Changes in Red Color Sensitivity over the Spawning Cycle of Female Three-spined Stickleback (Gasterosteus aculeatus).

Male red nuptial coloration is a primary mating signal for three-spined sticklebacks (Gasterosteus aculeatus), and the retinae of both sexes are especially sensitive to red during the breeding season. Red sensitivity is an important aspect of female mate choice in this species, but only when they are ready to spawn and not over the entire breeding period. Here, we aimed to determine if the red sensitivity of female sticklebacks change over their repeat spawning cycle. To this end, we assessed retinal opsin mRNA levels and behavioral red sensitivity in females over this cycle. Both methods indicated that females were more sensitive to red during spawning than in the inter-spawning intervals. Relative expression levels of red color opsin genes (lws) and optical motor sensitivity were high during spawning, decreased after the spawning period, and then increased again 72-96 h later when they were ready to spawn again. Thus, female sticklebacks altered their color sensitivity according to need, but the underlying mechanism remains unclear.

Ovarian fluid in the three-spined stickleback Gasterosteus aculeatus: effects of egg overripening and sex steroid treatment.

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), 17,20ß-dihydroxypregn-4-en-3-one (17,20ß-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 17,20ß-P increased the amount of ovarian fluid and the fluid protein concentration was increased by 17,20ß-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.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

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 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). 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-ß) and luteinizing hormone (lh-ß) 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 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß 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.

Sexual maturation and changes in water and salt transport components in the kidney and intestine of three-spined stickleback (Gasterosteus aculeatus L.).

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, -3a, -8aa, -8ab, -10a, and -10b, two Na(+),K(+)-ATPase alpha-1 subunit isoforms (nka547, nka976), Na(+),K(+),2Cl(-)-, and Na(+),Cl(-)-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.

GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

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.

Androgens increase lws opsin expression and red sensitivity in male three-spined sticklebacks.

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 (lws) 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.

Androgen feedback effects on LH and FSH, and photoperiodic control of reproduction in male three-spined sticklebacks, Gasterosteus aculeatus.

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.

Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

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-ß and LH-ß 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-ß and LH-ß were higher in infected males than in uninfected males. Also in females, FSH-ß mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-ß 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.

Androgen receptor-beta mRNA levels in different tissues in breeding and post-breeding male and female sticklebacks, Gasterosteus aculeatus.

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 hyperosmo-regulation 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.

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar).

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.

Effects of castration and androgen-treatment on the expression of FSH-beta and LH-beta 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.

Stickleback sperm saved by salt in ovarian fluid.

Sperm of the three-spined stickleback Gasterosteus aculeatus display a prolonged motility in the presence of ovarian fluid. The ovarian fluid prolongs sperm motility in freshwater from approximately 1 min to several hours, a trait that possibly gives the stickleback its unusual ability to spawn in waters of all salinities. The aim of the study was to look for factor(s) within the ovarian fluid responsible for prolonging sperm motility as well as to investigate the possible biological importance of the ovarian fluid under natural conditions. To that end, we measured the ionic content (Na(+), Cl(-), Ca(2+) and K(+)) of the ovarian fluid and prepared ionic artificial ovarian fluids. We also prepared a mannitol solution with the same osmolality as the ovarian fluid in order to distinguish between the ionic and osmotic effect. We found that the ionic artificial fluids were equally effective as the natural ovarian fluid in prolonging sperm motility and survival over a range of concentrations, whereas the mannitol solution was far less effective. By insertion of natural ovarian fluid or ovarian fluid from which macromolecules had been removed by ultra filtration in nests it was found that macromolecules help by retaining ions. We also found that ovarian fluid in water, at concentrations as low as 0.75 and 1.56%, prolonged sperm motility and that the concentration of ions (Na(+)) present in the nest 15 min after spawning corresponded to at least 3% ovarian fluid. Previous fertilisation experiments have shown that it takes at least 15 min for stickleback sperm to fertilise all eggs in a batch. This indicates that the role of ovarian fluid in prolonging the sperm motility is biologically relevant and that the effect is exerted by the fluid's ionic content.

Seasonal changes in expression of LH-beta and FSH-beta in male and female three-spined stickleback, Gasterosteus aculeatus.

In teleost fishes, like in other vertebrates, the gonadal development is stimulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). To achieve a better understanding of the role of gonadotropins in teleost reproduction; expression of LH-beta and FSH-beta mRNA and the status of gonads and secondary sexual characters were analyzed over the annual cycle in male and female three-spined sticklebacks, a species in which the development of male secondary sexual characters and spermatogenesis are separated in time. The kidney in the male stickleback hypertrophies during the breeding season and produces a glue used when building nests. Kidney weights, as well as levels of 11-ketotestosterone (11KT), reached a peak in May. Both testosterone (T) levels and the gonadosomatic index (GSI, gonad weight/body weight x 100) in females started to increase in April, and peaked in May as well. Later in summer, after the breeding season, these features declined. In females, LH-beta expression followed the GSI and T levels closely, levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-beta expression peaked earlier, in January and declined slowly over spring. In males, LH-beta expression peaked in May. During June-September, when spermatogenesis was active, LH-beta levels were very low. FSH-beta expression peaked in January, earlier than LH-beta expression did, and reached the lowest levels in July. Thus, when spermatogenesis started at the end of summer, the expression of both GTH-beta mRNAs, and circulating 11KT, displayed their lowest levels.

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-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 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.

Cloning and sequencing of the FSH-beta and LH beta-subunit in the three-spined stickleback, Gasterosteus aculeatus, and effects of photoperiod and temperature on LH-beta and FSH-beta mRNA expression.

The aim of this study was to characterize the FSH-beta and LH-beta subunit in the three-spined stickleback, a fish used extensively in experimental studies, and to use the cloned cDNAs as probes for measuring FSH-beta and LH-beta mRNA expression in sticklebacks treated with different photoperiods and temperatures. A first strand cDNA was prepared from 10 pituitaries from male sticklebacks, and cDNA fragments were amplified by PCR using degenerated primers based on highly conserved regions of known teleost FSH-beta and LH-beta cDNA sequences. To obtain full-length cDNAs of FSH-beta and LH-beta, RACE amplifications were performed. The cDNA of FSH-beta was 540 bp long, encoding a protein of 122 amino acids and LH-beta cDNA was 568 bp long, encoding a protein of 148 amino acids. Of gonadotropin (GTH) beta-subunits published so far, those most similar to stickleback GTHs are found among other percomorph fishes, with amino acid similarities of 46-55% for FSH-beta and 75-77% for LH-beta subunits.The cloned cDNAs were used as probes to analyze LH and FSH mRNA expression in pituitaries from sticklebacks exposed to different photoperiods and temperatures. Two hundred males were divided into four groups and kept under different photoperiods and temperatures; light:dark (LD) 16:8, 20 degrees C; LD 16:8, 7 degrees C; LD 8:16, 20 degrees C, and LD 8:16, 7 degrees C. Red breeding colors and a marked kidney hypertrophy, androgen-dependent male secondary sexual characters in the stickleback, appeared in the group kept under LD 16:8 at 20 degrees C, but not in the other groups. The kidney epithelium height (KEH) was significantly lower in the LD 8:16, 20 degrees C group than in all other groups, and this was also the only group with active spermatogenesis. The LD 8:16 20 degrees C had significantly lower expression of both FSH-beta and LH-beta than all other groups. Thus, both GTHs displayed their lowest expression when spermatogenesis was active and when low KEH indicated that androgens levels were at their lowest.

Androgen and behavior in the male three-spined stickleback, Gasterosteus aculeatus. II. Castration and 11-ketoandrostenedione effects on courtship and parental care during the nesting cycle.

Courtship declines and ceases while parental care increases in the presence of developing eggs during the nesting cycle of the male three-spined stickleback, Gasterosteus aculeatus. Furthermore, circulating 11-ketotestosterone (11KT) levels are higher during the initial "courtship phase" than during the later "parental phase," similar to that found in other paternal fishes. This study aimed to investigate a possible functional relationship between changes in 11KT levels and changes in reproductive behavior during the nesting cycle. To this end, groups of nonspawned and spawned male sticklebacks were sham-operated, castrated, or castrated and treated with 11-ketoandrostenedione (11KA), and the effects of the treatments on courtship and parental care were studied. Castration removed circulating 11KT, while 11KA replacement prevented the natural decline in 11KT during the parental phase (11KA converts to 11KT extratesticularly), as assessed by radioimmunoassay. Regardless of treatment, parental care remained low and courtship was present in all nonspawned males, even at the end of the experiment. However, courtship did eventually decline in castrated nonspawned males compared to the other two nonspawned groups. In all treatments of spawned males there was a drastic decline in courtship and an increase in parental care. In castrated spawned males, however, the decline in courtship came earlier than in the other two spawned groups. 11KA treatment did not prevent the natural decline in courtship/increase in parental care in spawned males, indicating that the natural decline in 11KT is not responsible for the main portion of the rapid changes in these behaviors over the stickleback's nesting cycle. The limited effects of castration also exclude other gonadal hormones from being responsible for most of these changes.

Androgen and behavior in the male three-spined stickleback, Gasterosteus aculeatus I.--changes in 11-ketotestosterone levels during the nesting cycle.

Circulating 11-ketotestosterone (11KT) levels are higher during the courtship phase than during the later parental phase in a number of male teleosts. The present study describes the temporal changes in 11KT levels and their relationships to changes in courtship behavior, after different number of spawnings, over the nesting cycle of the male three-spined stickleback, Gasterosteus aculeatus, a small teleost showing pronounced paternal behavior. Plasma 11KT levels, measured by radioimmunoassay, were approximately 34 times higher during the initial courtship phase than at the end of the following parental phase in spawned males. In addition, males that spawned with three or more females on a single day showed an earlier decline in 11KT levels and in courtship behavior compared to males that were only allowed to spawn with a single female. Plasma 11KT levels remained high in males not allowed to spawn.