Matthijs A. Zandbergen

Evolutionary development of three gonadotropin-releasing hormone (GnRH) systems in vertebrates

Gonadotropin-releasing hormone (GnRH) is the neuropeptide that links the brain to the reproductive system. Most vertebrate species express two forms of GnRH, which differ in amino acid sequence, localization, distribution, and embryological origin. The GnRH system in the ventral forebrain produces a species-specific GnRH form and projects toward the gonadotropic cell in the pituitary. The GnRH neurons of this system originate from the olfactory placode and migrate into the brain during early development. The other GnRH system is localized in a nucleus in the midbrain, where large cells express chicken-GnRH-II, of which the function is still unclear. In modern teleosts, a third GnRH system is present in the terminal nerve, which contains salmon GnRH. The three GnRH systems appear at different times during fish evolution. Besides the two accepted lineages in GnRH evolution (of conserved chicken GnRH-II in the midbrain and of mammalian GnRH or species-specific GnRH in the hypophysiotropic system), we propose a third lineage: of salmon GnRH in the terminal nerve.

Expression and distribution of two gonadotropin-releasing hormones in the catfish brain.

The expression of prepro-catfish GnRH mRNA and prepro-chicken GnRH-II mRNA was investigated by means of in situ hybridization. The differential distribution of cells expressing the respective mRNAs was compared with the distribution of cells immunoreactive for (1) catfish (cf) GnRH and chicken (c) GnRH-II and (2) both GnRH-associated peptides (GAPs). It was found that the prepro-cfGnRH mRNA expressing cells were located in the ventral forebrain, with a similar distribution of the cfGnRH- and cfGAP-immunoreactive perikarya. The prepro-cGnRH-II mRNA expressing cells were exclusively located in the midbrain tegmentum, at the same position as a group of large cGnRH-II- and CIIGAP-immunoreactive perikarya. It was concluded that the peptidergic neurons in the ventral forebrain contain cfGnRH, whereas cGnRH-II perikarya are restricted to the midbrain. The proximal pars distalis of the pituitary, containing the gonadotropin cells, is innervated by fibers immunoreactive for both cfGnRH and cfGAP and originating from the cfGnRH neurons in the ventral forebrain. We could, however, not detect fibers innervating the pituitary that were immunoreactive for cIIGAP.

Human-like brain hemispheric dominance in birdsong learning

Unlike nonhuman primates, songbirds learn to vocalize very much like human infants acquire spoken language. In humans, Broca’s area in the frontal lobe and Wernicke’s area in the temporal lobe are crucially involved in speech production and perception, respectively. Songbirds have analogous brain regions that show a similar neural dissociation between vocal production and auditory perception and memory. In both humans and songbirds, there is evidence for lateralization of neural responsiveness in these brain regions. Human infants already show left-sided dominance in their brain activation when exposed to speech. Moreover, a memory-specific left-sided dominance in Wernicke’s area for speech perception has been demonstrated in 2.5-mo-old babies. It is possible that auditory-vocal learning is associated with hemispheric dominance and that this association arose in songbirds and humans through convergent evolution. Therefore, we investigated whether there is similar song memory-related lateralization in the songbird brain. We exposed male zebra finches to tutor or unfamiliar song. We found left-sided dominance of neuronal activation in a Broca-like brain region (HVC, a letter-based name) of juvenile and adult zebra finch males, independent of the song stimulus presented. In addition, juvenile males showed left-sided dominance for tutor song but not for unfamiliar song in a Wernicke-like brain region (the caudomedial nidopallium). Thus, left-sided dominance in the caudomedial nidopallium was specific for the song-learning phase and was memory-related. These findings demonstrate a remarkable neural parallel between birdsong and human spoken language, and they have important consequences for our understanding of the evolution of auditory-vocal learning and its neural mechanisms.

Memory in the making: localized brain activation related to song learning in young songbirds

Songbird males learn to sing their songs from an adult ‘tutor’ early in life, much like human infants learn to speak. Similar to humans, in the songbird brain there are separate neural substrates for vocal production and for auditory memory. In adult songbirds, the caudal pallium, the avian equivalent of the auditory association cortex, has been proposed to contain the neural substrate of tutor song memory, while the song system is involved in song production as well as sensorimotor learning. If this hypothesis is correct, there should be neuronal activation in the caudal pallium, and not in the song system, while the young bird is hearing the tutor song. We found increased song-induced molecular neuronal activation, measured as the expression of an immediate early gene, in the caudal pallium of juvenile zebra finch males that were in the process of learning to sing their songs. No such activation was found in the song system. Molecular neuronal activation was significantly greater in response to tutor song than to novel song or silence in the medial part of the caudomedial nidopallium (NCM). In the caudomedial mesopallium, there was significantly greater molecular neuronal activation in response to tutor song than to silence. In addition, in the NCM there was a significant positive correlation between spontaneous molecular neuronal activation and the strength of song learning during sleep. These results suggest that the caudal pallium contains the neural substrate for tutor song memory, which is activated during sleep when the young bird is in the process of learning its song. The findings provide insight into the formation of auditory memories that guide vocal production learning, a process fundamental for human speech acquisition.

Maturational gonadotropin from the African catfish, Clarias gariepinus : purification, characterization, localization, and biological activity

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

The effects of aromatizable androgens, non-aromatizable androgens, and estrogens on gonadotropin release in castrated African catfish, Clarias gariepinus (Burchell)

SummaryTo study the feedback mechanism of gonadal hormones on GTH secretion in male African catfish, the effects of castration and steroid replacement on GTH release, pituitary GTH content, and ultrastructural appearance of gonadotropes were investigated.Castration resulted in an increase in plasma GTH levels, a decrease in pituitary GTH content, and a degranulation of many gonadotropes. The aromatizable androgens testosterone and androstenedione were able to abolish the castration-induced increase in plasma GTH. This was accompanied with a restoration of pituitary GTH content and a regranulation of gonadotropes. The non-aromatizable androgens 5α-dihydrotestosterone and 11β-hydroxyandros tenedione did not have these effects. Replacement with estrone or estradiol resulted in an increase in pituitary GTH, however, without abolishing the elevated plasma GTH levels; ultrastructurally, many gonadotropes showed a welldeveloped granular endoplasmic reticulum together with a regranulation.The results of the present study indicate the significance of androgen aromatization in the feedback mechanism of gonadal steroids on the brain-pituitary axis.

Regulation of Steady-State Luteinizing Hormone Messenger Ribonucleic Acid Levels, De Novo Synthesis, and Release by Sex Steroids in Primary Pituitary Cell Cultures of Male African Catfish, Clarias gariepinus

Abstract Primary pituitary cell cultures from sexually mature adult male African catfish, Clarias gariepinus, were used to study the regulation of LH biosynthesis by sex steroids. The cell cultures were exposed to testosterone (T), estradiol (E2), or 5α-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11β-hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E2 elevated glycoprotein α (GPα) and LHβ steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E2 on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GPα and LHβ mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [3H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish.

Gonadal steroids and the maturation of the species-specific gonadotropin-releasing hormone system in brain and pituitary of the male African catfish (Clarias gariepinus).

The effect of testosterone (T), 11-ketotestosterone (KT) and estradiol (E(2)) on the development of the catfish gonadotropin-releasing hormone system (cfGnRH) of male African catfish (Clarias gariepinus), at the onset of puberty [between 10 and 12 weeks post hatching (ph)] was investigated. The cfGnRH neurons, located in the ventral forebrain, were visualized by immunofluorescence and their numbers were determined and the amounts of cfGnRH-associated peptide (cfGAP) in the pituitary were measured by RIA. Steroid treatments did not significantly alter the numbers of immunoreactive GnRH neurons. However, T and E(2) caused an increase in the amount of GnRH, demonstrated by the intensity of the immunostaining of GnRH neurons and fibers in the brain and the amount of cfGAP in the pituitary. Treatment with KT, the main circulating androgen in adult male catfish, neither changed the number of cfGnRH neurons, nor elevated the cfGnRH content in the pituitary. In previous experiments with younger, prepubertal fish (2-6 weeks ph), T caused an elevation of the number of cfGnRH neurons to the same level as present in pubertal fish of 12-14 weeks. We conclude that the onset of puberty in the male African catfish coincides with the completion of the steroid-dependent structural maturation of the cfGnRH system in the brain. T and/or E(2), however, are still able to exert a positive influence on the amounts of cfGnRH during the later stages of pubertal development, thus still playing a role in the control of the cfGnRH system.

Development of three distinct GnRH neuron populations expressing two different GnRH forms in the brain of the African catfish (Clarias gariepinus).

The early development of both the catfish gonadotropin‐releasing hormone (cfGnRH)‐ and the chicken GnRH‐II (cGnRH‐II) system was investigated in African catfish by immunocytochemistry by using antibodies against the GnRH‐associated peptide (GAP) of the respective preprohormones. Weakly cfGnRH‐immunoreactive (ir) neurons and fibers were present at 2 weeks after hatching (ph) but only in the ventral telencephalon and pituitary. Two weeks later, cfGnRH fibers and neurons were also observed in more rostral and in more caudal brain areas, mainly in the preoptic area and hypothalamus. Based on differences in temporal, spatial, and morphologic appearance, two distinct cfGnRH populations were identified in the ventral forebrain: a population innervating the pituitary (ventral forebrain system) and a so‐called terminal nerve (TN) population. DiI tracing studies revealed that the TN population has no neuronal connections with the pituitary. The cGnRH‐II system is present from 2 weeks ph onward in the midbrain tegmentum and only their size and staining intensity increased during development. Based on the comparison of GnRH systems amongst vertebrates, we hypothesize that during fish evolution, three different GnRH systems evolved, each expressing their own molecular form: the cGnRH‐II system in the midbrain, a hypophysiotropic GnRH system in the hypothalamus with a species‐specific GnRH form, and a salmon GnRH‐expressing TN population. This hypothesis is supported by phylogenetic analysis of known GnRH precursor amino acid sequences. We hypothesize, because the African catfish is a less advanced teleost species, that it contains the cfGnRH form both in the ventral forebrain system and in the TN population.J. Comp. Neurol. 437:308–320, 2001.

Testicular responsiveness to gonadotropic hormonein vitro and Leydig and Sertoli cell ultrastructure during pubertal development of male African catfish (Clarias gariepinus)

The gonadotropin (GTH)-stimulated testicular androgen secretionin vitro and the ultrastructure of Leydig and Sertoli cells was studied during the pubertal development in male African catfish. Testicular weight increased from less than 1 mg in the ninth week of age to nearly 600 mg in the 28th week. Immature testes (stage I: spermatogonia) were highly sensitive to GTH and secreted very high amounts of androgens per mg of tissue. The secretion per mg tissue decreased gradually in stages II (spermatogonia and spermatocytes) and III (spermatogonia, spermatocytes, and spermatids), but precipitously in stage IV (all germ cell stages, including spermatozoa). However, due to the testicular weight gain, the total androgen output per pair of testes increased slightly in stage III and strongly in stage IV. The sensitivity to GTH decreased with the appearance of haploid germ cells in stage III. Leydig cells but not Sertoli cells showed the ultrastructural characteristics of steroid producing cells. Leydig cell morphology did not change in stages I–III, while in stage IV, more smooth endoplasmic reticulum was present. The ultrastructural characteristics of Sertoli cells did not change prominently. Thus, spermatogonial multiplication and spermatocyte formation takes place when the testicular steroidogenic system is highly active and responsive to GTH; whereas the differentiation of haploid germ cells is accompanied by a reduced responsiveness to GTH and by the secretion of several-fold lower androgen amounts per mg of tissue.