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Relationship between brain gonadotrophin-releasing hormone (GnRH) and seasonal reproductive cycle of “caribe colorado,” Pygocentrus notatus
GENERAL
AND
COMPARATIVE
Relationship between (GnRH) and Seasonal FRANCA GENTILE, Department
of Physiology,
64,239-245
ENDOCRINOLOGY
(1986)
Brain Gonadotrophin-Releasing Reproductive Cycle of “Caribe Pygocentrus notatus OFELIA LIRA, AND DAYSSI MARCANO-DE
“J. M. Apartado
Vargas” 47633,
Medical Caracas
School, Universidad 1041-A, Venezuela
Central
Hordes Colora COTTER de Venezuela,
Accepted May 13; 1986 Immunoreactive gonadotrophic hormone-releasing hormone (ir-GnRH) was detected in hypothalamic and telencephalic extracts of the Venezuelan freshwater fish “caribe colorado,” Pygocentrus notatus. Hypothalamic ir-GnRH from female fish demonstrated displacement curves parallel to those of synthetic mammalian luteinizing hormone-releasing hormone (LHRH). The content of hypothalamic and telencephalic ir-GnRH from female fish was more than four-fold greater than that of male animals. Also, fluctuations that depended on the reproductive state and environmental conditions (rainfall) occurred in females but not in males. Thus, ir-GnRH levels were higher in hypothalamic and telencephalic extracts from sexually mature females than in those from fish sampled outside the climatically determined breeding season. 0 1986 Academic Press. Inc.
The “‘caribe colorado,” Pygocentvus notutus, is a seasonal breeder widely distributed in rivers, lagoons, and ponds of the Venezuelan plains. The fish usually reach maturity in their third year and have an annual gonadal cycle closely related to changes in the environmental conditions of the Venezuelan plains (Mago-Leccia, 1970; Gonzalez, 1980), which are characterized by a dry season (December-April) followed by a rainy season (May-November) . The annual gonadal cycle of this species shows a pattern of gonadal maturation starting a few months before the beginning of the rainy season. Thus, the process of gonadal recrudescense shows a positive correlation with the dry season, in which the maximal gonosomatic index is achieved (May). The caribe Colorado then awaits the onset of the rainy season, which provides favorable environmental conditions, for the triggering of spawning in the female and r To whom correspondence should be sent.
and reprint
requests
spermiation in the male. Spawning, which is completed in a very short time, is followed by a period of sexual quiescence during the ‘wet season. Studies on various mammalian species have shown that gonadotrophic activity in the pituitary glan is influenced by the neuropeptide, gonadotrophic hormone-releasing hormone (GnRH), derived from specific brain regions. Similarly, in teleosts, gonadotrophic activity is regulated by releasing factors produced in the thalamus and other parts of the brain et al., 1978; Peter and Grim, 1979; 1981). Recently, the presence in tele GnRH that is similar to luteinizi mone-releasing hormone (LHRH) was demonstrated, and GnRH irnrnu~or~ac~~v~ perikarya and axons have been fo~~~~ in the brain of several teleost species (for reviews see Crim et al., 1978; Crim and V 1983; Peter, 1983; Demski, 1984). In tion, a functional role for GnRIl in teleost fish has been proposed (Goos and Mu anoglu, 1977; Schreibman et al”., 1979; et al., 1982; Mtinz et al., 1982), and &ret239 0016-64X0/86 $1.50 Copyright All rights
Q 1986 by Academic Press. Inc. of reproduction in any lorm reserved.
240
GENTILE,
LIRA, AND MARCANO-DE
studies have established a correlation between the secretory activity of some hypothalamic nuclei with the annual reproductive cycle in various teleosts (Zambrano, 1972). Using radioimmunoassay procedures, we have demonstrated in the caribe colorado, P. not&us, the presence of a substance with GnRH-like immunoreactivity in different brain regions. Also, the immunoreactive-GnRH (ir-GnRH) content was shown to correlate with environmental conditions and with the gonosomatic index. This provides a basis for studies on the regulation of reproduction in this species. MATERIALS
AND METHODS
Adult male and female P. notorus were collected, in groups of approximately 20, over 5 months (MarchJuly) from lagoons and ponds of the Guarico River and its tributaries (Guarico and Apure states) and transported in aerated tanks to the laboratory (some 18 km south of the recollection area). The fish were kept in aerated tap water held in fiberglass tanks and maintained at a temperature of 28” for a period of 12 hr. Several measurements (total length, standard length, body and gonad weights) were recorded for each fish. Animals were sacrificed by decapitation. The brains were immediately removed and the tissues were weighted, frozen, and maintained at -40” until the hormone extraction. At the same time, the gonads were extirpated and weighed, and the gonosomatic index (GSI) was calculated: GSI =
weight of gonad weight of fish
x 100%
Tissue dissection and extraction procedures. Following removal of the brain, the pituitary (P) was set aside and the brains were then dissected. Both the whole hypothalamus (H) and remaining forebrain, comprised of telencephalon and olfactory bulb (TEL), were homogenized separately for the extraction of the hormone. Each region from two animals was pooled, and two determinations were made for each brain region analyzed (hypothalamus, telencephalon, and pituitary gland). After weighing, the brain tissues were homogenized in ice-cold 2 N acetic acid (at a final tissue concentration not exceeding 50 mg/ml) and centrifuged at 5OOOgfor 15 min, and the supernatant was lyophilized (King and Millar, 1980). Lyophilized extracts were resuspended in 250 ~1 of phosphosaline buffer (0.04 M phosphate, pH 7.4,0.6% NaCl, and 1% serum albumin) and 50 ~1 was used for assay of GnRH immunoreactivity.
COTTE
Radioimmunoassay of GnRH. This assay was performed using an antiserum (generously provided by Dr. N. White, Chelsea Hospital for Woman, London) at a final dilution of l:lO,OOO. The sensitivity of the assay was 30 pgiml and the standard curve was linear between 30 and 1000 pgiml. Synthetic LHRH (Peninsula Laboratories, San Carlos, CaIif.) was iodinated using the chloramine-T method and [lZ51]LHRH was separated from free iodine using cellulose (CF-11, Whatman Inc., England). Details of the specificity of the antibody used to measure LHRH have been described elsewhere (Jeffcoate et al., 1974). The antiserum does not recognize the peptide fragments produced after cleavage at the Glys-Leu7 bond, but it does cross-react with LHRH after removal of the COOH-terminal glycinamide. Hypothalamic extracts obtained from female fish, collected in April, were assayed in serial dilutions in triplicate and an inhibition assay was performed to establish the identity of the GnRH-like immunoreactive material. Extrahypothalamic brain extracts were assayed in triplicate at a single dilution. Statistical analysis. Data for all parameters were expressed as the means I SEM and statistical comparisons were made using Student’s t test.
RESULTS Gonosomatic Index (GSI)
During 1983, maximal precipitation (100 mm) occurred in the recollection area from April to November. No marked variations in air temperatures (about 25”) occurred during this period. The body and gonad weights of female and male caribe Colorado are shown in Tables 1 and 2. The fish underwent gonadal maturation from March 11 to May 4, and spawning and spermiation occurred between June 7 and July 27. The GSI for females increased from 0.92 2 TABLE BODY
WEIGHT FEMALE
AND
Pygocentr-us notatus
PERIODICALLY
DURING
March April May June July
N 11 14
4 7 27
Nore. number
9 10 6 12 I
Values are of animals.
INDEX (GSI) SAMPLED
A REPRODUCTIVE Body
Date
1
GONOSOMATIC
424.2 380.5 382.6 555.6 319.4 expressed
CYCLE
weight k) i 2 i +F
GSI mJ)
141.39 16.82 41.60 25.51 30.90
as means
OF
0.92 4.08 6.01 2.97 0.26 t
SEM
-c 5 ir k i
0.13 0.48 0.43 0.33 0.03
of the
BRAIN GnRH AND FISH REPRODUCTIVE TABLE BODY MALE
CYCLE
2
AND GONOSOMATIC INDEX (GSI) OF Pygocentvus notatus SAMPLED PERIODICALLY DURING A REPRODUCTIVE CYCLE WEIGHT
Body weight
GSI
N
ld
(%I
i
299.1 c 20.08
April 14
May 4 June 7 July 27
1;
Date
March 11
I
380.2 422.5 422.1 306.2
Note. Values are expressed number of animals.
i f ? i
16.14 42.93 34.00 30.30
as means
0.14 t 0.03
i ? -t
0.37 0.49 0.29 0.07
0.06 0.03 0.03
k 0.01
c SEM
of the
0.13% on March 11 to 5.01 2 0.43% on May 4, and declined sharply by June 7 when in most cases ovulation was incomplete. By July 27, all fish captured had spawned (Table 1). In males the increase and decrease of the GSI were less marked (Table 2). Characterization
of Hypothalamic
GnRH
Serial dilutions of tissue extracts from hypothalami caused a dose-dependent decrease in the binding of labeled LHRH to its specific antibody. The binding curve was parallel to that given by the addition of increasing concentrations of unlabeled synthetic LHRH. Relation between Regional Brain Concentrations and Season OY Sex Immunoassayable GnRH was present in extracts of the hypothalamus and the telencephalon in all fish. However, significant differences were noted according to the sex or the physiological condition of the fish. In females, the ir-GnRH content of the hypothalamus and the telencephalon changed significantly during the year. No such changes, however, were observed in male tissues (Figs. 1 and 2). In females, lower ir-GnRH levels were found in hypothalamic extracts in March compared to April and May. The ir-GnRH content in the hypothalamus was highest in April, a month before the maximal GSI was obtained. There was also a decrease of ir-
,i FIG. 1. Sex and seasonal differences in the content of ir-GnRH in hypothalamic extracts from female f and male (A) PygocentrL*s notatm in relation to annual changes in male (A) and female (0) GS% during 1983. Values are expressed as means f SEM of tbe number of determinations shown. **P < 0.001 when compared with March, May. June. and July. *P < 0.005 when compared with March and Juiy. *P < 0.001 when compared with June.
GnRH activity in hypothalamic extracts after spawning. In addition, significant sex differences were seen in the ir-GnRH content during April and May. In April, the irGnRH content of the female hypotbalam~s was approximately four times greater than that of males (Fig. 1). In the telence~~a~~~, ir-GnRH levels increased just before spawning (May), paralleling the G creases, and then decreased by the time of spawning. Again, sex differences in the content of ir-GnRH in this brain region were observed, with a five-fold increase in the ir-GnRH content in female compared to male telencephalon (Fig. 2). In p$uitary extracts of caribe Colorado, ir-GnRH was not present in either female or male specimens .
Our results suggest the presence of a substance related to synthetic LHR hypothalamic and telencephalic extracts of P. noaatus. The distribution of the irnrn~~o~
242
GENTILE,
LIRA,
AND MARCANO-DE
?6-
2
257 -1
MARCH APRIL
MAY
JUNE
JULY
-0
FIG. 2. Sex and seasonal differences in the content of ir-GnRH in telencephalic extracts from female (Of and male (a) Pygocentrus notatus in relation to annual changes in male (A) and female (0) GSI during 1983. Values are expressed as means f SEM of the number of determinations shown. **P < 0.001 when compared with March, April, June, and July *P < 0.005 when compared with March and July.
reactive material seems to be identical in both male and female fish. However, the levels of ir-GnRH showed fluctuations according to the reproductive state in females but not in males. The occurrence of GnRH in this species has not previously been investigated, but the present study clearly demonstrates the presence of immunoreactive GnRH in this teleost. GnRH-immunoreactive systems have been studied in several teleosts. Immunoreactive cell bodies have been demonstrated in the ventral parts of the olfactory nerve and bulb in the goldfish (Stell et al., 1984; Kah et al., 1984), in the ventrolateral parts of the anterior nucleus preopticus periventricularis and in the posterior nucleus lateralis tuberis in mature goldfish (Kah et al., 1984), and in the nucleus preopticus in carp (Pan et al., 1979) and platyfish (Schreibman et al., 1982). Immunoreactive GnRH has also been demonstrated within telencephalic perikarya of Salmo gairdneri (Goos and Murathanoglu, 1977) and goldfish (Kah et al., 1984). How-
COTTE
ever, Deery (1974) failed to detect immunoassayable GnRH in one elasmobranch (dogfish) and another teleost (goldfish). The similarity in the slope of the displacement curves in competitive inhibition assays with mammalian LHRH suggests that an immunologically similar LHRH is present in P. notatus. Previous studies have suggested that the teleost GnRH is different from the mammalian peptide (King and Millar, 1979), and recently a salmon GnRH was characterized as being a decapeptide which differs from mammalian LHRH at positions 7 and 8 (Sherwood et al., 1983). Our demonstration that GnRH P. notatus seems to be immunologically similar to that of mammals disagrees with these findings. However, reports of the occurrence of GnRH in teleosts have been conflicting, this possibly being due to seasonal fluctuations in the hypothalamic content of ir-GnRH in these fish. In this study, high concentrations of ir-GnRH were detected in hypothalamic and telencephalic extracts from sexually mature female P. notatus captured during the dry season (April-May) where the maximal gonadal maturation was observed, and the content of ir-GnRH was four- to six-fold lower in extracts from specimens captured outside the breeding season. Similar results were found by King and Millar (1980) in gravid Tilapia, where immunoreactive GnRH was present but was absent from brain sampled during the nonbreeding season. Also, immunoreactive cell bodies were located in the posterior nucleus lateralis tuberis only in mature goldfish (Kah er al., 1984). Perhaps the most significant finding to emerge from the present work is the marked difference in the content of irGnRH between the sexes and its relationship with the various stages of the reproductive cycle. Thus, male fish had significantly less GnRH than females in periods of maximal gonadal maturation. In female P. notatus, seasonal changes in ir-GnRH content appeared to vary independently in
BRAIN GnRH AND FISH REPRODUCTIVE
two of the regions assayed (hypothalamus and telencephalon) . No decrease in binding of labeled LHRH in the competitive radioimmunoassay used was caused by extracts of the pituitary gland from this fish. This suggests that an immunologically reactive LHRH is absent, or that the amount present is below the detection limit of the assay. These results are in disagreement with previous reports on other species. GnRH immunoreactive fibers have been described in the proximal pars distalis in several species (Dubois et al., 1979; Schreibman et al., 1979; Nozaki and Kobayashi, 1979; Miinz et al., 1981; Kah et al., 1984). Regions which show changes in GnRH content dependent on the gonosomatic index are known to be involved in teleostean reproduction. Regulation of gonadotrophic secretion seems to be centered in the nucleus lateralis tuberis of the hypothalamus (Peter and Crim, 1979) and lesions of the posterior nucleus lateralis tuberis resulted in a decreased gonosomatic index (Peter, 1982). On the other hand, the telencephalon has been implicated in the control of sexual behavior, nest building, spawning, and sperm release (Davis et al., 1976; Schwagmeyer et al., 1977) and recent lesion studies showed that destruction of the medial nuclei of the ventral telencephalon results in impairment of spawning behavior in male goldfish (Kyle and Peter, 1982). Furthermore, GnRH immunoreactive ceils have been seen in the ventromedial telencephalon (Goos and Murathanoglu, 1977) and in the posterior parts of the area dorsalis telencephali (Kah et al., 1984). It is significant that a progressive increase in the female GnRH content from March to May is consistent with the expected pattern of gonadal development culminating in spawning. During spawning, which occurs principally in June in this species, the hypothalamic and telencephalic GnRH content is higher than in July when the fish are reproductively quiescent.
CYCLE
It is interesting to note that in contrast to the hypothalamus where the conc~~trati~~ of ir-GnRH was highest a month before the maximal GSI, the highest concentration of ir-GnRH in the telencephalic extracts was found to parallel the increase in the GSI. This further suggests that this brain region could be important in aspects of the reproductive physiology in addition to gonadotrophin secretion, such as triggering of spawning or the control. of sexual behavior related to spawning. In mammals, GnRH has been impIicate~ in some aspects of sexual behavior ( and Carol, 1979; De Wied and G&pen, 1977). Another possibility is that the tide is acting as a neuromodulator, fying the activity of the other neural pathways as has been suggested for ot vertebrates. In summary, we have shown that in P. notatus high levels of ir-GnRH are related to changes in the reproductive cycle and in environmental conditions. The results suggest several functional and anatomical correlations which are likely to reflect mechanisms controlling reproductive function in teleosts. Marked sex and season ences in brain regions in which G concentrated further suggests that in the central nervous system is rel the reproductive physiology.
This investigation was supported by CONICIT Grant Sl-1623. We are grateful to Dr. N. White (Chelsea Hospital for Women, London) for the generous gift of anti-LHRH, and to Mr. Otto Castillo and the other members of FONAIP for their valuable help in the collection of the caribe Colorado. We also thank Mr. Javier Bricefio for generous help in the drawing of graphs.
REFERENCES Ball, J. N. (1981). Hypothalamic control of the pars distal6 in fishes, amphibians, and reptiles. Gen. Comp. Endocrinol. 44, 135-170. Borg, B., Goes, I-I. J. Th., and Terlou, M. (P982).
244
GENTILE,
LIRA, AND MARCANO-DE
LHRH-immunoreactive cells in the brain of the three-spined stickleback, Gasterosteus aculeatus L. (Gasterosteidae). Cell. Tissue. Res. 226, 695-699. Crim, J. W., and Vigna, S. R. (1983). Brain, gut and skin peptide hormone in lower vertebrates. Amer. Zool. 23, 621-638. Crim, J. W., Dickhoff, W. W., and Gorbman, A. (1978). Comparative endocrinology of piscine hypothalamic hypophysiotropic peptides: Distribution and activity. Amer. 2001. 18, 41 l-424. Davis, R. E., Kassel, J., and Schwagmeyer, P. (1976). Telencephalic lesions and behavior in the teleost, Macropodus opercularis: Reproduction startle reaction and operant behavior in the male. Behav. Biol.
18, 16.5-177.
Deery, D. J. (1974). Determination by radioimmunoassay of the luteinising hormone releasing hormone (LHRH) content of the hypothalamus of the rat and some lower vertebrates. Gen. Comp. Endocrinol.
24, 280-285.
Demski, L. (1984). The evolution of neuroanatomical substrates of reproductive behaviour: Sex steroid and LHRH-specific pathways including the terminal nerve. Amer. 2001. 24, 809-830. De Wied, C., and Gispen, W. H. (1977). Behavioral effects of peptides. In “Peptides in Neurobiology” (H. Gainer, ed.), pp. 397-448. Plenum, New York. Dubois, M. P., Billard, R., Breton, B., and Peter, R. E. (1979). Comparative distribution of somatostatin, LHRH, neurophysin, and endorphin in the rainbow trout: An immunocytochemical study. Gen.
Camp.
Endocrinol.
37, 220-232.
Gonzalez, J. A. (1980). “Reproduction y Crecimiento de1 “Caribe Colorado” Serrasalmus notutus Lutken, 1974 (Teleostei, Characiformes, Characidae) en Los Llanos Venezolanos.” Trabajo especial de Grado, Escuela de Biologia, Facultad de Ciencias, UCV Caracas, Venezuela. Goos, H. J. Th., and Murathanoglu, 0. (1977). Localisation of gonadotropin releasing hormone (GRH) in the forebrain and neurohypophysis of the trout (Salmo gairdneri). Cell. Tissue. Res. 181, 163-168. Jeffcoate, S. L., Holland, D. T., Fraser, H. M., and Gunn, A. (1974). Preparation and specificity of antibodies to the decapeptide, luteinizing hormone releasing hormone (LHRH). Zmmunochemistry 11, 75-77. Kah, O., Chambolle, P., Dubourg, P., and Dubois, M. I? (1984). Immunocytochemical localization of luteinizing hormone releasing hormone in the brain of goldfish Carassius auratas. Gen. Comp. Endocrinol. 53, 107-115. King, J. A., and Millar, P. R. (1979). Heterogeneity of
COTTE
vertebrate luteinizing hormone releasing hormone. Science 206, 67-69. King, J. A., and Millar, P. R. (1980). Comparative aspects of luteinizing hormone releasing hormone, structure and function in vertebrate phylogeny. Endocrinology 106, 707-717. Kyle, A. L., and Peter, R. E. (1982). Effects of forebrain lesions on spawning behavior in the male goldfish. Physiol. Behav. 28, 1103- 1109. Mago-Leccia, E (1970). Estudios preliminares sobre la ecologia de 10s peces de 10s llanos de Venezuela. Acta Biol. Venez. 7, 71-102. Moss, R. L., and Carol, A. (1979). Sexual function and brain peptide. In “Brain Peptides: A New Endocrinology” (A. M. Goot Jr., E. J. Peck Jr., and A. E. Boyd III, eds.), pp. 325-346. Elsevier, Amsterdam. Mtinz, H., Class, B., Stumpf, W. E., and Jennes, L. (1982). Centrifugal innervation of the retina by luteinising hormone releasing hormone (LHRH)immunoreactive telencephalic neurons in teleostean fishes. Cell. Tissue Res. 222, 313-323. Miinz, H., Stumpf, W. E., and Jennes, L. (1981). LHRH-systems in the brain of platyfish. Brain Res. 221, I-13. Nozaki, M., and Kobayashi, H. (1979). Distribution of LHRH like substance in the vertebrate brain as revealed by immunocytochemistry. Arch. Histol. Japan. 42, 201-219. Pan, C. H., Feng, M. Q., Ling, N. C., Pao, S., Xu, W. Q., Xu, G. X., and Shen, R. C. (1979). Immunocytochemical studies on gonadotropin releasing hormone (GRH) secretory nucleus of the carp (Cyprinus carpio). Acta Biol. Exp. Sin. 12, 305-310.
Peter, R. E. (1982). Neuroendocrine contro1 of reproduction in teleosts. Canad. J. Fish Aqaat. Sci. 39, 48-55. Peter, R. E. (1983). Evolution of neurohormonal regulation of reproduction in lower vertebrate. Amer. Zool.
23, 685-695.
Peter, R. E., and Crim, L. W. (1979). Reproductive endocrinology of fishes: Gonadal cycles and gonadotropin in teleost. Annu. Rev. Physiol. 41, 323-335. Schreibman, M. P., Halpern, L. R., Goos, H. J. Th., and Margolis-Kazan, H. (1979). Identification of luteinising hormone-releasing hormone (LHRH) in the brain and pituitary gland of a fish by immunocytochemistry. J. Exp. Zool. 210, 153-160. Schreibman, M. P., Margolis-Kazan, H., and Halpem-Sebold, L. (1982). Immunoreactive gonadotropin and luteinising hormone releasing hormone in the pituitary gland of neonatal platyfish. Gen. Comp. Endocrinol. 37, 385-391. Schwagmeyer, I?, Davis, R. E., and Kassel, J. (1977). Telencephalic lesions and behavior in the teleost
BRAIN GnRH AND FISH REPRODUCTIVE opercularis (L): Effects on telencephalon and olfactory bulb ablation on spawning and foamnest building. Behav. Biol. 20, 463-470. Sherwood, N., Eiden, L., Brownstein, M., Spiess, J., Rivier, J., and Vale, W. (1983). Characterization of a teleost gonadotropin-releasing hormone. Macropodus
Proc.
Nat/.
Acad.
Sci. USA
80, 2794-2798.
CYCLE
245
Stell, W. K., Walker, S. E., Chohan, K. S., and Ball, A. K. (1984.). The goldfish nerves terminalis: An LHRH- and FMRF amide-immunoreactive oifactoretinal pathway. Proc. Nati. Acad. Sci. USA 81, 940-944.
Zambrano, D. (1972). Innervation of the teleost pituitary. Gen. Comp. Endocrinoi. 3 (suppl.), 22-31.
AND
COMPARATIVE
Relationship between (GnRH) and Seasonal FRANCA GENTILE, Department
of Physiology,
64,239-245
ENDOCRINOLOGY
(1986)
Brain Gonadotrophin-Releasing Reproductive Cycle of “Caribe Pygocentrus notatus OFELIA LIRA, AND DAYSSI MARCANO-DE
“J. M. Apartado
Vargas” 47633,
Medical Caracas
School, Universidad 1041-A, Venezuela
Central
Hordes Colora COTTER de Venezuela,
Accepted May 13; 1986 Immunoreactive gonadotrophic hormone-releasing hormone (ir-GnRH) was detected in hypothalamic and telencephalic extracts of the Venezuelan freshwater fish “caribe colorado,” Pygocentrus notatus. Hypothalamic ir-GnRH from female fish demonstrated displacement curves parallel to those of synthetic mammalian luteinizing hormone-releasing hormone (LHRH). The content of hypothalamic and telencephalic ir-GnRH from female fish was more than four-fold greater than that of male animals. Also, fluctuations that depended on the reproductive state and environmental conditions (rainfall) occurred in females but not in males. Thus, ir-GnRH levels were higher in hypothalamic and telencephalic extracts from sexually mature females than in those from fish sampled outside the climatically determined breeding season. 0 1986 Academic Press. Inc.
The “‘caribe colorado,” Pygocentvus notutus, is a seasonal breeder widely distributed in rivers, lagoons, and ponds of the Venezuelan plains. The fish usually reach maturity in their third year and have an annual gonadal cycle closely related to changes in the environmental conditions of the Venezuelan plains (Mago-Leccia, 1970; Gonzalez, 1980), which are characterized by a dry season (December-April) followed by a rainy season (May-November) . The annual gonadal cycle of this species shows a pattern of gonadal maturation starting a few months before the beginning of the rainy season. Thus, the process of gonadal recrudescense shows a positive correlation with the dry season, in which the maximal gonosomatic index is achieved (May). The caribe Colorado then awaits the onset of the rainy season, which provides favorable environmental conditions, for the triggering of spawning in the female and r To whom correspondence should be sent.
and reprint
requests
spermiation in the male. Spawning, which is completed in a very short time, is followed by a period of sexual quiescence during the ‘wet season. Studies on various mammalian species have shown that gonadotrophic activity in the pituitary glan is influenced by the neuropeptide, gonadotrophic hormone-releasing hormone (GnRH), derived from specific brain regions. Similarly, in teleosts, gonadotrophic activity is regulated by releasing factors produced in the thalamus and other parts of the brain et al., 1978; Peter and Grim, 1979; 1981). Recently, the presence in tele GnRH that is similar to luteinizi mone-releasing hormone (LHRH) was demonstrated, and GnRH irnrnu~or~ac~~v~ perikarya and axons have been fo~~~~ in the brain of several teleost species (for reviews see Crim et al., 1978; Crim and V 1983; Peter, 1983; Demski, 1984). In tion, a functional role for GnRIl in teleost fish has been proposed (Goos and Mu anoglu, 1977; Schreibman et al”., 1979; et al., 1982; Mtinz et al., 1982), and &ret239 0016-64X0/86 $1.50 Copyright All rights
Q 1986 by Academic Press. Inc. of reproduction in any lorm reserved.
240
GENTILE,
LIRA, AND MARCANO-DE
studies have established a correlation between the secretory activity of some hypothalamic nuclei with the annual reproductive cycle in various teleosts (Zambrano, 1972). Using radioimmunoassay procedures, we have demonstrated in the caribe colorado, P. not&us, the presence of a substance with GnRH-like immunoreactivity in different brain regions. Also, the immunoreactive-GnRH (ir-GnRH) content was shown to correlate with environmental conditions and with the gonosomatic index. This provides a basis for studies on the regulation of reproduction in this species. MATERIALS
AND METHODS
Adult male and female P. notorus were collected, in groups of approximately 20, over 5 months (MarchJuly) from lagoons and ponds of the Guarico River and its tributaries (Guarico and Apure states) and transported in aerated tanks to the laboratory (some 18 km south of the recollection area). The fish were kept in aerated tap water held in fiberglass tanks and maintained at a temperature of 28” for a period of 12 hr. Several measurements (total length, standard length, body and gonad weights) were recorded for each fish. Animals were sacrificed by decapitation. The brains were immediately removed and the tissues were weighted, frozen, and maintained at -40” until the hormone extraction. At the same time, the gonads were extirpated and weighed, and the gonosomatic index (GSI) was calculated: GSI =
weight of gonad weight of fish
x 100%
Tissue dissection and extraction procedures. Following removal of the brain, the pituitary (P) was set aside and the brains were then dissected. Both the whole hypothalamus (H) and remaining forebrain, comprised of telencephalon and olfactory bulb (TEL), were homogenized separately for the extraction of the hormone. Each region from two animals was pooled, and two determinations were made for each brain region analyzed (hypothalamus, telencephalon, and pituitary gland). After weighing, the brain tissues were homogenized in ice-cold 2 N acetic acid (at a final tissue concentration not exceeding 50 mg/ml) and centrifuged at 5OOOgfor 15 min, and the supernatant was lyophilized (King and Millar, 1980). Lyophilized extracts were resuspended in 250 ~1 of phosphosaline buffer (0.04 M phosphate, pH 7.4,0.6% NaCl, and 1% serum albumin) and 50 ~1 was used for assay of GnRH immunoreactivity.
COTTE
Radioimmunoassay of GnRH. This assay was performed using an antiserum (generously provided by Dr. N. White, Chelsea Hospital for Woman, London) at a final dilution of l:lO,OOO. The sensitivity of the assay was 30 pgiml and the standard curve was linear between 30 and 1000 pgiml. Synthetic LHRH (Peninsula Laboratories, San Carlos, CaIif.) was iodinated using the chloramine-T method and [lZ51]LHRH was separated from free iodine using cellulose (CF-11, Whatman Inc., England). Details of the specificity of the antibody used to measure LHRH have been described elsewhere (Jeffcoate et al., 1974). The antiserum does not recognize the peptide fragments produced after cleavage at the Glys-Leu7 bond, but it does cross-react with LHRH after removal of the COOH-terminal glycinamide. Hypothalamic extracts obtained from female fish, collected in April, were assayed in serial dilutions in triplicate and an inhibition assay was performed to establish the identity of the GnRH-like immunoreactive material. Extrahypothalamic brain extracts were assayed in triplicate at a single dilution. Statistical analysis. Data for all parameters were expressed as the means I SEM and statistical comparisons were made using Student’s t test.
RESULTS Gonosomatic Index (GSI)
During 1983, maximal precipitation (100 mm) occurred in the recollection area from April to November. No marked variations in air temperatures (about 25”) occurred during this period. The body and gonad weights of female and male caribe Colorado are shown in Tables 1 and 2. The fish underwent gonadal maturation from March 11 to May 4, and spawning and spermiation occurred between June 7 and July 27. The GSI for females increased from 0.92 2 TABLE BODY
WEIGHT FEMALE
AND
Pygocentr-us notatus
PERIODICALLY
DURING
March April May June July
N 11 14
4 7 27
Nore. number
9 10 6 12 I
Values are of animals.
INDEX (GSI) SAMPLED
A REPRODUCTIVE Body
Date
1
GONOSOMATIC
424.2 380.5 382.6 555.6 319.4 expressed
CYCLE
weight k) i 2 i +F
GSI mJ)
141.39 16.82 41.60 25.51 30.90
as means
OF
0.92 4.08 6.01 2.97 0.26 t
SEM
-c 5 ir k i
0.13 0.48 0.43 0.33 0.03
of the
BRAIN GnRH AND FISH REPRODUCTIVE TABLE BODY MALE
CYCLE
2
AND GONOSOMATIC INDEX (GSI) OF Pygocentvus notatus SAMPLED PERIODICALLY DURING A REPRODUCTIVE CYCLE WEIGHT
Body weight
GSI
N
ld
(%I
i
299.1 c 20.08
April 14
May 4 June 7 July 27
1;
Date
March 11
I
380.2 422.5 422.1 306.2
Note. Values are expressed number of animals.
i f ? i
16.14 42.93 34.00 30.30
as means
0.14 t 0.03
i ? -t
0.37 0.49 0.29 0.07
0.06 0.03 0.03
k 0.01
c SEM
of the
0.13% on March 11 to 5.01 2 0.43% on May 4, and declined sharply by June 7 when in most cases ovulation was incomplete. By July 27, all fish captured had spawned (Table 1). In males the increase and decrease of the GSI were less marked (Table 2). Characterization
of Hypothalamic
GnRH
Serial dilutions of tissue extracts from hypothalami caused a dose-dependent decrease in the binding of labeled LHRH to its specific antibody. The binding curve was parallel to that given by the addition of increasing concentrations of unlabeled synthetic LHRH. Relation between Regional Brain Concentrations and Season OY Sex Immunoassayable GnRH was present in extracts of the hypothalamus and the telencephalon in all fish. However, significant differences were noted according to the sex or the physiological condition of the fish. In females, the ir-GnRH content of the hypothalamus and the telencephalon changed significantly during the year. No such changes, however, were observed in male tissues (Figs. 1 and 2). In females, lower ir-GnRH levels were found in hypothalamic extracts in March compared to April and May. The ir-GnRH content in the hypothalamus was highest in April, a month before the maximal GSI was obtained. There was also a decrease of ir-
,i FIG. 1. Sex and seasonal differences in the content of ir-GnRH in hypothalamic extracts from female f and male (A) PygocentrL*s notatm in relation to annual changes in male (A) and female (0) GS% during 1983. Values are expressed as means f SEM of tbe number of determinations shown. **P < 0.001 when compared with March, May. June. and July. *P < 0.005 when compared with March and Juiy. *P < 0.001 when compared with June.
GnRH activity in hypothalamic extracts after spawning. In addition, significant sex differences were seen in the ir-GnRH content during April and May. In April, the irGnRH content of the female hypotbalam~s was approximately four times greater than that of males (Fig. 1). In the telence~~a~~~, ir-GnRH levels increased just before spawning (May), paralleling the G creases, and then decreased by the time of spawning. Again, sex differences in the content of ir-GnRH in this brain region were observed, with a five-fold increase in the ir-GnRH content in female compared to male telencephalon (Fig. 2). In p$uitary extracts of caribe Colorado, ir-GnRH was not present in either female or male specimens .
Our results suggest the presence of a substance related to synthetic LHR hypothalamic and telencephalic extracts of P. noaatus. The distribution of the irnrn~~o~
242
GENTILE,
LIRA,
AND MARCANO-DE
?6-
2
257 -1
MARCH APRIL
MAY
JUNE
JULY
-0
FIG. 2. Sex and seasonal differences in the content of ir-GnRH in telencephalic extracts from female (Of and male (a) Pygocentrus notatus in relation to annual changes in male (A) and female (0) GSI during 1983. Values are expressed as means f SEM of the number of determinations shown. **P < 0.001 when compared with March, April, June, and July *P < 0.005 when compared with March and July.
reactive material seems to be identical in both male and female fish. However, the levels of ir-GnRH showed fluctuations according to the reproductive state in females but not in males. The occurrence of GnRH in this species has not previously been investigated, but the present study clearly demonstrates the presence of immunoreactive GnRH in this teleost. GnRH-immunoreactive systems have been studied in several teleosts. Immunoreactive cell bodies have been demonstrated in the ventral parts of the olfactory nerve and bulb in the goldfish (Stell et al., 1984; Kah et al., 1984), in the ventrolateral parts of the anterior nucleus preopticus periventricularis and in the posterior nucleus lateralis tuberis in mature goldfish (Kah et al., 1984), and in the nucleus preopticus in carp (Pan et al., 1979) and platyfish (Schreibman et al., 1982). Immunoreactive GnRH has also been demonstrated within telencephalic perikarya of Salmo gairdneri (Goos and Murathanoglu, 1977) and goldfish (Kah et al., 1984). How-
COTTE
ever, Deery (1974) failed to detect immunoassayable GnRH in one elasmobranch (dogfish) and another teleost (goldfish). The similarity in the slope of the displacement curves in competitive inhibition assays with mammalian LHRH suggests that an immunologically similar LHRH is present in P. notatus. Previous studies have suggested that the teleost GnRH is different from the mammalian peptide (King and Millar, 1979), and recently a salmon GnRH was characterized as being a decapeptide which differs from mammalian LHRH at positions 7 and 8 (Sherwood et al., 1983). Our demonstration that GnRH P. notatus seems to be immunologically similar to that of mammals disagrees with these findings. However, reports of the occurrence of GnRH in teleosts have been conflicting, this possibly being due to seasonal fluctuations in the hypothalamic content of ir-GnRH in these fish. In this study, high concentrations of ir-GnRH were detected in hypothalamic and telencephalic extracts from sexually mature female P. notatus captured during the dry season (April-May) where the maximal gonadal maturation was observed, and the content of ir-GnRH was four- to six-fold lower in extracts from specimens captured outside the breeding season. Similar results were found by King and Millar (1980) in gravid Tilapia, where immunoreactive GnRH was present but was absent from brain sampled during the nonbreeding season. Also, immunoreactive cell bodies were located in the posterior nucleus lateralis tuberis only in mature goldfish (Kah er al., 1984). Perhaps the most significant finding to emerge from the present work is the marked difference in the content of irGnRH between the sexes and its relationship with the various stages of the reproductive cycle. Thus, male fish had significantly less GnRH than females in periods of maximal gonadal maturation. In female P. notatus, seasonal changes in ir-GnRH content appeared to vary independently in
BRAIN GnRH AND FISH REPRODUCTIVE
two of the regions assayed (hypothalamus and telencephalon) . No decrease in binding of labeled LHRH in the competitive radioimmunoassay used was caused by extracts of the pituitary gland from this fish. This suggests that an immunologically reactive LHRH is absent, or that the amount present is below the detection limit of the assay. These results are in disagreement with previous reports on other species. GnRH immunoreactive fibers have been described in the proximal pars distalis in several species (Dubois et al., 1979; Schreibman et al., 1979; Nozaki and Kobayashi, 1979; Miinz et al., 1981; Kah et al., 1984). Regions which show changes in GnRH content dependent on the gonosomatic index are known to be involved in teleostean reproduction. Regulation of gonadotrophic secretion seems to be centered in the nucleus lateralis tuberis of the hypothalamus (Peter and Crim, 1979) and lesions of the posterior nucleus lateralis tuberis resulted in a decreased gonosomatic index (Peter, 1982). On the other hand, the telencephalon has been implicated in the control of sexual behavior, nest building, spawning, and sperm release (Davis et al., 1976; Schwagmeyer et al., 1977) and recent lesion studies showed that destruction of the medial nuclei of the ventral telencephalon results in impairment of spawning behavior in male goldfish (Kyle and Peter, 1982). Furthermore, GnRH immunoreactive ceils have been seen in the ventromedial telencephalon (Goos and Murathanoglu, 1977) and in the posterior parts of the area dorsalis telencephali (Kah et al., 1984). It is significant that a progressive increase in the female GnRH content from March to May is consistent with the expected pattern of gonadal development culminating in spawning. During spawning, which occurs principally in June in this species, the hypothalamic and telencephalic GnRH content is higher than in July when the fish are reproductively quiescent.
CYCLE
It is interesting to note that in contrast to the hypothalamus where the conc~~trati~~ of ir-GnRH was highest a month before the maximal GSI, the highest concentration of ir-GnRH in the telencephalic extracts was found to parallel the increase in the GSI. This further suggests that this brain region could be important in aspects of the reproductive physiology in addition to gonadotrophin secretion, such as triggering of spawning or the control. of sexual behavior related to spawning. In mammals, GnRH has been impIicate~ in some aspects of sexual behavior ( and Carol, 1979; De Wied and G&pen, 1977). Another possibility is that the tide is acting as a neuromodulator, fying the activity of the other neural pathways as has been suggested for ot vertebrates. In summary, we have shown that in P. notatus high levels of ir-GnRH are related to changes in the reproductive cycle and in environmental conditions. The results suggest several functional and anatomical correlations which are likely to reflect mechanisms controlling reproductive function in teleosts. Marked sex and season ences in brain regions in which G concentrated further suggests that in the central nervous system is rel the reproductive physiology.
This investigation was supported by CONICIT Grant Sl-1623. We are grateful to Dr. N. White (Chelsea Hospital for Women, London) for the generous gift of anti-LHRH, and to Mr. Otto Castillo and the other members of FONAIP for their valuable help in the collection of the caribe Colorado. We also thank Mr. Javier Bricefio for generous help in the drawing of graphs.
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GENTILE,
LIRA, AND MARCANO-DE
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COTTE
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