Regulation of salmon gonadotrophin-releasing hormone gene expression by sex steroids in rainbow trout brain.

Salmon gonadotrophin-releasing hormone (sGnRH) is the major form of gonadotrophin-releasing hormone in the brain of Salmonids and is encoded by two different genes: sGnRH1 and sGnRH2. In the present study, we examined the expression patterns of these two genes during development and throughout the reproductive cycle of the female rainbow trout (Oncorhynchus mykiss), and also investigated the feedback action of sex steroids on brain mRNA levels. Both genes are expressed as early as 14 days postfertilisation and show a similar expression pattern during early life stages. In the adult female, sGnRH1 and sGnRH2 mRNAs are both present in neurones located in the ventral forebrain. This gene expression in the brain appears to be low during early vitellogenesis, and increases during oocyte maturation to reach a maximum after ovulation. The expression of sGnRH1 was not modified by in vivo steroid treatments in any experiment; however, testosterone and 5alpha-dihydrotestosterone down-regulate brain sGnRH2 gene in immature and adult ovariectomised females. Oestradiol treatment decreases sGnRH2 mRNA levels in the brain of adult ovariectomised females only. In the triploid fish brain, none of the steroids affect brain sGnRH mRNA levels. Our results suggest that, unlike sGnRH1, the sGnRH2 gene is under a strongly androgenic inhibitory control in the immature and adult female rainbow trout.

Differential regulation of tyrosine hydroxylase and estradiol receptor expression in the rainbow trout brain.

In numerous fish species, dopamine has been found to strongly inhibit gonadotropin release. Among the enzymes that regulate dopamine turnover, tyrosine hydroxylase (TH), the rate-limiting anabolic enzyme, could be a target for endocrine feedback regulation. Since dopamine turnover is stimulated by estradiol in rainbow trout, we have investigated the effect of estradiol on TH and estradiol receptor expression. In situ hybridization was used to quantify mRNA levels in the brain of ovariectomized female rainbow trout implanted or not with estradiol pellets. We demonstrated that preoptic TH and estradiol receptor mRNA levels are greatly decreased by gonadectomy during vitellogenesis. For TH expression, this effect was reversed in part by estradiol supplementation. We have also confirmed the existence of an inhibitory gonadal feedback on FSH secretion, mediated by estradiol. The stimulating effect of estradiol on TH expression found in this study could be a pathway involved in gonadal feedback on gonadotropin release.

Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain.

Two different precursor genes encoding the decapeptide salmon GnRH (sGnRH) are present in most salmonid species. In rainbow trout, a precedent Southern blot study revealed the existence of two different sGnRH genes and, recently, two different genes and their complementary DNAs that encode the identical peptide sGnRH were isolated from ovary and testis. Our study confirms the existence of two different mRNAs encoding sGnRH (sGnRH mRNA-I and sGnRH mRNA-II) in the brain of rainbow trout and, for the first time, full-length complementary DNA sequences are given. Central and peripheral distributions of the two messengers are described and seem to indicate different regulation of their expression. sGnRH mRNA-I is found essentially in the olfactory bulbs and telencephalon, whereas sGnRH mRNA-II is more widely expressed in the brain. Our observations allow speculation on the respective roles of two genes encoding the same decapeptide.

Expression of sGnRH mRNA in gonads during rainbow trout gametogenesis.

The salmon gonadotropin-releasing hormone (sGnRH) is the major form of GnRH decapeptide expressed in the salmonid brain and it acts as a gonadotropin releaser. In rainbow trout, sGnRH-1 and sGnRH-2 mRNA forms were found in brain and gonads. We analyzed the expression of both forms in trout gonads at different stages of gametogenesis. Northern blot demonstrated that sGnRH-2 mRNA was the major sGnRH form in testis and ovary. In testis but not in ovary, brain or pituitary, alternatively spliced sGnRH-2 transcripts which coded for prepro-sGnRH with a truncated GnRH-associated peptide due to a premature stop codon in retained intron 2 were detected. In testis, sGnRH mRNA was highly expressed before the onset of spermatogenesis, it disappeared at stage II and then increased progressively up to stage VI. In ovary, the expression of sGnRH was high in immature pre-vitellogenic fish and progressively decreased throughout vitellogenesis. At ovulation it reached its maximum and came down again after stripping. The decrease of sGnRH mRNA expression during the period of active spermatogonial proliferation in testis and increase during meiosis occurrence in testis and ovary suggest an anti-proliferative and meiosis-stimulating effect of sGnRH during rainbow trout gametogenesis.

Stage-dependent and alternative splicing of sGnRH messengers in rainbow trout testis during spermatogenesis.

The gonadotropin releasing hormone (GnRH) has long been considered as a neuropeptide involved in the control of the reproductive cycle. However, the presence of GnRH and its receptors in various tissues, including ovary and testis, suggests a role as autocrine/paracrine factor. In the present study, we report the expression of the sGnRH-1 and sGnRH-2 genes encoding salmon GnRH in rainbow trout testis throughout testicular development and spermatogenesis. We demonstrate that both sGnRH mRNA are expressed prior of sexual differentiation. In adult, northern blot analysis indicates that sGnRH-2 transcripts are expressed in the testis at higher levels than sGnRH-1 messengers. Moreover, we observed that the expression of sGnRH-2, and not sGnRH-1, messengers was stage-dependent. sGnRH-2 mRNA expression decreases at the onset and progressively rebounds at the end of spermatogenesis. In addition, we demonstrate that a complex stage-dependent and differential splicing of the sGnRH-2 messengers occurs throughout spermatogenesis. We isolated five transcripts corresponding to sGnRH-2 messengers. Two of them may encode a novel and shortened GnRH-associated peptide containing 18 residues instead of 46. Our data provide new insight in the putative role of GnRH and GAP peptides as autocrine/paracrine factors of spermatogenesis.

Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon.

A recombinant vector containing antisense DNA complementary to Atlantic salmon (Salmo salar) sGnRH cDNA driven by specific promoter Pab derived from a corresponding sGnRH gene was introduced into rainbow trout (Oncorhynchus mykiss) eggs. This resulted in transgenic animals that had integrated one copy of the transgene into their genome and transmitted it through the germline. Antisense-sGnRH mRNA (AS) was expressed mainly in the brain of transgenic AS(+) fish. Levels of sGnRH endogenous mRNA in the brain were lower in 11-month-old AS(+) fish compared with nontransgenic AS(-) individuals from the same F2 progeny. sGnRH levels significantly decreased in the pituitary of transgenic males and females around the maturation period and in the brain of AS(+) immature females compared with controls. No reliable statistical difference was found in the levels of FSH and LH between AS(+) and AS(-) groups either in immature or mature fish. The majority of transgenic fish reached maturity at the same time as did nontransgenic individuals, although the maturation of AS(+) animals seemed to be more asynchronous. For the first time, the influence of antisense messengers on endogenous mRNA in transgenic fish and the corresponding protein is described.

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus.

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.

Identification of potential sites of cortisol actions on the reproductive axis in rainbow trout.

The full length cDNA encoding a rainbow trout glucocorticoid receptor (rtGR) has been obtained from rainbow trout liver and intestine libraries. Northern blot analysis showed that the corresponding messengers are detected in the brain of trout with a size 7.5 kb similar to the size of rtGR mRNA in other target tissues. The distribution of the rtGR mRNA and protein was studied in the forebrain of the trout by means of both in situ hybridization and immunohistochemistry and compared with that of the oestrogen receptor (rtER). The GR and ER mRNAs and proteins were detected with a strong overlapping mainly in the: (a) preoptic region; (b) mediobasal hypothalamus; and (c) anterior pituitary, confirming their implication in the neuroendocrine control of pituitary functions. In both diencephalon and pituitary, the peptidergic phenotype of some neuron or cell categories expressing either type of receptors could be determined by double staining. Furthermore, double staining studies have demonstrated colocalization of the two receptors in the same neurons or pituitary cells. The rtER and rtGR were found to be co-expressed in the dopaminergic neurons inhibiting GTH2 secretion and in pituitary cells of the anterior lobe--notably the gonadotrophs. Given that the promoter of the ER gene contains several potential glucocorticoid-responsive elements (GRE) and that cortisol inhibits the oestradiol-stimulated ER expression in the liver, the possibility exists for modulation of ER gene expression by GR in the hypothalamo-pituitary complex. This could explain some of the well documented effects of stress on the reproductive performance in salmonids.

Characterization of a novel alpha-amidated decapeptide derived from proopiomelanocortin-A in the trout pituitary.

Two complementary DNAs encoding distinct forms of POMC have been characterized in the trout pituitary. One of the POMC variants (POMC-A) possesses a C-terminal extension of 25 amino acids, which has no equivalent in other POMCs described to date. This C-terminal peptide contains three pairs of basic amino acids, suggesting that it may be the precursor of multiple processed peptides. In addition, the presence of a C-terminal glycine residue suggests that some of the processing products may be alpha-amidated. To characterize the molecular forms of the peptides generated from the C-terminal domain of trout POMC-A, we have developed specific antibodies against the C-terminal pentapeptide YHFQG and its alpha-amidated derivative YHFQ-NH2. Immunocytochemical labeling of pituitary sections with antibodies against YHFQ-NH2 revealed the presence of numerous immunoreactive cells in the pars intermedia and the rostral pars distalis. In contrast, the antibodies against YHFQG produced only weak immunostaining. HPLC analysis combined with RIA detection revealed that extracts of the pars intermedia and pars distalis contain several peptides derived from the C-terminal extension of trout POMC-A, with the predominant molecular form exhibiting the same retention time as ALGERKYHFQ-NH2. Tryptic digestion of this major form produced a peptide that coeluted with YHFQ-NH2. These data indicate that the processing of the C-terminal extension of trout POMC-A generates several novel peptides including the decapeptide amide ALGERKYHFQ-NH2.

Distribution and expression of glucocorticoid receptor mRNA in the forebrain of the rainbow trout.

The expression and distribution of glucocorticoid receptor mRNA was studied in the forebrain of mature female and immature undifferentiated rainbow trout (Oncorhynchus mykiss) by means of Northern blotting and in situ hybridization. A single mRNA species of 7.5 kb was detected in mRNA polyA+ prepared from the anterior brain. In situ hybridization was carried out using a 35S-labelled riboprobe corresponding to the A/B-domain (between nucleotides 1224 and 1763) of the recently cloned rainbow trout glucocorticoid receptor cDNA. Comparison of adjacent sections hybridized with the sense and antisense probes allowed detection of a specific signal with a similar distribution pattern in all animals studied. In the telencephalon, a specific hybridization was detected in scattered cells of the dorsal telencephalic hemisphere, but the stronger signal was consistently observed in the dorsal nucleus, and to a lesser degree in the ventral nucleus of the ventral telencephalon. Heavy hybridization staining was consistently observed in all subdivisions of the preoptic nucleus and the nucleus lateralis tuberis, which are the main hypophysiotrophic regions in fish. A weaker signal was detected in the nucleus anterioris periventricularis, nucleus suprachiasmaticus and thalamic region. The presence of a strong signal in virtually all magnocellular neurons of the nucleus preopticus, known for producing vasotocin, isotocin and corticotropin-releasing factor favors a direct function of cortisol in regulating its own secretion.

Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distributions of estrogen receptor-immunoreactive cells and gonadotrophin-releasing hormone-immunoreactive neurons on the same sections of the brains of adult male and female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the ventral and lateral telencephalon, the preoptic region, the mediobasal hypothalamus, and the ventromedial thalamic nucleus. Gonadotrophin-releasing hormone-immunoreactive perikarya were detected in the olfactory bulbs, the ventral telencephalon, the preoptic area, and the mediobasal hypothalamus. Double-staining studies showed that, although some estrogen receptor-positive cells were in close proximity to gonadotrophin-releasing hormone-immunoreactive perikarya, careful examination of 550 gonadotrophin-releasing hormone-positive cells from five adult females and two adult males failed to demonstrate any evidence that gonadotrophin-releasing hormone neurons coexpress estrogen receptor in the brain of the rainbow trout. The present study provides, for the first time in teleosts, morphological evidence that gonadotrophin-releasing hormone neurons do not represent major direct targets for estradiol, suggesting that the positive feedback effects of estradiol onto the gonadotrophin-releasing hormone system are likely to be conveyed via other cell populations.

Distribution of estrogen receptor-immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss).

Using antibodies against the hormone binding domain of the trout estrogen receptor (ER), the distribution of ER-immunoreactive (ER-IR) cells was studied in the brain of maturing diploid and triploid female rainbow trout using a streptavidin-biotin-peroxidase method followed by a nickel-intensified diaminobenzidine reaction. This technique resulted in an excellent signal/background ratio allowing unambiguous identification of positive cells. In all animals, ER-IR cells were consistently located in three brain regions, the ventral telencephalon, the anterior ventral preoptic region, and the mediobasal hypothalamus. About 250 ER-IR cells were observed in the ventral and dorsal parts of the ventral telencephalon. In the anterior nucleus preopticus periventricularis, about 2400 ER-IR cells were observed surrounding the preoptic recess. In the posterior hypothalamus, approximately 2700 ER-IR cells were located in the anterior, posterior and inferior divisions of the nucleus lateralis tuberis and in the nucleus saccus vasculosus. In these regions cell nuclei exhibiting different densities of staining were observed and absolutely no labeling of cytoplasmic processes was detected. These results are in partial agreement with those obtained either after injection of tritiated-estradiol in other teleots species or in situ hybridization of ER mRNAs in trout. In particular, no immunoreactivity was observed in the thalamic region nor in the nucleus posterioris periventricularis. These data indicate that target cells for estradiol are essentially located in brain regions involved in the neuroendocrine control of pituitary functions and having direct connections with the hypophysis.

Localization of salmon gonadotropin-releasing hormone mRNA and peptide in the brain of Atlantic salmon and rainbow trout.

The decapeptide gonadotropin-releasing hormone (GnRH) is a key hormone for the central regulation of reproduction. The distribution of salmon GnRH (sGnRH), which is the major form in salmonids, has been studied in different fish species by immunocytochemistry. Discrepancies in data concerning the distribution of sGnRH perikarya led us to investigate this problem in two species, the Atlantic salmon and the rainbow trout, with in situ hybridizaiton of sGnRH messenger, a highly specific molecular tool. By Northern blot analysis, the rainbow trout sGnRH messenger appears to be about 500 bases in length, which is close to those isolated from Atlantic salmon or masu salmon and characterized previously. In situ hybridization with riboprobes generated with Atlantic salmon sGnRH cDNA demonstrated that sGnRH perikarya are restricted to the ventral part of olfactory bulbs, telencephalon, and preoptic area. They are distributed on a nearly continuous line extending from the olfactory bulbs to the preoptic area in both salmonid species studied. Despite the presence of GnRH-like immunoreactivity in the preoptic magnocellular nucleus (NPOm) and in the tegmentum of the midbrain (MT), the sGnRH mRNA is not present in these two structures. Stained cells in NPOm could be target cells for GnRH and immunoreactive neurons in MT are likely to be chicken GnRH-II containing cells. Our study not only gives a precise distribution of the sGnRH system in two salmonids, Atlantic salmon and rainbow trout, but also clarifies the ambiguous data published up to now in rainbow trout.

Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry.

Complementary DNA fragments that encode central and C-terminal domains of rainbow trout estrogen receptor (rtER) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). Both fusion proteins were induced by IPTG and could readily be detected as a 53-55 kDa band in crude extracts or in insoluble fraction after polyacrylamide gel electrophoresis and Coomassie blue staining. These recombinant proteins were solubilized and partially purified (ca. 60-75%) using centrifugation and different concentrations of urea. Gel mobility shift assays revealed that the hybrid protein containing ER central domain forms a specific complex with a synthetic estrogen-response-element. Similarly, we showed by steroid-binding assays that the hybrid protein containing the ER C-terminal domain binds specifically estrogen and not other steroids. These hybrid receptors were further isolated by electroelution after electrophoresis and used to immunize rabbits. Polyclonal antibodies from each antiserum were purified using GST-rtER fusion proteins. The specificity of these purified antibodies was confirmed by Western blot analysis using extracts from yeast and COS-1 cells transfected with rtER cDNA expression vectors. In these cells, rtER level was about 300-500 fmol/mg of protein, and the receptor was found as a single band migrating as a 65 kDa polypeptide. Interestingly, Western blot analysis with both purified antibodies directed against central or C-terminal regions of rtER revealed two receptor forms in trout liver nuclear extracts: a major form migrating as 65 kDa protein also observed in transfected cells, and a minor band at 71 kDa specific to the liver. Both receptor form levels were strongly induced by estradiol whereas they were virtually undetectable in untreated male trout livers. Immunocytochemistry performed on brain and pituitary of female trout revealed the presence of rtER in neurons located in the ventral telencephalon, preoptic area and mediobasal hypothalamus, as well as cells in the proximal pars distalis of the pituitary.

The catecholaminergic system of the quail brain: immunocytochemical studies of dopamine beta-hydroxylase and tyrosine hydroxylase.

The distribution of dopamine beta-hydroxylase and tyrosine hydroxylase, two key enzymes in the biosynthesis of catecholamines, was investigated by immunocytochemistry in the brain of male and female Japanese quail. Cells or fibers showing dopamine beta-hydroxylase and tyrosine hydroxylase immunoreactivity were considered to be noradrenergic or adrenergic, while all structures showing only tyrosine hydroxylase immunoreactivity were tentatively considered to be dopaminergic. The major dopaminergic and noradrenergic cell groups that have been identified in the brain of mammals could be observed in the Japanese quail, with the exception of a tuberoinfundibular dopaminergic group. The dopamine beta-hydroxylase-immunoreactive cells were found exclusively in the pons (locus ceruleus and nucleus subceruleus ventralis) and in the medulla (area of the nucleus reticularis). The tyrosine hydroxylase-immunoreactive cells had a much wider distribution and extended from the preoptic area to the level of the medulla. They were, however, present in larger numbers in the area ventralis of Tsai and in the nucleus tegmenti pedunculo-pontinus, pars compacta, which respectively correspond to the ventral tegmental area and to the substantia nigra of mammals. A high density of dopamine beta-hydroxylase- and tyrosine hydroxylase-immunoreactive fibers and punctate structures was found in several steroid-sensitive brain regions that are implicated in the control of reproduction. In the preoptic area and in the region of the nucleus accumbens-nucleus stria terminalis, immunonegative perikarya were completely surrounded by immunoreactive fibers forming basket-like structures. Given that some of these cells contain the enzyme aromatase, these structures may represent the morphological substrate for a regulation of aromatase activity by catecholamines. The dopamine beta-hydroxylase-immunoreactive fibers were also present in a larger part of the preoptic area of females than in males. This sex difference in the noradrenergic innervation of the preoptic area presumably reflects the sex difference in norepinephrine content in this region.

Effects of neurochemical lesions of the preoptic area on male sexual behavior in the Japanese quail.

Two experiments were carried out during which the noradrenergic neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP) was applied to the brain of quail in order to evaluate the role of the noradrenergic system in the control of male copulatory behavior. In the first experiment, the ICV injection of 5-ADMP slightly enhanced the sexual behavior observed in testosterone (T)-treated castrated male quail. This brings additional support to the notion that norepinephrine tonically inhibits male copulatory behavior in quail. In the second experiment, 5-ADMP implanted directly into the preoptic area disrupted the restoration by T of copulatory behavior in castrated quail and, at the same time, produced a brain lesion that partly destroyed the sexually dimorphic medial preoptic nucleus, a previously established site of T action on behavior. These lesions produced by a high (presumably too high) concentration of neurotoxin provided an independent confirmation of effects previously observed after electrolytic lesions. Correlation analyses also confirmed that the medial part of the POM just rostral to the anterior commissure is more closely associated with copulatory behavior and may, therefore, represent a key center for steroid action on this behavior.

Catecholamine synthesis in the rainbow trout (Oncorhynchus mykiss) brain: modulation of tyrosine hydroxylase activity.

Levels of catecholamines and tyrosine hydroxylase (TH) activity were measured in brain homogenates from female rainbow trout. In triploid fish or in diploid fish in ovarian recrudescence, the patterns of catecholamine content expressed as a function of in vitro TH activity vary in different areas of the brain. Km for the pterin cofactor is lower in the telencephalon than in the hypothalamus. Dopamine (DA) and 2-hydroxyestradiol (20HE2) inhibit TH activity (by competitive and non-competitive interaction respectively).The K1 for DA were different in the telencephalon and the hypothalamus and this could explain the different patterns of catecholamine levels and TH activity for these two structures. 20HE2 inhibits TH activity in vitro; a catechol moiety is required since estradiol (E2) is notinhibitory. However, the exact mechanism of inhibition remains unclear. The rapid effect of 20HE2 cannot explain the previously reported activation of catecholamine synthesis by E2 in vivo.

Effects of estradiol on brain aminergic turnover of the female rainbow trout (Oncorhynchus mykiss) at the beginning of vitellogenesis.

Brain serotonin and dopamine (DA) turnovers in the female rainbow trout were studied at the beginning of the vitellogenesis and related to blood estradiol (E2) levels; pituitary and plasma gonadotropin (GtH) were also assayed. Ovariectomy did not modify brain aminergic turnover. E2 replacement on ovariectomized fish increased hypothalamic DA turnover (increased DA and increased DA metabolites). E2 stimulated GtH synthesis (positive feedback) but did not enhance GtH release; hypothalamic E2-mediated aminergic inhibition upon release was suspected. Individual relations between blood E2 levels and catecholaminergic neurotransmitters were determined. A linear positive correlation (r = 0.82) was found for the hypothalamus, but not for the pituitary, the preoptic area, or the telencephalon. These data suggest that an activation of hypothalamic tyrosine hydroxylase (the limiting step of catecholamines synthesis) by E2 could develop as vitellogenesis proceeds.

Serotonin and dopamine turnover in the female rainbow trout (Oncorhynchus mykiss) brain and pituitary: changes during the annual reproductive cycle.

Brain serotonin (5HT) and dopamine (DA) turnover were studied at various stages of the reproductive cycle of the female rainbow trout by simultaneous determination by HPLC of neurotransmitters and major related metabolites. An increase of 5HT turnover in telencephalon and hypothalamus and a decrease of DA turnover in pituitary and hypothalamus were observed during the periovulatory period. Some changes also occurred during vitellogenesis: decreased 5HT metabolite in telencephalon and preoptic area and increased DA content in preoptic area. These data suggest that physiological fluctuations of biogenic amines could be involved in both ovarian recrudescence and ovulation, with major effects on the hypothalamo-hypophysial complex during the periovulatory period.