Effects of temperature on the final stages of sexual maturation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14–16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17β-oestradiol (E₂) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2–3 weeks after the first appearance of expressible milt, while levels of 17,20β-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20β-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20β-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20β-P and prevented the normal decline of T and E₂ levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.     

Diurnal rhythm of steroid biosynthesis in the testis of terminal phase male of protogynous wrasse, Pseudolabrus sieboldi, a daily spawner

The wrasse, Pseudolabrus sieboldi, is a diandric protogynous labrid fish. Spawning is performed by a terminal phase (TP) male and an initial phase (IP) female between 6:00 and 9:00 h daily during two-month-long spawning season. In the present study, to investigate the roles of steroid hormones in the diurnal spermatogenesis of the P. sieboldi TP male, all steroid hormones produced in the testis were identified and the synthetic pathways of these steroids were determined. Furthermore, the circulating levels of the major steroids produced were analyzed throughout a day at 3-hour intervals during spawning season. In the testis, 11-ketotestosterone (11-KT), estradiol-17β (E2), 17,20β-dihydoxy-4-pregnane-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were synthesized as the major metabolites. In vitro steroid biosynthesis experiments showed similar results to the circulation profiles of the major steroids. This study is the first to clarify the complete steroidogenic pathways in the gonads of a diandric protogynous species throughout its life, when combined with the results of the steroidogenesis in the ovarian follicles. This is also the first report of a clear diurnal rhythm of the steroid production corresponding to the spermatogenic process in the testis of a male teleost.     

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C₂₁ steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10⁻¹² M. The male tench were equally sensitive to glucuronidated 17,20β-P (10^−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11^−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F_2α (PGF_2α) and 15-keto PGF_2α (11^−11.5 and 10^−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E₂), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plasma sex steroid hormonal profile and gonad histology during the annual reproductive cycle of river catfish Hemibagrus nemurus (Valenciennes, 1840) in captivity

Plasma sex steroid hormonal profile and gonad histology were correlated to study the annual reproductive cycle of Hemibagrus nemurus. Hormones were measured by Enzyme Linked Immunosorbent Assay. Gonad tissues were observed by using light microscopy. The highest testosterone (T) value for male was observed in November and that of female was in October. 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels were highest in June and November, respectively. Hormonal profiles of T, 11-KT and E2 showed several peaks which indicated a non-seasonal pattern. There were significant differences (p < 0.05) in the monthly levels of T, 11-KT and E2. Gonadosomatic index of H. nemurus ranged from 1.14 ± 0.02 % to 7.06 ± 1.40 %, and high gonadosomatic indices were recorded in May, August and November. Gonad histology revealed that spermatozoa were always present in the testes which implied continuous spermatogenesis and asynchronous ovarian development pattern was observed in the ovaries. The annual reproductive cycle of H. nemurus did not show a seasonal pattern and this indicate that H. nemurus is a non-seasonal breeder with several spawning cycles and can be referred to as indeterminate batch spawner. The major significances of this study are annual sex steroid hormonal profile and asynchronous ovarian development of H. nemurus. This information will contribute to our knowledge of reproductive biology of H. nemurus.     

Molecular control mechanisms of fish spermatogenesis

Fish spermatogenesis, from spermatogonial stem-cell renewal to sperm maturation, is controlled by the sex steroid hormones. Mitotic divisions of spermatogonia can be categorized by spermatogonial stem cell renewal and spermatogonial proliferation. Spermatogonial renewal is regulated by estradiol-17β (E₂; the natural estrogen in vertebrates), and spermatogonial proliferation toward meiosis is promoted by 11-ketotestosterone (11-KT), the main androgen in teleost. The action of E2 and 11-KT is mediated by other factors produced by Sertoli cells; E2 is mediated by spermatogonial stem-cell renewal factor and 11-KT is mediated by spermatogenesis preventing substance and activin B. Although 11-KT also induce meiosis and spermiogenesis, the control mechanisms of these processe are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation. Sperm maturation is regulated by 17α,20β-dihydroxy-4-pregnen-3-one (DHP), which is progestin in teleost. The DHP acts directly on spermatozoa to active the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.     

Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost,Stizostedion vitreum

The annual reproductive cycle of walleye (Stizostedion vitreum) was characterized by documenting changes in gonadal development and serum levels of estradiol-17β (E₂), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), and 11-ketotestosterone (11-KT) in wild fish captured from upper midwestern lakes and rivers throughout the year. Fish from the populations used in this study spawn annually in early- to mid-April. Walleye showed group synchronous ovarian development with exogenous vitellogenesis beginning in autumn. Oocyte diameters increased rapidly from ∼ 200 μm in October to ∼ 1,000 μm in November, and reached a maximum of 1,500 μm just prior to spawning. Changes in gonadosomatic indices (GSIs) paralleled changes in oocyte diameters. Serum E₂ levels in females increased rapidly from low values in October (< 0.1 ng ml⁻¹) to peak levels of 3.7 ng ml⁻¹ in November, coinciding with the period of the most rapid ovarian growth. Subsequently, E₂ levels decreased from December through spawning. Serum T levels exhibited a bimodal pattern, increasing to 1.6 ng ml⁻¹ in November, and peaking again at 3.3 ng ml⁻¹ just prior to spawning. We detected 11-KT in the serum of some females at concentrations up to 5.6 ng ml⁻¹, but no seasonal pattern was apparent. In this study (unlike our results in a related study) 17,20-P was not detected. In males, differentiation of spermatogonia began in late August, and by January the testes were filled (> 95% of germ cells) with spermatozoa. Mature spermatozoa could be expressed from males from January through April. GSIs ranged from 0.2% (post-spawn) to 3.2% (pre-spawn). Serum T levels rose from undetectable levels in post-spawn males to 1.6 ng ml⁻¹ by November, remained elevated throughout the winter, and peaked at 2.8 ng ml⁻¹ I prior to spawning. Levels of 11-KT in males remained low (< 10 ng ml⁻¹, from post-spawning through January, then increased significantly by March and peaked just prior to spawning at 39.7 ng ml⁻¹. Our results indicate that vitellogenesis and spermatogenesis are complete or nearly so, in walleye by early winter, and suggest that it may be possible to induce spawning in this species several months prior to the normal spawning season by subjecting fish to relatively simple environmental and hormonal treatments.     

Endocrine changes during the annual reproductive cycle of the red porgy, Pagrus pagrus (Teleostei, Sparidae)

Changes in 17-estradiol (E2), estrone (E1), testosterone (T), 11-ketotestosterone (11KT), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels were correlated to changes in gonadosomatic index (GSI), vitellogenin concentration (Vg), ovarian and testicular histology during the annual reproductive cycle of the red porgy, Pagrus pagrus. The production of E2, E1, T and 17,20-P was confirmed by analysis of the steroidogenic activity of ovaries. In females, the average concentration of E2 was lower than 2 ng ml^–1. E2 values first increased significantly at the stage of endogenous vitellogenesis and remained high during exogenous vitellogenesis. E1 levels were lower values than E2 (less than 300 pg ml^–1), but they increased at the beginning of exogenous vitellogenesis. Estrogens concentrations followed similar pattern to Vg and were significantly correlated. Mean levels of T were mostly lower than 1 ng ml^–1. They followed a pattern similar to that of E2 except for a further increase observed at the stage of final maturation. T and E2 levels were significantly correlated. The concentration of 11KT did not change significantly. The levels of 17,20-P ranged between 0.22 and 1.22 ng ml^–1 but changes were not related to gametogenesis. In males, the concentrations of T and 11KT fluctuated significantly during the sexual maturity stages, showing a similar pattern and were significantly correlated to GSI changes. T levels increased during spermiogenesis and spermiation stages to reach about 3 ng ml^–1. 11KT levels stayed about half those of T. The levels of estrogens showed no significant changes. Level of 17,20-P showed no significant variation related to male maturity. Results are discussed in relation to changes in plasma steroid levels during gametogenesis of other multiple spawner species.     

Seasonality of reproduction in male spotted murrel <Emphasis Type="Italic">Channa punctatus</Emphasis>: correlation of environmental variables and plasma sex steroids with histological changes in testis

The present study was undertaken to develop a comprehensive understanding of how environmental cues and sex steroids relate with cyclic changes in spermatogenesis in freshwater spotted snakehead Channa punctatus that is nutritious and economically important. The seasonal histological changes in testis and annual profile of gonadosomatic index (GSI) of C. punctatus delineated the testicular cycle into four phases: regressed (December–March), preparatory (April–June), spawning (July and August) and postspawning (September–November). Among environmental variables, correlation and regression analyses exhibited an important relationship between photoperiod and testicular weight while role of rainfall was seen confined to spawning. The seasonal profile of plasma sex steroids when correlated with cyclic changes in spermatogenesis in spotted snakehead, testosterone (T) seems to be involved in controlling the major events of spermatogenesis from renewal of stem cells to spawning of spermatozoa. Another important androgen prevalent in teleosts, 11-ketotestosterone (11-KT), was high during preparatory phase, suggesting that 11-KT in addition to T plays an important role in progression of spermatogenesis and spermiation in C. punctatus. However, 11-KT was not seen to be associated with milt production and release of spermatozoa during spawning. Plasma profile of estradiol-17β (E₂) during different reproductive phases revealed the involvement of E₂ in repopulation of stem cells during postspawning phase and in maintaining quiescence of testis during regressed phase.     

Diethylstilbestrol arrested spermatogenesis and somatic growth in the juveniles of yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>), a fish with sexual dimorphic growth

In fish, spermatogenesis and somatic growth are mainly regulated by hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-somatic (HPS) axes, respectively. Xenoestrogens have been reported to impair spermatogenesis in some fishes, and arrest somatic growth in some others, whereas, whether xenoestrogens are capable of disrupting spermatogenesis and somatic growth simultaneously in fish that exhibits sexual dimorphic growth is little known, and the underlying mechanisms remain poorly understood. In this study, male juveniles of yellow catfish (Pelteobagrus fulvidraco), which exhibits a sexual dimorphic growth that favors males, were exposed to diethylstilbestrol (DES) for 28 days. After exposure, DES significantly disrupted the spermatogenesis (decreased gonadal-somatic index (GSI) and germ cell number) and arrested the somatic growth (declined body weight) of the catfish juveniles. Gene expression and plasma steroid analyses demonstrated the suppressed mRNA levels of genes in HPG axis (gnrh-II, fshβ, and lhβ in the brain and dmrt1, sf1, fshr, cyp17a1, cyp19a1a, and cyp11b2 in the testis) and decreased 17β-estrodial (E2) and 11-ketotestosterone (11-KT) levels in plasma. Further analysis revealed the arrested germ cell proliferation (cyclin d1), meiosis (dmc1, sycp3), and enhanced apoptosis (decreased bcl-2 and elevated bax/bcl-2 ratio) in the testis. Besides, DES also suppressed the mRNA levels of genes in HPS axis (ghrh, gh, and prl in the brain and ghr, igf1, igf2a, and igf2b in the liver). The suppressed HPG and HPS axes were thus supposed to disturb spermatogenesis and arrest somatic growth in yellow catfish. The present study greatly extended our understanding on the mechanisms underlying the toxicity of DES on spermatogenesis and somatic growth of fish.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Steroid synthesis in follicular cells and induction of germinal vesicle breakdown in spotted wolffish (Anarhichas minor) oocytes

Incubation of follicular cells from postvitellogenic spotted wolffish ovaries with tritiated steroid precursors revealed that granulosa cells were able to convert 17-hydroxyprogesterone (17-P) to 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and androstenedione. Theca cells had limited ability to synthesise additional steroids from 17-P but converted 17,20β-P to 17,20β-P sulphate. Neither of the two cell layers was able to synthesise 5β-pregnane-3α,17,20β-triol-20-sulphate (3α,17,20β-P-5β-S) which is found in high concentrations in plasma. 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), 17,20β-dihydroxy-5β-pregnan-3-one (17,20β-P-5β) and 17,20β-P were most potent in inducing germinal vesicle breakdown (GVBD). Sulphation of 17,20β-P resulted in loss of GVBD inducing activity.     

Differentiation and development of Leydig cell, and induction of spermatogenesis during testicular differentiation in the Japanese eel, Anguilla japonica

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight^-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.     

Testicular development and serum profiles of steroid hormone levels in captive male Pacific herring Clupea pallasii during their first maturational cycle

The present study investigates the relationship between testicular development and serum steroid hormone levels in captive Pacific herring Clupea pallasii during the first reproductive cycle. The maturity of the testis was divided into five periods based on histological observation. These are early spermatogenic stage (April to July), mid-spermatogenic stage (August to November), late spermatogenic stage (December to March), functional maturation stage (early April) and spent stage (late April). The pattern of seasonal change in gonadosomatic index (GSI) clearly reflected testicular maturity. 11-Ketotestosterone (11-KT) levels increased from October to a peak level (6.58 ± 1.87 ng/mL) in January, and were maintained at this level until March. In contrast, testosterone levels were consistently low, less than 1 ng/mL, at all times. These results suggest that 11-KT is the predominant androgen that controls spermatogenesis in this species. 17,20β-Dihydroxy-4-pregnen-3-one (DHP) showed a single sharp peak (3.38 ± 0.35 ng/mL) in early April of the second year, suggesting that milt production is induced by DHP as in some other teleost species.     

Serum steroid profiles in artificially maturing female Japanese eel,Anguilla japonica

To investigate whether steroid profiles in salmon pituitary homogenate (SPH)-induced artificially maturing Japanese eel, Anguilla japonica, resemble those in other, naturally maturing fishes, the daily changes in 11 steroids were analyzed for a 70-day period (average time needed to reach the maturational phase). Concentrations of most steroids were low and changed on a weekly basis, with maximum values 2–5 days after an SPH injection. Thus, pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, 17α,20 β-dihydroxy-4-pregnen-3-one, androstenedione and estrone levels were barely or not detectable in serum throughout the experimental period, which is largely in keeping with what is known about oogenesis-related steroids in other fishes. In contrast, serum testosterone (T) levels were high, but fluctuated considerably with each SPH injection (about 0.3–8.3 ng/ml). The serum estradiol-17β (E₂) levels increased after SPH injections and gradually rose throughout the experiment, peaking at the end of the experimental period (about 0.2–7.8 ng/ml). Serum levels of 11-ketotestosterone (11-KT) before SPH treatment were higher (approximately 2 ng/ml) than those of the other steroid hormones (less than 0.5 ng/ml). 11-KT levels increased gradually over the experimental period, and, like E₂, levels peaked towards the end of the experimental period (about 15 ng/ml). The observed patterns for T, E₂ and 11-KT are unlike those in other fishes. Furthermore, the consistent elevations in levels of 11-KT, both before and after SPH treatment, are suggestive of an important role for this steroid in controlling oocyte growth.     

Disruption of gonadal maturation in cultured Senegalese sole Solea senegalensis Kaup by continuous light and/or constant temperature regimes

A major problem in the development of Senegalese sole Solea senegalensis intensive culture is the poor control on reproduction, in part due to the lack of knowledge on the precise role of photoperiod and temperature. Thus, gonadal maturation was evaluated by assessing the sequential changes in plasma levels of 17β-estradiol (E₂), 11-ketotestosterone (11-KT), and testosterone (T) in both female and male cultured Senegalese sole (F1 generation) exposed to various combinations of constant or naturally-fluctuating daylength and water temperature. Under natural photoperiod (NP; 36° N), exposure to constant temperature (t0; 18-20 °C) disrupted gonadal development, as indicated by a lower incidence (in comparison with naturally-fluctuating water temperature; 14-24 °C) of females at advanced maturation (from February to April: 12 vs. 33%) and running males (from February to May: 46% vs. 57%), and the reduced mean (± S.E.M.) sex steroid plasma levels (female peak E₂ levels: 2.9 ± 0.28 vs. 1.8 ± 0.3 ng ml^− 1; male peak T levels: 1.5 ± 0.14 vs. 0.9 ± 0.06 ng ml^− 1). Therefore, the onset and progression of gonadal development in this species seem to be strongly (“proximally”) influenced by fluctuating water temperature. When compared to NP and t0, exposure to continuous light (LL) under t0 significantly reduced steroid production (female peak E₂ levels: 1.8 ± 0.28 vs. 0.5 ± 0.05 ng ml^− 1; male peak 11-KT levels: 9.4 ± 1.06 vs. 5.4 ± 1.33 ng ml^− 1) and subsequently gonadal development (lower proportions of females at intermediate [46 vs. 6%] and advanced maturation [12 vs. 0%] from February to April and of RM [46 vs. 33%] from February to May). Thus, the seasonal changes of daylength would be crucial for normal gonadal development, being its cueing effects of higher magnitude than those of water temperature. The present report constitutes the first systematic study focused on the environmental control of reproductive events in Senegalese sole.     

Plasma levels of immune factors and sex steroids in the male seahorse <Emphasis Type="Italic">Hippocampus erectus</Emphasis> during a breeding cycle

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11β-OHT), 17α-methyltestosterone (17α-MT), 17β-estradiol (E2), and 17α-hydroxy-20β-dihydroprogesterone (17α-20β-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11β-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20β-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20β-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.     

Estradiol-17β suppresses testicular development and stimulates sex reversal in protandrous black porgy,Acanthopagrus schlegeli

Two year old black porgy (Acanthopagrus schlegeli) fed a diet containing 4.0 mg kg^–1 of estradiol-17 (E₂) for 5 months had significantly lower GSI than the control group during the spawning season. E₂ suppressed testicular development, spermiation and plasma testosterone (T) and 11-ketotestosterone (11-KT) and stimulated ovarian development, vitellogenesis and sex reversal. Spermiation in the control group occurred in January and February with the concentrations of 1.08–1.36 × 10¹⁰ sperm ml^–1 of milt. Higher plasma T and 11-KT, but lower E₂ levels were detected in the spermiating fish (control group). Higher plasma E₂ levels were detected in the sex reversing black porgy during the pre-spawning season. A sharp rise in plasma 11-KT and a drop in T levels were detected in spermiating fish (control group) from January to February. Plasma 11-KT levels correlated with the testicular development and spermiation. The data suggest that E₂ plays an important role in controlling the sex reversal of black porgy.to whom correspondence should be addressed.     

Involvement of sex steroids,luteinizing hormone and thyroid hormones in upstream and downstream swimming behavior of land-locked sockeye salmon <Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>

The involvement of testosterone (T), estradiol-17β (E₂), 11-ketotestosterone (11-KT), 17,20β-dihydroxy-4-pregnene-3-one (DHP), luteinizing hormone (LH), thyroxine (T₄), and triiodothyronine (T₃) in the regulation of downstream and upstream movement (swimming behavior) was investigated in land-locked sockeye salmon Oncorhynchus nerka, using an artificial raceway. During the downstream migratory period, T implant resulted in high plasma T levels and inhibited the occurrence of downstream swimming behavior (negative rheotaxis) in yearling (1+) immature smolts. In terms of upstream behavior, 2-year-old (2+) males exhibited high plasma T and 11-KT levels, while 2+ females had elevated T and DHP levels. In 1+ immature fish, a T implant induced upstream swimming behavior (positive rheotaxis). In experiments 1 and 3, the plasma T₄ and T₃ levels of non-migrants tended to be higher than those of migrants. In contrast, no marked changes in plasma and pituitary LH were found in both downstream and upstream migrants. These results suggest that sex steroids, such as T, play significant roles in the regulation of downstream and upstream swimming behaviors in land-locked sockeye salmon.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.