Production of 11-Oxygenated Androgens by Testicular Adrenal Rest Tumors.

CONTEXT: Testicular adrenal rest tumors (TART) are a common complication in males with classic 21-hydroxylase deficiency (21OHD). TART are likely to contribute to the androgen excess in 21OHD patients, but a direct quantification of steroidogenesis from these tumors has not been yet done. OBJECTIVE: We aimed to define the production of 11-oxygenated 19-carbon (11oxC19) steroids by TART. METHODS: Using liquid chromatography-tandem mass spectrometry, steroids were measured in left (n = 7) and right (n = 4) spermatic vein and simultaneously drawn peripheral blood (n = 7) samples from 7 men with 21OHD and TART. For comparison, we also measured the peripheral steroid concentrations in 5 adrenalectomized patients and 12 age- and BMI-matched controls. Additionally, steroids were quantified in TART cell- and adrenal cell-conditioned medium, with and without adrenocorticotropic hormone (ACTH) stimulation. RESULTS: Compared with peripheral blood from 21OHD patients with TART, the spermatic vein samples displayed the highest gradient for 11ß-hydroxytestosterone (11OHT; 96-fold) of the 11oxC19 steroids, followed by 11-ketotestosterone (47-fold) and 11ß-hydroxyandrostenedione (11OHA4; 29-fold), suggesting production of these steroids in TART. TART cells produced higher levels of testosterone and lower levels of A4 and 11OHA4 after ACTH stimulation compared with adrenal cells, indicating ACTH-induced production of testosterone in TART. CONCLUSION: In patients with 21OHD, TART produce 11oxC19 steroids, but in different proportions than the adrenals. The very high ratio of 11OHT in spermatic vs peripheral vein blood suggests the 11-hydroxylation of testosterone by TART, and the in vitro results indicate that this metabolism is ACTH-sensitive.

A quantitative analysis of total and free 11-oxygenated androgens and its application to human serum and plasma specimens using liquid-chromatography tandem mass spectrometry.

Bioactive 11-oxygenated C19 adrenal-derived steroids (11-oxy C19) are potentially relevant in diverse endocrine and metabolic contexts. We report the development and validation of a liquid chromatography electrospray ionization tandem mass spectrometric method (LC-ESI-MS/MS) for the simultaneous quantification of seven 11-oxy C19 using 200 µL of plasma or serum. Sample preparation involved chemical derivatization using hydroxylamine after liquid-liquid extraction to improve specificity and sensitivity. The method allowed the quantitation of total 11-oxy C19 (free + sulfate and glucuronide conjugates) following enzymatic hydrolysis. This included the abundant precursor 11-hydroxyandrostenedione (11OHA4) and the most potent androgenic derivatives 11-keto-testosterone (11KT) and 11-keto-dihydrotestosterone (11KDHT), their abundant metabolites 11-hydroxyandrosterone (11OHAST) and 11-keto-androsterone (11KAST) potentially feeding back into the pool of potent androgens, in addition to 11-keto-androstenedione (11KA4) and 11-hydroxytestosterone (11OHT). Stable isotopes were used as internal standards, and calibrators and quality controls were prepared in the same matrix as the study samples. Performance was validated against the Food and Drug Administration Criteria. The method was sensitive with lower limit of quantification (LLOQ) values of 10 and 20 pg/mL for free and total 11-oxy C19, respectively. The applicability was demonstrated in men and women adult donors that showed sex-differences. All steroids were quantified well above LLOQ, except 11KDHT that remained undetectable suggesting interfering endogenous molecules present in non-derivatized samples in which a peak was observed. By providing accurate and reliable quantitative data, this method will permit to evaluate how profiling of 11-oxy C19 will be most informative as diagnostic, prognostic and/or theranostic tools.

Sex Differences in 11-Oxygenated Androgen Patterns Across Adulthood.

CONTEXT: The gonads are the major source of sex steroids during reproductive ages. The gonadal function declines abruptly in women and gradually in men. The adrenals produce 11-oxygenated androgens (11-oxyandrogens), which start rising during adrenarche. Following menopause, 11-oxyandrogens levels remain similar to reproductive ages. OBJECTIVE: To compare the circulating 11-oxyandrogen concentrations in men and women across adult ages. METHODS: We used mass spectrometry to measure testosterone (T), androstenedione (A4), 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 and cytochrome b5 in adrenal tissue from 19 men, age 23-78 years. RESULTS: We included 590 patients (319 men), aged 18 to 97 years, and 84% white. 11KT and 11KA4 were stable across ages in women, but they declined in men (0.21 and 0.06 ng/dL/year, respectively; P < 0.05). 11OHA4 and 11OHT increased modestly with age in women (0.6 and 0.09 ng/dL/year, respectively; P < 0.01), and both remained stable across ages in men. As body mass index (BMI) increased, 11KA4 decreased in women, and 11KT increased in men, both suggesting higher 17ß-hydroxysteroid dehydrogenase activity in obese individuals. A4 and T declined with age and A4 with BMI in both sexes; T declined with BMI in men. Adrenal androgenic enzyme expressions in aging men were similar to those observed in women. CONCLUSIONS: In contrast with traditional androgens, the production of 11OHA4 and 11OHT is sustained with aging in both sexes. The bioactive androgen 11KT declines in aging men but not in women.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of compound SFDAC by intranasal administration of multiple escalating dose in healthy male subjects.

A novel corticosteroid compound (short form of IUPAC name: SFDAC) has been discovered by Sun Pharma Advanced Research Company (SPARC) Ltd. A randomized, observer-blind, active-controlled, parallel-groups, intranasal multiple escalating dose study was conducted in healthy male subjects to assess safety, tolerability, pharmacokinetics, and pharmacodynamics of compound SFDAC formulated as an aqueous suspension for intranasal administration. Intranasal sprays of SFDAC, active control fluticasone propionate (FP) and placebo were administered once in a day for 14 days as per randomization. Various clinical evaluations including 24-hour serum cortisol and urinary free cortisol (UFC) profiles were carried out. Blood samples were collected at pre-defined time-points and analyzed using a validated chromatographic method for estimation of SFDAC and its metabolite. The results of the study indicate that multiple dose of SFDAC intranasal spray upto 3,200 µg is safe and tolerated. Clinically significant suppression of hypothalamic pituitary adrenal (HPA) axis was not observed. The plasma concentration of SFDAC was found to be below the lower limit of quantification (LLQ) at most time-points for all subjects. SFDAC M1 metabolite was detected only at picogram level in plasma. The safety and pharmacokinetic characteristics of SFDAC observed in this study support further clinical development of the SFDAC nasal spray.

Dose-response relationship of 15alpha-hydroxylated sex steroids to gonadotropin-releasing hormones and pituitary extract in male sea lampreys (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is one of the earliest extant vertebrates for which the hypothalamic-pituitary-gonadal (HPG) axis has been shown to control and regulate reproduction in a similar fashion to gnathostome vertebrates. While the two forms of gonadotropin-releasing hormones in the sea lamprey (GnRH I and GnRH III) have been studied extensively, their in vivo effect on synthesis of 15alpha-hydroxytestosterone (15alpha-T) and 15alpha-hydroxyprogesterone (15alpha-P) have only been partially characterized. In the present study, plasma concentrations of 15alpha-T and 15alpha-P were measured in prespermiating sea lampreys that were given a single injection of either GnRH I or GnRH III in doses ranging from 5 to 100 microg/kg, or of pituitary extract (as a source of gonadotropin). Plasma was sampled at 1-6h and 6-48 h post-injection, in separate experiments, in order to characterize the peak and duration of responses. 15alpha-T plasma concentrations increased slightly in response to all three treatments, but not in a dose-dependent manner, and the timing of peak concentrations varied between doses. However, 15alpha-P plasma concentrations showed a greater range of response (between 1 and 100 ng/ml) and were clearly correlated with the injection dose. Plasma concentrations of 15alpha-P also responded to far lower doses of GnRH I and GnRH III than any other steroid previously investigated in lampreys. The plasma concentrations of 15alpha-P peaked at 6h after injection for all three treatments, and levels reached a mean of 53.1 ng/ml. In female lampreys that were injected twice with 50 microg/ml GnRH I or III, 15alpha-T concentrations did not exceed 0.5 ng/ml and 15alpha-P concentrations did not exceed 1 ng/ml. These results lend further support to the hypothesis that 15alpha-P plays an important role in the reproductive endocrinology of male lampreys.

Moderate coffee and alcohol consumption improves the estrogen metabolite profile in adjuvant treated breast cancer patients: a pilot study comparing pre- and post-operative levels.

Most, but not all, studies have found that women with a high urinary 2-hydroxyestrogen (2OHE) to 16alpha-hydroxyestrone (16alphaOHE1) ratio are at reduced risk for breast cancer and have a better prognosis. The aim was to identify factors associated with the pre-operative 2OHE to 16alphaOHE1 ratio and factors that predicted the change in the ratio between the pre-operative visit and first follow-up visit three to six months post-operatively among 59 women with primary ER positive breast cancer tumors. Body measurements, questionnaires and blood samples for measurements of the 2OHE and 16alphaOHE1 plasma levels and CYP1A2 *1F genotyping were collected at both visits. Post-operatively, 15 women received tamoxifen, 30 women tamoxifen and radiotherapy concomitantly, and 14 women radiotherapy. The pre-operative ratio was not correlated with tumor characteristics, but was significantly higher in women who consumed three or more cups of coffee daily (p = 0.009). The number of CYP1A2 *1F C-alleles was correlated with a lower ratio at both visits (p = 0.13 and p = 0.02, respectively). The ratio increased between the two visits in 69.5% of the women. The factors associated with a significant increase in the ratio were concomitant tamoxifen and radiotherapy (p = 0.006), increasing alcohol consumption (p = 0.006), and a high coffee consumption (p = 0.03), but not age or CYP1A2 *1F genotype. In this pilot study, breast cancer patients who started tamoxifen during radiotherapy and who had a moderate coffee and alcohol consumption demonstrated a significant improvement in their estrogen metabolite profile between the pre- and post-operative visits.

Is it necessary to measure free testosterone to assess hyperandrogenemia in women? The role of calculated free and bioavailable testosterone.

Hirsutism in women is defined as excessive facial and/or body terminal hairs showing a masculine distribution; the condition affects approximately 7% of women of reproductive age, and chronic anovulation is a common problem for infertile couples, with a rate of 20-25%. There is a general consensus that these women should be evaluated endocrinologically, as many are found to have an androgen excess (AE) disorder. Free testosterone (FT) is the most prevalent marker in women with androgen excess, but the reference measurement procedures for FT are time-consuming and complex manual procedures that are not routinely practicable in large laboratories. Recently, models have been developed for calculating FT from total testosterone (TT), sex hormone binding globulin (SHBG), and albumin. These calculated values have been found to correlate closely with values estimated using the reference measurement procedures. This study compared measured endocrinological parameters--TT, free testosterone (aFT) by analogue ligand immunoassay method, dihydrotestosterone (DHT), dehydroepiandrosterone sulfate (DHEAS), (SHBG), And calculated parameters--calculated free testosterone (cFT), calculated bioavailable testosterone (cBT), and the free androgen index (FAI) in hirsute women and women with polycystic ovary syndrome (PCOS)--with the values in control individuals. A modified Ferriman-Gallwey score was use to describe the hirsutism pattern. No differences were observed when the measured hormone parameters were compared, while the calculated parameters were significantly increased in women in the hirsutism and PCOS groups in comparison with the values in the control group. Calculate parameters mat be more appropriate markers for assessing hyperandrogenemia in women in comparison with measured values of simple enzyme immuno-assays. These calculated values may be capable of replacing the values estimated using reference measurement procedures, so that time-consuming and complex manual procedures for measuring free testosterone with the reference methods may be dispensable in clinical practice.

Aging may be associated with concentrations of tamoxifen and its metabolites in breast cancer patients.

BACKGROUND: Although tamoxifen (TAM) is the predominant adjuvant therapy for estrogen receptor positive (ER(+)) breast tumors, 50% of breast cancer patients do not respond positively to this therapy, or they experience adverse side effects. This variability in TAM responsiveness may be due to differences in TAM metabolism that stem from differences in race, age, and body mass index (BMI). Thus, the purpose of this study was to test the hypothesis that race, age, and BMI are associated with the metabolism of TAM to two primary metabolites, N-desmethyltamoxifen (N-DMT) and 4-hydroxytamoxifen (4-OHT). METHODS: The study design was cross-sectional, and data were analyzed using independent sample t tests and multiple linear regression models. Breast cancer patients (n = 99) taking TAM for at least 30 days were recruited from a local hospital clinic. Each participant provided informed consent, completed a questionnaire, and donated a blood sample. The questionnaire was used to ascertain race, age, and BMI. The blood samples were used to measure plasma concentrations of TAM, N-DMT, and 4-OHT. RESULTS: Plasma concentrations of TAM, N-DMT, and 4-OHT differed among individual patients. Age, but not race and BMI, was positively associated with plasma concentrations of TAM and N-DMT, even after adjustment for potential confounders (p = 0.02 for TAM and p = 0.03 for N-DMT). CONCLUSIONS: This study suggests that aging may alter the metabolism of TAM. As increased levels of TAM and TAM metabolites may provide a possible explanation for why older women taking TAM are at increased risk for adverse side effects, future studies should determine whether age-related differences in the concentrations of TAM and TAM metabolites are associated with differences in TAM toxicity or responsiveness.

15 alpha-Hydroxytestosterone produced in vitro and in vivo in the sea lamprey, Petromyzon marinus.

Prior research has shown that the testes of lampreys are able to synthesize 15-hydroxylated steroid hormones in vitro. Here we show that testes of the sea lamprey Petromyzon marinus L. are able to convert tritiated testosterone into tritiated 15alpha-hydroxytestosterone (15alpha-T) in high yield. The identity of the tritiated 15alpha-T has been confirmed by: co-elution with standard 15alpha-T on high performance liquid chromatography (HPLC); co-elution on thin layer chromatography (TLC); co-elution of acetylated tritiated and standard 15alpha-T on TLC; and strong binding to an antiserum developed against 15alpha-T. The strong reaction between the tritiated 15alpha-T and the antiserum has been used to develop a radioimmunoassay (RIA). The RIA operates over the range of 500-2pg per tube; and can be applied directly to plasma samples. This assay has been used to demonstrate that 15alpha-T is present in blood plasma of the sea lamprey. The concentrations of 15alpha-T in captive lamprey were found to be as follows (pg/ml; mean+/-SEM, n): parasitic stage (reproductively immature), <20, n=7; pre-ovulatory females, 156+/-30, n=8; ovulated females, 62+/-9, n=5; pre-spermiating males, 275+/-19, n=8; spermiating males, 216+/-48, n=8. When spermiating male plasma was fractionated on HPLC, immunoreactivity was found exclusively in the expected elution position of 15alpha-T. The biological significance of this steroid has yet to be established.

Sex steroid levels in Porichthys notatus, a fish with alternative reproductive tactics, and a review of the hormonal bases for male dimorphism among teleost fishes.

Plasma levels of 11-ketotestosterone, 11-beta-OH-testosterone, testosterone, and 17-beta-estradiol were measured in reproductive Porichthys notatus, a teleost fish with two male morphs and alternative reproductive tactics. The two male types had contrasting androgen profiles. 11-Ketotestosterone was the predominant androgen in the Type I male morph which acoustically courts females, excavates nests, and guards eggs. Yet testosterone was predominant in the plasma of the Type II male morph which neither courts females nor nests, but instead parasitizes Type I males with sneak or satellite spawning tactics. The Type I-Type II male divergence in reproductive tactics and androgen levels is paralleled by dimorphisms in the vocal system, in body size, and in relative testis size. A review of endocrine data from six different species with male dimorphism shows consistent differences between morphotypes and a striking pattern: 11-ketotestosterone levels are uniformly elevated in each "courting male" morphotype relative to its "noncourting" conspecific. This cross-species pattern may reflect the behavioral, gonadal, or morphological differences which characterize the two morphotypes. At this point, the morphological interpretation is favored.

Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo.

19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 x 10(6) cells/flask) with androstenedione (8.75 mumol/l) or [3H]androstenedione (5 x 10(6) c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile:water, 37:63 (v/v) and methanol:water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxytestosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.

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.

Effect of gonadectomy and hypophysectomy on plasma steroid levels in male and female lampreys (Lampetra fluviatilis, L.).

Testosterone, estradiol, and dehydroepiandrosterone (T, E2, and DHA) were measured by RIA in plasma samples from three groups of sexually immature male and female (male, female) river lampreys: (I) intact, (II) gonadectomized, or (III) with pro- and mesoadenohypophysis removed (hypophysectomized). T levels were around 0.1 ng/ml in both I male and I female and increased significantly in all operated groups, especially in II male, to 1.2 ng/ml. E2 levels were between 0.7 and 1 ng/ml in I male and I female and were significantly elevated only in II male to 2.0 ng/ml. DHA levels showed significant decreases, from around 0.9 ng/ml in I male and I female, in both operated groups. It is suggested that T and E2 are secreted from a nongonadal source in both sexes and then converted to biologically active sex hormones in the gonads. Therefore T and E2 do not decrease, and may even accumulate, in the blood after gonadectomy. Since extirpation of the pro- and mesoadenohypophysis was not followed by a decrease in T and E2, their secretion may be stimulated by a hormone from another part of the pituitary such as the metaadenohypophysis.

Serum androgens during the annual reproductive cycle of the male mummichog, Fundulus heteroclitus.

A technique for the simultaneous detection of serum concentrations of testosterone (T), 17 beta-hydroxy-4-androstene-3,11-dione (11-KT), and 11 beta, 17 beta-dihydroxy-4-androsten-3-one (11 beta-HT) was established using a combination of high performance liquid chromatography separation and radioimmunoassay. In the mummichog, Fundulus heteroclitus, serum concentrations of T, 11-KT, and 11 beta-HT varied from 11.3, 10.1, and 27.9 ng/ml, respectively, during the breeding season to 0.2, 0.3, and 0.9 ng/ml, respectively, during the nonbreeding season. The changes in serum androgen levels, determined weekly, did not coincide with changes in the gonosomatic index or measurement of sperm/gram testes. Increases in serum androgen levels did coincide with the rise in sperm index, but the decreases in serum concentrations of androgens preceded the fall in sperm index by more than a month. A model is proposed to explain the reproductive cycle of male mummichogs.

A new method for the simultaneous determination of androstenedione, testosterone, 11-oxotestosterone and 11 beta-OH-testosterone in fish plasma using combined techniques of Celite chromatography and radioimmunoassay.

A new method for the simultaneous determination of androstenedione, testosterone, 11-oxotestosterone and 11 beta-hydroxy-testosterone in teleost plasma has been developed. Steroids extracted from the plasma were first separated by Celite chromatography using ethanediol as the stationary phase and different concentrations of ethyl acetate in iso-octane as the eluting solvents. Androstenedione, testosterone, 11-oxotestosterone and 11 beta-OH-testosterone were eluted successively with 0, 10, 15 and 40% of ethyl acetate in iso-octane. The eluted steroid fractions were then quantatively determined by radioimmunoassay. Data on the accuracy, precision, and sensitivity were presented showing that this new assay system was precise and reproducible. As an illustration of its application, this method was used in the present study to determine the plasma androgen levels in Monopterus albus and in methyl-testosterone treated Tilapia mossambicus.

The measurement of 11 beta, 17 beta-dihydroxy-4-androsten-3-one (11 beta-hydroxytestosterone) by radioimmunoassay in human plasma.

A radioimmunoassay (RIA) is described for the measurement of 11 beta,17 beta-dihydroxy-4-androsten-3-one (11 beta-hydroxytestosterone) in human plasma. The reliability criteria of the new RIA were similar to those of other steroid hormone radioimmunoassays. The mean plasma 11 beta,17 beta-dihydroxy-4-androsten-3-one level for healthy young subjects was 1.31 +/- 0.32 nmol/l (means +/- SD) in males and 1.19 +/- 0.35 nmol/l in females at 8 a.m.; during the night, there was a marked decrease, and at 11 p.m. the recorded values were 0.41 +/- 0.15 nmol/l and 0.46 +/- 0.14 nmol/l, respectively. During the corticotropin stimulation test, 11 beta,17 beta-dihydroxy-4-androsten-3-one increased from 1.03 +/- 0.33 nmol/l to 1.31 +/- 0.41 nmol/l, while in dexamethasone suppression tests a decrease from 2.04 +/- 0.82 nmol/l to 0.25 +/- 0.05 nmol/l was seen. In contrast, chorionic gonadotropin administration on 3 consecutive days did not influence plasma concentrations of 11 beta,17 beta-dihydroxy-4-androsten-3-one.

Identification of nonecdysteroid steroids in hemolymph of both male and female Astacus leptodactylus (Crustacea) by gas chromatography-mass spectrometry.

The O-pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB) derivatives of hemolymph steroid extracts from both male and female Astacus leptodactylus were subjected to negative ion chemical ionization and capillary gas chromatography-mass spectrometry (NCI/GC-MS). Five nonecdysteroid steroids, namely pregnenolone, 17 alpha-hydroxypregnenolone, testosterone, cholesterol, and 6 beta-hydroxyprogesterone were identified. With selected ion monitoring (SIM), indications were found for the presence of four more steroids: androstenedione, 5 alpha-dihydrotestosterone, 11-ketotestosterone, and 11 beta-hydroxytestosterone. The presence of 6 beta-hydroxyprogesterone could only be demonstrated in the female hemolymph. With the technique used, dehydroepiandrosterone, progesterone, 17 alpha-hydroxyprogesterone, and estrogens could not be detected in male or female hemolymph extracts.

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood.

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.