Testicular SteroidogenesisTesticular steroidogenesis hormone GnIH is a neurohormone testicular steroidogenesis suppresses reproduction by acting at both the brain and pituitary levels. Testicular steroidogenesis addition to the brain, GnIH may also testicular steroidogenesis produced in gonads and can regulate steroidogenesis and gametogenesis. However, the function of GnIH in gonadal physiology take oral steroids before or after workout received testicular steroidogenesis attention in fish. The main objective of this study was to evaluate the effects of peripheral sbGnih-1 and sbGnih-2 implants on gonadal development and steroidogenesis during the reproductive cycle of male sea bass Dicentrarchus labrax. In February spermiationfish treated with sbGnih-1 and sbGnih-2 exhibited testicles with abundant type A spermatogonia and partial spermatogenesis. In addition, we determined the effects of peripheral Gnih implants on plasma follicle-stimulating hormone Fsh and luteinizing hormone Lh levels, as well tedticular on brain and pituitary expression of the main reproductive hormone genes and their receptors during the spermiation period February.
Testicular Steroidogenesis | SpringerLink
Gonadotropin-inhibitory hormone GnIH is a neurohormone that suppresses reproduction by acting at both the brain and pituitary levels.
In addition to the brain, GnIH may also be produced in gonads and can regulate steroidogenesis and gametogenesis. However, the function of GnIH in gonadal physiology has received little attention in fish. The main objective of this study was to evaluate the effects of peripheral sbGnih-1 and sbGnih-2 implants on gonadal development and steroidogenesis during the reproductive cycle of male sea bass Dicentrarchus labrax.
In February spermiation , fish treated with sbGnih-1 and sbGnih-2 exhibited testicles with abundant type A spermatogonia and partial spermatogenesis. In addition, we determined the effects of peripheral Gnih implants on plasma follicle-stimulating hormone Fsh and luteinizing hormone Lh levels, as well as on brain and pituitary expression of the main reproductive hormone genes and their receptors during the spermiation period February.
Treatment with sbGnih-2 increased brain gnrh2 , gnih , kiss1r and gnihr transcript levels. Whereas, both Gnihs decreased lhbeta expression and plasma Lh levels, and sbGnih-1 reduced plasmatic Fsh.
Finally, through behavioral recording we showed that Gnih implanted animals exhibited a significant increase in diurnal activity from late spermatogenic to early spermiogenic stages. Our results indicate that Gnih may regulate the reproductive axis of sea bass acting not only on brain and pituitary hormones but also on gonadal physiology and behavior. September 15, ; Accepted: October 12, ; Published: This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist. As in other vertebrates, reproduction in fish is controlled by a complex system of endocrine, paracrine and autocrine regulatory signals that interact along the pineal-brain-pituitary-gonadal axis [ 1 — 4 ].
The adequate functioning of this reproductive axis requires the synchronization of multiple hormonal systems, which is guaranteed by the presence of specific sensory organs and receptors that are able to perceive environmental, internal and social stimuli and transduce them into the secretion of neurohormones [ 5 ]. Teleost fish lack the median eminence, a vascular system characteristic of tetrapods that connects the hypothalamus with the pituitary gland. Therefore, hypothalamic neurohormones directly reach the cells of the anterior lobe of the adenohypophysis, and stimulate or inhibit the synthesis and secretion of pituitary gonadotropins, follicle-stimulating hormone Fsh and luteinizing hormone Lh that, in turn, regulate gametogenesis and gonadal steroidogenesis, as well as other processes involved in reproduction [ 6 — 10 ].
As in mammals, steroids play a key role in teleosts regulating gametogenesis and gonadal physiology. In male fish, the testis is the primary target organ for pituitary Fsh and Lh and, as in most vertebrates, spermatogenesis in teleosts is dependent on the action of Fsh, whereas the major role of Lh is to facilitate gamete maturation and spawning [ 11 , 9 ]. Plasma levels of steroid hormones exhibit remarkable variations during male gonad maturation.
Androgens such as testosterone T and ketotestosterone KT gradually increase their levels as spermatogenesis proceeds, decreasing at spermiation. Sexual steroids also play a role in transducing the sexual status to the pituitary and brain via short and long regulatory feedback, respectively.
The activation of these feedback mechanisms, with both positive and negative effects, depends on the phase of the development and of the reproductive cycle [ 12 , 3 ]. Since the identification of avian gonadotropin-inhibitory hormone GNIH [ 13 ], several studies have demonstrated that this family of neuropeptides also plays a key role in the regulation of reproduction in other vertebrates by decreasing the activity of GnRH-1 neurons and inhibiting gonadotropin synthesis and release in pituitary gonadotropes [ 14 — 18 ].
Nevertheless, the nature of Gnih actions in fish seems to vary depending on the species, the reproductive stage and the route of administration of this neuropeptide, and the mechanisms underlying these actions have yet to be fully elucidated.
Recently, studies in teleosts such as the tilapia Oreochromis niloticus reported that the Lpxrfa-2 peptide increased the release of Lh and Fsh both in vivo and in vitro [ 19 ].
Different studies have revealed that GNIH can modulate gonadal steroidogenesis in tetrapods [ 21 , 26 — 29 ], but evidence obtained in fish is much more scarce. Only a recent study performed in goldfish provides evidence that Gnih can be involved in male steroidogenesis, by increasing plasma T and Fsh and Lh receptor transcript levels in testicular cells [ 30 ].
The role of Gnih in the control of steroidogenesis and gametogenesis is far from being understood in this group of vertebrates. GNIH has also been implicated in the modulation of reproductive and feeding behavior in birds and mammals [ 31 ]. It has been suggested that GNIH may inhibit male socio-sexual behavior possibly by inhibiting GNRH neurons or increasing neuroestrogen concentration by stimulating the activity of brain cytochrome P aromatase.
Moreover, GNIH stimulates feeding behavior by modulating the activities of hypothalamic and central amygdala neurons and the release of different orexigenic and anorexigenic neuropeptides [ 31 ]. In fish, terminal nerve Gnrh-3 neurons show spontaneous pacemaker activity and appear responsible for controlling the motivational or arousal state of the animal, including sexual behavior [ 32 — 33 ].
Although RFRP-2 cannot be considered a true ortholog of Gnih, like RFRP-1 and RFRP-3, it has been shown recently that its synaptic release from hypothalamic neurons may inhibit the pacemaker activity of these GnRH-3 neurons by modulating the opening and closing of ionic channels, and acting as a negative motivational signal for sexual behavior [ 33 ].
The European sea bass Dicentrachus labrax is an important teleost species for marine aquaculture in Europe but still presents problems under farming conditions related to reproduction, such as early puberty and unbalanced sex proportions [ 34 ]. This species has also represented an interesting fish model for the study of environmental and endocrine control of reproduction [ 18 , 34 — 45 ].
Recently, we identified a gnih gene in sea bass encoding a prepro- gnih mRNA that gives rise to two different RFamide peptides, named as sbGnih-1 and sbGnih-2, which are produced by proteolytic processing from a single protein precursor [ 45 ]. In a previous study [ 18 ], we reported an inhibitory role of Gnih mainly sbGnih-2 in the reproductive axis of sea bass by acting at the brain and pituitary level.
The intracerebroventricular administration of Gnih down-regulated the brain expression of several genes of the Gnrh and kisspeptin systems, as well as pituitary gonadotropins mRNA levels and plasmatic Lh in sea bass [ 18 ]. In the present study, we aimed to evaluate whether Gnih may also play a role in the regulation of steroidogenesis and gonadal maturation in the European sea bass. For this purpose, we performed peripheral implants of sbGnih during critical periods of the reproductive cycle covering pre-spermatogenesis, spermatogenesis and spermiation stages from October to February , and analyzed its effects on plasma steroids and gonadotropins, as well as on the expression of different brain and pituitary reproductive hormone genes.
Finally, as the European sea bass has been reported to exhibit a phase inversion of its diel feeding and locomotor activity pattern, i. Animals were fed one time per day 9. Measures were taken to avoid the suffering of the animals. Based on available sea bass gonadotropin-inhibitory precursor sequence [GenBank accession no. The peptides were dissolved in coconut oil, which was also used as a vehicle in control animals. This preparation was injected as a warm fluid that solidifies within the fish to produce a slow releasing sbGnih emulsion.
In October , fifty-one specimens of European sea bass were distributed randomly among three tanks. Seventeen fish were assigned to each of three treatment groups: All the fish were subjected to the same routine and all procedures were carried out between 9: At the starting point of the experiment October 17 , all fish were anesthetized and weighed.
Each dose was injected intramuscularly one time per month, from October to January day 17 of each month. Injections were done alternately on the left or right sides of the fish body each month, at its rostral-dorsal pole.
In all groups, blood samples were collected 5 days post injection day 22 of each month from the caudal vessels using heparinized syringes and expelled into cold heparinized tubes. The gonads were removed and a small sample preserved in formalin for histological studies. Female specimens were not considered for the analysis. Tissues were homogenized in a mixer mill MM Retsch, Haan, Germany using 4—5 stainless steel spheres.
Primers for qPCR assay and amplicon sizes are shown in Table 1. INC, Japan as reported previously [ 18 ]. Briefly, PCR conditions were as follow: Duplicates of each sample were analyzed in the same test.
Standard curves were generated for each gene with fold serial dilutions of cDNA and all calibration curves exhibited slopes close to Melting curves were performed for each sample in order to confirm that a single product was amplified. The phases of testicular gametogenesis and the associated stages of testicular development were identified by light microscopy following the criteria established previously by Espigares et al. Tanks were each fitted with a photoswitch model E3S-AD62, Omron, Japan at the bottom of the tank 30 cm below the surface and 8 cm from the bottom.
Photoswitches were connected to a computer and worked by emitting a continuous infrared light beam. Interruptions in the beam caused by fish movements within 20 cm were recorded on the computer and organised into 10 min bins using specialised software DIO96USB, University of Murcia, Spain. Locomotor activity was continuously recorded for the whole duration of experiments.
Locomotor activity records and diurnalism were analyzed using the software package for chronobiological studies El Temps v 1. Statistical differences in gene expression, hormonal levels and diurnalism of locomotor activity were determined by ANOVA followed by Student-Newman-Keuls post hoc test, using Statgraphic Plus 5.
Before the analysis, data were checked for normality and homogeneity of variance, and the values were log-or square root- transformed when required. Control animals exhibited similar plasma profiles of testosterone T and KT along the 5-month experimental period, with lowest levels in October and November, a significant increase in December and January, when both androgens peaked, decreasing thereafter in February Fig 1A and 1D.
Vehicle implanted animals represent the control group A, D, G. Although no significant differences in gonadosomatic indexes were observed 1. In control fish only late meiotic and full spermiogenic testicles containing mostly sperm and some cysts of spermatocytes and spermatids were observed Fig 2A , Table 2. However, both sbGnih-1 and sbGnih-2 treated fish also exhibited a significant percentage of testicles containing abundant type A spermatogonia SgA and scattered isolated clusters of spermatozoids Fig 2B and 2C , Table 2 , which resulted in a partial spermatogenesis instead of complete, as observed in controls.
Full spermiating control fish A with lobules mostly filled with sperm inset. Gnih-1 B and Gnih-2 C treated fish testicles exhibiting only isolated clusters of sperm and abundant type A spermatogonia SgA, insets. We also analyzed the effects of Gnih on transcript levels of different genes of the Gnrh gnrh1 , gnrh2 , gnrh3 , gnrhr-II-2b , kisspeptin kiss1 , kiss2 , kiss1r , kiss2r and Gnih sbgnih , sbgnihr systems.
None of the brain reproductive genes studied elicited significant changes in their expression in sbGnih-1 treated animals Figs 3 and 4. Vehicle implanted animals represent the control group. Data show transcript levels of kiss1 A , kiss1r B , kiss2 C and kiss2r D in male sea bass specimens at the spermiation stage February. We also analyzed the effect of sbGnih-1 and sbGnih-2 implants on plasma Fsh and Lh levels at the spermiation stage February. A significant decrease in plasma Fsh levels was observed in animals treated with sbGnih-1 but not in sbGnih-2 implanted animals Fig 6A.
A, plasma levels of Fsh and B, plasma levels of Lh in male sea bass specimens at the spermiation stage February. The representative actograms and mean waveforms of locomotor activity of controls, sbGnih-1 and sbGnih-2 implanted animals are presented in S1 Fig and Fig 7. The analysis of actograms and mean waveforms revealed that in controls this activity was concentrated at the beginning of the day, with a secondary peak of activity at the end of the diurnal phase S1A Fig and Fig 7A.
Average diel profiles of locomotor activity mean waveforms of controls A , sbGnih B and sbGnih C implanted fish from October to February. The grey area in the waveform represents the mean values and the continuous line the SEM. Horizontal bars above graphs indicate day-time open bars and night-time solid bars.
In controls, the percentage of activity during the light phase increased from October to November from In contrast, both sbGnih-1 and sbGnih-2 implanted animals exhibited a markedly different pattern of locomotor activity as the reproductive cycle progressed, with sustained percentage of diurnal activity from October to November from Since its identification in the year , GNIH has been clearly established as a negative regulator of reproduction in birds and mammals by acting mainly at the brain and pituitary levels [ 13 , 56 — 60 ].
In fish, the mechanisms of actions of Gnih on gonadotropin release and the reproductive axis are not so clear and contradictory findings have been reported [ 16 , 18 , 19 , 45 , 61 , 62 ]. In addition, Gnih functions and implications in steroidogenesis and gametogenesis in teleosts have not been sufficiently assessed. In order to fill this gap, in the present study we performed a long term experiment aiming at identifying the actions of Gnih in steroidogenesis and gametogenesis in the testis of the European sea bass.