Brain-pituitary-gonad Axis Research Articles

Direct Feedback Regulation of E2, T, and hCG in the Brain–Pituitary–Gonad Axis of Japanese Eel (Anguilla japonica) during Artificial Maturation

The feedback regulatory effects of estrogen (E2) and androgen (T) on the gonadotropin-releasing hormone (GnRH) and gonadotropin (GtH) within the brain–pituitary–gonad (BPG) axis in eels with undeveloped ovaries were investigated through in vivo studies. However, the regulatory role of the BPG axis only became apparent during ovary development in the migratory stage. To further elucidate the direct feedback regulation of the BPG axis, female Anguilla japonica underwent artificial induction of vitellogenesis, and the regulation of BPG axis tissues by GtH (human chorionic gonadotropin, hCG), E2, and T was explored through in vitro exposure. The mRNA expression levels of GnRH (mGnRH), GtH (fshb and lhb), and steroid biosynthesis enzymes (cyp11a1, hsd3b, cyp17a1, and cyp17a2) in the diencephalon, pituitary, and ovary, respectively, were determined. The results showed that the expression level of mGnRH in the diencephalon was significantly downregulated by 0.1 IU/mL hCG but upregulated by both 1 nM E2 and higher concentrations of T, suggesting a direct positive feedback regulation of E2 on mGnRH. In the pituitary, the expression levels of fshb and lhb were upregulated by E2, while fshb was suppressed by T. In the ovaries, the expression of cyp11a1 and hsd3b was upregulated by 1 nM E2, whereas T exposure resulted in an opposite effect. Cyp17a1 mRNA levels did not differ significantly with E2 treatment but were upregulated by 1 nM T. These findings suggest that low concentrations of E2 exhibited positive feedback regulation on all three levels (diencephalon, pituitary, and ovary) of the BPG axis, while T showed weaker and differential feedback regulation in BPG axis tissues. Overall, this study’s results revealed the direct feedback regulation of hCG, E2, and T on the BPG axis in eels, a phylogenetic base of teleosts.

Chronological Changes in Gonadotropin-Releasing Hormone 1, Gonadotropins, and Sex Steroid Hormones along the Brain-Pituitary-Gonadal Axis during Gonadal Sex Differentiation and Development in the Longtooth Grouper, Epinephelus bruneus.

(1) Fshβ and Lhβ showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshβ and Lhβ at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E2 concentrations increased significantly at 174, 333, and 1435 dph, while T concentrations exhibited significant increases at 174 and 333 dph. These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis.

Bidirectional sex-change behavior and physiological aspects in the Gorgeous goby Lythrypnus pulchellus (Gobiidae).

The Gorgeous goby Lythrypnus pulchellus shows extreme sexual plasticity with the bidirectional sex-change ability socially controlled in adults. Therefore, this study describes how the hierarchical status affects hormone synthesis through newborn hormone waste products in water and tests the influence of body size and social dominance establishment in sex reversal duration and direction. The associated changes in behavior and hormone levels are described under laboratory conditions in male-male and female-female pairs of similar and different body sizes, recording the changes until spawning. The status establishment occurred in a relatively shorter time period in male and female pairs of different sizes (1-3 days) compared to those of similar size (3-5 days), but the earlier one did not significantly affect the overall time of sex change (verified by pair spawning). The changes in gonads, hormones, and papilla occurred in sex-changer individuals, but the first one was observed in behavior. Courtship started at 3-5 days in male pairs and from 2 h to 1 day in female pairs of both groups of different and similar sizes. Hormones did not gradually move in the new sexual phenotype direction during the sex-change time course. Nonetheless, estradiol regulated sex change and 11-ketotestosterone enabled bidirectional sex change and was modulated by agonistic interactions. Cortisol is associated with status and gonadal sex change. In general, similar mechanisms underlie sex change in both directions with a temporal change sequence in phases. These results shed new light on sex-change mechanisms. Further studies should be performed to determine whether these localized changes exist in the steroid hormone synthesis along the brain-pituitary gonad axis during social and bidirectional sex changes in L. pulchellus.

Tissue-specific promoters regulate the transcription of cyp19a1 in the brain-pituitary-gonad axis of Japanese eel (Anguilla japonica)

Aromatase is a key enzyme that catalyzes the biosynthesis of estrogens. Previous study indicated that putative tissue-specific promoters of the one aromatase gene (cyp19a1) may drive the differential regulatory mechanisms of cyp19a1 expression in Anguilla japonica. In the present study, for elucidating the transcription characteristics and the function of putative tissue-specific promoters of cyp19a1 in the brain-pituitary-gonad (BPG) axis during vitellogenesis, we investigated the transcriptional regulation of cyp19a1 by 17β-estrogen (E2), testosterone (T), or human chorionic gonadotropin (HCG) in A. japonica. The expression of estrogen receptor (esra), androgen receptor (ara), or luteinizing hormone receptor (lhr) was up-regulated as cyp19a1 in response to E2, T, or HCG, respectively in the telencephalon, diencephalon, and pituitary. The expression of cyp19a1 was also upregulated in the ovary by HCG or T in a dose-dependent manner. Unlike in the brain and pituitary, the expression of esra and lhr, rather than ara, was upregulated by T in the ovary. Subsequently, four primary subtypes of 5′-untranslated terminal regions of cyp19a1 transcripts and the corresponding two 5′ flanking regions (promoter P.I and P.II) were identified. The P.II existed in all BPG axis tissues, whereas the P.I with strong transcriptional activity was brain- and pituitary-specific. Furthermore, the transcriptional activity of promoters, the core promoter region, and the three putative hormone receptor response elements were validated. The transcriptional activity did not change when the HEK291T cells co-transfected with P.II and ar vector were exposed to T. These results suggested that the expression of cyp19a1 was upregulated indirectly through esra and lhr rather than ara by T in the ovary, whereas the expression of cyp19a1 was upregulated directly through androgen receptor and the downstream androgen response element of tissue-specific P.I in the brain and pituitary. The results of the study reveal the regulatory mechanisms of estrogen biosynthesis and provide a reference for optimizing the technology of artificially induced maturation in eels.

Identification of a New Insulin-like Growth Factor 3 (igf3) in Turbot (Scophthalmus maximus): Comparison and Expression Analysis of IGF System Genes during Gonadal Development

Fish reproduction is closely related to the regulation of the brain and liver, making it essential to identify the factors that control this process. The turbot (Scophthalmus maximus) is an economically significant species that has been successfully breeding through industrial aquaculture. Investigation of factors into the involvement of gonadal development is crucial for artificial breeding. In this study, a new insulin-like growth factor 3 igf3 gene was cloned and characterized. Additionally, all three types of turbot IGFs contain a distinct IGF domain, with IGF3 and IGF2 being grouped with other teleosts, demonstrating a closely related genetic relationship. The expression analysis showed that igf3 mRNA is predominantly expressed in the gonad and brain (specifically in the pituitary and hypothalamus), suggesting its effects at multiple levels in the brain–pituitary–gonadal axis. Furthermore, the mRNA levels of igfs during gonadal development were examined. In the gonad and liver of female turbots, the expression levels of igfs mRNA significantly increased from stage II to VI during the process of oogenesis, including maturation and degeneration. In the testis and liver of male turbots, igf3 mRNA maintained high expression levels during the proliferation of spermatogonia at stages II and III. In addition, the highest levels of igf1 and igf2 were observed at the beginning of spermatogenesis and during sperm production at stages V and VI. All the results suggest that the IGFs were closely related to the gonadal development in turbot and improve a better understanding of the IGF system in the regulation of gonadal development in teleost.

Constant High Temperature Promotes Early Changes in Testis Development Associated with Sexual Maturation in Male Atlantic Salmon (Salmo salar L.) Post-Smolts

Sexual maturation of post-smolts is a concern for Atlantic salmon producers, and its occurrence is dependent upon factors such as water temperature and energy availability, among others. The present study was designed to investigate the effect of different temperatures and feeding regimes on testis development and local regulation of spermatogenesis in salmon post-smolts. A 3 × 2 factorial design was used, with three temperatures (8 °C, 12.5 °C, and 18 °C) and two feed regimes (100% and 67% ration). Salmon (1800 parr, initial mean weight 23.1 ± 7.2 g) were reared from 28 October 2018 to 30 May 2019 in a freshwater flow-through system under continuous light (LD24:0), except for a 5-week winter signal (LD12:12) introduced on 4 February 2019. Testis histology, transcription of follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr), and transcription of testis proteins involved in spermatogenesis regulation such as gonadal-soma-derived factors 1 (gsdf1) and 2 (gsdf2), anti-Müllerian hormone (amh), and insulin-like growth factor-3 (igf3), were analyzed. Results showed that high temperature alone (18 °C), irrespective of the feed regime, promoted early presence of type B spermatogonia and reduced transcript levels of the proliferation-inhibitory factor amh in males still considered immature, of groups 18–100% and 18–67% that later matured in high proportion (~100%). This effect was also present to some degree in the group 12.5–100% (40% maturation), and absent in 12.5–67%, 8–100%, and 8–67% (groups with little or no maturation). Later, at onset of rapid testis growth, high temperature was linked to a pronounced downregulation of amh and of the self-renewal factors gsdf1 and gsdf2, as well as to a pronounced upregulation of the proliferation-stimulating factor igf3. Overall, the present findings demonstrate that rearing salmon at high temperatures can stimulate an early activation of the brain–pituitary–gonad axis before actual onset of rapid testis growth, enhancing and accelerating the mechanisms that control initiation and progression of maturation, while the feed regime has a minor impact. This poses a challenge for current salmon aquaculture practices that use sustained high temperature to maximize growth, since these practices increase the risk of early post-smolt maturation.

Interaction of temperature and feed ration on male postsmolt maturation of Atlantic salmon (Salmo salar L.)

Early maturation of Atlantic salmon male postsmolts is undesirable in aquaculture due to its negative impact on growth, welfare and seawater adaptation, however it is an increasing problem under intensive rearing conditions. The effect of temperature and feeding ration on sexual maturation during the induction of smoltification was tested with a 3 × 2 factorial design including three temperatures (8, 12.5 and 18 °C) and two feeding rations (100% and 67%), from 28 October 2018 to 30 May 2019. Fish in the six resulting treatments (1800 parr, initial mean weight 23.1 ± 7.2 g) were reared in flow-through under continuous light (LD24:0) except for a 5-week winter signal (LD12:12) introduced on 4 February 2019 to induce smoltification. A major effect of temperature determining the life history pattern of each group was observed. At 18 °C, a majority of the males were maturing in May under both rations (100% males in 18–100% and 88.9% males in 18–67%). Fish at 18 °C experienced an early activation of the BPG (Brain-Pituitary-Gonad) axis and displayed very poor signs of smoltification. At 12.5 °C, early maturation was also present, although at lower proportion than at 18 °C and more dependent upon feed ration (40% of males maturing in 12.5–100%, 8.6% in 12.5–67%). Salmon at 12.5 °C displayed the best growth within each feed regime, some early signs of smolting but subsequent low Na+, K+- ATPase activity after the winter signal. At 8 °C, salmon showed best signs of smoltification and lowest growth, while no males matured. The feed restriction had a minor effect on maturation at 18 °C but a greater impact at 12.5 °C, evidenced by significant differences between 12.5–100% and 12.5–67% in gonadosomatic index, plasma 11-ketotestosterone, insulin-like growth factor-I, and pituitary fshb (follicle stimulating hormone β-subunit gene) transcription. Onset of maturation was associated with the photoperiod change from short to long daylength, which probably also induced a slight activation of the BPG axis in non-maturing groups. Our results suggest that postsmolt producers must be cautious when using sustained high water temperatures, or the risk of early maturation and poor hypo-osmoregulatory performance will increase significantly. In addition, reducing the feeding ration will not help decrease incidence of maturation without significantly affecting growth.

Functional Characterization of Three GnRH Isoforms in Small Yellow Croaker Larimichthys polyactis Maintained in Captivity: Special Emphasis on Reproductive Dysfunction.

Simple SummarySmall yellow croaker is a popular marine fish in southeast Asian countries. Due to a decline in production in the wild, marine aquaculture has been initiated in Korea. Seed production is performed using captive-reared broodstock, which is known to undergo reproductive dysfunction. Reproductive dysfunction is closely linked to endocrinological dysfunction in the brain-pituitary-gonad (BPG) axis. Gonadotropin-releasing hormone (GnRH) acts as a central regulator in the BPG-axis. To determine the possible involvement of GnRHs in reproductive dysfunction of small yellow croaker reared in captivity, three GnRH isoforms were cloned and functional characterization has been performed. The expression of GnRH1 in the brain was significantly higher at the ripen stage in both sexes during gonadal development. Gonadotropin subunits (GPα, FSHβ, LHβ) were simultaneously increased in the pituitary at the ripen stage in both sexes. Interestingly, females showed significantly lower expression of GnRH1 and GtHs than males. Both in vivo and in vitro administration of GnRH1 showed that GtHs were increased significantly in the pituitary at high concentration. However, sex-steroids (E2 and MT) significantly inhibited the GnRH1 expression in the brain in a dose-dependent manner. Altogether, GnRH1 plays a key role in gonadal maturation, and a low level of GnRH1 and GtHs might be responsible for reproductive dysfunction in the female small yellow croaker.Fish reproduction is regulated by the brain–pituitary–gonad (BPG) axis where the gonadotropin-releasing hormone (GnRH) plays a central role. Seed production of small yellow croaker (Larimichthys polyactis) is performed using captive-reared broodstock known to undergo reproductive dysfunction, which is connected to endocrinological dysfunction. To determine the endocrinological mechanism of GnRHs in the BPG axis of small yellow croaker, full-length sequences of three GnRH isoforms encoding sbGnRH (GnRH1), cGnRH-II (GnRH2), and sGnRH (GnRH3) were cloned and characterized from brain tissue. qRT-PCR, in vivo, and in vitro experiments were performed for functional characterization. The mRNA expression of GnRH1 in the brain and gonadotropin subunits (GPα, FSHβ, and LHβ) in the pituitary were significantly higher at the ripen stage during gonadal development and GnRH1 at spawning stage during spawning events. Expression of both GnRH1 and GtH subunits was significantly lower in females than males. GtH subunits were induced at higher concentrations of GnRH1 in vivo and in vitro. Sex-steroids significantly inhibited the GnRH1 expression in vitro in a dose-dependent manner. Taken together, results indicated that GnRH1 plays a key role in gonadal maturation and sex-steroids induced negative feedback in the regulation of GnRH. A lower level of GnRH1 and GtHs might be responsible for reproductive dysfunction in a female small yellow croaker.

Impact of germ cell ablation on the activation of the brain-pituitary-gonadal axis in precocious Atlantic salmon (Salmosalar L.) males.

The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage. Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshβ messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

A cross-taxonomic perspective on the integration of temperature cues in vertebrate seasonal neuroendocrine pathways

The regulation of seasonality has been an area of interest for decades, yet global climate change has created extra urgency in the quest to understand how sensory circuits and neuroendocrine control systems interact to generate flexibility in biological timekeeping. The capacity of temperature to alter endogenous or photoperiod-regulated neuroendocrine mechanisms driving seasonality, either as a direct cue or through temperature-dependent effects on energy and metabolism, is at the heart of this phenological flexibility. However, until relatively recently, little research had been done on the integration of temperature information in canonical seasonal neuroendocrine pathways, particularly in vertebrates. We review recent advances from research in vertebrates that deepens our understanding of how temperature cues are perceived and integrated into seasonal hypothalamic thyroid hormone (TH) signaling, which is a critical regulator of downstream seasonal phenotypic changes such as those regulated by the BPG (brain-pituitary-gonadal) axis. Temperature perception occurs through cutaneous transient receptor potential (TRP) neurons, though sensitivity of these neurons varies markedly across taxa. Although photoperiod is the dominant cue used to trigger seasonal physiology or entrain circannual clocks, across birds, mammals, fish, reptiles and amphibians, seasonality appears to be temperature sensitive and in at least some cases this appears to be related to phylogenetically conserved TH signaling in the hypothalamus. Nevertheless, the exact mechanisms through which temperature modulates seasonal neuroendocrine pathways remains poorly understood.

Transcriptome characterization of BPG axis and expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine

BackgroundThe clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain–pituitary–gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae).ResultsRNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17β-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17β, while 3β-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly.ConclusionsThis is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.

Fish reproductive biology – Reflecting on five decades of fundamental and translational research

Driven by the broad diversity of species and physiologies and by reproduction-related bottlenecks in aquaculture, the field of fish reproductive biology has rapidly grown over the last five decades. This review provides my perspective on the field during this period, integrating fundamental and applied developments and milestones. Our basic understanding of the brain-pituitary–gonadal axis led to overcoming the failure of farmed fish to ovulate and spawn in captivity, allowing us to close the fish life cycle and establish a predictable, year-round production of eggs. Dissecting the molecular and hormonal mechanisms associated with sex determination and differentiation drove technologies for producing better performing mono-sex and reproductively-sterile fish. The growing contingent of passionate fish biologists, together with the availability of innovative platforms such as transgenesis and gene editing, as well as new models such as the zebrafish and medaka, have generated many discoveries, also leading to new insights of reproductive biology in higher vertebrates including humans. Consequently, fish have now been widely accepted as vertebrate reproductive models. Perhaps the best testament of the progress in our discipline is demonstrated at the International Symposia on Reproductive Physiology of Fish (ISRPF), at which our scientific family has convened every four years since the grandfather of the field, the late Ronald Billard, organized the inaugural 1977 meeting in Paimpont, France. As the one person who has been fortunate enough to attend all of these meetings since their inception, I have witnessed first-hand the astounding evolution of our field as we capitalized on the molecular and biotechnological revolutions in the life sciences, which enabled us to provide a higher resolution of fish reproductive and endocrine processes, answer more questions, and dive into deeper comprehension. Undoubtedly, the next (five) decades will be similarly exciting as we continue to integrate physiology with genomics, basic and translational research, and the small fish models with the aquacultured species.

Brain Control Reproduction by the Endocrine System of Female Blue Gourami (Trichogaster trichopterus).

Blue gourami belongs to the Labyrinithici fish and the Anabantiform order. It is characterized by a specific organ located above its gills for the respiration of atmospheric oxygen. This specific adaptation to low oxygen levels affects reproduction that is controlled by the brain, which integrates different effects on reproduction mainly through two axes—the gonadotropic brain pituitary gonad axis (BPG) and the hypothalamic-pituitary-somatotropic axis (HPS axis), including the interactions between them. This brain control reproduction of the Anabantoidei suborder summarizes information that has been published on the hormones involved in controlling the reproduction system of a model female blue gourami fish (Trichogaster trichopterus), including unpublished data. In the whole-brain transcriptome of blue gourami, 17 transcription genes change during vitellogenesis in the brain. The hormones involved in reproduction in blue gourami described in the present paper include: Kisspeptin 2 (Kiss 2) and its receptors 1 and 2 (KissR 1 and 2); gonadotropin-releasing hormone 1, 2 and 3 (GnRH1, 2 and 3); GnRH receptor; pituitary adenylate cyclase-activating polypeptide (PACAP) and its related peptide (PRP); somatolactin (SL); follicle-stimulating hormone (FSH); luteinizing hormone (LH); growth hormone (GH); prolactin (PRL), 17β-estradiol (E2); testosterone (T); vitellogenesis (VTL); and 17α,20β- dihydroxy-4-pregnen-3-one (17,20P). A proposed quality model is presented regarding the brain control oogenesis in blue gourami that has a Labyrinth organ about which relatively little information has been published. This paper summarizes the complex various factors involved in the interactions between external and internal elements affecting the brain of fish reproduction in the Anabantiform order. It is suggested to study in the future the involvement of receptors of hormones, pheromones, and genome changes in various organs belonging to the reproduction system during the reproduction cycles about which little is known.

Changes in expression of reproduction-related hormones in the brain and pituitary during early ovarian differentiation and development in the red spotted grouper Epinephelus akaara, with emphasis on FSHβ and LHβ

The brain-pituitary-gonadal (BPG) axis, an important endocrine system, is closely related to gonadal development in teleosts. This study aimed to investigate the regulation of reproduction-related hormones in the BPG axis during early ovarian differentiation and development from 30 to 160 days post hatching (dph) in the red spotted grouper. The immune signals of FSHβ and LHβ were detected by immunohistochemistry. kiss1, kiss2, gpr54, gnrh1, fshβ, and lhβ gene expression was analyzed using qRT-PCR. 17β-estradiol (E2) levels were analyzed by ELISA. The immune signals of FSHβ and LHβ were detected in the central, ventral, or marginal part of the proximal pars distalis area at 55, 80, 100, 120, and 160 dph, along with a few signals detected in the external border of the pars intermedia area at 55, 120, and 160 dph. As a result of histological observation, we determined that ovarian differentiation began at 80 dph and finished at 120 dph. At 30 dph before ovarian differentiation, only the FSHβ signal was detected but with weak reaction, suggesting the earlier appearance of FSHβ than LHβ after hatching. Comparison of the signal intensity and gene expression of FSHβ and LHβ indicated that LH had stronger signals and higher transcript levels at 80 dph while E2 levels also increased, suggesting that LH may play a more active role in the onset of ovarian differentiation. gnrh1 and the kisspeptin genes showed no clear changes, but gpr54 (kisspeptin receptor) significantly increased during the ovarian differentiation phase. These results suggest that the kisspeptin receptor GPR54 may be involved in ovarian differentiation in the red spotted grouper.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males

BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 °C, T10) for 2 weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest.

Mechanistic target of rapamycin (mTOR) implicated in plasticity of the reproductive axis during social status transitions

The highly conserved brain-pituitary-gonadal (BPG) axis controls reproduction in all vertebrates, so analyzing the regulation of this signaling cascade is important for understanding reproductive competence. The protein kinase mechanistic target of rapamycin (mTOR) functions as a conserved regulator of cellular growth and metabolism in all eukaryotes, and also regulates the reproductive axis in mammals. However, whether mTOR might also regulate the BPG axis in non-mammalian vertebrates remains unexplored. We used complementary experimental approaches in an African cichlid fish, Astatotilapia burtoni, to demonstrate that mTOR is involved in regulation of the brain, pituitary, and testes when males rise in rank to social dominance. mTOR or downstream components of its signaling pathway (p-p70S6K) were detected in gonadotropin-releasing hormone (GnRH1) neurons, the pituitary, and testes. Transcript levels of mtor in the pituitary and testes also varied when reproductively-suppressed subordinate males rose in social rank to become dominant reproductively-active males, a transition similar to puberty in mammals. Intracerebroventricular injection of the mTORC1 inhibitor, rapamycin, revealed a role for mTOR in the socially-induced hypertrophy of GnRH1 neurons. Rapamycin treatment also had effects at the pituitary and testes, suggesting involvement of the mTORC1 complex at multiple levels of the reproductive axis. Thus, we show that mTOR regulation of BPG function is conserved to fishes, likely playing important roles in regulating reproduction and fertility across all male vertebrates.

Daily rhythms in the reproductive axis of Nile tilapia (Oreochromis niloticus): Plasma steroids and gene expression in brain, pituitary, gonad and egg

Control of reproduction is one of the key issues to extend tilapia aquaculture. In most fish species, reproduction is a rhythmic process which ensures that reproduction timing matches the most favorable environmental conditions to maximize the survival of offspring. Although the existence of daily spawning rhythms has been reported for a number of fish species, the daily rhythms in neuroendocrine factors along the BPG (brain-pituitary-gonadal) axis remains mostly unknown. The objective of this paper was to investigate the existence of daily rhythms in BPG gene expression and plasma sex steroids in tilapia (Oreochromis niloticus). To this end, 36 males and 36 females were kept in separate tanks at 29 ± 0.5 °C and in a 12 h:12 h light/dark cycle. After 4 weeks of acclimation, male and female tilapia were mixed into six groups at a female:male ratio of 6:6. Two weeks later, fish were anesthetized and sampled every 4 h during a 24-hour cycle, and blood, brain, pituitary, gonad and eggs were collected for later analyses. The expression levels of the genes from the brain (gnrh1, gnrh2, gnrh3, kiss2, gnih), pituitary (fshβ and lhβ), gonad (star, cyp11a, cyp17, cyp19a1a and amh) and eggs (zp3b and fyna) were analyzed by qPCR. ELISA was used to analyze testosterone and estradiol in male and female plasma, respectively, and maturation-inducing hormone (MIH) in plasma in both sexes. The results revealed timely-coordinated daily rhythms along the BPG axis of tilapia. The expression of the brain genes showed that their acrophase was distributed throughout the LD cycle. However, a coordinated and harmonious pattern of daily rhythms appeared on the following levels of the BPG axis. The peaks in expression of the pituitary genes were reached at night, while the gonadal genes and plasma sex steroids peaked in the light phase. These findings can be useful for improving breeding protocols for tilapia aquaculture and for defining optimum timing for reproduction in captivity.

Daily rhythms of expression in reproductive genes along the brain-pituitary-gonad axis and liver of zebrafish

The brain-pituitary-gonadal (BPG) axis regulates the activation of the endocrine machinery that triggers reproduction, which is a typical rhythmic process. In this research we focused on investigating the daily expression rhythms of the key reproductive genes involved in the BPG axis and the liver of zebrafish. To this end, male and female zebrafish were subjected to a stimulating photoperiod with a 14 h light:10 h dark cycle. Brain, pituitary and gonads, as well as female liver samples, were taken every 4 h during a 24 h cycle. The results revealed that most genes exhibited statistically significant daily rhythms. Most of the brain reproductive genes (gnrh2, gnrh3, kiss1, kiss2 and gnrhr3) displayed a daily rhythm of expression with a nocturnal acrophase (between Zeitgeber Time [ZT] 14:34 h and ZT18:34 h, lights off at ZT = 14 h). The male kiss2 gene presented neither significant rhythms nor daily variations, while the male gnrh3 and female kiss2 genes exhibited diurnal peaks of expression at ZT06:34 h and ZT04:34 h, respectively. In contrast, the pituitary genes (fshβ, lhβ, gnrhr2) showed daily rhythms of expression with an acrophase during the light phase (between ZT02:10 h and ZT10:35 h). The female gnrhr3 gene exhibited neither significant rhythms nor daily variations. The male gnrhr3 gene presented a nocturnal acrophase (ZT14:32 h). The gonad genes (star, cyp17a1, 20βhsd, lhr, fshr, cyp19a1a, foxl2, amh, dmrt1 and 11βhsd) revealed statistically significant daily rhythms with nocturnal acrophases, except for female cyp17a1a (ZT06:21 h) and 20βhsd (ZT05:19 h). Lastly, the female liver genes presented daily rhythms with a maximum peak of expression around the transition phase from darkness to light (ZT01:00 h for erα and at ZT23:09 h for vtg2). These findings are consistent with the daily reproduction rhythms displayed by zebrafish, which are timed by the reproductive axis. Considering that reproductive success is critical for survival of the species, the knowledge of the rhythms of the endocrine BPG machinery provides useful information to understand the reproduction process and to establish optimal protocols and conditions for reproductive treatments.

In situ localization of vasotocin receptor gene transcripts in the brain-pituitary-gonadal axis of the catfish Heteropneustes fossilis: a morpho-functional study.

In the catfish Heteropneustes fossilis, three vasotocin (VT) receptor subtype genes, v1a1, v1a2, and v2a, were cloned and characterized previously. In the present study, using RNA probes, we localized the distribution of the gene transcripts in the brain-pituitary-gonadal (BPG) axis. The V1a-type receptor, v1a1 and v1a2, genes showed similar and overlapping distribution in the brain. The gene paralogs are distributed in the radial glial cells (RGCs) of the telencephalic ventricle and around the third ventricle in the hypothalamus and thalamus, olfactory tract, nucleus preopticus, nucleus lateralis tuberis, nucleus recessus lateralis and posterioris, nucleus saccus vasculosi, thalamic nuclei, habenular nucleus, habenular commissure, basal part of pineal stalk, accessory pretectal nucleus, optic tectum, corpus and valvula of the cerebellum, and facial and vagal lobes. The V2a receptor gene (v2a) has restricted distribution and is largely confined to the anterior subependymal region of the telencephalon. The localization pattern shows that the V1a-type receptors are distributed in major sensorimotor processing centers and the neuroendocrine/reproductive centers of the brain. In the pituitary, the receptor genes were localized differentially in the three divisions with the V1a-type receptor genes strongly expressed in the rostral pars distalis compared to the v2a paralog. In the ovary, the V1a-type receptor genes were localized in the follicular layer while v2a was localized in the oocyte membrane. In the testis, v1a2 and v2a are densely distributed in the interstitial tissue and seminiferous epithelium but the v1a1 is lowly expressed. The results suggest that the VT receptor genes have an extensive but differential distribution in the BPG axis. Future experimental studies are required to correlate the cellular localizations with specific functions of VT in the BPG axis.

Embryonic exposure to benzo(a)pyrene inhibits reproductive capability in adult female zebrafish and correlation with DNA methylation

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17β-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain–pituitary–gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish.