Evolution of Neurohormonal Regulation of Reproduction in Lower Vertebrates'

AMER. ZOOL., 23:685-695 (1983) Evolution of Neurohormonal Regulation of Reproduction in Lower Vertebrates' RICHARD E. PETER Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada SYNOPSIS. Immunological and chromatographic studies demonstrate that the gonadotropin (GtH) releasing hormone (GnRH) in the brain of chondrichthyes, teleosts, reptiles and birds is different from luteinizing hormone-releasing hormone (LH-RH) of mammals. LH-RH is present in brain extracts from amphibia. The alterations in the structure of LH-RH found in various of the non-mammalian vertebrates are at positions 7 and 8; the structure of chum salmon GnRH is Trp 7 -Leu 8 -LH-RH, and in chickens is Gln 8 -LH-RH. A response to injection of LH-RH or its agonistic analogues, in terms of increased blood levels of GtH or a gonadal response such asovulation indicative of increased GtH secretion, has been found in all classes of vertebrates, and the cephalochordate amphioxus. This suggests a basic similaritiy of the GnRH receptors throughout vertebrates, and that the ancestral origin of the system was in the invertebrate chordates. Immunohistochemical studies demonstrate that many lower vertebrates have perikarya containing LH-RH-like material in the preoptic and ventrobasal hypothalamic regions. Brain lesioning studies provide functional evidence for GnRH from both locations in amphibia and only the ventrobasal hypothalamus in teleosts. Brain lesioning studies on goldfish suggest the presence of a GtH release-inhibitory factor (GRIF). Dopamine has GRIF activity in goldfish and common carp to modulate the actions of LH-RH and spontaneous release of GtH. How widespread this system for dual neurohormonal regulation of GtH secretion is in vertebrates is not known. INTRODUCTION Discovery of the primary structure of mammalian luteinizing hormone-releasing hormone (pglu'-his 2 -Trp 3 -Ser 4 -Tyr 5 -Gly 6 - Leu 7 -Arg 8 -Pro 9 -Gly' -NH 2 : LH-RH; for review, Vale et al., 1977) was a milestone in neuroendocrine research. This greatly influenced research on the neuroendocrine regulation of gonadotropin (GtH) secretion in non-mammalian vertebrates in that it provided important tools for investigation in this area. In this review I shall discuss the structure of GtH releasing hormone (GnRH) in non-mammalian vertebrates. In addition, the responses of lower vertebrates to LH-RH, analogues of LH- RH, and the GnRHs of non-mammalian vertebrates will be compared to derive a basis for discussion of pituitary receptors for GnRH. Also, information on the brain locations of perikarya immunoreactive for LH-RH, and data indicating possible func- ' From the Symposium on Evolution of Endocrine Systems in Lower Vertebrates, A Symposium Honoring Professor Aubrey Corbman presented at the Annual Meeting of the American Society of Zoologists, 27-30 December 1982, at Louisville, Kentucky. 685 tional involvement of different brain regions in regulation of GtH secretion via GnRH in lower vertebrates will be reviewed. Recent studies on goldfish indicate the presence of a hypothalamic GtH releaseinhibitory factor (GRIF), and that dopamine has GRIF activity. The relative importance of GRIF in regulation of GtH secretion will be discussed. GONADOTROPIN RELEASING HORMONE Heterogeneity of structure of GnRH in vertebrates Antisera against LH-RH that are known to be specific for the central parts of the molecule, particularly positions 7 and 8, have poor crossreactivity with hypothalamic extracts from tilapia, Sarotherodon mossambkus (King and Millar, 1979, 1980), codfish, Gaclus morhua morhua (Barnett et al., 1982), the dogfish shark Poroderma africanum, the tortoise Chersine angulata, the lizard Mabuya capensis (King and Millar, 1979, 1980)', chickens, Gallus gallus ciomeshcus (King and Millar, 1979, 1980; Miyamoto et al., 1982), and pigeons, Columba hvia (King and Millar, 1979, 1980). This

686 RICHARD E. PETER is in contrast to the results with hypothalamic extracts from the frogs Rana pipiens, R. catesbeiana (Alpert et ai, 1976), and Xenopus laevis (King and Millar, 1979, 1980), and the toad Bufogariepensis (King and Millar, 1979, 1980), which have high crossreactivity with all antisera to LH-RH, and are parallel to synthetic LH-RH and hypothalamic extracts from the rat in all LH- RH radioimmunoassays. Hypothalamic extracts from teleosts, reptiles and birds have, on the other hand, high crossreactivity and parallelism with LH-RH standards when used with antisera specific for only positions 1 and 10 (King and Millar, 1979, 1980; Barnett et ai, 1982, codfish only). These results suggest that the GnRH of amphibia is identical to LH-RH, but that of birds, reptiles, teleosts and elasmobranchs has substitutions at positions 7 and 8. The similarity of the materials in tilapia, reptiles and birds was further demonstrated by similar migration patterns of the LH-RH-like immunoreactive materials in chromatography procedures; the immunoreactive materials in amphibian and rat hypothalamic extracts had migration patterns similar to LH-RH, and these patterns were different from the patterns for tilapia, reptiles and birds (King and Millar, 1979, 1980). Why amphibia have a LH- RH the same as mammals is not clear at this time. However, Eiden and Eskay (1980) have shown that sympathetic ganglia of bullfrogs, Rana catesbeiana, have a LH-RH molecule that is different from the brain material, indicating the presence of two such factors in amphibia. Brain extracts from codfish contain three immunoreactive LH-RH-like materials (Barnett et al., 1982). On the basis of chromatography studies, two of these materials are similar in size to LH-RH and the third is a larger molecule, suggested to be a precursor. Idler and Crim (1983) also have evidence for a GnRH fraction from winter flounder, Pseudopleuronectes americanus, that has a higher molecular weight than LH- RH, as well as one that is similar in size. The chromatography studies by these investigators also indicate that the teleost GnRH molecules are different in structure than LH-RH. King and Millar (1982a, b, c) have determined the primary structure of the GnRH of chickens. Extensive chromatography techniques were applied to the immunoreactive LH-RH-like material, and the crossreactivity of this material with five LH- RH antisera was tested. These studies narrowed the altered position in chicken GnRH to position 8 of LH-RH. Gln 8 -LH- RH was synthesized and was found to have identical chromatographic and immunological behavior as the GnRH in chicken hypothalami. This structure for chicken GnRH was confirmed by extraction of GnRH with this amino acid composition from 250,000 chicken hypothalami. Miyamoto et al. (1982) also concluded that Gln 8 - LH-RH was the most likely structure of chicken GnRH. These investigators purified and characterized a fraction containing GnRH biological activity, as detected by LH and follicle stimulating hormone (FSH) release from dispersed rat pituitary cells. Amino acid analysis of the purified product indicated substitution of-arg 8 - by Glu or Gin, the most plausible being Gin. Chicken GnRH has about 47c of the potency of LH-RH in stimulating LH and FSH release from rat pituitary cells (Miyamoto et al., 1982), whereas it has equal potency with LH-RH in stimulating LH release from chicken pituitary cells in vitro (King and Millar, 1982«). In an abstract it has been reported that the GnRH material in the brain of chum salmon, Oncorhxnchus keta, differs from LH- RH by amino acid substitutions at positions 7 and 8 (Sherwood et ai, 1982). The structure of chum salmon GnRH was reported to be Trp 7 -Leu 8 -LH-RH. Interestingly the authors also reported that this form of GnRH was chromatographically and immunologically similar to the LH-RH-like material in frog sympathetic ganglia. This supports that amphibians have at least two GnRHs, LH-RH (King and Millar, 1979, 1980) and the variant identified in chum salmon. Elucidation of the primary structure of chicken and chum salmon GnRH are clearly major ad\ances in this field. These discoveries will lead to interesting studies on the comparative actions of the different forms

of GnRH on GtH release in a variety of vertebrates, some preliminary results of which are cited below. Although the basic structure of LH-RH has been conserved during vertebrate evolution, it is apparent from the chromatographic and immunological data reviewed above that other variants of LH-RH probably remain to be discovered. At this point it is not possible to suggest a phylogeny for the family of LH- RH molecules in vertebrates, although immunological data suggest that the differences will probably all lie in substitutions at positions 7 and 8. It is also possible that GnRHs exist that are not crossreactive with antisera against LH-RH, and are therefore being missed in some of the extraction studies. However, this may not be a major problem since King and Millar (1982«, b, c) purified and characterized an immunoreactive chicken GnRH, whereas Miyamoto et al. (1982) purified and characterized biologically active chicken GnRH, and both sets of investigators ended with a similar conclusion, Gln 8 -LH-RH. GtH release-response to GnRH in lower vertebrates Since the basic structure of the LH-RH molecule has apparently been conserved throughout vertebrate evolution, the function of this peptide as a stimulator of gonadotropin release may also be a general phenomenon in vertebrates. That such a molecule with a similar function existed even earlier in phylogeny is supported by recent studies on amphioxus (Branchwstoma belcheri Gray, subphylum Cephalochordata; C.-Y. Chang et al., 1983). In this study it was found that injection of the superactive analogue Des-Gly' 0 [D-Ala 6 ]LH- RH ethylamide (LRH-A) into the body cavity of amphioxus caused increases in the amounts of progesterone, but not estradiol, in females, and testosterone in males that could be extracted from homogenates of the whole body. Injection of human chorionic gonadotropin (hcg) also caused increases in progesterone and testosterone, but hcg was less effective than LRH-A. Although the site of action of LRH-A remains to be determined, the authors of this work suggest that it may be on the LH NEUROHORMONES AND REPRODUCTION 687 immuno-positive cells in Hatschek's pit, a structure presumed to be, in part, homologous with anterior pituitary gland. Injection of LRH-A into adult sea lamprey, Petromyzon marinus, following a priming injection with salmon GtH induced ovulation earlier than in controls and increased plasma levels of estradiol (Sower et al., 1982a, b). In Pacific lamprey plasma levels of estradiol were increased by LRH- A injection in a similar experimental protocol. These studies indicate that the agnathans are responsive to LH-RH and are therefore likely to have a GnRH similar in structure to LH-RH. Preliminary studies on the dogfish, Sc\- liorhinus canicula, indicated that injection of LH-RH caused an increase in plasma GtH levels (Dodd, 1975). This was confirmed indirectly by more recent studies in which it was demonstrated that injection of LH-RH into dogfish caused increases in plasma levels of estrogen and androgen: crude dogfish hypothalamic extract was, however, more effective than LH-RH in this regard (Jenkins and Dodd, 1980). Sturgeons, members of the Chondrostei, respond to injection of LH-RH with a biphasic increase in plasma GtH, an early peak at 30 min and a longer term rise at 4 to 15 hr after injection (Barannikova elai, 1982). Ovulation was also induced. It is well documented that LH-RH and synthetic superactive analogues, such as LRH-A, are effective in stimulating GtH release in vivo and in vitro in teleost fishes, and are effective agents for inducing gonadal development and ovulation in certain species under specified conditions (for reviews: Peter, 1982«, b, 1983). Self-potentiation and self-suppression of the GtH release-response were each found to occur on occasion in goldfish treated with LH- RH and LRH-A under specified conditions (Peter, 1980). In mammals multiple injections or long-term exposure by perfusion of superactive analogues of LH-RH generally cause self-suppression of the GtH release-response to an extent that plasma levels of LH and FSH remain basal following further challenges with the analogue (Clayton and Catt, 1981; Vale et al., 1981). However, the self-suppression reported in

688 RICHARD E. PETER goldfish by Peter (1980) is not as marked. Landlocked Atlantic salmon, Salmo salar, implanted with a pellet giving continuous long-term release of a superactive analogue of LH-RH have a chronic increase in plasma GtH levels lasting at least 4 \vk and stimulation of gonadal activity, including ovulation (Crim et al., 1983), without indication of self-suppression of activity. This suggests that some fundamental difference exists in the response of teleost and mammalian GtH cells to LH-RH or LH- RH analogues: more specifically, that the teleost GtH cells are not as susceptible to down regulation of receptors. Alternately, this apparent difference could be attributed to the fact that the teleostean GnRH is different from LH-RH, and that the difference in response by the teleosts is due to use of a peptide that is unnatural for the teleostean GnRH receptors. Although this issue cannot be resolved until we have a selection of teleost GnRHs to test, initial experiments using Trp 7 -Leu 8 -LH-RH on goldfish indicate that it is about as effective as LH-RH in stimulating GtH release (R. Peter, C. S. Nahorniak and M. Sokolowska, unpublished results). Peter (1980, and unpublished results) in studies on in vivo GtH release by goldfish, and Crim et al. (1981) in in vivo and in vitro studies on GtH release in brown trout, Salmo trutta, reported no substantial difference in the response to LH-RH and "superactive" agonistic analogues, except for a more prolonged release-response to LRH-A in goldfish. Van Der Kraak et al. (1983) found that the in vivo response of coho salmon, Oncorh\nchus kisutch, to LRH-A was more sensitive and greatly prolonged compared to the response to LH- RH. Crim et al. (1981) reported that LH- RH analogues that are antagonistic in mammals are also antagonistic in brown trout. Thus, although there are species differences in responsiveness, the receptors on the GtH cells of teleosts can distinguish between LH-RH and various agonistic and antagonistic analogues of LH-RH, similar to mammals. Early studies indicated that amphibia are highh responsive to LH-RH. Thornton and Geschwind (1974) demonstrated that LH- RH and frog, Rana pipiens, hypothalamic extract both stimulated release of LH from frog pituitaries in vitro, as measured by maturation of oocytes in vitro from Xenopus laevis. Injection of LH-RH induced vitellogenesis and ovulation in Xenopus laevis (Thornton and Geschwind, 1974), and spermiation in the treefrog Hyla regilla (Licht, 1974). In crested newts, Triturus cristatus carnifex, daily injections of LH-RH in hypophysectomized animals bearing a pituitary autograft restored spermatogenesis (Mazzi et al., 1974); a single injection of LH-RH into intact female newts induced ovulation in a significant proportion of the animals (Vellano et al., 1974). More recently, Daniels and Licht (1980) found that injection of LH-RH into adult bullfrogs, Rana catesbeiana, caused a rapid increase in plasma levels of LH and FSH. The peak LH and FSK levels, reached at about 10 min after injection, were dose dependent in male bullfrogs; females were less responsive and did not show a clear dose response. At low dosages there was some suggestion of self-potentiation of the LH and FSH release-response. The male bullfrogs were more responsive to the superactive analogue (imbzl-d-his 6, Pro 9 - NEt)-LH-RH than to LH-RH, and the response to the analogue had a longer duration (McCreery et al., 1982). Daily injection of the superactive analogue into females caused daily peaks in LH and FSH to occur, usually with increments in the peak LH levels; ovulation occurred by the fourth day in 3 of 5 animals. Prior injection of an antagonistic analogue decreased the response to the superactive analogue. From these results it is apparent that the GtH cells of anurans distinguish between LH- RH, and superactive and antagonistic analogues, similar to mammals. Similar to teleosts, the Amphibia do not have any marked self-suppression of responsiveness to multiple applications of LH-RH or a superactive analogue. Reptiles apparently are relatively unresponsive to LH-RH. Callard and Lance (1977) found some increase in plasma LH and progesterone levels after injection of LH-RH in the painted turtle, Clinsemw piita. In the green turtle, Chelumn wvla\.

Licht (1980) reported that it was necessary to use a high dose of 1 mg to induce an increase in plasma LH levels. Licht et al. (1982) were not able to detect a change in LH levels in response to large dosages or multiple injections of LH-RH or a superactive analogue in the olive ridley sea turtle, Lepidochelys olivacea. Thus, the lack of sensitivity of the reptiles to LH-RH or its analogues presents the enigma of whether the receptors on the GtH cells are specific to reptilian GnRH and do not recognize LH-RH, which is very unusual since all other vertebrates do respond to it, or whether there is some other explanation, such as rapid enzymatic degradation of GnRH in the peripheral circulation. Although a full discussion of the responses of birds to LH-RH and analogues is somewhat beyond the purview of this paper, I shall briefly review salient points in this area. Early studies demonstrated that various bird species have increased LH release following treatment with LH-RH (Bayle, 1980). Williams and Sharp (1978) found no difference in the response to LH-RH, in terms of the time course of changes in plasma LH levels, of young versus old laying hens (Gallus gallus domesticus). However, there is a decrease in responsiveness to LH-RH as hens proceed through sexual maturation to the.egg laying stage (Wilson and Sharp, 1975). Male chickens have increased plasma LH levels after injection of a LH-RH agonistic analogue (Pethes et al., 1980). Aside from the report that LH-RH and Gln 8 -LH-RH are equipotent in stimulating LH release from dispersed chicken pituitary cells (King and Millar, 1982a), apparently no comparisons have been made on the effects of LH-RH and different analogues of LH-RH on LH and FSH release in birds. However, from the data available it seems reasonable to speculate that the receptors on GtH cells in birds will distinguish agonistic and antagonistic analogues of LH-RH similar to mammals, but the degree of responsiveness to the so-called superactive agonists remains open. With the exception of certain reptiles, the lower vertebrates in general have been found to respond to LH-RH or agonistic NEUROHORMONES AND REPRODUCTION 689 analogues of LH-RH with an increase in blood levels of GtH, or exhibited a response such as an increase in blood levels of sex steroids indicative of increased blood GtH levels. The response by amphioxus to LRH-A indicates that the presence of such receptors extends to chordates ancestral to the vertebrates. The apparent similarity of receptors throughout the lower vertebrates is supported by our recent preliminary findings that goldfish respond with an increase in serum GtH of comparable magnitude to LH-RH, Trp 7 -Leu 8 -LH-RH, Gln 8 -LH-RH and D-Trp 6 -Gln 8 -LH-RH, whether these are given alone or after injection of pimozide, a dopamine receptor antagonist (R. Peter, C. S. Nahorniak and M. Sokolowska, unpublished results). Furthermore, the results of King and Millar (1982a) that LH-RH and Gln 8 -LH-RH are equally effective in stimulating LH release from chicken pituitary cells add credence to the idea that GnRH receptors throughout non-mammalian vertebrates recognize and respond to similar molecules. Mammals, on the other hand, apparently have receptor specialization for LH-RH, because the rat was relatively unresponsive to chicken GnRH. Also, certain other aspects of receptor-ligand interactions seem to differ between mammals and lower vertebrates, namely the apparent resistance of teleosts and amphibia to down regulation of receptors for GnRH. Anatomical and functional evidence for brain areas involved in GnRH LH-RH-like material was found in perikarya of the nucleus preopticus (NPO) of adult Pacific lamprey, Entosphenus tridentata (Crim et al., 1979a) and the western brook lamprey, Lampetra nchardsoni (Crim et al., 19796). The reactive perikarya were usually located laterally in the NPO and were separate from those cells staining with aldehyde fuchsin, the classical neurosecretory stain. LH-RH immunoreactive material was found in the neurohypophysis, along with the aldehyde fuchsin stained material. Similar results for the lamprey Entosphenus japonica were reported by Nozaki and Kobayashi (1979). No LH-RH immunoreactive materials have been found

690 RICHARD E. PETER in the brain of hagfish (Crim et al., 1979a; Nozaki and Kobayashi, 1979). Without any experimental data, it is difficult to place any functional significance on these observations. Nozaki and Kobayshi (1979) did not detect LH-RH immunoreactive material in the pituitary and no reactive perikarya were found in the brain of the dogfish Triakis scyllia. There are no studies available indicating that some brain region in chondrichthyes supports gonadal activity via secretion of GnRH. LH-RH immunoreactive fibers and perikarya have been found in the brain, and in the neurohypophysis of the proximal pars distalis in several teleost species (for reviews: Ball, 1981; Peter 1982rt, b, 1983), and will be selectively reviewed here. In platyfish (Xiphophorus maculatus, X. helleri, and X. sp.) (Schreibman et al., 1979, 1982; Miinz et al., 1981) and goldfish (Kah et al., 1982) reactive perikarya were found in the nucleus lateral tuberis (NLT), along the lateral extensions of the preoptic recess in the ventral-lateral nucleus preopticus periventricularis (NPP), and either in the anterior-ventral telencephalon (platyfish) or bordering the olfactory bulbs and olfactory nerves (goldfish; O. Kah, personal communication). LH-RH reactive perikarya bordering the olfactory bulbs have been described in many teleost species (Miinz et al., 1982). Platyfish also have reactive perikarya in the dorsal midbrain just posterior to the posterior commissure (Miinz et al., 1981). In the three-spined stickleback, Gasterosteus aculeatus, reactive perikarya were found in the pars magnocellularis portion of the NPO, in the dorsal thalamus (nucleus dorsomedialis thalami and nucleus ventromedialis thalami), the nucleus posterioris periventricularis in the ventral-posterior thalamus, and in the midbrain tegmentum just posterior of the posterior commissure (Borg et al., 1982). Goos and Murthanoglu (1977) found reactive perikarya in the dorsal-medial telencephalon of rainbow trout. Immunoreactive fibers are widely distributed in the brain of teleosts, with concentrations particularly in the ventral and lateral preoptic regions, the NLT region, the dorsal thalamic region, and the dorsal telencephalon (for review, Peter, 1982fl, b, 1983). In brain lesioning studies on goldfish, destruction of the NLT, particularly in the pituitary stalk region and posterior to the pituitary stalk, caused gonadal regression or block of gonadal recrudescence (for review, Ball, 1981; Peter, 1982«, b, 1983). Such lesions caused the daily cycle in serum GtH levels, normally found in goldfish undergoing ovarian recrudescence, to disappear (Peter, 19826). These results correspond with the observation by Kah et al. (1982) of LH-RH immunoreactive perikarya in the posterior portion of the NLT of goldfish. Brain lesioning studies on Atlantic salmon (Dodd et ai, 1978) and killifish Fundulus heteroditus (Pickford et al., 1981), also demonstrated that destruction of a part of the NLT caused gonadal regression. Peter and Crim (1978) found ovarian regression in goldfish after placing large lesions in the preoptic region. This suggests involvement of the preoptic region in GnRH secretion in goldfish, consistent with localization of LH-RH in immunoreactive perikarya in this region. However, the effects of preoptic lesions were not confirmed in other studies on goldfish (see discussions by Peter, 19826, 1983). Thus, the functional evidence available on teleosts supports only the NLT as being the source for GnRH involved in regulation of GtH secretion. Although this is the conclusion at this time, further studies are still necessary to determine if the LH-RH immunoreactive perikarya in the preoptic region, or other areas are functionally involved in regulation of GtH release in goldfish or other species. A large literature exists on the immunocytochemical localization of LH-RH reactive material in the brain of amphibia (for review, Ball, 1981), and will be only briefly reviewed here. In Rana temporaria reactive perikarya were found in an unpaired nucleus in the medial septal region, just anterior to the preoptic recess (Goos et al., 1976). Paired tracts emerging from this nucleus were traced to the median eminence. A similar distribution of LH- RH immunoreactive perikarya and pathways has been described in R. pipien ^ (Alpert

et ai, 1976), R. catesbeiana (Alpert et ai, 1976; Nozaki and Kobayashi, 1979), Xenopus laevis and Cpiops pyrrhogaster (Nozaki and Kobayashi, 1979), and other species (for review, Ball, 1981). In Xenopus lan'is and Tnturus mannoratus immunoreactive perikarya were also found in the ventral infundibular region (Doerr-Schott and Dubois, 1978; Ball, 1981). These data fit well with the results available from brain lesioning experiments. A series of experiments on R. temporaria, involving cuts at various brain levels and isolations of the ventrobasal hypothalamus, demonstrated that the ventrobasal hypothalamus (area ventralis tuberis cinerei, or infundibular nucleus) is necessary for normal seasonal gonadal development (Dierickx, 1974). However, connection with an anterior center is needed for ovulation. The precise location of this ovulation center, and whether it serves as a source for GnRH, or neural input to the infundibular region, is not clear. Mazzi (1978) found that insertion of a permanent barrier between the preoptic region and tuberal hypothalamus blocked gonadal development in the newt Triturus cristatus. No lesioning studies are available in which blood levels of LH and FSH have been measured. Nevertheless, the results available indicate involvement of GnRH producing centers in the ventrobasal hypothalamus and the preoptic-septal region in regulation of pituitary activity. The contrasting data available on Triturus and Rana suggest that the relative importance of these two centers can vary between species. In the snake Elaphe climacophora LH-RH immunoreactive perikarya were found in the medial septal region and the adjacent medial preoptic region, and a few were scattered in the bed nucleus of the hippocampal commissure (Nozaki and Kobayashi, 1979); no reactive perikarya were found in the ventrobasal hypothalamus. Fiber tracts from these areas coursed to the median eminence. No reactive perikarya were found in studies on other species of reptiles (Nozaki and Kobayashi, 1979), except for a few scattered cell bodies in the dorsal telencephalon of the lizard Lacerta muralis (Doerr-Schott and Dubois, 1978). NEUROHORMONES AND REPRODUCTION 691 Brain lesioning studies on the lizard Anolis carolinensis demonstrated that lesions in the anterior hypothalamic-preoptic area, but not more anteriorly in the septal region, caused testicular regression (Wheeler and Crews, 1978; Crews, 1979). Without information on the distribution of LH-RH immunoreactive perikarya in Anolis it is difficult to reconcile the data from these lesioning experiments with the immunohistochemistry data on the snake, which places the emphasis on the septal region as the potential source of GnRH. However, this may not be a major problem as the septal region does grade into the preoptic region; clearly, further studies on reptiles are necessary. In mammals (for review, Sharp and Fraser, 1978) and birds (for review, Bayle, 1980) the anterior preoptic region and the infundibular region (arcuate nucleus) have been established by LH-RH immunohistochemical studies and lesioning studies as sources for GnRH. In mammals there are differences between species in the role of the suprachiasmatic-preoptic region versus the arcuate nucleus in the regulation of tonic release of LH-RH for maintenance of gonadal activity and the ovulatory surge of LH-RH. In some mammals LH-RH perikarya may not be present in the arcuate nucleus. A similar binary distribution of GnRH perikarya appears in many of the so-called lower vertebrates. Accordingly, there can be shifts in the relative importance of these two centers in lower vertebrates, as is apparent in anurans. However, the original location for GnRH perikarya may be preoptic, if the agnathans are accepted as an indicator of the condition in ancestral vertebrates. GONADOTROPIN RF.LEASE-I NH1BITORY FACTOR Lesions in the anterior and lateral regions of the hypothalamus of the dogfish Scyliorhinus canicula caused an increased frequency of ovulation and oviposition (unpublished results cited by Dodd, 1975). This was suggested as evidence for a hypothalamic GtH release-inhibitory factor, although other interpretations such as

692 RICHARD E. PETER neural inhibition of GnRH neurons are also possible. A GtH release-inhibitory factor (GRIF) was interpreted to be present in goldfish on the basis of brain lesioning experiments (Peter etai, 1978); large lesions in the NLT region caused a marked prolonged increase in serum GtH levels and ovulation in gravid female goldfish, indicative that the lesions abolished a tonic inhibition on GtH release. Further studies by Peter and Paulencu (1980) demonstrated that lesions in the NLT region that destroyed the pituitary stalk caused the marked increase in serum GtH release, and that lesions restricted to the NLT either had no effect if placed anteriorly or caused gonadal regression if placed posteriorly. Lesions placed in various hypothalamic regions indicated that GRIF originates in the preoptic region in the anterior-ventral NPP, and courses to the pituitary via bilateral pathways in the lateral preoptic and anterior hypothalmic regions. Recently J. P. Chang et al, (1983) found that drugs that inhibited dopamine synthesis caused an increase in serum GtH levels in goldfish, whereas those that blocked synthesis of norepinephrine had no effects; clonidine, an a-adrenergic receptor agonist, stimulated serum GtH levels. These results suggest that dopamine has an inhibitory action on GtH release in goldfish. Further studies demonstrated that dopamine and apomorphine, a dopamine receptor agonist, can decrease the high spontaneous release of GtH in goldfish with lesions in the preoptic region, and also modulate the release of GtH stimulated by LRH-A, or block the action of LRH-A (Chang and Peter, 1983«). This indicates that dopamine has GRIF activity directly on GtH cells. Furthermore, blocking the GRIF activity of dopamine by injection of pimozide, a dopamine receptor antagonist, greatly potentiates the GtH release-response to LRH-A (Chang and Peter, 19836). The combination of treatment with pimozide and LRH-A is a highly effective means for inducing ovulation in goldfish (Sokolowska?/«/., 1982: Chang and Peter, 19836), and common carp, Cyprinus carpio (Billard et «/., 1983). The brain origin of the dopamine that influences GtH cell activity in goldfish is not known. There are no data available from other groups of lower vertebrates indicating that dopamine has GRIF activity. However, in certain mammals dopamine has similar GRIF activity (for literature see Chang and Peter, 1983a). In chickens Knight et al. (1982) found evidence for an inhibitory effect of dopamine on LH release. Although the authors interpreted these actions to be via central effects of dopamine to influence GnRH release, the results could in part be due to direct GRIF activity of dopamine. The studies on goldfish establish a dual neuroendocrine control of GtH release, consisting of counterbalancing stimulatory and inhibitory components. This is similar to the neuroendocrine regulation system for other vertebrate anterior pituitary hormones, e.g., growth hormone secretion is regulated by a releasing hormone and the inhibitory hormone somatostatin. The function of the releasing hormone is obvious in such a system. The role of the inhibitory hormone is to modulate spontaneous release of the pituitary hormone and the action of the releasing hormone, the GRIF actions of dopamine in goldfish being such an example. The distribution in vertebrates of a similar dual neuroendocrine regulation system for GtH will be an interesting question for future research. ACKNOWLEDGMENTS I wish to thank J. P. Chang for l "viewing this manuscript. Unpublished results reported herein were supported by grants A6371 and G0838 from the Natuial Sciences and Engineering Research Council of Canada. A travel grant from the Alberta Heritage Foundation for Medical Research is gratefully acknowledged. REFERENCES Alpert, L. C.J. R. Brawer, I. M. D.Jackson, and S. Reichlin. 1976. Localization of LHRH in neurons in frog brain (Rana pipiens and Rana catesbeiana). Endocrinology 98:910-921. Ball, J. N. 1981. Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles. Gen. Comp. Endocrinol. 44:135-170. Barnett, F. H., J. Sohn, S. Reichlin, and I. M. D. Jackson. 1982. I hree lutemizing hormone-

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