Gonadotropins in the Russian Sturgeon: Their Role in Steroid Secretion and the Effect of Hormonal Treatment on Their Secretion.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and β-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique β-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%). The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity. Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.publishedVersio

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshβα, mdLhβα, tiFshβα, tiLhβα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshβα was able to activate the mdLhr, and mdLhβα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhβα, tiFshβα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshβα, tiLhβα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshβα, mdLhβα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.publishedVersio

Gonadotropins in the Russian Sturgeon: Their Role in Steroid Secretion and the Effect of Hormonal Treatment on Their Secretion.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and β-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique β-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels

Data on Western blot and ELISA analysis of medaka (Oryzias latipes) follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) using recombinant proteins expressed with Pichia pastoris

The gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play essential roles in vertebrate reproduction. This article presents data on molecular weight validation of recombinant medaka (Oryzias latipes) (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) generated by Pichia pastoris, as well as data on a validation of produced antibodies against Fshβ and Lhβ by Western blot analysis. Furthermore, the article includes data on Fsh and Lh protein levels in male medaka pituitaries using recombinant mdFshβα and mdLhβα within enzyme-linked immunosorbent assays (ELISAs), in which protein amounts were analyzed related to body weight and age of the fish. This dataset is associated with the research article entitled “Medaka Follicle-stimulating hormone (Fsh) and Luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression” (Burow et al., in press). Keywords: Enzyme-linked immunosorbent assay, Follicle-stimulating hormone, Luteinizing hormone, Oryzias latipes, Recombinant gonadotropins, Western blot analysi

Data on Western blot and ELISA analysis of medaka (Oryzias latipes) follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) using recombinant proteins expressed with Pichia pastoris

The gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play essential roles in vertebrate reproduction. This article presents data on molecular weight validation of recombinant medaka (Oryzias latipes) (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) generated by Pichia pastoris, as well as data on a validation of produced antibodies against Fshβ and Lhβ by Western blot analysis. Furthermore, the article includes data on Fsh and Lh protein levels in male medaka pituitaries using recombinant mdFshβα and mdLhβα within enzyme-linked immunosorbent assays (ELISAs), in which protein amounts were analyzed related to body weight and age of the fish. This dataset is associated with the research article entitled “Medaka Follicle-stimulating hormone (Fsh) and Luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression” (Burow et al., in press)

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%). The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity. Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshβα, mdLhβα, tiFshβα, tiLhβα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshβα was able to activate the mdLhr, and mdLhβα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhβα, tiFshβα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshβα, tiLhβα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshβα, mdLhβα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia

Effect of hormonal treatments on E2, 11-KT, FSH and LH plasma level in 6-year-old Russian sturgeon females.

<p>The control group was injected with 0.2 mL/kg saline. The first treatment group was injected (IM) with salmon GnRH analog ([D-Arg⁶,Pro⁹- NEt]-salmon GnRH; sGnRHa) in slow-release implants (Evac) up to a final dose of 10 μg/kg. Fish from the second group received testosterone (T) as food supplements (60 mg/kg food; 0.4% BW food per day). The third group received both GnRH implants and T as food supplements, at the above concentrations. Each bar represents the mean ± SEM. Different letters indicate that treatments have significantly different effects (ANOVA followed by Newman–Keuls test, (p<0.05).</p