Joseph G. Dulka

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

SummaryThe organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.

Direct evidence that 17α,20β-dihydroxy-4-pregnen-3-one functions as a goldfish primer pheromone: Preovulatory release is closely associated with male endocrine responses

Abstract This study directly tested the hypothesis that 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) is a goldfish preovulatory pheromone (pheromone released at peak levels during oocyte final maturation) which increases blood gonadotropin (GtH) and milt volume in males. During spontaneous ovulation, GtH and 17,20β-P in female blood and 17,20β-P released to the water increased dramatically 7–10 hr prior to ovulation, peaked 1–4 hr prior to ovulation, and then rapidly declined. Males held with these females, or exposed to their odors, had increased GtH levels and milt volumes at approximately the time when increased 17,20β-P release by ovulatory females commenced. Although these findings strongly support the hypothesis that 17,20β-P is a preovulatory female sex pheromone in goldfish which stimulates male GtH levels and milt production prior to spawning, the milt increases occurred earlier than predicted, suggesting either that preovulatory 17,20β-P release begins earlier than the data indicate or that other steroids known to have pheromonal activity are released before 17,20β-P.

Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldfish: existence of a dopaminergic preoptico-hypophyseal pathway.

To investigate the existence of a dopaminergic preoptico-hypophyseal pathway in the goldfish, electrolytic lesions were placed in the rostral preoptic area and their effects on gonadotrophin levels and pituitary innervation examined. In a first experiment, the fish were sacrificed 2 days after surgery and the pituitary studied by electron microscopy. Numerous exocytosis profiles were observed in the gonadotrophs, confirming the large increase in serum gonadotrophin levels measured in the animals. In addition, type A and B degenerating fibers were detected in the neurohypophysis and the pars distalis, in particular at the level of the gonadotrophs. In the second experiment, the distribution of tyrosine hydroxylase-immunopositive fibers was studied in the pituitary of controls and lesioned animals. It was found that lesioning the anterior ventral preoptic region resulted in the disappearance of all positive fibers in the pars distalis, while those in the neurointermediate lobe appeared unaffected. The presence of a large group of catecholaminergic perikarya in the destroyed area was confirmed in control animals. These results and other data strongly support the existence of a dopaminergic preoptico-hypophyseal pathway, providing a morphological support for the inhibitory effect of dopamine on the release of anterior pituitary hormones in teleosts, in particular gonadotrophin.

Extreme olfactory specificity of male goldfish to the preovulatory steroidal pheromone 17α,20β-dihydroxy-4-pregnen-3-one

Summary1.To determine the specificity of the goldfish (Carassius auratus) olfactory system to the reproductive pheromone 17α,20β-dihydroxy-4-pregnen-3-one (17α,20βP), and to determine if related sex steroids might also function as pheromones, electro-olfactogram (EOG) responses were recorded from mature male goldfish.2.Of the 24 steroids tested, 17α,20βP was the most stimulatory. It had a detection threshold of 10−12M and at a concentration of 10−8M elicited an EOG response 3 times that elicited by 10−5M L-serine3.17α,20β,21-triol-4-pregnen-3-one, a metabolite of 17α,20βP, was the only other highly stimulatory steroid. Its threshold was 10−11M.4.In cross-adaptation experiments EOG responses to all 21-carbon steroids were inhibited during adaptation to 17α,20βP: responses to this pheromone are transduced by a single receptor/transduction mechanism.5.To verify the biological relevance of EOG recording whole animal responsiveness was determined by measuring blood gonadotropin. When goldfish were placed into homogeneous steroid solutions endocrine responsiveness strongly correlated with EOG recording. However, when steroids were added to aquaria containing fish, responses were less specific indicating that transient wisps of steroids trigger endocrine responses.6.Although the extreme sensitivity and specificity of the goldfish olfactory system to 17α,20βP gives it the potential to serve as a highly specific cue, realization of this potential is probably determined by the dynamics of pheromone exposure.

Amino acid neurotransmitters and dopamine in brain and pituitary of the goldfish: involvement in the regulation of gonadotropin secretion.

Abstract: An isocratic high–performance liquid chromatographic technique was developed to measure levels of γ–aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3–mercaptopropionic acid or the GABA transaminase inhibitor γ–vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isetheonic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with α–methyl–p–tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content. The increase in serum gonadotropin concentrations in response to a gonadotropin–releasing hormone analogue was potentiated by pretreatment with monosodium glutamate. This article demonstrates that procedures causing elevation in GABA and taurine concentrations stimulate gonadotropin release in a teleost fish.

Relationship between serum growth hormone levels and the brain and pituitary content of immunoreactive somatostatin in the goldfish, Carassius auratus L

In this study, the relationships between endogenous brain and pituitary immunoreactive somatostatin (irSRIF) and circulating growth hormone (GH) levels in the goldfish were examined using two approaches. First, the amount of irSRIF in extracts of the pituitary gland and various brain regions was measured by radioimmunoassay several times throughout the year and was compared to serum GH levels at each time. The amounts of irSRIF in extracts of the pituitary gland, hypothalamus, and telencephalon were found to be inversely related to seasonal changes in serum GH levels, such that irSRIF was highest in these regions when serum GH levels were lowest (November and February). Conversely, irSRIF in these regions was lower in May, June, and July when serum GH levels were highest. These results suggest that endogenous irSRIF in the pituitary and forebrain may participate in the regulation of seasonal changes in serum GH levels in the goldfish. In extracts from other brain regions (thalamus + midbrain and cerebellum + medulla), some changes in the amount of irSRIF were observed among the various sample times, but these variations were not related to changes in serum GH levels. In a second set of experiments, the origin of irSRIF fibers innervating the goldfish pituitary gland was examined by using brain lesioning techniques to destroy regions of the forebrain known to contain irSRIF perikarya and fibers, and subsequently measuring the amount of irSRIF in the pituitary gland. Lesions in the preoptic area of the forebrain resulted in increased serum GH levels concomitant with a decrease in pituitary irSRIF content. This provides direct evidence that the preoptic area is the origin of a somatostatinergic projection inhibiting GH secretion from the goldfish pituitary. Lesions centered in the nucleus lateral tuberis (NLT) pars anterioris did not influence serum GH levels or the pituitary content of irSRIF. In contrast, more posterior lesions centered in the NLT pars posterioris (NLTp) resulted in a dramatic reduction in the amount of irSRIF in the pituitary. This suggests that the majority of irSRIF projections to the goldfish pituitary pass through the area destroyed by the lesion centered in the NLTp; it is also possible that perikarya within this area may be the origin of at least some of the irSRIF-containing fibers in the goldfish pituitary. Together, results from the present study provide evidence of a functional relationship between circulating levels of GH and endogenous brain and pituitary irSRIF in the goldfish.

Periovulatory female goldfish release three potential pheromones: 17α,20β-Dihydroxyprogesterone, 17α,20β-dihydroxyprogesterone glucuronide, and 17α-hydroxyprogesterone

Abstract Our previous studies have shown that 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) produced by preovulatory female goldfish functions both as a hormone promoting oocyte final maturation and as a primer sex pheromone stimulating rapid reproductive endocrine responses in the male. In the present study, the amounts of free and glucuronated 17,20β-P as well as free 17α-hydroxyprogesterone (17P) released to the holding water by female goldfish throughout the periovulatory period were determined. Compared to nonovulating female goldfish, ovulating goldfish released very high levels of each of these steroids. This study confirmed that 17,20β-P is released to the water by ovulating fish in sufficient amounts to have pheromonal activity and indicated that 17P may also function as a pheromone. Although considerable quantities of 17,20β-P glucuronide were also released, its physiological actions are unknown.

A reduction in pituitary dopamine turnover is associated with sex pheromone-induced gonadotropin secretion in male goldfish

In goldfish, the gonadal steroid, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), functions as a potent preovulatory female sex pheromone which stimulates rapid elevations in serum gonadotropin (GtH) levels and subsequent increases in milt production in males. GtH secretion in goldfish is known to be regulated by the stimulatory actions of gonadotropin-releasing hormone (GnRH) and the inhibitory actions of dopamine (DA). This study specifically examined whether the 17,20 beta-P-induced elevation in male GtH is caused by pheromone-mediated changes in DA inhibition at the level of the pituitary. First, we have demonstrated that dihydroxyphenylacetic acid (DOPAC) is the primary metabolite of DA catabolism in the brain and pituitary gland of goldfish. Second, we measured changes in circulating levels of GtH and changes in pituitary content of DA and its metabolite, DOPAC, as well as possible alterations in DA turnover rate (DOPAC/DA ratio) following short-term exposure of male goldfish to water-borne 17,20 beta-P. Water-borne 17,20 beta-P consistently increased serum GtH levels in males within 20 min of exposure and maintained elevated levels for up to 120 min. Although changes in pituitary DA content were not observed during periods of high GtH release, coincident reductions in pituitary levels of DOPAC were measured within 45 min of exposure to the pheromone. More importantly, there was a significant decrease in the rate of DA turnover in the pituitary, as assessed by comparing the ratio of DOPAC to DA present, at 20, 45, and 120 min of exposure. Since the reduction of DA turnover in the pituitary is inversely correlated with periods of increased GtH release, the present results suggest that water-borne 17,20 beta-P causes an abatement of DA release to the pituitary. Based on the latency of the GtH response to water-borne 17,20 beta-P, a rapid reduction of DA turnover in the pituitary appears to be at least part of the neuroendocrine trigger for 17,20 beta-P-induced GtH release in male goldfish.