Nilli Zmora

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 genes 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.

The medio-basal hypothalamus as a dynamic and plastic reproduction-related kisspeptin-gnrh-pituitary center in fish.

Kisspeptin regulates reproductive events, including puberty and ovulation, primarily via GnRH neurons. Prolonged treatment of prepubertal striped bass females with kisspeptin (Kiss) 1 or Kiss2 peptides failed to enhance puberty but suggested a gnrh-independent pituitary control pathway. Kiss2 inhibited, but Kiss1 stimulated, FShβ expression and gonadal development, although hypophysiotropic gnrh1 and gnrh receptor expression remained unchanged. In situ hybridization and immunohistochemistry on brains and pituitaries revealed a differential plasticity between the 2 kisspeptin neurons. The differences were most pronounced at the prespawning phase in 2 regions along the path of gnrh1 axons: the nucleus lateralis tuberis (NLT) and the neurohypophysis. Kiss1 neurons appeared in the NLT and innervated the neurohypophysis of prespawning males and females, reaching Lh gonadotropes in the proximal pars distalis. Males, at all reproductive stages, had Kiss2 innervations in the NLT and the neurohypophysis, forming large axonal bundles in the former and intermingling with gnrh1 axons. Unlike in males, only preovulatory females had massive NLT-neurohypophysis staining of kiss2. Kiss2 neurons showed a distinct appearance in the NLT pars ventralis-equivalent region only in spawning zebrafish, indicating that this phenomenon is widespread. These results underscore the NLT as important nuclei for kisspeptin action in 2 facets: 1) kisspeptin-gnrh interaction, both kisspeptins are involved in the regulation of gnrh release, in a stage- and sex-dependent manner, especially at the prespawning phase; and 2) gnrh-independent effect of Kiss peptides on the pituitary, which together with the plastic nature of their neuronal projections to the pituitary implies that a direct gonadotropic regulation is plausible.

A Novel Hormone Is Required for the Development of Reproductive Phenotypes in Adult Female Crabs

The crustacean male-specific androgenic hormone is widely accepted as a key factor in sexual differentiation and in the development of secondary sex characteristics. However, the mechanism by which the plethora of different reproductive strategies are controlled and executed in crustaceans is not known. We discovered in the blue crab, Callinectes sapidus, a hitherto unknown neurohormone, named crustacean female sex hormone (CFSH), in distinct neurosecretory cells in the eyestalk ganglia. CFSH is highly expressed in females but weakly in males, and its crucial role in developing adult female phenotypes has now been established. CFSH cDNA encodes a 225-amino acid (aa) novel protein composed of a 23-aa predicted signal peptide, 33-aa precursor-related peptide and 167-aa mature protein that did not match any other sequence in GenBank. CFSH RNA interference knockdown by multiple administrations of double-stranded RNA at the prepubertal stage causes abnormal development of brooding and mating systems upon puberty. These systems include a pair of gonopores and an egg attachment system for brooding, comprised of an enlarged semicircular abdomen and ovigerous setae. The ovigerous setae in CFSH knocked-down females were fewer and 50% shorter and the gonopores were either significantly smaller than those of controls, misplaced, or absent. We also identified CFSH in the green crab, Carcinus maenas, a species that shares a similar reproductive strategy with C. sapidus. Together, our data provide the first evidence for the presence of a female hormone in crustaceans and its importance in positively controlling anatomic features associated with brooding and mating systems. From an evolutionary standpoint, the endocrine control supporting a female-specific reproductive strategy, as previously described for many vertebrate species, has now been demonstrated for the first time in crustaceans.

Clearance of 17α-ethynyltestosterone from muscle of sex-inversed tilapia hybrids treated for growth enhancement with two doses of the androgen

Rothbard, S., Zohar, Y., Zmora, N., Levavi-Sivan, B., Moav, B. and Yaron, Z., 1990. Clearance of 17a-ethynyltestosterone from muscle of sex-inversed tilapia hybrids treated for growth enhancement with two doses of the androgen. Aquaculture., 89: 365-376. Seven- to ten-month-old tilapia hybrids (Oreochromis niloticus~ 0. aureus), treated for sex inversion early in their ontogeny, were fed daily with pellets containing 17cu-ethynyltestosterone (ET) at either 2 or 60 mg/kg for 2 or 11 weeks, respectively. One-g samples of the dorsal musculature were homogenized and extracted with chloroform: methanol (2 : 1; v/v), washed with water and the organic phase was removed and dried. The extract was dissolved in 80% methanol and the steroid cleaned to some extent on a SEP-PAK Cl8 cartridge by elution with 3 ml methanol 751. The isolated fraction was evaporated and aliquots were redissolved in a mixture of acetonitrile (CH&N) 17% or 46% containing 0. II trifluoroacetic acid (TFA) in various proportions and analyzed by reversed phase high-performance liquid chromatography (HPLC) at 254 nm. Muscle samples of fish treated for 11 weeks with 60 mg/kg contained detectable amount of the steroid only on the first day after the termination of feeding with ET. Samples taken on the 3rd, 5th and 7th days did not differ from the untreated controls and ET concentrations were below the detectable level of 50 rig/g.. The concentrations of the androgen in muscle samples of fish fed with the lower dose of ET were below the level of detection even on the day that feeding was terminated. It may be concluded that the synthetic androgen fed to tilapia even at a high dose, comparable to that used for sex inversion, reaches levels of less than 50 rig/g within 3 days. Residues of the androgen in fish maintained for several months on an androgen-free diet are expected to be negligible. Nevertheless, the possibility of contamination of the holding facilities and the environment with the synthetic androgen remains to be studied.

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

ABSTRACT 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.

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

Abstract 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. Summary Sentence Lpxrfa regulates reproduction in the zebrafish brain-pituitary axis through inhibitory effects on gonadotropins and Gnrh3, and Lpxrfa elicits these effects by utilizing Lpxrf receptors, as well as receptors of other reproductive neuropeptides.

Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass

Kisspeptin and neurokinin B (NKB) are neuropeptides co-expressed in the mammalian hypothalamus and coordinately control GnRH signaling. We have found that Nkb and kisspeptin neurons are distinct in the teleost, striped bass (STB) and capitalized on this phenomenon to study the mode of action of Nkb and its related neuropeptide-F (Nkf), both of which are encoded by the tac3 gene. In vitro brain slices and in vivo administration studies revealed that Nkb/f consistently downregulated kiss2, whereas antagonist (AntD) administration restored this effect. Overall, a minor effect was noted on gnrh1 expression, whereas Gnrh1 content in the pituitaries was reduced after Nkb/f treatment and increased with AntD. Concomitantly, immunostaining demonstrated that hypothalamic Nkb neurons border and densely innervate the largest kiss2 neuronal population in the hypothalamus, which also coexpresses Nkb receptor. No expression of Nkb receptor or Nkb neuronal projections was detected near/in Gnrh1 soma in the preoptic area. At the level of the pituitary, however, the picture was more complex: both Nkb/f and AntD upregulated lhb and fshb expression and Lh secretion in vivo Together with the stimulatory effect of Nkb/f on Lh/Fsh secretion from pituitary cells, in vitro, this may indicate an additional independent action of Nkb/f within the pituitary, in which the hypothalamic pathway is more dominant. The current study demonstrates that Nkb/f utilizes multiple pathways to regulate reproduction in the STB and that in the brain, Nkb mainly acts as a negative modulator of kiss2 to regulate the release of Gnrh1.

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 (fshβ and lhβ) from the pituitaries of sexually mature male and female common snook were analyzed using a heterologous ELISA and quantitative RT-PCR, respectively. The fshβ and lhβ 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 fshβ and lhβ 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, lhβ mRNA expression in males peaked at the mid germinal epithelium stage as for testosterone and 11-KT in the blood while fshβ 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.

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

Abstract 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. Summary Sentence Knockout of Gnrh2 and Gnrh3 in zebrafish does not impede reproduction, but invokes a potential compensatory response, including the upregulation of several reproductive and feeding factors, neuronal plasticity of Scg2, and downregulation of Agrp1.