In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gon... more In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gonadal steroidogenic system, subsequent development of secondary sex characteristics and reproductive behavior. To explore if the absence of germ cells affects puberty or growth in Atlantic salmon, germ cell-free (GCF), dnd knockout and wild type (WT) postsmolts were stimulated to enter puberty. No GCF fish entered puberty, whereas 66.7% (males) and 30% (females) WT fish completed or entered puberty, respectively. Expression of genes related to steroidogenesis (star, cyp17a1, cyp11β, cyp19a1a), gonadal somatic cells (insl3, amh, igf3), oocytes (bmp15), gonadotropin receptors (fshr, lhcgr), and pituitary gonadotropic cells (fshb, lhb, gnrhr4) showed an immature status and failure to up-regulate gonadal sex steroid production in male and female GCF fish was also reflected in low or undetectable plasma sex steroids (11-ketotestosterone, estradiol-17β and testosterone). A gender difference (high in females, low in males) was found in the expression of star and cyp17a1 in GCF fish. No clear difference in growth was detected between GCF and immature WT fish, while growth was compromised in maturing WT males. We demonstrate for the first time in a vertebrate that germ cells are required for pubertal activation of the somatic steroidogenic cells.
INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in th... more INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (A und) but not of type A differentiating (A diff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating A und. Since the area occupied by A und decreases and that of A diff increases, we conclude that hINSL3 recruits A und into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single A und spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fshstimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit A und spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.
Biochemical and Biophysical Research Communications, Aug 1, 1992
Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the ... more Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the African catfish, Clarias gariepinus, using reversephase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of African catfish GnRH were determined using Edman degradation after digestion with pyroglutamyl aminopeptidase. In addition, both GnRHs were studied by mass spectrometry. The primary structure of African catfish GnRH I is identical to Thai catfish GnRH I, pGlu-His-Trp-Ser-His-Gly'Leu-Asn-Pro-Gly-NH 2, and the primary structure of African catfish GnRH II is identical to the widely distributed and highly conserved chicken GnRH II, pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH 2. ~ 1992 Academio P ..... ~no. It is well established that in vertebrates gonadotropin secretion is under control of hypothalamic peptides, the gonadotropin-releasing hormones (GnRHs). The structure of six GnRHs has been identified: all are decapeptides. The mammalian GnRH, commonly referred to as luteinizing hormone releasing hormone (LHRH), was isolated and chemically characterized for the first time in porcine and ovine hypothalamus tissue (1). Two more GnRHs were identified from chicken hypothalamus (chicken GnRH I and chicken GnRH II)(2,3), one from salmon brain (4), one from lamprey brain (5) and one from the brain of the Thai catfish (Clarias macrocephalus)(6). In mammals, to date only one form of GnRH has been isolated. In contrast, in teleost fish, multiple forms of GnRH are present within brain tissue of a single species. Salmon GnRH has been found in salmon brain tissue in combination with a GnRH identical to chicken GnRH II, and in other species also a number of unidentified GnRH-like peptides have been detected (7-10). In the European eel, GnRHs identical to chicken GnRH II and mammalian GnRH are present (11).
Glycoprotein hormone receptors contain large Nterminal extracellular domains (ECDs) that distingu... more Glycoprotein hormone receptors contain large Nterminal extracellular domains (ECDs) that distinguish these receptors from most other G proteincoupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N-and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which -strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R)
The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the hum... more The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the -strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/ hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptor's responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two -strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R -strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R -strand 6, rather than the introduction of its corresponding hLH-R -strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R -strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn 104 , a positive LH-R determinant, and Lys 179 a negative FSH-R determinant) that are situated on the C-terminal ends of -strands 3 and 6, respectively.
General and Comparative Endocrinology, Sep 1, 2013
In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone... more In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone beta (fshb) transcript levels were transiently elevated one year before spawning, re-increased in February, and remained high during spawning in November and in post-ovulatory fish in December. The first increase in plasma 17b-estradiol (E2), testosterone (T) and gonadosomatic index (GSI) was recorded in January; E2 rose up to one month prior to ovulation, while T and GSI kept increasing until ovulation. Pituitary luteinizing hormone beta (lhb) transcript levels peaked at the time of ovulation. Except for transient changes before and after ovulation, ovarian follicle stimulating hormone receptor (fshr) transcript amounts were relatively stable at a high level. By contrast, luteinizing hormone receptor (lhcgr) transcript levels started out low and increased in parallel to GSI and plasma E2 levels. Exposure to continuous light (LL) induced a bimodal response where maturation was accelerated or arrested. The LL-arrested females showed previtellogenic oil droplet stage follicles or primary yolk follicles only, and fshb and E2 plasma levels collapsed while fshr increased. The LL-accelerated females showed elevated lhb transcript levels and slightly elevated E2 levels during early vitellogenesis, and significantly elevated lhcgr E2 and GSI levels in late vitellogenesis. We conclude that Fsh-dependent signaling stimulates recruitment into and the sustained development through vitellogenesis. Up-regulation of lhcgr gene expression during vitellogenesis may reflect an estrogenic effect, while elevated fshr gene expression following ovulation or during LL-induced arrestment may be associated with ovarian tissue remodeling processes.
Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment rec... more Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3-to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGn-RHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of downregulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH 3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10 ؊13-10 ؊9 M). GH 3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10 ؊13-10 ؊7 M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins. (Molecular Endocrinology 12: 161-171, 1998)
In mammals, the specificity of FSH-FSH receptor (FSHR), LH-LH receptor (LHR) and TSH-TSH receptor... more In mammals, the specificity of FSH-FSH receptor (FSHR), LH-LH receptor (LHR) and TSH-TSH receptor couples is such that no cross-activation occurs under normal physiological conditions. The interactions between fish gonadotropins and their receptors, however, appear to be less discriminatory. For example, the catfish FSHR is highly responsive to both catfish LH and catfish FSH, while the catfish LHR is specific for its cognate LH. Comparative structure-function studies aimed at elucidating the molecular basis of ligand promiscuity (in fish) and ligand selectivity (in mammals) are described in this paper.
Background: Our understanding of the molecular mechanisms implementing pubertal maturation of the... more Background: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. Conclusions: This study has-for the first time-linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.
The gene and cDNA encoding a putative follicle-stimulating hormone  subunit (cfFSH) from Africa... more The gene and cDNA encoding a putative follicle-stimulating hormone  subunit (cfFSH) from African catfish (Clarias gariepinus) were cloned. Similar to other FSH genes, the cfFSH gene consisted of three exons interrupted by two introns. The cfFSH cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone  subunits were positionally conserved in cfFSH. The cfFSH mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSH transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSH mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSH mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSH transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.
The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hF... more The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their ϳ350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human folliclestimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeatcontaining subdomain (LRR subdomain) flanked by Nand C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel -strands. Receptors in which individual -strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R -strands 3 and 6. A mutant receptor in which the hFSH-R -strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most -strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.
Precocious male maturation causes reduced welfare and increased production costs in Atlantic salm... more Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases folliclestimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonadosomatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes,~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/ steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17b-estradiol (E 2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates
General and Comparative Endocrinology, May 1, 2008
Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognat... more Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognate hormones. In contrast, the follicle-stimulating hormone receptor of the African catfish (cfFSHR) is promiscuously activated by catfish luteinizing hormone (cfLH). Glycoprotein hormones bind to the concave site of the cusp-shaped N-terminal GpHR exodomain, which is formed by 9-10 parallel b-strands. Hence, hormone selectivity of each GpHR for its cognate ligand is defined by amino acid sequence divergence in these b-strands between different GpHRs. To identify the molecular determinants that allow promiscuous activation of the cfFSHR by cfLH, b-strands were systematically exchanged between the cfFSHR and the human FSHR. Both gain-of-function and loss-of-function mutational approaches revealed that b-strand 2 of the cfFSHR contains determinants that contribute to the receptor's responsiveness to cfLH.
The goal of this paper is to establish Japanese medaka (Oryzias latipes) as a model for relaxin f... more The goal of this paper is to establish Japanese medaka (Oryzias latipes) as a model for relaxin family peptide research, particularly for studying the functions of RLN3 and INSL5, hormones playing roles in neuroendocrine regulation. Medaka, like other teleosts, retained duplicate copies of rln3, insl5 and their rxfp3/4-type receptors following fish-specific whole genome duplication (WGD) and paralogous copies of these genes may have subfunctionalised providing an intuitive model for teasing apart the pleiotropic roles of the corresponding genes in mammals. To this end, we provide experimental evidence for the expression of the relaxin family genes in medaka that had previously only been identified in-silico, confirm the gene structure of five of the ligand genes, characterise gene expression across multiple tissues and during embryonic development, perform in situ hybridization with anti-sense insl5a on embryos and in adult brain and intestinal samples, and compare these results to the data available in zebrafish. We find broad similarities but also some differences in the expression of relaxin family genes in zebrafish versus medaka, and find support for the hypothesis that the rln3a/rln3b and insl5a/insl5b paralogues have been subfunctionalized. Given that medaka has a suite of relaxin family genes more similar to other teleosts, and has retained the gene for rxfp4 (which is lost in zebrafish), our results suggest that O. latipes may be a good model for delineating the ancestral function of the relaxin family genes involved in neuroendocrine regulation.
Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, ... more Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cystforming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.
Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain... more Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.
The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea st... more The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and-B preprohormones. Here, we report the isolation and structural characterization of a~, a 2 and /3 peptides from dissected neurons VD1 and RPD2. The a I and a 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas 13 peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the a 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic a I and o~ z peptides on isolated auricle preparations of the Lymnaea heart were examined. The two a peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, though non-identical, sets of peptides.
In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gon... more In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gonadal steroidogenic system, subsequent development of secondary sex characteristics and reproductive behavior. To explore if the absence of germ cells affects puberty or growth in Atlantic salmon, germ cell-free (GCF), dnd knockout and wild type (WT) postsmolts were stimulated to enter puberty. No GCF fish entered puberty, whereas 66.7% (males) and 30% (females) WT fish completed or entered puberty, respectively. Expression of genes related to steroidogenesis (star, cyp17a1, cyp11β, cyp19a1a), gonadal somatic cells (insl3, amh, igf3), oocytes (bmp15), gonadotropin receptors (fshr, lhcgr), and pituitary gonadotropic cells (fshb, lhb, gnrhr4) showed an immature status and failure to up-regulate gonadal sex steroid production in male and female GCF fish was also reflected in low or undetectable plasma sex steroids (11-ketotestosterone, estradiol-17β and testosterone). A gender difference (high in females, low in males) was found in the expression of star and cyp17a1 in GCF fish. No clear difference in growth was detected between GCF and immature WT fish, while growth was compromised in maturing WT males. We demonstrate for the first time in a vertebrate that germ cells are required for pubertal activation of the somatic steroidogenic cells.
INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in th... more INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (A und) but not of type A differentiating (A diff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating A und. Since the area occupied by A und decreases and that of A diff increases, we conclude that hINSL3 recruits A und into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single A und spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fshstimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit A und spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.
Biochemical and Biophysical Research Communications, Aug 1, 1992
Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the ... more Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the African catfish, Clarias gariepinus, using reversephase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of African catfish GnRH were determined using Edman degradation after digestion with pyroglutamyl aminopeptidase. In addition, both GnRHs were studied by mass spectrometry. The primary structure of African catfish GnRH I is identical to Thai catfish GnRH I, pGlu-His-Trp-Ser-His-Gly'Leu-Asn-Pro-Gly-NH 2, and the primary structure of African catfish GnRH II is identical to the widely distributed and highly conserved chicken GnRH II, pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH 2. ~ 1992 Academio P ..... ~no. It is well established that in vertebrates gonadotropin secretion is under control of hypothalamic peptides, the gonadotropin-releasing hormones (GnRHs). The structure of six GnRHs has been identified: all are decapeptides. The mammalian GnRH, commonly referred to as luteinizing hormone releasing hormone (LHRH), was isolated and chemically characterized for the first time in porcine and ovine hypothalamus tissue (1). Two more GnRHs were identified from chicken hypothalamus (chicken GnRH I and chicken GnRH II)(2,3), one from salmon brain (4), one from lamprey brain (5) and one from the brain of the Thai catfish (Clarias macrocephalus)(6). In mammals, to date only one form of GnRH has been isolated. In contrast, in teleost fish, multiple forms of GnRH are present within brain tissue of a single species. Salmon GnRH has been found in salmon brain tissue in combination with a GnRH identical to chicken GnRH II, and in other species also a number of unidentified GnRH-like peptides have been detected (7-10). In the European eel, GnRHs identical to chicken GnRH II and mammalian GnRH are present (11).
Glycoprotein hormone receptors contain large Nterminal extracellular domains (ECDs) that distingu... more Glycoprotein hormone receptors contain large Nterminal extracellular domains (ECDs) that distinguish these receptors from most other G proteincoupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N-and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which -strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R)
The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the hum... more The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the -strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/ hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptor's responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two -strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R -strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R -strand 6, rather than the introduction of its corresponding hLH-R -strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R -strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn 104 , a positive LH-R determinant, and Lys 179 a negative FSH-R determinant) that are situated on the C-terminal ends of -strands 3 and 6, respectively.
General and Comparative Endocrinology, Sep 1, 2013
In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone... more In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone beta (fshb) transcript levels were transiently elevated one year before spawning, re-increased in February, and remained high during spawning in November and in post-ovulatory fish in December. The first increase in plasma 17b-estradiol (E2), testosterone (T) and gonadosomatic index (GSI) was recorded in January; E2 rose up to one month prior to ovulation, while T and GSI kept increasing until ovulation. Pituitary luteinizing hormone beta (lhb) transcript levels peaked at the time of ovulation. Except for transient changes before and after ovulation, ovarian follicle stimulating hormone receptor (fshr) transcript amounts were relatively stable at a high level. By contrast, luteinizing hormone receptor (lhcgr) transcript levels started out low and increased in parallel to GSI and plasma E2 levels. Exposure to continuous light (LL) induced a bimodal response where maturation was accelerated or arrested. The LL-arrested females showed previtellogenic oil droplet stage follicles or primary yolk follicles only, and fshb and E2 plasma levels collapsed while fshr increased. The LL-accelerated females showed elevated lhb transcript levels and slightly elevated E2 levels during early vitellogenesis, and significantly elevated lhcgr E2 and GSI levels in late vitellogenesis. We conclude that Fsh-dependent signaling stimulates recruitment into and the sustained development through vitellogenesis. Up-regulation of lhcgr gene expression during vitellogenesis may reflect an estrogenic effect, while elevated fshr gene expression following ovulation or during LL-induced arrestment may be associated with ovarian tissue remodeling processes.
Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment rec... more Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3-to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGn-RHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of downregulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH 3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10 ؊13-10 ؊9 M). GH 3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10 ؊13-10 ؊7 M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins. (Molecular Endocrinology 12: 161-171, 1998)
In mammals, the specificity of FSH-FSH receptor (FSHR), LH-LH receptor (LHR) and TSH-TSH receptor... more In mammals, the specificity of FSH-FSH receptor (FSHR), LH-LH receptor (LHR) and TSH-TSH receptor couples is such that no cross-activation occurs under normal physiological conditions. The interactions between fish gonadotropins and their receptors, however, appear to be less discriminatory. For example, the catfish FSHR is highly responsive to both catfish LH and catfish FSH, while the catfish LHR is specific for its cognate LH. Comparative structure-function studies aimed at elucidating the molecular basis of ligand promiscuity (in fish) and ligand selectivity (in mammals) are described in this paper.
Background: Our understanding of the molecular mechanisms implementing pubertal maturation of the... more Background: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. Conclusions: This study has-for the first time-linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.
The gene and cDNA encoding a putative follicle-stimulating hormone  subunit (cfFSH) from Africa... more The gene and cDNA encoding a putative follicle-stimulating hormone  subunit (cfFSH) from African catfish (Clarias gariepinus) were cloned. Similar to other FSH genes, the cfFSH gene consisted of three exons interrupted by two introns. The cfFSH cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone  subunits were positionally conserved in cfFSH. The cfFSH mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSH transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSH mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSH mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSH transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.
The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hF... more The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their ϳ350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human folliclestimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeatcontaining subdomain (LRR subdomain) flanked by Nand C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel -strands. Receptors in which individual -strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R -strands 3 and 6. A mutant receptor in which the hFSH-R -strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most -strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.
Precocious male maturation causes reduced welfare and increased production costs in Atlantic salm... more Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases folliclestimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonadosomatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes,~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/ steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17b-estradiol (E 2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates
General and Comparative Endocrinology, May 1, 2008
Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognat... more Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognate hormones. In contrast, the follicle-stimulating hormone receptor of the African catfish (cfFSHR) is promiscuously activated by catfish luteinizing hormone (cfLH). Glycoprotein hormones bind to the concave site of the cusp-shaped N-terminal GpHR exodomain, which is formed by 9-10 parallel b-strands. Hence, hormone selectivity of each GpHR for its cognate ligand is defined by amino acid sequence divergence in these b-strands between different GpHRs. To identify the molecular determinants that allow promiscuous activation of the cfFSHR by cfLH, b-strands were systematically exchanged between the cfFSHR and the human FSHR. Both gain-of-function and loss-of-function mutational approaches revealed that b-strand 2 of the cfFSHR contains determinants that contribute to the receptor's responsiveness to cfLH.
The goal of this paper is to establish Japanese medaka (Oryzias latipes) as a model for relaxin f... more The goal of this paper is to establish Japanese medaka (Oryzias latipes) as a model for relaxin family peptide research, particularly for studying the functions of RLN3 and INSL5, hormones playing roles in neuroendocrine regulation. Medaka, like other teleosts, retained duplicate copies of rln3, insl5 and their rxfp3/4-type receptors following fish-specific whole genome duplication (WGD) and paralogous copies of these genes may have subfunctionalised providing an intuitive model for teasing apart the pleiotropic roles of the corresponding genes in mammals. To this end, we provide experimental evidence for the expression of the relaxin family genes in medaka that had previously only been identified in-silico, confirm the gene structure of five of the ligand genes, characterise gene expression across multiple tissues and during embryonic development, perform in situ hybridization with anti-sense insl5a on embryos and in adult brain and intestinal samples, and compare these results to the data available in zebrafish. We find broad similarities but also some differences in the expression of relaxin family genes in zebrafish versus medaka, and find support for the hypothesis that the rln3a/rln3b and insl5a/insl5b paralogues have been subfunctionalized. Given that medaka has a suite of relaxin family genes more similar to other teleosts, and has retained the gene for rxfp4 (which is lost in zebrafish), our results suggest that O. latipes may be a good model for delineating the ancestral function of the relaxin family genes involved in neuroendocrine regulation.
Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, ... more Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cystforming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.
Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain... more Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.
The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea st... more The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and-B preprohormones. Here, we report the isolation and structural characterization of a~, a 2 and /3 peptides from dissected neurons VD1 and RPD2. The a I and a 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas 13 peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the a 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic a I and o~ z peptides on isolated auricle preparations of the Lymnaea heart were examined. The two a peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, though non-identical, sets of peptides.
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Papers by Jan Bogerd