Final Report Summary - NEUROBASS (Acquiring competencies in neuroendocrinology by an aquaculture researcher through investigating Gonadotropin-inhibitory hormone & Kisspeptin as mediators between environmental cues & fish reproduction)
NEUROBASS has been highly successful in developing an aquaculture researcher’s career as an investigator of the fundamental study of reproduction. The researcher has gained new state-of-the-art experience, competencies and skills in neuroanatomy and neuroendocrinology. This repertoire is urgently required and essential for the applied optimisation of rearing environment and control of reproduction for commercially important fish species. The ultimate driving force being to promote the sustainability of European aquaculture.
NEUROBASS represents a cutting-edge research study. It was hypothesised that GnIH and kisspeptin systems can act as mediators of the effects of environmental factors on the neuroendocrine and endocrine systems governing reproduction. The overall objective was to test this hypothesis in the European sea bass (Dicentrarchus labrax) through the means of two research sub-projects: 1) Sub-Project 1 investigated the potential interaction of the environmental synchroniser, light, and its transducer neurohormone, melatonin, with the GnIH and kisspeptin neuroendocrine systems, 2) Sub-Project 2 investigated the potential interaction of the GnIH and kisspeptin systems with GnRH, gonadotropic and gonadal systems. A multi-targeted approach, combining neuroanatomical, ex vivo, in vivo and molecular studies was employed within each Sub-Project.
Firstly for Sub-Project 1, neuroanatomical mapping revealed the presence of the GnIH system in key areas of the brain related to reproduction (terminal nerve, ventral telencephalon, preoptic and hypothalamic regions) and in the rostral mesencephalic tegmentum, these areas can be linked to photic information reception as they are connected with the retina (terminal nerve ganglion cells) and have bidirectional connections with the pineal organ (rostral tegmental region). Indeed, it was demonstrated that both the retina and pineal organ receive GnIH fibers. Kisspeptin findings were in accordance with previous work, with the main kiss2 population localized around the lateral recess. Kiss1, is located in the habenula, which is described as the pinealofugal terminal region and exhibits coursing axons that enter the pineal.
An ex vivo study was performed to investigate the direct effect of melatonin treatment on brain explants. A significant reduction in GnIH receptor (sblpxrfa-r) and kisspeptin receptor (gpr54-2b) expression was observed under a high dose of melatonin (100 nM). In contrast, an up-regulation of midbrain tegmental GnRH (gnrh2) expression was noted.
In vivo work included two short-term (one week) studies of pinealectomy (Px, removal of the pineal gland) on adult sea bass, in the resting (July) and reproductive (March) seasons. Pinealectomy in the resting season significantly increased kiss2 expression. No clear effect on GnIH (sblpxrfa) or kiss1 was identified in either season. An important observation however was the strong seasonal difference in GnIH expression, with high levels during the resting season and a steep reduction during the reproductive season. This difference was mirrored by gnrh2 but to a lesser extent. An effect of pinealectomy was also noted for gnrh1 in the reproductive season.
A corresponding longer-term (3 month) study investigating the effect of sustained pinealectomy, including the additional treatments of ophthalmectomy (Ox, enucleation of the eyes) and melatonin implant (M), revealed effects on both the GnIH and kisspeptin systems. These included significantly higher GnIH (sblpxrfa) expression in pinealectomised (Px) individuals. A reduction in GnIH receptor (sblpxrfa-r) expression was observed in ophthalmectomised (Ox), pinealectomised + ophthalmectomised (PxOx) and PxOxM individuals. For kiss2, expression increased in Px, Ox and PxOx animals. Likewise kiss1 expression was significantly higher in Ox individuals. The locomotor activity and feeding behaviour of animals was also affected by pinealectomy, ophthalmectomy and/or melatonin implant.
Sub-project 2 investigated the interaction of GnIH and kisspeptin systems with downstream neuroendocrine systems. GnIH results were of particular interest, double immunohistochemistry with GnRH3 indicated that GnIH immunoreactivity almost completely overlapped GnRH3 immunoreactivity in the olfactory bulb/terminal nerve region and confocal microscopy confirmed that these two peptides were localized in the same cell and packaged in separate vesicles. Also of importance was the direct contact of GnRH1 cells by GnIH fibers, an interaction that appeared to be influenced by season as more GnIH fibers and contact was evident in the resting versus the reproductive season. Also of interest, double immunohistochemistry revealed that kiss2 cells located in the nucleus of the lateral recess of the brain were seen to be in close proximity with GnIH fibers and in some cases potential contact was evident. At the level of the pituitary, on analysis of GnIH fibers with FSH and LH cells, again there appeared to be potential interaction albeit seasonally or reproductive stage specific. Conspicuous GnIH fibers and potential contact with FSH and LH cells were seen in February. These morphofunctional evidences could be on the basis of the inhibitory effects of GnIH on the main neuroendocrine and endocrine reproductive systems reported in the host laboratory.
At the level of the gonads, GnIH receptor and kisspeptin receptor expression were confirmed. An ex vivo experiment was performed which found that the GnIH peptide sblpxrfa2, stimulated the production of the maturation inducing steroid (MIS), a sex steroid involved in a shift towards the final stages of spermatogenesis and spermiation. There was no clear effect of kisspeptin, specifically kiss2, during the same season. The effect of GnIH on gonad physiology was also studied in vivo and preliminary results, based on gonadosomatic data, suggested that the sblpxrfa1 peptide affected gonadal physiology.
This project has confirmed our hypothesis that the GnIH and kisspeptin systems can act as mediators between the input of environmental light and reproduction. It has provided evidence for the integration of both systems in the complex photoneuroendocrine axis, deduced from neuroanatomical localisation with structures involved in light sensing such as the retina and pineal organ and from the effects of removing these main structures in vivo and by melatonin treatment ex vivo. Importantly, with the seasonal differences observed in the GnIH system and to a lesser extent the kisspeptin system, it is most likely the combined function of both of these neuropeptide systems that contribute to transducing the seasonal shift in photoperiod to a physiological change in reproduction. Neuroanatomical evidence pointing to the contact of GnRH, FSH/LH and kisspeptin cells by GnIH fibers strongly supported the downstream role of these systems in the reproductive axis. Moreover, evidence of the effects of GnIH in the gonads, suggests that at least GnIH, if not kisspeptin too, is playing a role at all three levels of the reproductive axis: the brain, pituitary and gonads. In addition, neuroanatomical evidence points the role of GnIH system in feeding, gustatory, sensory and behavioral processes. Could the GnIH system be linking light, reproduction, feeding and behavior?
Important applications for the research findings of NEUROBASS include the optimisation of the photic environment for: promoting reproduction or, vice versa, for the inhibition of precocious puberty/reproduction where maturation results in a great decrease in a somatic growth and a potential release of gametes to the wild. Both scenarios have highly significant economic consequences. In this respect, an important research step would be to develop technologies for the administration of GnIH in vivo. Additional complementary studies in the host lab, not only with GnIH implants but also by intracerebroventricular injection have indicated the efficacy of GnIH. It is of great interest to develop methodologies for the nanoencapsulation of GnIH for a less invasive and environmentally friendly administration.
Also importantly, this project has been performed in a marine perciform, Dicentrarchus labrax, and may potentially be applied to other commercially important perciformes including gilthead seabream, tuna, wrasse, cobia, meagre. The GnIH system is a hot topic for marine perciformes because the commonly believed functional antagonist to reproduction, dopamine, has not shown clear inhibitory action in this evolved group of teleosts.
In a broader sense, NEUROBASS has built upon an essential platform for EU research and excellence: the generation of detailed scientific knowledge for application in answering key reproductive issues in the aquaculture industry. Taken together, the maturation of an individual researcher’s career and importantly the welcome and integration of the researcher by the host scientist in charge/research group, NEUROBASS has yielded a strong lasting collaboration, doors have been opened, this platform of research can only grow stronger. The candidate plans to continue this fruitful collaboration in this topic by applying to the Call of Starting Grants from ERC Program in the near future.
NEUROBASS represents a cutting-edge research study. It was hypothesised that GnIH and kisspeptin systems can act as mediators of the effects of environmental factors on the neuroendocrine and endocrine systems governing reproduction. The overall objective was to test this hypothesis in the European sea bass (Dicentrarchus labrax) through the means of two research sub-projects: 1) Sub-Project 1 investigated the potential interaction of the environmental synchroniser, light, and its transducer neurohormone, melatonin, with the GnIH and kisspeptin neuroendocrine systems, 2) Sub-Project 2 investigated the potential interaction of the GnIH and kisspeptin systems with GnRH, gonadotropic and gonadal systems. A multi-targeted approach, combining neuroanatomical, ex vivo, in vivo and molecular studies was employed within each Sub-Project.
Firstly for Sub-Project 1, neuroanatomical mapping revealed the presence of the GnIH system in key areas of the brain related to reproduction (terminal nerve, ventral telencephalon, preoptic and hypothalamic regions) and in the rostral mesencephalic tegmentum, these areas can be linked to photic information reception as they are connected with the retina (terminal nerve ganglion cells) and have bidirectional connections with the pineal organ (rostral tegmental region). Indeed, it was demonstrated that both the retina and pineal organ receive GnIH fibers. Kisspeptin findings were in accordance with previous work, with the main kiss2 population localized around the lateral recess. Kiss1, is located in the habenula, which is described as the pinealofugal terminal region and exhibits coursing axons that enter the pineal.
An ex vivo study was performed to investigate the direct effect of melatonin treatment on brain explants. A significant reduction in GnIH receptor (sblpxrfa-r) and kisspeptin receptor (gpr54-2b) expression was observed under a high dose of melatonin (100 nM). In contrast, an up-regulation of midbrain tegmental GnRH (gnrh2) expression was noted.
In vivo work included two short-term (one week) studies of pinealectomy (Px, removal of the pineal gland) on adult sea bass, in the resting (July) and reproductive (March) seasons. Pinealectomy in the resting season significantly increased kiss2 expression. No clear effect on GnIH (sblpxrfa) or kiss1 was identified in either season. An important observation however was the strong seasonal difference in GnIH expression, with high levels during the resting season and a steep reduction during the reproductive season. This difference was mirrored by gnrh2 but to a lesser extent. An effect of pinealectomy was also noted for gnrh1 in the reproductive season.
A corresponding longer-term (3 month) study investigating the effect of sustained pinealectomy, including the additional treatments of ophthalmectomy (Ox, enucleation of the eyes) and melatonin implant (M), revealed effects on both the GnIH and kisspeptin systems. These included significantly higher GnIH (sblpxrfa) expression in pinealectomised (Px) individuals. A reduction in GnIH receptor (sblpxrfa-r) expression was observed in ophthalmectomised (Ox), pinealectomised + ophthalmectomised (PxOx) and PxOxM individuals. For kiss2, expression increased in Px, Ox and PxOx animals. Likewise kiss1 expression was significantly higher in Ox individuals. The locomotor activity and feeding behaviour of animals was also affected by pinealectomy, ophthalmectomy and/or melatonin implant.
Sub-project 2 investigated the interaction of GnIH and kisspeptin systems with downstream neuroendocrine systems. GnIH results were of particular interest, double immunohistochemistry with GnRH3 indicated that GnIH immunoreactivity almost completely overlapped GnRH3 immunoreactivity in the olfactory bulb/terminal nerve region and confocal microscopy confirmed that these two peptides were localized in the same cell and packaged in separate vesicles. Also of importance was the direct contact of GnRH1 cells by GnIH fibers, an interaction that appeared to be influenced by season as more GnIH fibers and contact was evident in the resting versus the reproductive season. Also of interest, double immunohistochemistry revealed that kiss2 cells located in the nucleus of the lateral recess of the brain were seen to be in close proximity with GnIH fibers and in some cases potential contact was evident. At the level of the pituitary, on analysis of GnIH fibers with FSH and LH cells, again there appeared to be potential interaction albeit seasonally or reproductive stage specific. Conspicuous GnIH fibers and potential contact with FSH and LH cells were seen in February. These morphofunctional evidences could be on the basis of the inhibitory effects of GnIH on the main neuroendocrine and endocrine reproductive systems reported in the host laboratory.
At the level of the gonads, GnIH receptor and kisspeptin receptor expression were confirmed. An ex vivo experiment was performed which found that the GnIH peptide sblpxrfa2, stimulated the production of the maturation inducing steroid (MIS), a sex steroid involved in a shift towards the final stages of spermatogenesis and spermiation. There was no clear effect of kisspeptin, specifically kiss2, during the same season. The effect of GnIH on gonad physiology was also studied in vivo and preliminary results, based on gonadosomatic data, suggested that the sblpxrfa1 peptide affected gonadal physiology.
This project has confirmed our hypothesis that the GnIH and kisspeptin systems can act as mediators between the input of environmental light and reproduction. It has provided evidence for the integration of both systems in the complex photoneuroendocrine axis, deduced from neuroanatomical localisation with structures involved in light sensing such as the retina and pineal organ and from the effects of removing these main structures in vivo and by melatonin treatment ex vivo. Importantly, with the seasonal differences observed in the GnIH system and to a lesser extent the kisspeptin system, it is most likely the combined function of both of these neuropeptide systems that contribute to transducing the seasonal shift in photoperiod to a physiological change in reproduction. Neuroanatomical evidence pointing to the contact of GnRH, FSH/LH and kisspeptin cells by GnIH fibers strongly supported the downstream role of these systems in the reproductive axis. Moreover, evidence of the effects of GnIH in the gonads, suggests that at least GnIH, if not kisspeptin too, is playing a role at all three levels of the reproductive axis: the brain, pituitary and gonads. In addition, neuroanatomical evidence points the role of GnIH system in feeding, gustatory, sensory and behavioral processes. Could the GnIH system be linking light, reproduction, feeding and behavior?
Important applications for the research findings of NEUROBASS include the optimisation of the photic environment for: promoting reproduction or, vice versa, for the inhibition of precocious puberty/reproduction where maturation results in a great decrease in a somatic growth and a potential release of gametes to the wild. Both scenarios have highly significant economic consequences. In this respect, an important research step would be to develop technologies for the administration of GnIH in vivo. Additional complementary studies in the host lab, not only with GnIH implants but also by intracerebroventricular injection have indicated the efficacy of GnIH. It is of great interest to develop methodologies for the nanoencapsulation of GnIH for a less invasive and environmentally friendly administration.
Also importantly, this project has been performed in a marine perciform, Dicentrarchus labrax, and may potentially be applied to other commercially important perciformes including gilthead seabream, tuna, wrasse, cobia, meagre. The GnIH system is a hot topic for marine perciformes because the commonly believed functional antagonist to reproduction, dopamine, has not shown clear inhibitory action in this evolved group of teleosts.
In a broader sense, NEUROBASS has built upon an essential platform for EU research and excellence: the generation of detailed scientific knowledge for application in answering key reproductive issues in the aquaculture industry. Taken together, the maturation of an individual researcher’s career and importantly the welcome and integration of the researcher by the host scientist in charge/research group, NEUROBASS has yielded a strong lasting collaboration, doors have been opened, this platform of research can only grow stronger. The candidate plans to continue this fruitful collaboration in this topic by applying to the Call of Starting Grants from ERC Program in the near future.