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Background: Both gonadotropin-releasing hormone (GnRH) and kisspeptin have a critical role in the photoperiodic control of sexual maturation in mammals. Question: What are the roles of GnRH and kisspeptin in the photoperiodic control of sexual maturation in the threespine stickleback, Gasterosteus aculeatus? Method: If kisspeptin and GnRH trigger the photoperiodic induction of maturation in fishes, they would be expected to be activated prior to, or at least no later than, the gonadotropins. The mRNA levels of brain gnrh2, gnrh3, kiss2 and its receptor, gpr54, as well as of pituitary lh-beta and fsh-beta, were measured in males under standard laboratory conditions [16 hours light/8 hours dark (L16/D8) or 8 hours light/16 hours dark (L8/D16)] after 3, 10, 19, and 29 days at 20 degrees C. Results: From day 19, males in the long-day photoperiod (L16/D8) condition were visibly mature with bright breeding coloration. Compared with short-day photoperiod (L8/D16) fish, L16/D8 fish had higher lh-beta mRNA levels at days 10, 19, and 29, and higher fsh-beta mRNA levels at day 10 only. Furthermore, L16/D8 fish had higher gnrh3 mRNA levels at days 10 and 29, and higher gnrh2 mRNA levels at day 29, than L8/D16 fish. Finally, kiss2 and gpr54 mRNA levels of L16/D8 fish were significantly higher than those of L8/D16 fish at days 19 and 29, respectively. Conclusions: At the onset of sexual maturation, the levels of lh-beta, fsh-beta, and gnrh3 rose earlier than those of kiss2 and gpr54. These results are consistent with GnRH3 having a role in the early phases of maturation, but do not support a role for kisspeptin in photoperiodic initiation of maturation in male stickleback.

The ovarian fluid properties of the three-spined stickleback Gasterosteus aculeatus were studied in overripe and non-overripe ovulated female sticklebacks and in females that were implanted with Silastic capsules containing testosterone (T), oestradiol (E2), 17,20-dihydroxypregn-4-en-3-one (17,20-P) or progesterone (P4) into the abdominal cavity. Overripe females had less ovarian fluid than non-overripe females, but with higher dry mass, higher protein concentration and lower viscosity. T and 17,20-P increased the amount of ovarian fluid and the fluid protein concentration was increased by 17,20-P. 1-D sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that ovarian fluid contains several proteins, with high individual variability but with no consistent differences between groups. Some of the ovarian fluid proteins appeared to correspond to proteins from the eggs. The results suggest that secretion of ovarian fluid may be influenced by steroid hormones and that changes in its properties are related to the overripening of ovulated eggs. In at least some respects it appears that the changes in the ovarian fluid is a result, rather than the cause of overripening.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72 h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72 hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-beta) and luteinizing hormone (lh-beta) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72 hps. Plasma T levels showed peaks at 24 and 48 hps and decreased at 72 hps, while E2 levels increased already at 6 hps and remained at high levels up to 48 hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-beta mRNA levels showed a peak at 48 hps, while fsh-beta did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Selective Serotonin Re-uptake Inhibitors (SSRIs) are a class of psychotropic drugs used to treat depression in both adolescents and pregnant or breast-feeding mothers as well as in the general population. Recent research on rodents points to persistent behavioural effects of pre- and perinatal exposure to SSRI which last into adulthood. To study effects of developmental exposure in fish, three-spine sticklebacks were exposed to 1.5 µg/l of the SSRI citalopram in the ambient water for 30 days, starting two days post-fertilisation. After 100 days of remediation in clean water the fish were put through an extensive test battery. Feeding behaviour was tested as the number of bites against a piece of food and found to be increased in the exposed fish. Aggression levels were measured as the number of bites against a mirror image during 10 minutes and was also found to be significantly increased in the exposed fish. Novel tank behaviour and locomotor activity was tested in an aquarium that had a horizontal line drawn half-way between the bottom and the surface. Neither the latency to the first transition to the upper half, nor the number of transitions or the total time spent in the upper half was affected by treatment. Locomotor activity was significantly reduced in the exposed fish. The light/dark preference was tested in an aquarium where the bottom and walls were black on one side and white on the other. The number of transitions to the white side was significantly reduced in the exposed fish but there was no effect on the latency to the first transition or the total time spent in the white half. The results in the current study indicate that developmental SSRI exposure causes persistent behavioural effects in fish and contribute to the existing knowledge about SSRIs as environmental pollutants., As manuscript in dissertation. with title: Developmental exposure to the SSRI citalopram causes persistent behavioural effects in the three-spined stickleback (Gasterosteus aculeatus)

Leptin modulates all levels of the reproductive endocrine axis in mammals, and in turn, both leptin and the leptin receptor are regulated by sex steroids. The aim of this study was to investigate if sex steroids regulate the leptin system also in fish. Immature one-year old male Atlantic salmon parr were implanted with Silclear capsules that were either empty or filled with 11-ketoandrostenedione (11 KA) or testosterone (T) and the effects of 35-days treatment were investigated on measures of maturation, gene expression of leptin (lepa1, lepa2), leptin receptor (lepra1) and circulating plasma leptin. Both 11-KA and T stimulated the reproductive axis by increasing testes weight and up-regulated pituitary lh-beta mRNA levels and for T also fsh-beta. T up-regulated transcription levels of lepa1 and lepra1 in the pituitary, while 11-KA had no effect. Leptin receptor expression in the testis was unaltered by either androgen. T up-regulated lepa1 mRNA levels significantly also in the liver, but had no effect on lepa2, and 11 KA did not affect hepatic gene expression of either lepa1 or lepa2. Plasma leptin levels did not differ significantly between treatments. The results indicate that androgens regulate gene expression of leptin and the leptin receptor in different tissues in fish and that the effects of leptin might be tissue specific considering plasma levels remained unaltered. Overall, the results suggest a role for leptin in fish reproduction, where sex steroids are able to regulate components of the leptin system differentially in liver and important tissues of the reproductive axis.

Background: Female threespine sticklebacks, Gasterosteus aculeatus, are batch spawners. As in most teleosts, the ovulated eggs are kept in the ovarian cavity until spawning. If spawning or spontaneous release of the eggs does not take place, they can become overripe and harden, and in most cases remain in the ovary. The overripe eggs are lost for reproduction and also block further spawnings. Reproductive hormones regulate egg production and may be involved in the mechanism of overripening. Question: What are the reproductive endocrinological parameters characterizing overripening of ovulated eggs in the threespine stickleback? Organism: Wild-caught adult threespine sticklebacks from the southern Baltic at Skare in southern Sweden and the island of Asko in northwestern Baltic Proper in Sweden. Experiments: We collected blood samples for hormone measurements, as well as pituitaries and brains for measurement of mRNA from both sexually mature non-overripe (non-ovulated and/or ovulated) and overripe (egg-bound) females. For the Skare fish, sexual maturation was induced under laboratory conditions by exposure to a long photoperiod and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. The Asko fish were sampled directly from nature, during the natural summer breeding season and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. Methods: In the fish collected from Skare, we used radioimmunoassay to measure the plasma levels of four steroids: testosterone, estradiol, 17,20 beta-dihydroxypregn-4-en-3-one (17,20 beta-P), and 17,20 beta, 21-trihydroxypregn-4-en-3-one (17,20 beta,21-P). We also measured the mRNA levels of gonadotropins [GTHs: follicle-stimulating hormone (fsh-beta) and luteinizing hormone (lh-beta)] in the pituitary, and of gonadotropin-releasing ho

In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T + POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T + POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response., AuthorCount:6

Mature three-spined stickleback males use spiggin threads secreted from their kidney to glue together nest material. This requires strongly hypertrophied renal proximal tubular cells, which compromises renal osmoregulatory function during the breeding period. Experimental evidence suggests that the intestine takes over hypotonic fluid secretion at that stage but the mechanism is unexplored. To unravel the molecular mechanism we analyzed and compared transcript levels of several membrane proteins involved in water and salt transport in intestinal and renal tissues, in non-mature males (NM), mature males (MM), and mature females (MF). Aquaporin paralogs aqp1a, -30, -8aa, -8ab, -10a, and -10b, two Na+,K+-ATPase alpha-1 subunit isoforms (nka547, nka976), Na+,K+,2C1(-)-, and Na+,CI--cotransporters (nkcc1a, nkcc2,ncc), the cystic fibrosis transmembrane conductance regulator (cftr) and two claudin isoforms (cldn2, cldn15a) were expressed in the intestine and kidney in all groups. There were no differences in aqp and cldn expression between intestines of NM and MM; nkcc2 was lower and nka levels tended to be higher in intestines of MM than in NM. In the kidney, aqp1 and aqp8ab levels were lower in MM than in NM, whereas aqp3a, nkcc1a, cldn15a, and spiggin were markedly elevated. This was accompanied by marked hypertrophy of kidney tubules in MM. The data support an altered kidney function in terms of water handling in mature males, whereas there was no support for modified trans-epithelial water permeability or salt-secretory activity in the intestine of mature males. Salt-absorptive activity in the intestine may, however, be down-regulated during male maturation.

Optomotor studies have shown that three-spined sticklebacks (Gasterosteus aculeatus) are more sensitive to red during summer than winter, which may be related to the need to detect the red breeding colour of males. This study aimed to determine whether this change of red light sensitivity is specifically related to reproductive physiology. The mRNA levels of opsin genes were examined in the retinae of sexually mature and immature fish, as well as in sham-operated males, castrated control males, or castrated males implanted with androgen 11-ketoandrostenedione (11 KA), maintained under stimulatory (L16:D8) or inhibitory (L8:D16) photoperiods. In both sexes, red-sensitive opsin gene (Iws) mRNA levels were higher in sexually mature than in immature fish. Under L16:D8, lws mRNA levels were higher in intact than in castrated males, and were up-regulated by 11 KA treatment in castrated males. Moreover, electroretinogram data confirmed that sexual maturation resulted in higher relative red spectral sensitivity. Mature males under L16:D8 were more sensitive to red light than males under L8:D16. Red light sensitivity under L16:D8 was diminished by castration, but increased by 11 KA treatment. Thus, in sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression. This is the first study to demonstrate that sex hormones can regulate spectral vision sensitivity., AuthorCount:7

Sexual maturation in the stickleback is controlled by photoperiod. The aim of this study was to find out whether changes in feedback effects exerted by sex steroids could mediate the photoperiodic effect, which is regarded to be of an all-or-nothing character. To that end, males were castrated and treated with different doses of testosterone (T) and in one experiment also with the aromatase inhibitor fadrozole (AI) and kept under different photoperiods. In control fish, long day (LD 16:8) stimulated maturation, associated with more hypertrophied kidneys (a secondary sexual character) and higher levels of pituitary lhb and fshb mRNA than under short day conditions (LD 8:16). Under LD 8:16, low doses of T suppressed both lhb and fshb mRNA levels. However, with the use of high doses of T and/or longer photoperiods the inhibitory effects on lhb and fshb mRNA levels became less clear or instead positive effects were observed. Under intermediate photoperiod conditions, the negative feedback effect of a low dose of T on fshb was more prominent with shorter photoperiods, whereas no such shift was observed for lhb mRNA. The inhibitory effect of the low dose of T on lhb mRNA levels under LD 8:16 was abolished by AI, whereas the stimulatory effect of the high dose of T was not. The negative feedback effects were more marked under short days than under long days, whereas positive feedback effects were more marked under long days. The suppression of both fshb and lhb mRNA levels by low androgen levels, especially under short days, may inhibit maturation completely unless a rise of androgens above threshold levels would allow complete maturation., AuthorCount:6

Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks. Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-beta and LH-beta and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-beta and LH-beta were higher in infected males than in uninfected males. Also in females, FSH-beta mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-beta expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

Background: Androgens induce male characters by activating androgen receptors (AR). Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined. Methods: AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR. Results: Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy) and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmoregulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males. Conclusion: The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback., AuthorCount:5

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.

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-beta 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 upregulate androgen receptor mRNA level or increase receptor abundance, suggesting that autoregulation is not involved in differential ligand activation. However, comparison of the transactivation 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.