good example of this has recently been provided by MacKenzie et al., (1978) who showed than an amphibian (bullfrog) luteinizing hormone (LH) which in the homologous species was gonadotrophic, not only functioned as a thyroid stimulating hormone (TSH) in a number of heterologous (reptilian) species but was much more potent in this respect than bullfrog TSH. Thus we cannot be certain that the molecule possessing gonadotrophic activity in our heterologous bioassays also functions as a gonadotrophin in H. colliei, though it would be very surprising if it was not either a gonadotrophin or TSH. However, support for the view that the BL is, in fact, producing a gonadotrophin functionally active in H. colliei, comes from the data presented in Fig. 3. These clearly demonstrate that the BL to body weight ratio is not constant throughout the life cycle, but increases in both male and female fish as, and after, they attain sexual maturity. To date, the reproductive physiology of the Holocephali is virtually unexplored, though Dean (1906) reports that H. colliei reproduces throughout the year, with a possible peak of activity in late summer and early autumn; our own preliminary studies support this view. This means that between April and mid-July, the period during which the data presented in Fig. 3 were collected, the adult fish were probably at the nadir of their reproductive activity, and least likely to exhibit sexually associated differences in BL weight. In S. canicula, which also has an extended breeding period, and shows a marked annual cycle,
IN
Hydrolagus
colliei
BUCCAL
LOBE
179
significant changes in VL size (Dodd et al., unpublished), and gonadotrophin content (Sumpter and Dodd, 1979), have been demonstrated, both being greatest in female fish in the middle of their breeding season, and least in immature fish (Sumpter, 1977). ACKNOWLEDGMENTS This research was supported by grants from the Science Research Council and from the Browne Fund of the Royal Society to J.M.D., and by postgraduate studentships from the Natural Environment Research Council and Science Research Council to J.P.S. and N.J., respectively. Thanks are also due to Dr. William Marshall for assistance in collecting Hydrolagus glands, the National Institutes of Health for the gifts of ovine LH, and Dr. T. J. Pitcher for the statistical analysis. The gland material on which this paper is based was collected at the Friday Harbor Marine Laboratories of the University of Washington and the Bamfield Marine Station, British Columbia, Canada, and we wish to acknowledge the generous cooperation of the Directors.
REFERENCES Baumgartner, E. A. (1915). The development of the hypophysis in Squalus acanthias. J. Morphol. 26, 391-446. Bliss, C. I. (1952). “Statistics of Bioassay.” Academic Press, New York. Dean, B. (1906). “Chimaeroid Fishes and Their Development.” Carnegie Inst. of Washington, Washington, D.C., Publ. 32. Dent, J. N., and Dodd, J. M. (1961). Some effects of mammalian thyroid stimulating hormone, elasmobranch pituitary extracts and temperature on thyroidal activity in newly hatched dogfish (Scyliorhinus canicula). J. Endocrinol. 22, 395 -402. Dodd, J. M. (1963). The pituitary complex. In “Techniques in Endocrine Research” (P. Eckstein, and F. Knowles, eds.), pp. 161-185. Academic Press, New York. Dodd, J. M., Evenett, P. J., and Goddard, C. K. (1960). Reproductive endocrinology in cyclostomes and elasmobranchs. Syrup. 2001. Sot. London 1, 77-103. Follett, B. K., and Farner, D. S. (1966). Pituitary gonadotrophin in the Japanese quail (Coturnix coturnix japonica) during photoperiodically induced gonadal growth. Gen. Conp. Endocrinol. 7, 125-131. Fujita, T. (1963). Uber das Zwischenhim-Hypophy-
180
DODD
sensystem von Chimaera monstrosa. Z. Zetl60, 147-162. Holmes, R. L., and Ball, J. N. (1974). “The Pituitary Gland.” Cambridge Univ. Press, London/New York. Honma, Y. (1969). Some evolutionary Aspects of the Morphology and Role of the adenohypophysis in Fishes. Gunma Symp. Endocrinol. 6, 19-37. Jackson, R. G., and Sage, M. (1973). A comparison of the effects of mammalian TSH on the thyroidal glands of the teleost Gateichthys felis and the elasmobranch Dasyatis sabina. Comp. Biochem. Physiol. 44A, 867-870. Jenkins, N., Sumpter, J. P., and Follett, B. K. (1978). The effects of vertebrate gonadotrophins on androgen release in vitro from testicular cells of Japanese quail, and a comparison with their radioimmunoassay activities. Gen. Camp. Endocrinol. 35, 309321. MacKenzie, D. S., Licht, P., and Papkoff, H. (1978). Thyrotrophin from amphibian (Rana catesbeiana) pituitaries and evidence for heterologous activity of bullfrog luteinizing hormone in reptiles. Gen. forsch.
Comp.
Endocrinol.
36, 566-574.
Maung, 2. W., and Follett, B. K. (1977). Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testes. Gen. Camp. Endocrinoi.
33, 242-253.
Meurling, P. (1967). The vascularization of the pituitary in elasmobranchs. Sarsia 28, l-104. Norris, H. W. (1941). “The Plagiostome Hypophysis,
ET
AL.
General Morphology and Types of Structure.” Frinnell, Iowa. Sathyanesan, A. G. (1965). The hypophysis and hypothalamohypophyseal system in the chimaeroid fish Hydrolagus colliei (Lay and Bennett) with a note on their vascularisation. J. Morphol. 116, 413-450. Sumpter, J. P. (1977). “Biochemical and Physiological Studies on the Reproductive Hormones of the Dogfish.” Ph.D. thesis, Univ. of Wales. Sumpter, J. P., and Dodd, .I. M. (1979). The annual reproductive cycle of the female lesser spotted dogfish, Scyliorhinus canicula L. and its endocrine control. .I. Fish Biol. 15, 687-695. Sumpter, J. P., Follett, B. K., Jenkins, N., and Dodd, J. M. (1978a). Studies on the purification and properties of gonadotrophin from ventral lobes of the pituitary gland of the dogfish (Scyliorhinus canicula L.). Gen. Comp. Endocrinol. 36, 264-274. Sumpter, J. P., Jenkins, N., and Dodd, J. M. (1978b). Gonadotrophic hormone in the pituitary gland of the dogfish (Scyliorhinus canicula L.): distribution and physiological significance. Gen. Camp. Endocrinol.
36, 275-285.
Sumpter, J. P., Jenkins, N., Duggan, R. T., and Dodd, J. M. (1980). The steroidogenic effects of pituitary extracts from a range of elasmobranch species on isolated testicular cells of quail and their neutralisation by a dogfish anti-gonadotrophin. Gen. Comp.
Endocrinol.
40, 331.