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Our understanding of the hormonal mechanisms underlying parental care mainly stems from research on species with uniparental care. Far less is known about the physiological changes underlying motherhood and fatherhood in biparental caring species. Here, using two biparental caring cichlid species (Neolamprologus caudopunctatus and Neolamprologus pulcher), we explored the relative gene-expression levels of two genes implicated in the control of parental care, galanin (gal) and prolactin (prl). We investigated whole brain gene expression levels in both, male and female caring parents, as well as in non-caring individuals of both species. Caring males had higher prl and gal mRNA levels compared to caring females in both fish species. Expression of gal was highest when young were mobile and the need for parental defense was greatest and gal was lowest during the more stationary egg tending phase in N. caudopunctatus. The onset of parenthood was associated with lower expression of prl and higher expression of gal in N. pulcher, but this pattern was not observed in N. caudopunctatus. Our study demonstrates that gal gene expression is correlated with changes in parental care in two biparental cichlid species and extends both knowledge and taxonomic coverage of the possible neurogenetic mechanisms underlying parental care. FWF: P 27461, UID/Multi/04326/2019, DL57/2016/CT3751 info:eu-repo/semantics/publishedVersion

Somatostatin (SST) plays important roles in growth and development. In teleost fishes six SST encoding genes (sst1 to sst6) have been identified although few studies have addressed their function. Here we aim to determine the function of the teleost specific sst4 in the zebrafish. A CRISPR/Cas9 sst4 zebrafish mutant with loss of function (sst4−/−) was produced which grew significantly faster and was heavier at the onset of gonadal maturation than the wild type (WT). Consistent with their faster growth, liver igf1, igf2a and igf2b expression was significantly upregulated in the sst4−/− fish compared to the WT. Histological examination of the ovaries and testis indicated that sst4−/− fish had slightly delayed testicular gametogenesis compared to the WT. Significantly lower expression of igf3, amh, insl3, hsd17b3, hsd11b2, hsd20b, cyp11b and cyp17 was consistently observed in the sst4−/− testis. In contrast, the ovaries had lower expression of igf1, igf2a and cyp19a1a but increased expression of igf2b and hsd20b. The gonadotrophin beta subunits (fshb and lhb) in the brain were downregulated indicating the brain-pituitary-gonadal axis was downregulated in the sst4−/− fish and suggesting that the steroid production is compromised in the maturing gonads. In addition, analysis of sst1 and sst3 mRNA levels in sst4−/− fish suggests a dosage compensation effect of sst1 in the brain and liver. Altogether, the results from the zebrafish sst4−/− line support the idea that sst4 is involved in the regulation of igf signalling, somatic growth and reproduction since steroidogenesis and gametogenesis at pubertal onset were compromised. This research was supported by institutional funds from Shanghai Ocean University and Portuguese national funds from FCT - Foundation for Science and Technology through project UID/Multi/04326/2019. info:eu-repo/semantics/publishedVersion

Galanin (Gal) is a neuropeptide with multiple functions that is widely expressed in the central and peripheral nervous systems of vertebrates. Anatomical and functional evidence suggests a possible role in regulating reproduction in fishes. To test this possibility, we have isolated and characterized two gal alternative transcripts in European sea bass (Dicentrarchus labrax) that encode two prepropeptides, respectively of 29 (gal_MT853221) and 53 (gal_MT853222) amino acids. The two gal transcripts are highly expressed in brain, pituitary and gonads, and appear to be differentially regulated in males and females. In males, gal_MT853222 in the hypothalamus and gal_MT853221 in the pituitary were downregulated with the progression of spermatogenesis (stages I-III). Both transcripts are downregulated in testicles of 1-year (precocious) and 2-year spermiating males compared to immature fish of the same age. Gal peptides and receptors are expressed throughout ovarian development in the hypothalamic-pituitary-gonadal (HPG) axis of females. In the testis, immunoreactive Gal-29 and Gal-53 peptides were detected in blood vessels and Leydig cells during the spermatogenesis stages I-III but Gal immunostaining was barely undetected in more advanced stages. In the ovary, both peptides localized in interstitial cells and blood vessels and in theca cells surrounding the maturing oocytes. The immunolocalization of galanin in Leydig and theca cells suggests a possible role in steroid production regulation. The different pattern of gal expression and Gal localization in the testis and ovary may suggest the possibility that androgens and estrogens may also regulate Gal gene transcription and translation. Altogether, this study showed evidence for the possible involvement of locally produced Gal in gametogenesis and that its production is differentially regulated in male and female gonads., This research was funded by the Portuguese national funds from FCT (Foundation of Science and Technology, I.P.) through projects UIDB/04326/2020 and PTDC/MAR-BIO/3890/2012 and by the European Union Seventh Framework Programme AQUAEXCEL (Grant Agreement 262336, TNA project 318). AG and GM were supported by Spanish MINECO (AGL2015-67477-C2-1-R) and CSIC (201640E073). R.S.T.M and P.I.S.P were supported by FCT under contracts of Norma transitória (DL 57/2016/CP1361/CT0021 and DL57/2016/CP6321/CT0015, respectively) with Universidade do Algarve. We acknowledge Zélia Velez for the help during sampling, Deborah Power for IHC advice and Victoria Alvarado and Alicia Felip for providing hypothalamus and pituitary RNA panels.

Chemical communication is widespread in the animal kingdom and olfaction constitutes a powerful channel for social and environmental cues. In fish, olfactory stimuli are known to influence physiological processes, including reproduction. Here we investigate the effects of olfaction on puberty in European sea bass Dicentrarchus labrax males. Intact sea bass coming to first maturity (puberty) are able to smell conspecific odours. However, induced anosmia during most of the spermatogenesis period had no effect on the sex ratio, gonad maturation state or gonado-somatic index at the time of reproduction. Furthermore anosmia decreased mRNA expression of brain KISS2 and pituitary LHb and FSHb, but not brain GnRH1 and GnRH3. Thus, although anosmia seems to modify gene expression of key reproduction related genetic factors, it seems to be insufficient to stop or delay growth or gonadal development and maturation.

In the present study the calcitonin receptor (CTR) sub-family of family B G-protein coupled receptors (GPCRs) in teleosts is evaluated and put in the context of the families overall evolution from echinodermates to vertebrates. Echinodermates, hemichordates, cephalochordates and tunicates have a single gene that encodes a receptor that bears similarity to the vertebrate calcitonin receptor (CTR) and calcitoninlike receptor (CTR/CLR). In tetrapods one gene encodes the calcitonin receptor (CALCR) and another gene the calcitonin receptor-like receptor (CALCRL). The evolution of CALCR has been under strong conservative pressure and a single copy is also found in fishes and high conservation of gene organisation and synteny exits from teleosts to human. A teleost specific CTR innovation that occurred after their divergence from holostei is the presence of several HBDs in the N-terminus. CALCRL had a different evolutionary trajectory from CALCR and although a single gene copy is present in tetrapods the sarcopterygii fish, the coelacanth, has 1 copy of CALCRL but also a fish specific form CALCRL3. The ray-finned fish, the spotted gar, has 1 copy of CALCRL and 1 of CALCRL3 but the teleost specific whole genome duplication has resulted in a CALCRL1 and CALCRL2 in addition to the fish specific CALCRL3. Strong conservation of CALCRL gene structure exists from human to fish. Promoter analysis in silico reveals that the duplicated CALCRL genes in the teleosts, zebrafish, takifugu, tetraodon and medaka, have divergent promoters and different putative co-regulated gene partners suggesting their function is different. We are grateful to Professor Adelino Canário for providing the sequence of sea bass EST clones. This study was supported by the European Regional Development Fund (ERDF) COMPETE – Operational Competitiveness Programme and Portuguese funds through FCT – Foundation for Science and Technology, under the project ‘‘PEst-C/MAR/LA0015/2013’’ and by FCT PTDC/BIA-BCM//73597/ 2010. RM (SFRH/BPD/66742/2009) and FV (SFRH/BPD/73597/ 2010) were in receipt of a post-doctoral grant from FCT, Portugal.

Paragangliomas are rare neuroendocrine tumors that arise in the sympathetic or parasympathetic nervous system. Sympathetic paragangliomas are mainly found in the adrenal medulla (designated pheochromocytomas) but may also have a thoracic, abdominal, or pelvic localization. Parasympathetic paragangliomas are generally located at the head or neck. Knowledge concerning the familial forms of paragangliomas has greatly improved in recent years. Additionally to the genes involved in the classical syndromic forms:VHLgene (von Hippel-Lindau),RETgene (Multiple Endocrine Neoplasia type 2), andNF1gene (Neurofibromatosis type 1), 10 novel genes have so far been implicated in the occurrence of paragangliomas/pheochromocytomas:SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, EGLN1, HIF2A,andKIF1B. It is currently accepted that about 35% of the paragangliomas cases are due to germline mutations in one of these genes. Furthermore, somatic mutations ofRET, VHL, NF1, MAX, HIF2A,andH-RAScan also be detected. The identification of the mutation responsible for the paraganglioma/pheochromocytoma phenotype in a patient may be crucial in determining the treatment and allowing specific follow-up guidelines, ultimately leading to a better prognosis. Herein, we summarize the most relevant aspects regarding the genetics and clinical aspects of the syndromic and nonsyndromic forms of pheochromocytoma/paraganglioma aiming to provide an algorithm for genetic testing.

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17β-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function. © 2014 Elsevier Inc., The research leading to these results was funded by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 222719 – LIFECYCLE and by the Foundation for Science and Technology of Portugal (FCT), through projects PTDC/MAR-BIO/3890/2012 and PEst-C/MAR/LA0015/2011 and fellowships to PISP (SFRH/BPD/25247/2005) and RSTM (SFRH/BPD/66742/2009).

The Ca(2+)-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca(2+) and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80microM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80microM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20alpha-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11beta-hydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca(2+)-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca(2+)-dependent CaM and that possibly Ca(2+)-CaM determines the shift in steroidogenesis from C21 steroids to androgens.

Steroid P450 11beta-hydroxylase, encoded by the CYP11B gene, is a key mitochondrial enzyme for the production of 11-oxygenated androgens, which have been shown to be potent masculinising steroids in several fish species. In this study we have isolated a CYP11B cDNA of 1903 base pairs from the testis of European sea bass (Dicentrarchus labrax) encoding a predicted protein of 552 amino acids. The amino acid identities to other vertebrate 11beta-hydroxylase proteins ranged from 66% to 72% to other fish; 45% to amphibian and 35-39% to mammalian. Southern blot indicated that a single CYP11B gene is present. Northern blot analysis detected two transcripts in testis and head kidney, one of the same size of the isolated clone and the other of 3.9 kb. Reverse transcriptase-polymerase chain reaction showed abundant mRNA expression only in testis and head kidney, being residual in a range of other tissues. Expression of CYP11B and CYP19A (which encodes for ovarian aromatase) was detected from at least 4 days post-hatching and did not appear to be affected by rearing temperature (15 and 20 degrees C) during the first 60 days, a period in which high temperatures promote masculinisation in European sea bass. Throughout, gonadogenesis (60-300 dph), a highly dimorphic pattern of CYP11B expression was consistent with a role of this gene in testicular development.

Background DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level. Methods A full length European sea bass DAX1 cDNA (sbDAX1) was isolated by screening a testis cDNA library. The structure of the DAX1 gene was determined by PCR and Southern blot. Multisequence alignments and phylogenetic analysis were used to compare the translated sbDAX1 product to that of other vertebrates. sbDAX1 expression was analysed by Northern blot and relative RT-PCR in adult tissues. Developmental expression of mRNA levels was analysed in groups of larvae grown either at 15°C or 20°C (masculinising temperature) during the first 60 days, or two groups of fish selected for fast (mostly females) and slow growth. Results The sbDAX1 is expressed as a single transcript in testis and ovary encoding a predicted protein of 301 amino acids. A polyglutamine stretch of variable length in different DAX1 proteins is present in the DNA binding domain. The sbDAX1 gene is composed of two exons, separated by a single 283 bp intron with conserved splice sites in same region of the ligand binding domain as other DAX1 genes. sbDAX1 mRNA is not restricted to the brain-pituitary-gonadal axis and is also detected in the gut, heart, gills, muscle and kidney. sbDAX1 mRNA was detected as early as 4 days post hatching (dph) and expression was not affected by incubation temperature. Throughout gonadal sex differentiation (60–300 dph) no dimorphic pattern of expression was observed. Conclusion The sbDAX1 gene and putative protein coding region is highly conserved and has a wide pattern of tissue expression. Although gene expression data suggests sbDAX1 to be important for the development and differentiation of the gonads, it is apparently not sex specific.