Anna Hellqvist

Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

Molecular Cloning and Characterization of Spiggin AN ANDROGEN-REGULATED EXTRAORGANISMAL ADHESIVE WITH STRUCTURAL SIMILARITIES TO Von Willebrand FACTOR-RELATED PROTEINS

One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called “spiggin,” which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (α, β, and γ). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.

Mechanisms in the photoperiodic control of reproduction in the stickleback

In sticklebacks, sexual maturation is stimulated by long photoperiods but not by short photoperiods, even at high temperatures. Extra-retinal photoreception can mediate this response, and appears to be more important thanretinal photoreception. Although plasma melatonin levels are high at night and low during the day, experiments using melatonin administration via the water indicate that melatonin is of no or little importance for the photoperiodic response. Androgens can be aromatised to estrogens in the stickleback brain. Treatment with aromatase inhibitors stimulates maturation of males also under short photoperiod, suggesting that aromatase is involved in the suppressive actions of short photoperiod. Expression of both follicle stimulating hormone (FSH)-β and luteinizing hormone (LH)-β is higher under long than under short photoperiod. FSH-β is controlled by a negative steroid feedback on the brain-pituitary-gonad axis under short photoperiod and by a positive steroid feedback under long photoperiod. It is suggested that the former can suppress reproduction under short photoperiod and the latter can stimulate breeding under long photoperiod.

Effects of castration and androgen-treatment on the expression of FSH-β and LH-β in the three-spine stickleback, gasterosteus aculeatus—Feedback differences mediating the photoperiodic maturation response? ☆

In many animals, including the three-spine stickleback (Gasterosteus aculeatus), photoperiod strongly influences reproduction. The aim of this study was to investigate if feedback mechanisms on the brain-pituitary-gonadal axis play a role in mediating the photoperiodic response in the stickleback. To that end, stickleback males, exposed to either non-stimulatory short photoperiod (light/dark 8:16) or under stimulatory long photoperiod (LD 16:8), were subjected to either sham-operation, castration, castration combined with treatment with the androgens 11-ketoandrostenedione (11KA) and testosterone (T), and the effects on levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)-beta mRNA were analyzed. During breeding season the kidney of the stickleback male hypertrophies and produces a glue used for building nests. Kidney weight and expression of both LH-beta and FSH-beta were higher in sham-operated fish kept under long than under short photoperiod. Under both photoperiods, LH-beta mRNA levels were lower in castrated males compared to sham-operated males and treatment with 11KA and T increased expression, indicating a positive feedback. A positive feedback was also found on FSH-beta expression under long photoperiod, where castration decreased, and androgen replacement restored FSH-beta mRNA expression. On the contrary, castration under short photoperiod instead increased FSH-beta levels whereas treatment with 11KA and T decreased FSH-beta expression, indicating a negative feedback on FSH-beta under these conditions. The positive feedback on FSH-beta expression under stimulatory photoperiod may accelerate maturation, whereas the negative feedback under inhibitory photoperiod may suppress maturation. This could be part of the mechanisms by which photoperiod controls maturation.

LH-β and FSH-β mRNA expression in nesting and post-breeding three-spined stickleback, Gasterosteus aculeatus, and effects of castration on expression of LH-β, FSH-β and spiggin mRNA

The mRNA expression of the LH-β and FSH-β subunits were measured in nesting and post-breeding male three-spined sticklebacks, Gasterosteus aculetaus, as well as in castrated and sham-operated nesting males. Furthermore, expression of an androgen induced kidney protein, spiggin, and 11-ketotestosterone (11KT) levels, were measured in the castrated and sham-operated males. Nesting males had significantly higher levels of both LH-β and FSH-β mRNA expression compared to post-breeding males. Furthermore, sham-operated males had significantly higher levels of LH-β mRNA and spiggin mRNA expression than the castrated fish. Expression of FSH-β, on the other hand, did not differ between castrated and sham-operated males. There were strong positive individual correlations between circulating levels of 11KT on the one hand and expressions of LH-β and spiggin mRNA, whereas the correlation between 11KT levels and FSH-β mRNA was weak. The negative effect of castration on β-LH mRNA indicates that gonadal hormones stimulate this expression, whereas this was not the case for β-FSH. The observed decline in β-LH expression after the end of the breeding season may be the result of cessation of the gonadal stimulation of the pituitary. On the other hand, it is not likely that this can explain the decline in FSH-β expression.

Effects of hemi-castration on plasma steroid levels in two teleost fishes; the three-spined stickleback, Gasterosteus aculeatus, and the Atlantic salmon, Salmo salar

In order to study the possible homeostatic regulation of gonadal steroids in fishes, plasma steroid levels were measured in hemi-castrated and sham-operated nesting male three-spined sticklebacks, Gasterosteus aculeatus, and in mature 2-year old male Atlantic salmon, Salmo salar. Hemi-castration significantly suppressed androgen levels in both species. In sticklebacks, plasma levels of 11-ketotestosterone (11KT) were 56% and levels of testosterone (T) 55% of those found in sham-operated males. In hemi-castrated salmon the levels of 11KT were 63%, and the levels of T were 75% of the levels in sham-operated males. In contrast, levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) in salmon (not measured in sticklebacks) were not different between hemi-castrated and sham-operated males. The results suggest that, although levels of the steroid 17,20-P were compensated in hemi-castrated salmon, the androgen levels in fish males in full spawning condition are not closely regulated by negative feedbacks.

Cloning and sequencing of FSH-β and LH-β subunits and changes in their expression during the reproductive cycle in the three-spined stickleback, Gasterosteus aculeatus

To achieve a better understanding of the role of gonadotropins (GTHs) in the stickleback we have cloned the full-length cDNAs of the β-subunits of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and analysed the expression during the seasonal cycle. In females, LH-β levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-β expression peaked earlier, in January and declined spring. In males, LH-β expression peaked in May. During June–September, when spermatogenesis occurs, LH-β levels were very low. FSH-β expression peaked earlier, in January, and reached the lowest levels in July. Thus, when spermatogenesis starts, the expression of both GTH-β mRNAs display their lowest levels.