Plasma steroid hormone levels in female flounder Platichthys flesus and the influence of fluctuating hydrostatic pressure.

The reproductive cycle in teleosts is timed to guarantee that eggs hatch in the right place at the right time, with environmental factors playing important roles in entraining and controlling the entire process. The effects of some environmental factors, like temperature and photoperiod, are now well understood. There are only a few studies regarding the effects of hydrostatic pressure (HP) on the reproductive cycle, in spite of its importance as a ubiquitous factor in all biological environments and affecting all living organisms. Hydrostatic pressure is of particular importance in fish because they can also experience rapid and cyclic changes in HP due to vertical movements in the water column. The aim of the present research was to investigate the effects of vertical migrations on the reproductive steroids of maturing female flounder. After a 14 day exposure to cyclic hydrostatic pressure (with a period of 12.4h and with a maximum peak of 800 kPa of absolute hydrostatic pressure), fish showed significantly lower plasmatic concentrations of "5ß,3α" steroids, metabolites of the putative maturation-inducing steroid in flounder (17α,20ß-dihydroxy-4-pregnen-3-one). Results indicate that environmentally realistic cyclic changes of hydrostatic pressure can influence the metabolism of reproductive steroids. This suggests a physiological role of tidally-associated vertical migrations, affecting oocyte maturation and retarding the reproductive cycle in this species until the spawning ground is attained.

Detection of 17alpha-hydroxyprogesterone caproate in equine plasma by gas chromatography/tandem mass spectrometry.

A method was developed for the analysis of the synthetic progestin 17alpha-hydroxyprogesterone caproate in equine plasma following its administration by intramuscular injection. The method employed a reversed-phase solid-phase extraction followed by enol-trimethylsilylation and analysis by gas chromatography/tandem mass spectrometry. The intact ester was detectable in the plasma for up to 2 weeks after a single therapeutic dose, and was found to be stable in equine whole blood for at least 2 months.

The effect of combined oestrogen and progesterone replacement on the renal responses to oxytocin and vasopressin in ovariectomized rats.

OBJECTIVE: Renal responsiveness to the neurohypophyseal hormones, oxytocin and vasopressin, has been shown in the rat to vary during pregnancy and lactation. A study was performed to determine whether ovarian steroids could contribute to the observed changes. DESIGN: Using a previously validated method, fluid excretion during infusion of oxytocin or vasopressin was monitored in ovariectomized animals with and without chronic administration of oestrogen and progesterone. METHODS: After 14 days treatment with vehicle or 12.5 mg hydroxyprogesterone caproate and 0.25 mg oestradiol valerate injected every 3 days, rats were infused with 0.077 mol/l NaCl for an equilibration period of approximately 2.5h. Timed urine collections for the determination of volume and electrolytes were then made during a control period of at least 45 min and for 60 min while the infusate was supplemented with vasopressin (40 fmol/min) or oxytocin (50 fmol/min). Further observations were made for a final 90 min of hypotonic saline infusion. In control infusions saline alone was given. RESULTS: Treatment with ovarian steroids did not affect the volume of urine excreted during hormone infusion. Electrolyte excretion, however, was affected with lower concentrations of sodium and chloride on oxytocin infusion being seen in the steroid-treated animals. During vasopressin infusion, peak electrolyte concentrations were also achieved later in this group of animals. CONCLUSION: The increased circulating concentrations of oestrogen and progesterone seen during pregnancy could contribute to variations in the natriuretic response to neurohypophyseal hormones observed in the rat.

Plasma and urine levels of C18, C19 and C21 steroids in an asynchronous fish, the tilapia Oreochromis mossambicus (Teleostei, Cichlidae).

Female and male tilapia, Oreochromis mossambicus, were treated with luteinizing hormone-releasing hormone analogue (LH-RHa) and pimozide (PIM) or with human chorionic gonadotropin (hCG) to stimulate gonadal development and sexual maturation. Plasma (both sexes) and urine (males) samples were collected periodically for steroid analysis by radioimmunoassay. Plasma levels of estradiol-17 beta (3-6 ng/ml) and testosterone, higher in female (up to 25 ng/ml) than in male (6-13 ng/ml; P < 0.05), were in the range of those established in other tilapia species. Plasma levels of the established teleost oocyte maturation-inducing steroids (MIS), that is 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (17,20 beta,21-P) were low (1-9 ng/ml) and were not different between treated and control fishes at 8, 12, 16, 24, 48, 72 and 96 hr after injection. Furthermore, in male O. mossambicus, 17,20 beta,21-P was undetectable. Plasma levels of 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (3,17,21-P-5 beta) were very high in both sexes (up to 700 ng/ ml), mostly in hormone-treated groups, whose levels were higher than controls (P < 0.05). Urine levels of conjugated 17,20 beta,21-P (glucuronides and sulphates) were not detectable, but those of 17, 20 beta-P (up to 25 ng/ ml) and 3,17,21-P-5 beta (up to 1 microgram/ml) were higher than free 17,20 beta-P and 3,17,21-P-5 beta measured in the plasma of the same animals (P < 0.05). Both LH-RHa + PIM and hCG induced sexual maturation of O. mossambicus (histological data); nevertheless, during that period all measured steroids, either in plasma or urine, almost did not fluctuate. Thus, this study does not make any comment about the MIS of tilapia. Nevertheless, the high levels of conjugated 3,17,21-P-5 beta and 17,20 beta-P in urine suggest a probable pheromone role for these steroids in this species.

11 beta-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 alpha-hydroxyprogesterone.

The 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11 beta HSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11 beta HSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 +/- 3.1 nM, and that for NAD+ was approximately 8 microM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity. 11 alpha-Hydroxyprogesterone (11 alpha OH-P) was an order of magnitude more potent a competitive inhibitor of the 11 beta HSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 = 0.9 vs. 15 nM). 11 beta OH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5 alpha-pregnandione and 5 beta-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11 alpha OH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11 alpha OH-P was not metabolized by 11 beta HSD-2. We were unable to demonstrate the presence of 11 alpha OH-P in human urine. In conclusion, a cell line stably transfected with the rat 11 beta HSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11 alpha OH-P was found to be a potent relatively specific inhibitor of the 11 beta HSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11 beta HSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.

Excretion of free and conjugated steroids in rainbow trout (Oncorhynchus mykiss): evidence for branchial excretion of the maturation-inducing steroid, 17,20 beta-dihydroxy-4-pregnen-3-one.

The goal of this study was to identify excretory routes of three main steroids produced by sexually mature male and female rainbow trout: 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), sulfated 17,20 beta-P (17,20 beta-P-S), and testosterone glucuronide (TG). Spermiating males or maturing trout were cannulated via the dorsal aorta and urinary bladder and injected with tritiated steroids. Blood, water, and urine were sampled over the next 12 hr when the fish were killed and bile was collected. The identities of the excreted products were determined by anion-exchange chromatography, reverse-phase high-performance liquid chromatography, enzyme hydrolysis, acid solvolysis, microchemical modification, and thin-layer chromatography. Following the injection of tritiated 17,20 beta-P, 25% of the radioactivity rapidly appeared unmodified in the water; 15% appeared slowly in the urine, mainly as 17,20 beta-P-S; and 40% was recovered in the bile, mainly as 17,20 beta-P-glucuronide. 17,20 beta-P was shown to be released into the water via the gills. Over the 12-hr sampling period, 20% of the 17,20 beta-P released into the water was taken up again by the fish (also branchially). A mathematical analysis showed that 40% of the 17,20 beta-P would have been released into the water in the absence of uptake. Following the injection of tritiated 17,20 beta-P-S, 63% appeared very rapidly, in an unmodified form, in the urine, and 15% was recovered in the bile. Following the injection of tritiated TG, 9% appeared slowly, mainly untransformed, in the urine, and 59% was recovered in the bile. These results show that the three types of steroids are released into the water by three different routes: free steroids, gills; sulfated steroids, urine; and glucuronidated steroids, bile.

Serum and urinary markers of exogenous testosterone administration.

In an attempt to find optimal markers of exogenous testosterone (T) administration in male athletes, a number of compounds were measured in 11 healthy men before and after 3, 6 and 9 months of weekly administration of 250 mg i.m. T enanthate and in age-matched untreated controls. The following variables were measured in serum: T, 17 alpha-hydroxyprogesterone (17-OHP), sex hormone-binding globulin (SHBG), estradiol-17 beta, estrone (free + conjugated) and luteinizing hormone (LH). The following variables were measured in urine: T glucuronide (urinary T), epitestosterone glucuronide (urinary epiT), estrone (free + conjugated) and LH. Serum T, serum T/17-OHP ratio, serum T/LH ratio, serum T/SHBG ratio, serum and urinary estrogens, urinary T/creatinine-, T/epiT- and T/LH ratios increased whereas serum 17-OHP, SHBG and LH and urinary epiT/creatinine- and LH/creatinine-ratios decreased significantly during treatment. Levels above the upper reference limit were found in all subjects at 3, 6 and 9 months for serum T/17-OHP and serum and urinary T/LH ratios and at 6 months for the urinary T/epiT ratio. Levels below the lower reference limit were found in all subjects at 3, 6 and 9 months for serum LH and the urinary LH/creatinine ratio, at 3 months for the urinary epiT/creatinine ratio and at 9 months for serum 17-OHP. No other variable showed abnormal values in all subjects at the same occasion. Despite significant changes during treatment, steroid concentrations as such are poor indicators of T doping. Serum and urinary LH levels, T/LH ratios and serum T/17-OHP ratios seem to be the most reliable markers of exogenous T administration in males.

Menstrual disturbance and hypersecretion of progesterone in women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

OBJECTIVE: While menstrual disturbance is often quoted as a feature of congenital adrenal hyperplasia (CAH), little is known about the mechanism of this symptom. We set out to determine the relationship between menstrual pattern and biochemical characteristics of women with CAH due to 21-hydroxylase deficiency. PATIENTS AND DESIGN: All 21 female patients with classic CAH attending the adult endocrinology clinics at The Middlesex Hospital were reviewed. Their ages at menarche and menstrual pattern were recorded and blood samples were taken in the follicular phase of the menstrual cycle when on their usual maintenance therapy. MEASUREMENTS: Measurements of serum LH, FSH, progesterone, 17 alpha-hydroxyprogesterone, testosterone, androstenedione and plasma renin activity were recorded. Urinary steroid profiles were obtained by gas chromatography and mass spectrometry. Molecular genetic analysis of the 21-hydroxylase gene was performed on leucocyte DNA. RESULTS: In the 18 patients who had spontaneous menarche the degree of menstrual disturbance and progesterone excess was related to the effectiveness of adrenal suppressive therapy. Three out of 21 patients, however, failed to experience menarche on standard medical therapy. These patients with primary amenorrhoea were characterized by reduced endometrial thickening, by non-suppressible serum progesterone concentrations despite suppression of 17 alpha-hydroxyprogesterone levels and by the presence of progesterone metabolites in urinary steroid profiles. Molecular genetic analysis did not differentiate between patients with raised progesterone concentrations and those without. CONCLUSION: A subgroup of women with congenital adrenal hyperplasia have the triad of non-suppressible serum progesterone of adrenal origin, primary amenorrhoea and infertility due to failure of endometrial thickening. The characteristic urinary steroid profile best distinguishes this subgroup of women from others with congenital adrenal hyperplasia and menstrual disturbance due to inadequate adrenal suppression.

Urinary 17 alpha-hydroxyprogesterone in management of 21-hydroxylase deficiency.

OBJECTIVES: The study was designed to assess the reliability of measurement of 24-hour urinary 17 alpha-hydroxyprogesterone (17-OHP) by radio-immunoassay (RIA) as an alternative biochemical assessment for monitoring the treatment of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) and to assess the need for sample purification by column chromatography to improve assay specificity. METHODOLOGY: Morning serum 17-OHP was measured using RIA and 24-hour urinary pregnanetriol using gas chromatography. Twenty-four-hour urinary 17-OHP was measured in samples from 17 prepubertal patients with CAH due to 21-OHD, and 20 normal prepubertal children as controls. In 24 urine samples, RIA of 17-OHP was performed with and without column chromatography. RESULTS: There was a good correlation between 24-hour urinary 17-OHP and 24-hour urinary pregnanetriol (r = 0.962, P < 0.01) and between 24-hour urinary 17-OHP and morning serum 17-OHP (r = 0.955, P < 0.01). There was no significant difference in the RIA of the urine samples with and without purification by column chromatography. CONCLUSIONS: The measurement of 24-hour urinary 17-OHP is a reliable alternative for the biochemical monitoring of 21-OHD, and RIA specificity is unaffected by omission of column chromatography.

Serum and urinary steroids in girls with precocious pubarche and/or hirsutism due to mild 3-beta-hydroxysteroid dehydrogenase deficiency.

To obtain data on the correlation of serum and urinary steroids in nonclassical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency, 9 girls with precocious pubarche and 33 adolescent girls with mild to severe hirsutism were studied. Urinary steroid profiles were analyzed by capillary gas chromatography. Serum 17-OH-pregnenolone (17-OHPreg) and 17-OH-progesterone (17-OHP) were determined by RIA after column-chromatographic separation. One out of 9 girls with precocious pubarche and 4/33 girls with hirsutism had elevated ratios of 17-OHPreg to 17-OHP after ACTH stimulation in serum and elevated urinary excretion of 5-ene steroids under basal conditions. These patients were defined to have decreased adrenal 3 beta-HSD activity. Basal and ACTH-stimulated serum 17-OHPreg levels in patients with mild 3 beta-HSD deficiency overlapped those of healthy controls and peripubertally virilized female patients without enzyme deficiency. Post-ACTH 17-OHPreg/17-OHP ratios in serum discriminated patients with and without 3 beta-HSD deficiency using a cutoff value of 13 instead of mean + 2 SD for age-related control values (6.7 and 11.6 for girls with Tanner stage II-III and IV-V, respectively). Sums of urinary 5-ene steroids in patients with 3 beta-HSD deficiency overlapped those in patients without enzyme deficiency. Results showed that an abnormal post-ACTH serum 17-OHPreg/17-OHP ratio may not be associated with elevated urinary 5-ene steroid excretion, and vica versa. In conclusion, patients with simultaneous elevation of post-ACTH serum 17-OHPreg/17-OHP ratio and basal urinary 5-ene steroid excretion are supposed to have mild 3 beta-HSD deficiency.

17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr which have been pre-exposed to the urine of ovulated females.

Electrophysiological recordings from the olfactory epithelium have shown that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate (17,20 beta-P-sulphate; a conjugate of the oocyte-maturation-inducing steroid in teleosts) is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr. However, the olfactory epithelium of these fish only appeared to be responsive to the steroid after stimulation with the urine of ovulated female Atlantic salmon. Immature fish did not respond at any time. Stimulation with urine from immature and precocious male Atlantic salmon parr did not make the olfactory epithelium of precocious male salmon parr responsive to the steroid. 17,20 beta-P-sulphate was found in the urines of ovulated females, precocious male parr and mature male Atlantic salmon. The findings are discussed in relation to the possible role of 17,20 beta-P-sulphate in the physiology of Atlantic salmon.

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.

[Determination of urinary 17 alpha-hydroxyprogesterone excretion using ELISA--evaluation of normal subjects and patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency].

The utility of urinary 17 alpha-hydroxyprogesterone (U-17-OHP) in the diagnosis and management of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) was evaluated in 16 patients with 21-OHD. The normal values for U-17-OHP in relation to age and other physiological conditions were investigated in 96 normal subjects and 7 low birth weight infants. Levels of U-17-OHP, serum 17-OHP (S-17-OHP) and urinary free cortisol (U-FC) were simultaneously determined using enzyme-linked immunosolvent assay (ELISA) combined with fractionation by high performance liquid chromatography (HPLC). Pregnanetriol (PT) levels in the same urine specimens were determined using glass capillary gas chromatography (GC). The results were as follows: 1) Normal subjects and low birth weight infants. A significant correlation between U-17-OHP excretions corrected for body surface area (BSA) in 2-h urine specimens and S-17-OHP concentrations at the midpoint of the urine sampling period was observed in normal subjects (r = 0.768, p less than 0.01). Circadian U-17-OHP excretion in 6 adult males was synchronous with that of U-FC. Age-related changes in actual U-17-OHP excretions (Mean +/- SD ng/day) were as follows: neonates: 31.9 +/- 10.3, children aged 2 to 4 years old: 29.1 +/- 14.5, 5 to 8 years old: 68.6 +/- 29.9, 9 to 11 years old: 151.3 +/- 50.0, 12 to 15 years old: 222.7 +/- 82.0, adult males: 400.1 +/- 62.5, adult females (luteal phase): 339.1 +/- 109.7, and adult females (follicular phase): 185.6 +/- 72.3, respectively. Correlation between U-17-OHP and PT excretions in 24-h urine specimens from normal subjects greater than 2 years of age was highly significant (r = 0.871, p less than 0.01). Although U-17-OHP values were measurable in neonates, those of PT were not detectable by GC in the same specimens because of low conversion of 17-OHP to PT. 2) Cases of 21-OHD. In 4 cases of 21-OHD diagnosed in the neonatal period (aged 11 to 15 days, all were of the salt-losing type), the U-17-OHP concentration in a single urine specimen was significantly higher than that of age-matched controls, whereas the PT concentration in one case was low and therefore had no diagnostic value. In 12 patients with 21-OHD receiving suppressive therapy, correlation between S-17-OHP concentrations and 24-h U-17-OHP excretions corrected for BSA was significant (r = 0.847, p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Serum levels of 5-androstene-3 beta,17 beta-diol sulphate, 5 alpha-androstane-3 alpha, 17beta-diol sulphate and glucuronide, in late onset 21-hydroxylase deficiency.

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-DIOL-G) and unconjugated androstenedione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI) and 17 alpha-hydroxyprogester-one (17OHP) were measured by specific radioimmunoassays (RIA) in 14 women with late-onset 21-hydroxylase deficiency (LOCAH), and in normal women (n = 73). The diagnosis of LOCAH was made on the finding of a (17OHP) response level greater than 30 nmol/l following ACTH stimulation, and/or an elevation of urinary metabolites of 17OHP. Mean values for serum concentrations of all steroids measured and the free androgen index (100 X T nmol/l divided by SHBG nmol/l) were significantly elevated, and SHBG levels depressed in patients with LOCAH. These studies show that in LOCAH, in addition to the unconjugated steroids AD and T, the sulphoconjugated steroids DHEA-S, 5-ADIOL-S and 3 alpha-DIOL-S are increased, as is the glucuronide conjugate 3 alpha-DIOL-G and the index of bioavailable testosterone (FAI), and that mean SHBG levels are depressed. These data suggest that as well as AD, 5-ADIOL-S and DHEA-S may act as pro-hormones for more potent steroids (T and 5 alpha-dihydrotestosterone) in peripheral tissues, while 3 alpha-DIOL-S and 3 alpha-DIOL-G may both reflect peripheral androgen metabolism in patients with LOCAH.

Analysis of corticosteroids in urine by HPLC and thermospray LC/MS.

The method for simultaneous determination and confirmation of nine corticosteroids in urine by high-performance liquid chromatography with a diode array detector and thermospray liquid chromatography-mass spectrometry was studied. HPLC was performed on a C18 column with a gradient mobile phase system of water and acetonitrile. The calibration curve was linear from 20 ng/mL to 1.0 micrograms/mL for each corticosteroid, and the detection limit was 10 ng/mL in 5 mL of urine. The extraction recovery of each corticosteroid from the spiked urine was equal to or greater than 85% by liquid-liquid extraction (LLE) at pH 9 using diethyl ether and approximately 79% or more by solid-phase extraction (SPE) with a Sep-Pak C18 cartridge. The mass spectra obtained with the positive ion mode showed protonated molecular species [M + H]+, ammonium adduct ion [M + NH4]+, [MH-60]+, [MH-30]+, and [MH-18]+. The LC/MS detection limits ranged from 10 to 50 ng in the scan mode and from 1 to 5 ng in the selected ion monitoring (SIM) mode.

Conjugates of ovarian steroids, including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (maturation-inducing steroid), accumulate in the urine of a marine teleost (plaice; Pleuronectes platessa).

Free and conjugated 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), 17 alpha,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol) and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (3 alpha,17,21-P-5 beta) were measured by radioimmunoassay in matching blood plasma and urine samples from plaice (Pleuronectes platessa) females at several ovarian maturity stages: post-vitellogenesis (IV), final oocyte maturation (V), and ovulation (VI). Free steroids were generally low in all samples. Conjugated steroids were up to 2 orders of magnitude higher in urine than in plasma samples. Conjugated 17,20 beta-P was higher in stage V than in stage IV or VI females. Conjugated 11-deoxycortisol was higher in stage IV and V females. Conjugated 3 alpha,17,21-P-5 beta was higher in stage V and VI females. These results support earlier studies which indicated that 17,20 beta-P was the most likely maturation-inducing steroid (MIS) in plaice, and that the urine might be a vehicle for steroid pheromones synthesized by the gonads.

Urinary excretion of free progestins, androgens, and estradiol after injection of (1-24)ACTH in the Mongolian gerbil.

Injection of a "long-acting" synthetic adrenocorticotrophin [(1-24)ACTH, 20 IU/animal] into Mongolian gerbils resulted in a 3.1 fold increase of urinary free testosterone excretion over 2 days. It was accompanied by an elevation of urinary free progesterone (2.1 fold), 17-hydroxyprogesterone (2.5 fold), DHEA (2.8 fold) and androstenedione (3.0 fold) excretion. Similarly, administration of human chorionic gonadotrophin (HCG, 100 IU/animal) increased urinary excretion of free testosterone (2.3 fold), progesterone (4.1 fold), 17-hydroxyprogesterone (2.9 fold), DHEA (4.6 fold), androstenedione (5.4 fold) and of estradiol (2.9 fold). Data presented in this work show that the measurement of urinary free steroid excretion represents a reliable index for the secretory activity of the adrenal-gonadal-axis, and that it may in some aspects be more practicable than the measurement of steroid plasma levels, especially in small laboratory animals, enabling us to monitor the excretion of various steroids over longer time periods without stressing the animals by handling/or blood sampling.