Elisabeth Sambroni

Effects of acute versus sustained administration of GnRHa on GtH release and ovulation in the rainbow trout, Oncorhynchus mykiss

Abstract The effects of different forms of GnRHa (sustained vs. acute) on the stimulation of gonadotropin secretion and the rate of ovulation were investigated in the rainbow trout, Oncorhynchus mykiss . A biodegradable sustained release form of DTrp 6 LH-RH induced a progressive rise in the gonadotropin plasma levels in a dose-dependent manner. The stimulation of gonadotropin secretion was maintained for 3 weeks. All fish ovulated within 5 days after injection using a high dosage (50 μg/kg body weight) whereas with the lower dosages (25 and 12.5 μg/kg body weight) 100% ovulation was reached after 8 days. In contrast, acute injections of GnRHa (20 μg/kg body weight) induced a transient stimulation of gonadotropin secretion. Ovulation rates were first accelerated within 8–10 days and then remained stable but they did not reach 100% 26 days after the injection. At that time the rate of ovulation in the control group had increased to 70%. These results are discussed, along with a comparison of variability of egg quality with treatments.

Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line.

In vertebrates, gonadotropins (GTHs) (FSH and LH) are two circulating pituitary glycoprotein hormones that play a major role in the regulation of gonadal functions, including gonadal cell proliferation/differentiation and steroidogenesis. In mammals, it is well known that their biological effects are mediated by highly specific membrane-bound receptors expressed preferentially on the somatic cells of the gonads. However, in fish, binding and functional studies have shown that cross-reactivity may occur in GTH receptors depending on the species. To understand the molecular mechanisms involved in GTH actions, functional characterization of trout GTH receptors and their gonadal gene expression pattern has been carried out. The present study describes the presence of two distinct GTH receptors in trout showing similarities with those of higher vertebrates but also differences in their structural determinants. In vitro functional studies demonstrate that rtLH specifically activates its cognate receptor (EC(50) = 117 ng/ml), whereas purified rainbow trout FSH (rtFSH) activates FSHR but also LHR at supraphysiological doses (EC(50) = 38 vs 598 ng/ml for FSHR and LHR respectively). The high doses of rtFSH required to activate LHR put into question the physiological relevance of this interaction. The use of heterologous chinook GTHs confirms the strong preference of each hormone for its cognate receptor. The gonadal expression pattern of the GTH receptor genes suggests that FSH may play an important role in regulating gonadal functions, not only at the early stages but also at the final stages of the male and female reproductive cycles, in addition to the LH pathway.

Effects of steroids on GTH I and GTH II secretion and pituitary concentration in the immature rainbow trout Oncorhynchus mykiss.

Using specific radio-immunoassays for rainbow trout GTH I and GTH II, the effects of testosterone and estradiol 17 beta have been studied or reinvestigated on the regulation of the secretion and the synthesis of the these two pituitary gonadotropins in the immature rainbow trout. After steroid implantation, the GTH II pituitary concentration is stimulated by testosterone and estradiol 17 beta for the entire period during which the plasma levels of these hormones are maintained to values comparable to those measured in the adult vitellogenic female rainbow trout. On the other hand, only testosterone induced a transient increase in the GTH I pituitary content 15 days after implantation, and estradiol provoked a decrease at day 30. The secretion of both GTH I and GTH II is stimulated by testosterone but not by estradiol 17 beta. Altogether, these results show that in the immature rainbow trout, testosterone preferentially modifies GTH I secretion, but not that of GTH II. They confirm that the stimulation of GTH II accumulation after testosterone or estradiol treatment would correspond to a stimulation of hormone synthesis. They evidence a differential action of both steroids on the synthesis of the two gonadotropins, especially a possible inhibition of GTH I synthesis by estradiol. They let suppose that the regulation of GTH I synthesis would involve factors other than steroids.

Fsh controls gene expression in fish both independently of and through steroid mediation.

The mechanisms and the mediators relaying Fsh action on testicular functions are poorly understood. Unlike in mammals, in fish both gonadotropins (Fsh and Lh) are able to efficiently stimulate steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, it is crucial to understand if Fsh effects are mediated through the production of steroids. To address this issue we performed transcriptome studies after in vitro incubations of rainbow trout testis explants in the presence of Fsh alone or in combination with trilostane, an inhibitor of Δ4- steroidogenesis. Trilostane significantly reduced or suppressed the response of many genes to Fsh (like wisp1, testis gapdhs, cldn11, inha, vt1 or dmrt1) showing that, in fish, important aspects of Fsh action follow indirect pathways and require the production of Δ4-steroids. What is more, most of the genes regulated by Fsh through steroid mediation were similarly regulated by Lh (and/or androgens). In contrast, the response to Fsh of other genes was not suppressed in the presence of trilostane. These latter included genes encoding for anti-mullerian hormone, midkine a (pleiotrophin related), angiopoietine-related protein, cyclins E1 and G1, hepatocyte growth factor activator, insulin-like growth factor 1b/3. A majority of those genes were preferentially regulated by Fsh, when compared to Lh, suggesting that specific regulatory effects of Fsh did not depend on steroid production. Finally, antagonistic effects between Fsh and steroids were found, in particular for genes encoding key factors of steroidogenesis (star, hsd3b1, cyp11b2-2) or for genes of the Igf system (igf1b/3). Our study provides the first clear evidence that, in fish, Fsh exerts Δ4-steroid-independent regulatory functions on many genes which are highly relevant for the onset of spermatogenesis.

Gonadotropin hormone (GtH) receptors in the ovary of the brown trout Salmo trutta L. in vitro studies

A method has been developed suitable for the study of the binding of salmon (Oncorhynchus tsawytscha) gonadotropic hormone (GtH) to brown trout (Salmo trutta) ovarian particulate fractions. Several proteolytic enzyme inhibitors were tested to preserve the binding capacity of particulate fractions (1000, 20,000, and 110,000g) from yolky oocytes. The best preservation was obtained with trypsin inhibitor. The effects on binding of the pH, time, and temperature of the incubation were investigated. Binding increased linearly with amount of particulate fraction, and is saturable. In both the 20,000 and 110,000g fractions, there are two classes of receptors: one with a high-affinity constant (3.14 and 0.95 10(10) M-1 for the 20,000 and 110,000g fractions, respectively) and a low capacity (1.76 and 0.63 fmol/g of ovary), the other with a higher-affinity constant (3.44 and 1.78 10(9) M-1) but a higher capacity (7.37 and 7.42 fmol/g of ovary). GtH binding was not affected by ovine luteinizing hormone (oHL), ovine follice-stimulating hormone (oFSH), and human chorionic gonadotropin (hCG), but was partially inhibited by 1 micrograms of bovine thyroid-stimulating hormone (bTSH) and salmon prolactin (sPRL). The results are discussed in relation to the biological specificity of fish GtH and to the unsolved problem concerning the number of gonadotropins in fish.

Development of a homologous radioimmunoassay for a gonadotropin of the gilthead seabream, Sparus aurata

Abstract A glycoproteic gonadotropin (GtH) was partially isolated from pituitaries of sexually mature Sparus aurata and used for the development of its radioimmunoassay (RIA). Two major chromatographic steps were used for the isolation of the GtH: affinity chromatography on Concanavalin A-Sepharose and gel filtration on Ultrogel ACA-54. The Sparus aurata GtH (SaGtH) was located using both a bioassay of in vitro production of 17α-hydroxy,20β-dihydroprogesterone (17α, 20β-OHP) by follicles of rainbow trout and a RIA for the β sub-unit of salmon GtH (sGtHβ). The isolated SaGtH was used for raising antibodies and as a standard. A highly purified preparation of SaGtH-125I was obtained by running the radiolabel through Sephadex G-50 and polyacrylamide gel electrophoresis (PAGE). The RIA was found to be sensitive (0.3 ng/ml), precise (intra-assay variability of 10–11%), and specific for GtH of Sparidae species (Sparus aurata and Sparus latus). It showed a poor cross-reactivity with pituitary extracts of several other marine fish. Using this RIA, a GnRH-induced SaGtH secretion was monitored both in vivo and in vitro.

Dosages radioimmunologiques heterologues de gonadotropines de type glycoproteiques maturantes de poissons teleosteens

Abstract The immunological specificity of fish gonadotropin does not generally allow the use of the homologous RIA systems prepared for salmon and carp GtH for the determination of gonadotropin in other species. Recently, an RIA using an antibody directed against the β c-GtH subunit has enabled GtH measurement in the eel. A similar approach using the s-GtH β subunit in homologous salmon systems and combinations between carp and salmon β systems has been developed. α and β subunits and the corresponding antibodies have been prepared from carp and salmon GtH. A study of the binding of the six antigens to each of the six antibodies confirms the species specificity of the α subunits. On the other hand, all the systems in which a β component is included, homologous or heterologous, for carp and salmon, are less specific and recognized c-GtH as well as s-GtH. The competition by pure antigens, partially purified pike and gilthead sea bream GtH, tilapia, milkfish and eel pituitary extracts, for the binding of s and β s-GtH, c and β c-GtH to the anti-serum directed against these four components has been studied. It was always possible to find one or several antigen-antibody combinations which were specifically competed for by each of the gonadotropic preparations. The assay has been validated for gilthead sea bream GtH by comparison of the results obtained after RIA determination and biological assay. Assays performed with perch show that it is also possible to use these systems for the GtH estimation in the plasma of this species.

Gdnf-Gfra1 Pathway Is Expressed in a Spermatogenetic-Dependent Manner and Is Regulated by Fsh in a Fish Testis

ABSTRACT What makes the spermatogonial stem cells (SSCs) self-renew or differentiate to produce spermatozoa is barely understood, in particular in nonmammalian species. Our research explores possible regulations of the SSC niche in teleost, locally by paracrine factors and peripherally by hormonal regulation. In the present study, we focus on the Gdnf-Gfra1 pathway that plays a major role in the regulation of SSC self-renewal in mammals. We describe a complex evolution of the genes encoding for Gdnf and Gfra1 proteins in trout with the emergence of three gdnf and two gfra1 paralogs. Using quantitative PCR measurements in isolated testicular cell populations, the gdnfb paralog was found expressed in A-spermatogonia and probably in another testicular cell type. In contrast, the transcript of gfra1a, the Gdnf receptor, was preferentially expressed in a population of undifferentiated A-spermatogonia (und A-Spg) separated by centrifugal elutriation. These und A-Spg also demonstrated high stemness potential in transplantation studies and preferentially expressed nanos2, a putative SSC marker in trout (Bellaiche et al., Biol Reprod 2014; 90:79). Flow cytometer experiments demonstrate that only a subfraction of und A-Spg express Gfra1. In trout, spermatogenesis develops along a strict annual cycle, and gdnfb and its receptor were expressed in a spermatogenetic activity-dependent manner. In particular, a dramatic increase of the gdnfb transcript coincided with the progressive cessation of rapid spermatogonial proliferation and of meiosis toward the end of the reproductive cycle. Together these results suggest that, in trout, Gdnfb is involved in the repression of und A-Spg differentiation. Fsh is an endocrine regulator of SSCs self-renewal through the up-regulation of Gdnf in rodents. We demonstrate that in trout, in vitro Fsh treatment stimulated the expression of the gfra1a1 but not of its ligand, gdnfb. Fsh treatment also stimulated the proliferation of und A-Spg cocultured with testicular somatic cells. Based on those results, the Gfra1-positive cells could correspond to the putative SSCs in rainbow trout, and we propose that the balance between SSC self-renewal and differentiation during the trout spermatogenetic cycle is under paracrine regulation by Gdnfb, which represses, and under peripheral regulation by Fsh via the control of gfra1a1 expression.

Delayed sexual maturation through gonadotropin receptor vaccination in the rainbow trout Oncorhynchus mykiss

In fish, gonadotropin hormones FSH-GTH1 and LH-GTH2 are less specific for their cognate receptors than in mammals. The respective reproductive functions of fish LH and FSH are thus difficult to establish. We aimed to study the effect of specific antagonists of the two gonadotropin receptors on trout sexual maturation in both sexes by targeting specific regions of LH and FSH receptors, Lhr and Fshr. Filamentous phages displaying Lhr specific or Fshr specific decapeptides from the extracellular hormone binding domain were engineered. Recombinant phages were used as receptor-specific antagonistic vaccines. Male and female trouts were immunized with anti-LHR, anti-FSHR, anti-FSHR+LHR or adjuvant alone, through multiple injections over 8-24 weeks, starting at different stages of sexual maturation. The consequences of immunization on gonadal development were evaluated by determining gonad growth, by histological analysis of testis and ovaries at the end of the vaccination period and by measuring blood plasma sex steroids using radioimmunoassay. We show for the first time in fish that the anti-receptor vaccinations could have specific antagonistic effects on the development of the reproductive functions; while the anti-FSHR affected the sexual maturation of prepubertal males and delayed sperm production, the anti-LHR blocked vitellogenesis in females. In maturing males, the combined anti-FSHR+LHR vaccine inhibited spermatogenesis and affected steroidogenesis. In that case, the effects of the vaccine on spermatogenesis were transient and reversible when immunization was stopped. Such an immunological strategy to specifically and transiently inhibit a receptor provides a promising approach for discovering their specific functions; it could also lead to a new technology for controlling the onset of puberty in aquaculture species.

New insights into the evolution, hormonal regulation, and spatiotemporal expression profiles of genes involved in the Gfra1/Gdnf and Kit/Kitlg regulatory pathways in rainbow trout testis

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20β progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.