Non-invasive measurement of 11-ketotestosterone, cortisol and androstenedione in male three-spined stickleback (Gasterosteus aculeatus)

The androgen 11-ketotestosterone (11-KT) plays an important role in reproductive physiology and behaviour in male teleosts. In the three-spined stickleback, Gasterosteus aculeatus, the plasma concentrations of 11-KT are related to the breeding status of the fish. Sticklebacks are relatively small (generally less than 1g) and in order to obtain sufficient plasma for assay of 11-KT, it has been necessary in the past to sacrifice the fish. In this paper, we report on the development of a non-invasive procedure for measuring 11-KT, cortisol and androstenedione (Ad) in the three-spined stickleback. Validation of the procedure included the demonstration that the rate of release of steroids into the water was correlated to their plasma concentrations. Ten males that were kept at a low temperature and short photoperiod were moved to high temperature and long photoperiod to initiate spermatogenesis and breeding. Every two to four days, for a total of 53 days, males were removed and placed in a beaker containing 50-ml water for 30 min. The water was then processed by solid phase extraction for radioimmunoassay. Males were presented with females on days 13/14, 18/19 and 44/45. 11-KT was originally undetectable but built up gradually to reach an average release rate of between 1 and 2.5 ng/g/h between days 16 and 45 and then started to decline (but non-significantly). Ad release reached a plateau of 1 ng/g/h about day 20. However, from days 44/45 to 51, there was a highly significant rise in the rate of release of Ad to 5 ng/g/h. On days 44/45, five of the males mated successfully and five did not. However, there were no significant differences in 11-KT or Ad release rates between the two groups. Cortisol release rates fluctuated with no pattern throughout the study. The results show that it is possible to make measurements on sex and stress steroid production in sticklebacks without recourse to anaesthesia, bleeding or sacrificing the fish. The procedure is potentially a powerful tool for the study of the link between steroids and behaviour in this useful sentinel species.     

Melatonin and maturation pace in female three-spined stickleback, Gasterosteus aculeatus

In many animals sexual maturation is controlled by the photoperiod. In mammals, the photoperiodic message is mediated via melatonin, but it is unclear whether this also applies to fishes. Administration of melatonin via the water on a schedule aimed at mimicking a short nonstimulatory photoperiod cycle has been found not to inhibit maturation in male three-spined sticklebacks, Gasterosteus aculeatus, kept under stimulatory long photoperiods. To study whether melatonin affects maturation pace, adult female sticklebacks kept under stimulatory photoperiodic regimes of Light:Dark (LD) 24:0 h (Exp. 1) or LD 16:8 h (Exp. 2) and 18 degrees were treated with melatonin (0, 20, or 80 microg/L water) via the water for 16 h per day. In addition, females were also kept under a nonstimulatory short photoperiod (LD 8:16). The time at which full maturation was achieved (running roe) was noted and the ovaries of nonovulated fish were studied histologically. Most fish under LD 24:0 and LD 16:8 matured (maturation rates in Exp. 1 and 2, respectively: control, 100 and 86%; low-dose melatonin, 83 and 93%; high-dose melatonin, 90 and 75%), whereas almost all females kept under LD 8:16 remained immature (maturation rates: 0% in Exp. 1 and 3% in Exp. 2). There was no difference in maturation pace, proportion of fish maturing, or relative ovarian weights between controls and melatonin-treated fish kept under LD 16:8. Furthermore, there was no difference in proportion of fish maturing or relative ovarian weights between controls and melatonin-treated fish kept under LD 24:0. However, LD 24:0 controls matured significantly earlier than fish receiving the high melatonin dose. Thus, there was an inhibitory effect of the high melatonin dose on maturation pace under LD 24:0. Nevertheless, this effect was small compared to the inhibitory effect of LD 8:16 treatment, suggesting that at least in this season, the major part of the photoperiodic effects in the stickleback is mediated via mechanisms other than circulating melatonin.     

Steroid metabolism in the testes of the breeding and nonbreeding three-spined stickleback, Gasterosteus aculeatus

Steroid metabolism in the testes of sticklebacks was studied in vitro by tissue incubations with [3H]pregnenolone or [3H]androstenedione as precursors. In males in full reproductive condition (nesting), [3H]pregnenolone was mainly converted via progesterone and 17 alpha-hydroxyprogesterone into androstenedione, 11 beta-hydroxyandrostenedione, and 11-ketoandrostenedione. The latter was the largest product formed. The main products from the [3H]androstenedione incubation, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione, confirm these findings. The rate of androgen synthesis, especially of 11-ketoandrostenedione, was much lower after the end of the breeding season.     

Characterization of gonadotropin-releasing hormone binding sites in the pituitary of the three-spined stickleback, Gasterosteus aculeatus

Binding sites for gonadotropin-releasing hormone (GnRH) in stickleback pituitary homogenates were characterized using an iodinated, superactive analog of salmon GnRH (sGnRH), D-Arg6-Pro9-sGnRH-NEt (sGnRHa). Binding of 125I-sGnRHa reached equilibrium after 60 min incubation at 4 degrees and was a function of tissue concentration. The specificity of 125I-sGnRHa binding was demonstrated by displacement with sGnRHa, sGnRH, and Buserelin [D-Ser(t-Bu)6-Pro9-GnRH-NEt]. Both Scatchard analyses of saturation data and displacement curves revealed a single class of high-affinity binding sites (Ka = 0.71 +/- 0.03 X 10(9) M-1, Bmax = 1087 +/- 165 fmol/mg protein).     

The effect of prolactin on fanning behavior in the male three-spined stickleback, Gasterosteus aculeatus L

The effect of administration of homologous prolactin on fanning behavior, an important aspect of parental care in sticklebacks and many other teleost fish, was studied. Prolactin was administered by implantation of an additional pituitary prolactin lobe in the dorsal body musculature of males with a nest without eggs. The low but rather constant level of fanning behavior is significantly increased from Day 6 to Day 12 after administration of the prolactin lobe. The implantation has no noticeable effects on cell and nuclear size or on the ultrastructure of the in situ prolactin cells of the recipient fish. Recovered prolactin lobe implants show two categories of prolactin cells. The major category consists of cells that are well developed and, similar to in situ prolactin cells, show structural signs of high secretory activity. The other prolactin cell category in the implants shows signs of cellular involution. Ten days after implantation, the first category predominates, and 16 days after implantation the second category. Prolactin lobe implantation increases the number of mucocytes in the epithelium of the skin, in particular at Day 10, and to a lesser extent at Day 16. This is considered evidence for a transient rise in blood prolactin levels in the recipient fish. We conclude that prolactin stimulates fanning behavior in male sticklebacks.     

Parental fanning behavior and prolactin cell activity in the male three-spined stickleback Gasterosteus aculeatus L

During the period that sexually mature male three-spined sticklebacks display parental fanning behavior, the rate of synthesis and release of the prolactin cells in the hypophysis, as estimated with quantitative electron microscopy, is enhanced considerably. This finding points to involvement of endogenous prolactin in the regulation of parental fanning behavior in this species.     

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.     

Temperature, but not photoperiod, influences gonadotropin-releasing hormone binding in the pituitary of the three-spined stickleback, Gasterosteus aculeatus.

Gonadotropin-releasing hormone (GnRH) binding characteristics in pituitaries of stickle-backs under different physiological conditions were studied using D-Arg6-Pro9-salmonGnRH-NEt as labeled ligand. Both males and females displayed marked seasonal changes in the capacity of high-affinity GnRH binding sites; there was a high content in the breeding season (summer) (800-1500 pmol/pituitary) and no detectable high affinity (< 150 pmol) binding in late winter-early spring. The binding capacity was lower in postbreeding fish (ca. 400 pmol/pituitary in females, ca. 900 pmol in males) than in breeding fish (females: ca. 1850, males ca. 1400 pmol). GnRH binding sites were also studied in fish exposed to long and short photoperiod in combination with high and low temperature in winter. Only long photoperiod in combination with high temperature stimulated sexual maturation. The capacity of the GnRH binding sites was similar in fish exposed to long (females 1550 pmol, males 1000 pmol) and short (females 1800, males 900) photoperiod in combination with high temperature. In fish exposed to low temperature, binding was nondetectable irrespective of the photoperiod.     

Seasonal variation of parasite infection of three-spined stickleback (Gasterosteus aculeatus L.) in the southern Baltic

Seasonal changes of infection were observed in the case of some species of ectoparasites: Trichodina domerguei domerguei, T. tenuidens, Gyrodactylus arcuatus, and Thersitina gasterostei, as well as for endoparasites: Glugea anomala, Schistocephalus solidus, Diphyllobothrium ditremum, and Proteocephalus filicollis. Same seasonal changes in frequency of developmental stages were observed in the case of females T. gasterostei. The highest level of infection with ectoparasites was observed in spring and summer and was probably an effect of increase of the reproductive activity with increase of temperature. Infection with tapeworms was generally low and was a result of feeding activity and fish diet.     

Ultrastructural evidence for two types of gonadotropic cells in the pituitary gland of the male three-spined stickleback,Gasterosteus aculeatus

Of the four secretory cell types observed in the proximal pars distalis of the pituitary gland of the male stickleback, two types probably have a gonadotropic function. The activity of the type 1 cells may be related to the development and maintenance of viable spermatozoa in the seminiferous tubules in the testes, whereas the activity of the type 2 cells seems to be correlated with Leydig cell development.     

Stimulatory effects of methyltestosterone on pituitary gonadotropic cells and testes leydig cells of the three-spined stickleback, Gasterosteus aculeatus L., in winter

Male three-spined sticklebacks were kept for a month in short photoperiod at ca. 20 degrees in winter. During this period methyltestosterone (MT), dissolved in the water (35 micrograms/liter), stimulated the appearance of breeding colors and kidney hypertrophy, characters which were both absent in the controls. MT furthermore prevented the onset of spermatogenesis. The gonadotropic cells of the MT-treated fish contained significantly more dilated endoplasmic reticulum and less granules than those of the controls. The nuclei of the testes Leydig cells were enlarged after treatment with MT. These findings suggest the existence of a positive feedback of androgen on the gonadal-pituitary axis of the male stickleback in winter.     

Seasonal changes in and effect of castration/androgen replacement on the plasma levels of five androgens in the male three-spined stickleback, Gasterosteus aculeatus L

The seasonal changes in plasma levels of the androgens 11-ketotestosterone (OT), testosterone (T), 11 beta-hydroxytestosterone (OHT), 11-ketoandrostenedione (OA), and 11 beta-hydroxyandrostenedione (OHA) were measured in the male three-spined stickleback (Gasterosteus aculeatus L). OT was the dominant plasma androgen in the breeding season in summer and was the only androgen that peaked during this period. The levels of OT correlated closely with the development of male secondary sexual characters and reproductive behavior. T and OHT were low in all seasons, whereas OHA and OA displayed the highest levels in early winter. During the postbreeding period, the time of active spermatogenesis, all measured steroids were low. Castration resulted in an almost complete loss of plasma OT and reduced T, whereas OHT, OHA, and OA were not reliably influenced. Androstenedione implants in castrated fish increased plasma T and OA implants increased plasma OT, suggesting a nontesticular site of conversion.     

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.     

Extraretinal photoreception involved in photoperiodic effects on reproduction in male three-spined sticklebacks, Gasterosteus aculeatus

The effects of human urinary erythropoietin (Ep), testosterone (TP), and l-thyroxine (T₄) were examined separately and in combination on tissue (muscle, kidney, and liver) oxidative metabolism and erythropoiesis of the spotted munia, Lonchura punctulata. The stimulatory influence of Ep, T₄, Ep + T₄, TP + T₄, and Ep + TP + T₄ on muscle, of T₄, Ep + T₄, and TP + T₄ on kidney, and of TP, TP + Ep, and TP + T₄ on liver oxygen uptake was observed. The number of red blood cells and the hemoglobin content were not influenced by T₄ or TP, but were increased by Ep and TP + T₄. The erythrocyte number was decreased by Ep + TP and Ep + TP + T₄.     

Effects of castration on pituitary gonadotropic cells of the male three-spined stickleback, Gasterosteus aculeatus L., under long photoperiod in winter: indications for a positive feedback

Male three-spined sticklebacks caught in winter were castrated or sham-operated and subsequently kept under long photoperiod at about 20 degrees for a month. With this treatment the sham-operated fish attained breeding condition. The gonadotropic cells of the sham-operated fish contained significantly more dilated endoplasmic reticulum and fewer granules than those of the castrated fish, indicating a higher secretory activity of the gonadotrops in the sham-operated fish. These findings suggest the existence of a physiological positive feedback within the gonadal-pituitary axis of the male stickleback when stimulated into its breeding condition by long photoperiod.     

Variation in brain arginine vasotocin (AVT) and isotocin (IT) levels with reproductive stage and social status in males of three-spined stickleback (Gasterosteus aculeatus)

Arginine vasotocin (AVT) and isotocin (IT) are fish nonapeptides synthesized in separate hypothalamic neurons from where they are transported to the neurohypophysis for storage and release into circulation. AVT is known to modulate aggression, courtship and parental care or social communication in many species, including fish, amphibians and birds. In this paper we examined a link between the level of AVT and IT in the brain and particular reproductive behavior in males of three-spined stickleback (Gasterosteus aculeatus). AVT and IT levels in whole brain of males of three-spined stickleback vary depending on specific breeding behavior of the individuals and their social status. These studies have shown the highest AVT levels in aggressive males that took care of the eggs. Brain AVT concentrations are also increased in nuptial colored subordinate males that fight to change their social status. On the other hand, IT is significantly higher in aggressive dominant males that defend their territory. IT may be also involved in courtship in three-spined stickleback. These findings highlight the importance of determination of "free", bioavailable neuropeptides' level in behavioral studies.     

Immunocytological localization and identification of the MSH-producing cells in the pituitary of the stickleback (Gasterosteus aculeatus L.)

The cellular origin of MSH in the pituitary of Gasterosteus aculeatus has been demonstrated with a bovine anti-aMSH serum. In the intermediate lobe only the lead hematoxylin-positive cells become fluorescent and may hence be considered the source of MSH. However, with bovine anti-βMSH serum both cell types are negative.     

The effect of estrogen on the gonads and on in vitro conversion of androstenedione to testosterone, 11-ketotestosterone, and estradiol-17beta in Sparus aurata (Teleostei, Sparidae).

The effects of estrogen on gonad morphology and steroidogenesis of sea bream, Sparus aurata, a protandrous hermaphrodite teleost, were investigated. Fish were treated in winter/spring for different periods with 17alpha-ethynylestradiol (ethE(2); experiment 1) and in summer with two doses of estradiol-17beta (E(2); experiment 2). Estrogen was more effective in summer. Its main effect on the gonad was inhibition of testicular growth and of male germ cell development beyond the spermatogonia stage, including mitosis. The effect of estrogen on ovarian development was slight and only apparent at the end of experiment 2 in the higher dose group. Gonadal fragments were incubated at different times during treatment with androstenedione and the output of testosterone (T), estradiol (E(2)), and 11-ketotestosterone (11KT) was measured by radioimmunoassay. T and E(2) production was inversely correlated with the proportion of testicular tissue (and positively with ovarian tissue) in the gonad in experiment 2. However, the production of 11KT was not correlated with any type of tissue, possibly because of further metabolism. Inhibition of testicular development by estrogen was also associated with higher output of steroid conjugates.