Hormones and reproduction in scleractinian corals

Most broadcast spawning scleractinian corals synchronously release gametes during a brief annual spawning period. In southern Taiwan, the mass spawning of scleractinians occurs in lunar mid-March. The exact cues triggering this annual phenomenon remain unclear. A scleractinian coral, Euphyllia ancora has been selected as a model for the hormones and reproduction studies. Testosterone (T) and estradiol (E2) in free and glucuronided forms were identified and consistently detected in coral polyps throughout the year. Peak levels of free E2, glucuronided E2 and T were obtained in the coral tissue just prior to spawning. The presence of specific aromatase activity was demonstrated in coral tissue. Higher concentrations of free E2 than glucuronided E2 were detected in the coral tissue throughout the year. In contrast, higher levels of glucuronided E2 than free E2 and glucuronided T were found in seawater during mass coral spawning. Furthermore, immunoreactivity and biological activity of immunoreactive gonadotropin-releasing hormone (irGnRH) was detected and quantified in coral tissue. Coral extracts (irGnRH) and mammalian (m)GnRH agonist had a similar dose-dependent effect on luteinizing hormone (LH) release in black porgy fish pituitary cells (in vitro). Peak levels of irGnRH were detected during the spawning period. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity, as well as the levels of T and E2 in free and glucuronided forms in coral. In conclusion, our data suggest that irGnRH and glucuronided E2 may play important roles in the control of reproduction and mass spawning in corals.     

Seasonal changes in serum steroid hormones in a protandrous teleost, the sobaity (Sparidentex hasta Valenciennes)

Serum concentrations of 11-ketotestosterone, 11β-hydroxytestosterone, testosterone, testoster-one glucuronide, oestradiol and 17,20β -dihydroxy-4-pregnen-3-one (17,20β -P) were measured in the sobaity at monthly intervals through their second breeding season. Concentrations of the 11-oxygenated androgens in the males and of oestradiol in the females peaked during the spawning season in January-February, while maximum levels of testosterone were found in the summer when these steroids were low. Testosterone glucuronide showed two peaks, one in the post-spawning period as oestradiol and the 11-oxygenated androgens were falling and the other coincident with the summer peak of testosterone. 17,20β -P was detectable in only one male and one female fish in February. Serum concentrations of 11-oxygenated androgens are more reliable than those of oestradiol for determining the sex of sobaity, and may also be used as indicators of the occurrence of sex reversal. The seasonal pattern of serum steroids correlated well with the changes of sexual status of the gonads during regression and recrudescence observed histologically and suggests that oestradiol may be involved in the sex inversion of this species.     

Estrone and estradiol-17 beta concentration in tissue of the scleractinian coral, Montipora verrucosa

Spawnings of scleractinian corals are affected by light, temperature, and other environmental cues, but no studies elucidate physiological mechanisms that regulate coral gametogenesis. We hypothesized that estrogens may act as bioregulators of coral reproduction. Estrone (E1) and estradiol-17 beta (E2) concentrations were measured in homogenates of tissue and skeleton from M. verrucosa. Tissue samples were collected monthly throughout the year, and more frequently in July and August around spawning. Steroids were extracted with diethyl ether, purified via celite chromatography and assayed with radioimmunoassay. Non-specific binding in coral tissue varied with sample weight and was elevated relative to standards. Monthly mean E1 ranged from 20-70 ng E1 g ash-free dry weight (AFDW)-1, with highest values in April. Smaller asynchronous peaks occurred in early July, prior to spawning. Monthly mean E2 ranged from 8-25 ng E2 g AFDW-1, with highest values in February and March. Peaks in E2 preceded peaks in E1, indicating metabolism of a pool of estrogen. E1 was positively correlated with protein concentration, which is consistent with a bioregulatory role of estrogens. Estrogen peaks in spring and prior to the July spawn corroborate our hypothesis that estrogens regulate coral gametogenesis and spawning.     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Influence of diet on plasma steroids and sex hormone-binding globulin levels in adult men

Several experimental studies have suggested that diet can alter the production and metabolism of steroids in men. The purpose of this study was to determine the levels of unconjugated steroids and steroid glucuronides as well as sex hormone-binding globulin (SHBG) among normal adult men who were either omnivorous or vegetarians. The participants were white volunteers ranging from 25-35 years of age and the blood samples were taken between 0900 h and 1000 h and between 1600 h and 1700 h for two consecutive days. No significant statistical change was found in plasma dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone and estradiol levels. Vegetarian group showed a higher levels of sex hormone-binding globulin (SHBG) while the free androgen index (FAI; calculated by the ratio testosterone/SHBG) was lower in this group. Although the concentrations of androsterone glucuronide were higher in vegetarian group, the vegetarians had a 25-50% lower level of androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide. Our data further indicate that both, androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide concentrations are significantly correlated with SHBG levels and with the FAI values. The increases in androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide levels in the omnivorous group are probably a consequence of the elevation of the FAI. Our data suggest that in a vegetarian group, less testosterone is available for androgenic action.     

Steroid regulation of brain aromatase expression in glia: female preoptic and vocal motor nuclei

Expression of the enzyme aromatase, which converts androgens to estrogens, is known to be regulated by gonadal steroids in brain areas linked to reproduction and related behaviors in several groups of vertebrates. Previously, we demonstrated in a vocal fish, the plainfin midshipman, that both males and females undergo seasonal changes in brain aromatase mRNA expression in the preoptic area (POA) and the dimorphic sonic/vocal motor nucleus (SMN) that parallel seasonal variation in circulating steroid levels and reproductive behavior. We tested the hypothesis that steroids are directly responsible for seasonal modulation of aromatase in females because they show the most dramatic fluctuations of testosterone (T) and 17beta-estradiol (E2) throughout the year. Adult female midshipmen were ovariectomized and administered T, E2, or blank (control) implants. We then quantified aromatase mRNA expression within the POA and SMN by in situ hybridization. Both T- and E2-treated females had elevated mRNA expression levels in both brain areas compared to controls. T affected aromatase expression in a level-dependent manner, whereas E2 showed a decreased effect at higher circulating levels. This study demonstrates that seasonal differences in brain aromatase expression in female midshipman fish may be explained, in part, by changes in levels of circulating steroids.     

Sex steroids relative to alternative mating behaviors in the simultaneous hermaphrodite Serranus subligarius (Perciformes: Serranidae)

This study is the first investigation of reproductive endocrinology in a simultaneously hermaphroditic teleost, the belted sandfish (Serranus subligarius). We address two questions: (1) Do steroid hormone levels vary during the spawning season or during the daily spawning cycle of sandfish? (2) Do hormone levels vary relative to an individual's phenotype-size, frequency of spawning and aggressive behaviors, and proportion of testis in the gonad? We analyzed circulating estradiol-17beta (E2), testosterone (T), 11-ketotestosterone (11KT), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20betaS), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) concentrations in a field population. Only E2 levels were significantly higher at the new and full moon, suggesting peak periods of vitellogenesis at these times. Naturally spawning sandfish were sampled every 2 h during the photophase of a 25-h period (12 pm to 1 pm the following day) and gonadosomatic index, degree of oocyte hydration and ovulation, and plasma levels of E2, T, DHP, and 20betaS were analyzed. E2 and T levels did not vary during photophase, suggesting continuous recruitment of oocytes into vitellogenesis. The 20betaS levels peaked around the time of final oocyte maturation. Since frequency of spawning behaviors changes with body size, we captured individuals of various sizes throughout the spawning season and analyzed circulating levels of hormones. 11KT and 20betaS levels increased significantly with body size. In 1992, we quantified frequency of spawning and aggressive behaviors, circulating T and 11KT levels and testicular mass relative to ovotestis mass in focal animals. 11KT levels tended to be positively correlated with frequency of courting male behavior, but were unrelated to the frequency of aggressive behavior or testis mass. Because hormone levels increased with size and frequency of each spawning behavior changes with size, we propose that sex steroids influence growth-related changes in spawning tactics of individuals.     

Sex steroids in plasma of lake whitefish Coregonus clupeaformis during spawning in Lake Erie

Lake whitefish, Coregonus clupeaformis, were collected from the Western basin of Lake Erie during spawning. Free and conjugated (sulfated and glucuronidated) steroids including testosterone (T), 11-ketotestosterone (11-kT), estradiol-17beta (E2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP) were measured in the plasma by radioimmunoassay. In males, the progression of spermiation was characterized by a significant decrease in plasma free steroids, whereas the levels of conjugated steroids remained similar and low, except for sulfated and glucuronidated testosterone. Plasma sex steroids did not correlate with the density or the motility of the spermatozoa. In females, the concentration of plasma T was significantly higher in preovulating than in ovulating females. The levels of E2 and 17,20betaP in ovulating lake whitefish exhibited large variations ranging from below detection limit to 0.9 ng ml(-1) and from 0.2 to 13 ng ml(-1), respectively. Analysis of conjugated steroids revealed high levels of glucuronidated and sulfated 17,20betaP and glucuronidated T in females ovulating in December. However, no significant differences in the proportion of the conjugated steroids were observed.     

Chemical signals in gametogenesis, spawning, and larval settlement and defense of the soft coral Sinularia polydactyla

 Mass spawning strategies of hard and soft corals on the Great Barrier Reef, Australia have been described in recent years. Nonetheless, the applicability of those studies to corals on other reef systems has not been well documented. Here we describe the mass spawning behavior of the soft coral Sinularia polydactyla on coral reefs surrounding Guam; specifically we describe the events in an annual gametogenic cycle including steroidogenesis, spawning, settlement and early life history defense. The gametogenic cycle of female colonies lasted 12 months while male colonies produced viable sperm within 9 months. Sinularia polydactyla exhibited a split spawn between March and June that correlated with a significant reduction in tissue concentrations of progesterone and testosterone. Estradiol was released into the water column, apparently by female colonies, just prior to spawning. There was a trend for preferential larval settlement in the presence of the crustose coralline algae Hydrolithon reinboldii rather than coral rubble, a natural biofilm, or filtered seawater. The defensive compounds pukalide and 11β-acetoxypukalide were found in eggs and larvae at adult level and three-fold lower than adult-level concentrations, respectively. These compounds provided some predator deterrent and antimicrobial protection against an ecologically relevant omnivorous fish Canthigaster solandri and a sympatric microbe Vibrio sp. Accepted: 10 September 1998     

Sex steroids in cervical mucus of spontaneous or induced ovulatory cycles

Evaluation of sex steroids in cervical mucus was performed at different phases of spontaneous or clomiphene-citrate-induced ovulatory cycles. To this end, 11 women with normal ovulatory cycles and 9 subjects with polycystic ovary syndrome of comparable age and body mass index were investigated. Serum and cervical mucus samplings were assessed for 17beta-estradiol (E2), progesterone, testosterone, and sex hormone binding globulin levels at the pre-, peri-ovulatory, and mid-luteal phases of the cycle. The cervical mucus maturation index also was estimated in all women. Measurable amounts of E2 were found in most mucus samples with a cyclic variation in all cases. The highest E2 and mucus maturation index values coincided, but both lagged by 24 h behind the serum mid-cycle peak of this steroid. Detectable amounts of progesterone were found in the luteal phase, testosterone was present at low levels throughout the cycle, but sex hormone binding globulin was undetectable in all cervical mucus samples. Differences between spontaneous or drug-induced ovulatory cycles were not found. It is concluded that sex steroids are present in human cervical mucus, showing variations similar to those in peripheral blood. The significance of these findings is not clear at present, but it is probably related to the cyclic changes of cervical epithelium and gland secretion. An important implication of the absence of measurable sex hormone binding globulin amounts in cervical mucus is that the free fraction of sex steroids present in that fluid are presumably higher, and therefore, expected to exert greater biologic activity than in peripheral blood.     

Effect of the aromatase inhibitor, 4 hydroxyandrostenedione, on the endotoxin-induced changes in steroid hormones in male rats.

The increase in circulating estrogen concentrations that follows injection of Escherichia coli endotoxin (Endo) may be due to increased aromatase activity. We have therefore analysed the effect of the aromatase inhibitor, 4 hydroxyandrostenedione (4OHA) on the steroid hormone response of male rats, particularly the dramatic increase in estrogens and decrease in androgens, induced by Endo. The concentrations of corticosterone (B), progesterone (P4), 17 alpha hydroxyprogesterone (17 alpha OHP4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) were determined 2 hours after injection of increasing doses of 4OHA with and without Endo. The increase in serum estrogen concentrations and drop in serum androgen levels in response to Endo were blocked by a single dose of 4OHA. The effect of 4OHA appeared to be dose dependent. Low doses (30 mg/kg and 50 mg/kg) induced significant changes in the estrogen and androgen responses, but the high dose (100 mg/kg) blocked all changes in sex steroids induced by Endo. 4OHA did not alter the Endo-induced changes in other steroids.     

Sex steroids, growth and condition of Arctic charr broodstock during an annual cycle

Changes in plasma concentrations of sex steroids, growth rate and condition of repeat spawning (3+) male and female Arctic charr were studied throughout an annual reproductive cycle. Individually marked fish (mean weight approx. 500 g) were held under conditions of liberal food supply, constant temperature (4° C) and simulated natural photoperiod (Tromsø, 70° N). Once each month fish were weighed, measured and blood samples taken for steroid analysis. Plasma concentrations of testosterone (T), 11-ketotestosterone (11-KT) and oestradiol-17β (E₂) were determined using radioimmunoassay (RIA). Both male and female fish displayed distinct seasonal changes in plasma concentrations of sex steroids, growth rate and condition. From February (minimal concentrations) to March all sex steroids increased slightly and these elevated concentrations were maintained until May. Thereafter, there was a second, and far more pronounced, increase in plasma steroid concentrations which culminated in peak steroid concentrations in September–October. There was then a rapid decline during the spawning period. In winter, growth rate and condition were generally low, then increased during the spring, reached a peak during the summer, and then declined with the onset of autumn. During spring (March–May), the frequency distributions of plasma testosterone concentrations in both male and female fish were bimodal. The fish of the upper modal group of the distribution had significantly higher growth rates and condition than those in the lower modal group. In summer and early autumn (June–September) the association between T and growth rate changed. Significant negative correlations between T and growth rates were observed in females. There was an increase in endocrine activity, indicated by elevated plasma sex steroid concentrations in March, 7–8 months prior to maturation. It is suggested that this may be one factor influencing the onset of spring growth and energy deposition among maturing charr.     

Coral spawning in the western Pacific Ocean is related to solar insolation: evidence of multiple spawning events in Palau

Coral reproductive schedules have long been related to concomitant increases in regional sea surface temperature (SST). Yet, corals also mass spawn in the tropics, where SSTs vary little throughout the year. Here we show that the rise toward and fall from solar insolation maxima, which is the electromagnetic energy incident on the surface of the earth, coincide with coral spawning events in tropical Palau. Two insolation maxima (a consequence of the vernal and autumnal equinoxes) in the tropics lead to multiple spawning events per year. SSTs are poor predictors of coral spawning in the tropics. Multiple spawning events increase the rate of genetic recombination, which in turn may facilitate the likelihood of speciation and explain, in part, the high coral diversity in the tropics. We also show that mass spawning coincides with near-maximum solar insolation (5.0–7.2 kWh/m²/day) events from 33°N to 30°S in the western Pacific Ocean, and should be considered as one of the primary variables driving coral reproductive cycles.     

Effect of elevated summer temperatures on gonadal steroid production, vitellogenesis and egg quality in female Atlantic salmon

Sex change in the coral-dwelling goby Gobiodon histrio was induced by placing two adult fish of the same sex on a coral colony. The sex change of individual fish was confirmed using histology, and whole-body concentrations of the gonadal steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-oestradiol (E₂) were examined. The results show that T, 11-KT and E₂ occurred in both female and male G. histrio . E₂ concentration in females was twice that in males, while concentrations of T did not differ between the sexes. Contrary to predictions, concentrations of T and E₂ did not differ between fish that changed sex and those that did not. Most samples had 11-KT concentrations below minimum levels of detection (  i.e. <0·15 ng ml⁻¹) and were therefore not analysed statistically. The results suggest that: (i) specific activation or de-activation of the T–E₂ (aromatase) pathway is a probable candidate for mediating serial adult sex change in G. histrio , and (ii) low levels of 11-KT may be important in allowing serial adult sex change in G. histrio .     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Acute sex steroid withdrawal increases cholesterol efflux capacity and HDL-associated clusterin in men

Exogenous androgens can lower HDL-cholesterol (HDL-C) concentrations, yet men with low serum testosterone have elevated rates of cardiovascular disease (CVD). HDL function may better predict CVD risk than absolute HDL-C quantity. We evaluated the acute effects of medical castration in men on HDL-C, cholesterol efflux capacity and HDL protein composition. Twenty-one healthy men, ages 18-55, received the GnRH antagonist acyline and one of the following for 28days: Group 1: placebo, Group 2: transdermal testosterone gel and placebo, Group 3: transdermal testosterone gel and an aromatase inhibitor. Sex steroids, fasting lipids, and cholesterol efflux to apoB-depleted serum were measured in all subjects. The HDL proteome was assessed in Group 1 subjects only. In Group 1, serum testosterone concentrations were reduced by >95%, and HDL-C and cholesterol efflux capacity increased (p=0.02 and p=0.03 vs. baseline, respectively). HDL-associated clusterin increased significantly with sex steroid withdrawal (p=0.007 vs. baseline). Testosterone withdrawal in young, healthy men increases HDL-C and cholesterol efflux capacity. Moreover, sex steroid deprivation changes HDL protein composition. Further investigation of the effects of sex steroids on HDL composition and function may help resolve the apparently conflicting data regarding testosterone, HDL-C, and CVD risk.     

Differential influence of peripheral and systemic sex steroids on skeletal muscle quality in pre- and postmenopausal women

Aging is associated with gradual decline of skeletal muscle strength and mass often leading to diminished muscle quality. This phenomenon is known as sarcopenia and affects about 30% of the over 60-year-old population. Androgens act as anabolic agents regulating muscle mass and improving muscle performance. The role of female sex steroids as well as the ability of skeletal muscle tissue to locally produce sex steroids has been less extensively studied. We show that despite the extensive systemic deficit of sex steroid hormones in postmenopausal compared to premenopausal women, the hormone content of skeletal muscle does not follow the same trend. In contrast to the systemic levels, muscle tissue of post- and premenopausal women had similar concentrations of dehydroepiandrosterone and androstenedione, while the concentrations of estradiol and testosterone were significantly higher in muscle of the postmenopausal women. The presence of steroidogenetic enzymes in muscle tissue indicates that the elevated postmenopausal steroid levels in skeletal muscle are because of local steroidogenesis. The circulating sex steroids were associated with better muscle quality while the muscle concentrations reflected the amount of infiltrated fat within muscle tissue. We conclude that systemically delivered and peripherally produced sex steroids have distinct roles in the regulation of neuromuscular characteristics during aging.