Chemogenetic Depletion of Hypophysiotropic GnRH Neurons Does Not Affect Fertility in Mature Female Zebrafish.

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.

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.

The gonadotropin-releasing hormones: Lessons from fish.

Fish have been of paramount importance to our understanding of vertebrate comparative neuroendocrinology and the mechanisms underlying the physiology and evolution of gonadotropin-releasing hormones (GnRH) and their genes. This review integrates past and recent knowledge on the Gnrh system in the fish model. Multiple Gnrh isoforms (two or three forms) are present in all teleosts, as well as multiple Gnrh receptors (up to five types), which differ in neuroanatomical localization, pattern of projections, ontogeny and functions. The role of the different Gnrh forms in reproduction seems to also differ in teleost models possessing two versus three Gnrh forms, Gnrh3 being the main hypophysiotropic hormone in the former and Gnrh1 in the latter. Functions of the non-hypothalamic Gnrh isoforms are still unclear, although under suboptimal physiological conditions (e.g. fasting), Gnrh2 may increase in the pituitary to ensure the integrity of reproduction under these conditions. Recent developments in transgenesis and mutagenesis in fish models have permitted the generation of fish lines expressing fluorophores in Gnrh neurons and to elucidate the dynamics of the elaborate innervations of the different neuronal populations, thus enabling a more accurate delineation of their reproductive roles and regulations. Moreover, in combination with neuronal electrophysiology, these lines have clarified the Gnrh mode of actions in modulating Lh and Fsh activities. While loss of function and genome editing studies had the premise to elucidate the exact roles of the multiple Gnrhs in reproduction and other processes, they have instead evoked an ongoing debate about these roles and opened new avenues of research that will no doubt lead to new discoveries regarding the not-yet-fully-understood Gnrh system.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

Nannochloropsis genomes reveal evolution of microalgal oleaginous traits.

Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and evolution of oleaginous traits remain largely unknown. Here we present a detailed phylogenomic analysis of five oleaginous Nannochloropsis species (a total of six strains) and one time-series transcriptome dataset for triacylglycerol (TAG) synthesis on one representative strain. Despite small genome sizes, high coding potential and relative paucity of mobile elements, the genomes feature small cores of ca. 2,700 protein-coding genes and a large pan-genome of >38,000 genes. The six genomes share key oleaginous traits, such as the enrichment of selected lipid biosynthesis genes and certain glycoside hydrolase genes that potentially shift carbon flux from chrysolaminaran to TAG synthesis. The eleven type II diacylglycerol acyltransferase genes (DGAT-2) in every strain, each expressed during TAG synthesis, likely originated from three ancient genomes, including the secondary endosymbiosis host and the engulfed green and red algae. Horizontal gene transfers were inferred in most lipid synthesis nodes with expanded gene doses and many glycoside hydrolase genes. Thus multiple genome pooling and horizontal genetic exchange, together with selective inheritance of lipid synthesis genes and species-specific gene loss, have led to the enormous genetic apparatus for oleaginousness and the wide genomic divergence among present-day Nannochloropsis. These findings have important implications in the screening and genetic engineering of microalgae for biofuels.

Seasonal expression of arginine vasotocin mRNA and its correlations to gonadal steroidogenic enzymes and sexually dimorphic coloration during sex reversal in the gilthead seabream (Sparus aurata).

Arginine vasotocin is a hormone produced in the hypothalamus of teleost fish that has been shown to regulate gonad development and sexual behavior. To study the role of arginine vasotocin in the gonadal cycle of the hermaphrodite gilthead seabream, Sparus aurata, we cloned the seabream arginine vasotocin (avt) complementary DNA (cDNA). We investigated the expression of brain avt throughout the gonad cycle using real-time quantitative PCR and compared its expression levels to the expression levels of two key gonadal steroidogenic enzymes, cyp19a1a and cyp11b2. In July, when the process of sex reversal is thought to begin, avt expression was elevated over the previous 2 months. Avt in the brain remained at or above the level of July until November then peaked again in December. There was no difference between males and females in the expression levels of brain avt throughout the year. However, only in ambisexual fish was the expression of the cyp19a1a gonadal aromatase correlated to the expression of avt in the brain. Cyp11b2 did not show any correlation to brain avt expression. We also found that females had more intense body coloration than males and that this intensity peaked prior to spawning. Avt expression and female coloration were positively correlated. The fact that brain avt expression was lowest during gonad quiescence, together with the observation of a correlation between brain avt with gonadal cyp19a1a and body coloration during that time suggests that avt may play a role during the process of sex reversal and spawning of the gilthead seabream.

Kisspeptin Antagonists Reveal Kisspeptin 1 and Kisspeptin 2 Differential Regulation of Reproduction in the Teleost, Morone saxatilis.

The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells. Pep 234 (a mammalian KISS1 antagonist) antagonizes Kiss1r signaling activated by Kiss1 and Kiss2, and pep 359 (a novel analog) antagonizes Kiss2 activation of both receptors. In vitro studies using brain slices demonstrated that only Kiss2 can upregulate the expression of the hypophysiotropic gnrh1, which was subsequently diminished by pep 234 and pep 359. In primary pituitary cell cultures, the two antagonists revealed a complex network of putative endogenous and exogenous regulation by kisspeptin. While both kisspeptins stimulate Fsh expression and secretion, Kiss2 predominately induces Lh secretion. Pep 234 and 359 treatment of spawning males hindered sperm production. This effect was accompanied with decreased brain gnrh1 and gnrh2 mRNA levels and peptide content in the pituitary, and increased levels of pituitary Lh, probably due to attenuation of Lh release. Strikingly, the mRNA levels of arginine-vasotocin, the neurons of which in the preoptic area coexpress kiss2r, were dramatically reduced by the antagonists. Our results demonstrate differential actions of Kiss1 and Kiss2 systems along the hypothalamic-pituitary axis and interactions with other neuropeptides, and further reinforce the importance of kisspeptin in the execution of spawning.

Production of reproductively sterile fish: A mini-review of germ cell elimination technologies.

As seafood consumption shifts from fisheries harvests to artificially propagated aquatic species, the increase of aquaculture activities poses a biological threat to our environment. Selectively bred, non-native and (eventually) genetically engineered farmed fish may escape from aquaculture operations, propagate and/or interbreed with wild stocks and subsequently alter the genetic makeup of populations in the environment. Thus, an effective strategy for bio-containment of farmed fish is critically needed. Farming reproductively sterile fish is the most environmentally sustainable approach to ensure complete bio-containment in large-scale aquaculture operations. Chromosome set manipulations to produce sterile fish, including polyploidy and hybridization, are currently the most common practices in the aquaculture industry. However, they do not always result in 100% sterility of the treated fish. Moreover, triploid fish typically do not perform as well as the non-manipulated diploids under commercial culture conditions. In the last half decade, several genetic engineering methods have been developed to produce sterile fish. In this review, we will address the latest technologies that use transgenic approaches to eliminate germ cells, resulting in the production of sterile fish. These latest advances also led us to the development of egg/embryo immersion methodologies to deliver and screen compounds that can be used to eliminate primordial germ cells and produce sterile fish. This emerging non-transgenic strategy for the production of reproductively sterile fish in aquaculture will also be discussed.

Influence of tidal cycles on the endocrine control of reproductive activity in common snook (Centropomus undecimalis).

The aim of our study was to confirm the role of tidal pattern on the coordination of oocyte maturation and spawning in common snook Centropomus undecimalis. To do so, we studied oocyte maturation during the spawning season in relation to the tidal pattern in both males and females by means of histology and hormonal profiling along the pituitary-gonadal axis. Plasma LH levels, as well as transcript levels of gonadotropin genes (fshß and lhß) from the pituitaries of sexually mature male and female common snook were analyzed using a heterologous ELISA and quantitative RT-PCR, respectively. The fshß and lhß cDNAs were isolated and phylogenetic analysis of the deduced amino acid sequences revealed strong identity with other teleosts (75-90%). A strong link was found between tide and follicular development irrespective of the time of the day: female snook sampled on the rising tide were all found to have oocytes in the Secondary Growth Stage whereas females sampled at high tide or on the falling tide had oocytes in the later stages of maturation and ovulation. In addition, LH plasma and mRNA levels of fshß and lhß increased during the later stages of vitellogenesis peaking at ovulation in females. Plasma estradiol and testosterone significantly increased in late vitellogenesis (Secondary Growth Stage) and oocyte maturation (Eccentric Germinal Vesicle Step) respectively. Among male common snook sampled, no correlation was identified between tide and gonadal development. In addition, lhß mRNA expression in males peaked at the mid germinal epithelium stage as for testosterone and 11-KT in the blood while fshß expression and plasma LH levels peaked at late germinal epithelium stage. This study confirms the role played by tidal cycle on the entrainment of the later stages of oogenesis of common snook and provides a better understanding of the link between environmental and endocrine control of reproduction in this species.

A new approach for eliminating off-flavors from RAS fish, as part of the normal grow-out period.

A new concept is presented for eliminating off-flavor from cold-water RAS-grown fish, while feeding, and as a part of the normal grow-out period. The technology is based on disconnecting the nitrification biofilter, and instead passing the water through an electrolysis system, which both oxidizes the ammonia and disinfects the water, while also removing the off-flavor compounds from the water, which thereby results in the purging of the fish. The purging period was expected to last up to 2 weeks and the fish are fed throughout it. Laboratory and pilot plant experiments were performed to prove the new concept. Lab experiments included quantification of the removal of MIB and geosmin by electrooxidation and stripping, together and separately, in the presence and absence of organic matter. A pilot plant experiment was performed using Rainbow trout to determine the rate at which the off-flavor compounds were removed from the water and the fish flesh (both skin and muscle were tested). The results show that the treatment process eliminated off-flavors in the water after ∼7 days and that the fish were below taste and odor threshold for geosmin and MIB after a maximum of 11 days. Detachment from the biofilter and the fact that the water was vigorously disinfected during the electrooxidation step guaranteed that no further off-flavor compounds would be generated during the operation. Aquacultural-management assessment indicates that RAS farms can increase both their annual production and their income by more than 10%, by implementing the suggested concept as part of the grow-out period.

Loss of function of Vasoactive-intestinal peptide alters sex ratio and reduces male reproductive fitness in zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess two isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely sub-fertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with WT males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely non-existent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of three key genes in the androgen synthesis chain in the testis, 3ß-hsd, 17ß-hsd1 and cyp11c1 (11ß-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

Zebrafish dmrta2 regulates the expression of cdkn2c in spermatogenesis in the adult testis.

The exact function of the doublesex and mab-3 related transcription factor-like family a2 gene (dmrta2) has remained largely unknown possibly because of its functional redundancy with dmrta1 in most vertebrates. In this study, dmrta1 was demonstrated to likely be absent in the zebrafish genome, which facilitated our functional analysis of dmrta2 in this model organism. To analyze its gene function in embryos and adults, we generated a mutant form of Dmrta2 (R106Q, Dmrta2(RQ)) with its in vitro DNA-binding capacity abolished and a transgenic line for the inducible expression of this mutant Dmrta2(RQ) upon doxycycline (Dox) treatment. Preferential dmrta2 expression was detected in the developing brain during embryogenesis and in the adult testis. During embryogenesis, Dmrta2(RQ) expression caused severe embryonic development defects and dramatic expression changes of two telencephalic marker genes, fibroblast growth factor 8a (fgf8a), and empty spiracles homolog 1 (emx1). In adults, the inducible Dmrta2(RQ) expression occurred specifically in the adult testis and recapitulated the endogenous dmrta2 expression in this organ. Intriguingly, adult males expressing dmrta2(RQ) showed normal spermatogenesis and were fertile, but the expression of cyclin-dependent kinase inhibitor 2C (cdkn2c), which is evolutionarily clustered with dmrta2, was significantly suppressed during spermatogenesis. Further protein-binding and promoter mutation analysis indicated that a putative Dmrta2-binding site on the cdkn2c promoter was required for sustaining the normal expression of cdkn2c during zebrafish spermatogenesis, suggesting that Dmrta2 might regulate the expression of cdkn2c.

Chronic kisspeptin administration stimulated gonadal development in pre-pubertal male yellowtail kingfish (Seriola lalandi; Perciformes) during the breeding and non-breeding season.

The kisspeptin system is now accepted as a key regulator of vertebrate reproductive function, particularly the onset of puberty. In teleosts, the stimulatory effect of exogenous kisspeptins has been demonstrated mainly at the hypothalamic and pituitary levels of the reproductive axis, with very limited information pertaining to gonadal response. We determined the effect of chronic peripheral administration of the conserved kisspeptin decapeptides (YNLNSFGLRY or Kiss1-10; and FNFNPFGLRF or Kiss2-10) on gonadal development of pre-pubertal yellowtail kingfish (Seriola lalandi), a Perciform teleost, during the breeding and non-breeding season. We utilized slow-release implants to chronically deliver the synthesized peptides, which were based on the yellowtail kingfish kiss1 and kiss2 cDNA sequences that we isolated. The expression level of kiss2r and gnrh1 in the brain or hypothalamus did not vary between treated and control groups. Pituitary expression of fshß and lhß was upregulated only with Kiss1-10 treatment regardless of the season. Based on histological evidence, gonadal development was stimulated in male fish with either Kiss1-10 or Kiss2-10, with Kiss2-10 being more effective during the non-breeding period. Overall, our results suggest that kisspeptins modulate the early gonadal development of male yellowtail kingfish, however that may vary with the breeding season.

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the gilthead seabream, Sparus aurata.

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts. In the first experiment, I measured the levels of GnRH-1, GnRH-2, and GnRH-3 mRNA throughout the gonad cycle. All three GnRH mRNAs increase around the peak of the spawning season (December). GnRH-3 mRNA expression is also elevated in August, which coincides with the beginning of gonad differentiation. All three GnRH mRNAs have the lowest expression levels in the month of September. There was no difference between males and females in the expression levels of any of the three GnRH mRNA. In the second experiment, I measured individual dominance ranks in six groups of fish, three during quiescence and three during spawning. GnRH-1 mRNA expression was positively correlated with dominance rank only during the quiescent period. The more dominant fish tended to have higher GnRH-1 mRNA expression. The existence of a quiescent-only correlation between GnRH-1 mRNA and dominance rank suggests a mechanism by which activation of gonad maturation could occur first in the most dominant ambisexual fish.

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone species.

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.

Novel bacterial isolate from Permian groundwater, capable of aggregating potential biofuel-producing microalga Nannochloropsis oceanica IMET1.

Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochloropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001.

Vasoactive Intestinal Peptide Indirectly Elicits Pituitary LH Secretion Independent of GnRH in Female Zebrafish.

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhß mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild-type and gnrh3-/- females at 1 hour posttreatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA's manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and 17ß-estradiol in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

A half century of fish gonadotropin-releasing hormones: Breaking paradigms.

The field of fish gonadotropin-releasing hormones (GnRHs) is also celebrating its 50th anniversary this year. This review provides a chronological history of fish GnRH biology over the past five decades. It demonstrates how discoveries in fish regarding GnRH and GnRH receptor multiplicity, dynamic interactions between GnRH neurons, and additional neuroendocrine factors acting alongside GnRH, amongst others, have driven a paradigm shift in our understanding of GnRH systems and functions in vertebrates, including mammals. The role of technological innovations in enabling scientific discoveries is portrayed, as well as how fundamental research in fish GnRH led to translational outcomes in aquaculture. The interchange between fish and mammalian GnRH research is discussed, as is the value and utility of using fish models for advancing GnRH biology. Current challenges and future perspectives are presented, with the hope of expanding the dialogue and collaborations within the neuroendocrinology scientific community at large, capitalizing on diversifying model animals and the use of comparative strategies.

Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropin-releasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a flatfish that exhibits severe reproductive dysfunctions in captivity. This study aimed at investigating the existence of a dopamine (DA) inhibitory tone on the reproductive axis of this species. Four groups of Senegalese sole breeders were treated with, saline (controls, CNT), the DA antagonist pimozide (PIM, 5 mg kg(-1)), gonadotropin-releasing hormone agonist (GnRHa, 40 µg kg(-1)) or a combination of PIM+GnRHa (COMB). Effects were evaluated on pituitary GnRH levels (ELISA), pituitary gonadotropin subunit transcript levels (qPCR), plasma levels of sex steroids and vitellogenin (ELISA), gonad development (histology), spermiation and egg production. The GnRHa treatment induced egg release and stimulated testis maturation. In males, PIM did not affect pituitary GnRH content, but enhanced GnRHa-induced pituitary GPα transcripts and modified plasma androgen levels; moreover, PIM stimulated spermatogenesis and milt production, both alone and combined with GnRHa. In females, PIM did not affect pituitary and plasma endocrine parameters and did not affect egg production and fertilization success of the broodstock, either alone or in the combined treatment. In conclusion, data indicated the existence of a DA inhibition in mature males, which would be absent or weakly expressed in females.