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

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

Effect of combined GnRH and testosterone treatment on gonadal staging.

<p>Effect of combined GnRH and testosterone treatment on gonadal staging.</p

Secretion of estradiol from sturgeon ovaries.

<p>Sturgeon ovaries containing white (A), yellow (B), gray (C) or black (D) oocytes from a mature female at 10-year age, in response to various doses of recombinant sturgeon FSHβα, rsLHβα, adenylate cyclase activator (Forskolin, FSK;E) or carp pituitary extract (CPE; F).</p

Competitive binding curves for Russian sturgeon FSH and LH standards.

<p>Typical standard curve for the measurement of Russian sturgeon FSH (A) and LH (B).</p

Characterization of Pichia-expressed recombinant stLHβ, stLHβα, stFSHβ and stFSHβα by Western-blot analysis and deglycosilation.

<p>Supernatants of transformed Pichia cultures were separated by 15% semi-native PAGE and immunoreacted with antibodies raised against anti-His (A) recombinant stLHβ (B) or recombinant stFSHβ (C). For deglycosylation analysis, denatured and reduced proteins were incubated with (lanes 2, 4, 6, 8 and 10) or without (lanes 1, 3, 5, 7 and 9) N-glycosidase F. stLHβ: lanes 1,2; stLHβα: lanes 3,4; stFSHβ: lanes 5,6; stFSHβα: lanes 7,8; sturgeon pituitary extract (diluted 1:100): lanes 9,10; PM-protein marker; HM-His-tag protein marker.</p

Parallelism between the standard curves of ELISA for the measurements of Russian sturgeon gonadotropins.

<p>Russian sturgeon FSH (A) or LH (B) and displacement curves obtained with serial dilutions of plasma from a male (Fish 1) and female (Fish 2). (C) Displacement curves obtained with serial dilutions of pituitary extract of Russian sturgeon FSH and (D) Pituitary extract of Russian sturgeon LH.</p

Secretion of 11-KT from sturgeon testes.

<p>Testes from pre-mature (A) or mature (B) sturgeon, in response to various doses of recombinant sturgeon FSHβα, rsLHβα, adenylate cyclase activator (Forskolin, FSK; C) or carp pituitary extract (CPE; D).</p