Quality assessment of compounded 17-hydroxyprogesterone caproate.

OBJECTIVE: The purpose of this study was to evaluate the quality of compounded 17-hydroxyprogesterone caproate (17-OHPC). STUDY DESIGN: Compounded 17-OHPC that was obtained from 15 compounding pharmacies throughout the United States was analyzed for potency, impurities, sterility, and pyrogen status. RESULTS: Eighteen samples were supplied by 15 compounding pharmacies. The concentration of 17-OHPC in all samples was within the specification limits, and all tested samples passed sterility and pyrogen testing. Only 1 of 18 samples was out of specification limits for impurities. CONCLUSION: Compounded 17-OHPC that was obtained from 15 pharmacies throughout the United States did not raise safety concerns when assessed for potency, sterility, pyrogen status, or impurities.

Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish.

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20ß-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen-antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml(-1). The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus.

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Piscine follicle-stimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles.

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.

Quality investigation of hydroxyprogesterone caproate active pharmaceutical ingredient and injection.

The purpose of this study was to investigate the quality of hydroxyprogesterone caproate (HPC) active pharmaceutical ingredient (API) sources that may be used by compounding pharmacies, compared to the FDA-approved source of the API; and to investigate the quality of HPC injection samples obtained from compounding pharmacies in the US, compared to the FDA-approved product (Makena(®)). Samples of API were obtained from every source confirmed to be an original manufacturer of the drug for human use, which were all companies in China that were not registered with FDA. Eight of the ten API samples (80%) did not meet the impurity specifications required by FDA for the API used in the approved product. One API sample was found to not be HPC at all; additional laboratory testing showed that it was glucose. Thirty samples of HPC injection obtained from compounding pharmacies throughout the US were also tested, and eight of these samples (27%) failed to meet the potency requirement listed in the USP monograph for HPC injection and/or the HPLC assay. Sixteen of the thirty injection samples (53%) exceeded the impurity limit set for the FDA-approved drug product. These results confirm the inconsistency of compounded HPC Injections and suggest that the risk-benefit ratio of using an unapproved compounded preparation, when an FDA-approved drug product is available, is not favorable.

Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden.

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20ß-diol-3-one (17,20ß-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.

Teleost maturation-inducing hormone, 17,20ß-dihydroxypregn-4-en-3-one, peaks after spawning in Tinca tinca.

During an eight month study of the reproductive cycle in two age groups, and in both sexes, of tench (Tinca tinca L.), it was found that plasma concentrations of the presumptive 'maturation inducing hormone (MIH)' 17,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) did not reach a peak during the spawning season, but as much as two months after spawning had ceased. The cessation of the spawning season was confirmed by histological examination of the gonads and by measurement of 11-ketotestosterone and 17ß-estradiol in the plasma of males and females, respectively. Measurements were also made of the 'alternative MIH' 17,20ß,21-trihydroxypregn-4-en-3-one in the older fish. However, this steroid did not show the same pattern as 17,20ß-P. An assessment was made of the prevalence of primary spermatocytes in the testes of post-spawned fish - to test an alternative hypothesis that 17,20ß-P might be involved in the stimulation of meiosis. However, there was no evidence for any increase in testis differentiation post-spawning. In fact the testes became increasingly undifferentiated as the autumn progressed. The role, if any, of this 'unseasonal' peak of 17,20ß-P production remains to be determined.

Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814).

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.

Studies on the influence of the structural modifications in the tracer on the immunoassay of progesterone.

The main objective of the present study was to examine the influence of different bridges in radioiodinated tracers on the assay performance of progesterone using antibodies. Three homologous and two heterologous immunoassay systems for the measurement of progesterone in human serum are described. Using an antiserum raised against progesterone-11alpha-hemisuccinate-bovine serum albumin (BSA), assays with homologous radioligands, namely progesterone-11alpha-hemisuccinate-125I-tyrosine methyl ester (TME) and progesterone-11alpha-hemisuccinate-125I-histamine, heterologous bridge radioligand, namely progesterone-11alpha-hemiphthalate-125I-TME, and a heterologous site radioligand namely progesterone-3-(O-carboxymethyl) oxime (CMO)-125I-histamine were optimized. A homologous assay system, using antiserum raised against progesterone-3-carboxymethyl oxime-BSA and progesterone-3-CMO-125I-histamine as the radioligand was also optimized to develop a radio-immunoassay (RIA) for serum progesterone. Amongst the two homologous radioligands, viz., progesterone-11alpha-hemisuccinate-125I-histamine and the corresponding TME conjugate tracer, the former yielded a standard curve with a higher slope (-0.6) as compared to the latter (-0.5). The heterologous bridge system with progesterone-11alpha-hemiphthalate-125I-TME resulted in a more sensitive assay (slope of -0.8) than the homologous tracers, whilst the heterologous site radioligand, viz., progesterone-3-CMO-125I-histamine gave the most sensitive assay (slope of -1.2). The homologous assay with antiserum against progesterone-3-CMO-BSA and progesterone-3-CMO-125I-histamine tracer gave a standard curve having a slope of -0.97. The two antibodies developed against progesterone, viz., progesterone-11alpha-hemisuccinate-BSA and progesterone-3-CMO-BSA were characterized for their titre, sensitivity, and specificity. Considering the slope, sensitivity, cross-reactivity, and the quality of tracer, the assay system using antiserum against progesterone-11alpha-hemisuccinate-BSA and progesterone-3-CMO-125I-histamine was found to be suitable for the development of RIA for serum progesterone. The bridges used in an immunogen for production of antibodies, as well as in the preparation of tracer, have a great influence on the assay characteristics.

Effects of gonadotrophin in vivo and 2-hydroxyoestradiol-17beta in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis.

An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20beta-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 microM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20beta-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 microM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20beta-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20beta-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.

Comparison of synthesis of 15 alpha-hydroxylated steroids in males of four North American lamprey species.

Recent studies have provided evidence that 15 alpha-hydroxytestosterone (15 alpha-T) and 15 alpha-hydroxyprogesterone (15 alpha-P) are produced in vitro and in vivo in adult male sea lampreys (Petromyzon marinus), and that circulatory levels increase in response to injections with gonadotropin-releasing hormone (GnRH). We examined four species from the Petromyzontidae family including silver lampreys (Ichthyomyzon unicuspis), chestnut lampreys (I. castaneus), American brook lampreys (Lethenteron appendix), and Pacific lampreys (Entosphenus tridentatus) to determine if these unusual steroids were unique to sea lampreys or a common feature in lamprey species. In vitro production was examined through incubations of testis with tritiated precursors, and 15 alpha-T and 15 alpha-P production was confirmed in all species through co-elution with standards on both high performance liquid chromatography (HPLC) and thin layer chromatography. In vivo production was proven by demonstrating that HPLC-fractionated plasma had peaks of immunoreactive 15 alpha-T and 15 alpha-P that co-eluted with standards through using previously developed radioimmunoassays for 15 alpha-T and 15 alpha-P. The possible functionality of 15 alpha-T and 15 alpha-P was further examined in silver and Pacific lampreys by investigating the effect of injection of either type of lamprey GnRH on plasma concentrations of 15 alpha-T and 15 alpha-P. Injections with exogenous GnRH did not affect circulatory levels of either steroid in silver lampreys, and only GnRH III elicited higher levels of both steroids in Pacific lampreys. The 15 alpha-hydroxylase enzyme(s) for steroids appeared to present in adult males of all species examined, but the question of whether 15 alpha-hydroxylated steroids are functional in these lamprey species, and the significance of the 15-hydroxyl group, requires further research.

Inhibitory effects of flavonoids on the reduction of progesterone to 20alpha-hydroxyprogesterone in rat liver.

The first aim of this study is to characterize the reduction of progesterone in rat liver. Progesterone was mainly reduced to 20alpha-hydroxyprogesterone in the cytosolic fraction of rat liver. The amount of 20alpha-hydroxyprogesterone formed from progesterone in the cytosolic fraction was significantly larger in the males than in the females and this enzyme reaction proceeded not only in the presence of NADPH, but also in the presence of NADH. Furthermore, we attempted to evaluate the inhibitory effects of 15 flavonoids on the NADPH-dependent reduction of progesterone to 20alpha-hydroxyprogesterone in liver cytosol of male rats. The order of the inhibitory potencies was luteolin>apigenin>quercetin>myricetin=fisetin=kaempferol. Other flavonoids exhibited lower inhibitory potencies. Energy-minimized molecular models demonstrated that a planar benzopyrone ring (A and C rings) with a coplanar phenyl ring (B ring) is a structural characteristic determining the inhibitory effects of flavonoids other than isoflavones.

15Alpha-hydroxyprogesterone in male sea lampreys, Petromyzon marinus L.

There is growing evidence that sea lampreys, Petromyzon marinus L., produce gonadal steroids differing from those of other vertebrates by possessing an additional hydroxyl group at the C15 position. Here we demonstrate that sea lamprey testes produce 15alpha-hydroxyprogesterone (15alpha-P) in vitro when incubated with tritiated progesterone, that 15alpha-P is present in the plasma of sea lampreys, and that plasma concentrations of immunoreactive (ir) 15alpha-P rise dramatically in response to injections of gonadotropin-releasing hormone (GnRH). The identity of the tritiated 15alpha-P produced in vitro was confirmed by co-elution with standard 15alpha-P on high performance liquid chromatography, co-elution with standard and acetylated 15alpha-P on thin layer chromatography, and specific binding to antibodies raised against standard 15alpha-P. The in vitro conversion was used to produce tritiated 15alpha-P label for a radioimmunoassay (RIA), which is able to detect 15alpha-P in amounts as low as 2 pg per tube. The RIA has been used to measure the plasma concentrations of 15alpha-P in males given two serial injections, 24 h apart, of either lamprey GnRH I or GnRH III (50, 100, or 200 microg/kg) or saline control, with plasma being sampled 8 and 24 h after the second injection. Plasma concentrations of ir-15alpha-P rose from < 1 to 36 ng/ml (mean of all treatments) 8 h after injection and declined within 24 h. This is the first time that an RIA has detected such high steroid concentrations in lampreys. This finding is suggestive of a role for 15alpha-P in control of reproduction in the sea lamprey.

Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model.

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.

High performance liquid chromatographic separation of steroids from ovarian follicles of fresh water perch Anabas testudineus: identification and characterization of the maturation-inducing hormone.

Accurate separation and identification of steroids from the postvitellogenic ovarian follicles of Indian climbing perch Anabas testudineus was performed by high-performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA). The steroids from such follicles, incubated in Cortland's saline with or without homologous fish pituitary extract (FPE), were extracted with dichloromethane and separated on a micro Bondapak C(18) column. Identification of the HPLC fractions was further confirmed by thin layer chromatography. As HPLC peaks for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T) were close, clear separation of these steroids and accurate measurement of their quantities was achieved by RIA of HPLC fractions using specific antibodies. Altogether, nine eluted fractions in the FPE-untreated and ten in FPE-treated samples were obtained. Of these, six were identified as: 5 beta-pregnan-3 alpha,17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P); DHP; T; 17 alpha-hydroxyprogesterone (17 alpha-P(4)); progesterone (P(4)); and androstenedione (AD). Three fractions from untreated and four from FPE-treated samples, however, remained unidentified. Of all the HPLC fractions examined for their relative maturational inducing (MI) potency on full grown (postvitellogenic) ovarian follicles of perch, the fraction identified as DHP was found to be the most effective inducer of germinal vesicle breakdown (GVBD) both at low and high concentrations. Fractions identified as 5 beta-3 alpha, 17 alpha, 20 beta-P and 17alpha-P(4) could induce only 32% and 20% GVBD at their highest concentration, while none of the unidentified fractions showed any MI activity. FPE caused increased production of DHP, testosterone, and 5 beta-3 alpha, 17 alpha, 20 beta-P. The qualitative differences between the fractions obtained from FPE-treated samples and those from FPE-untreated samples were only the appearance of a new polar metabolite of unknown function. The present study showed that, as a single steroid, DHP was the most potent MIH for A. testudineus.

Plasma steroids in mature common dentex (Dentex dentex) stimulated with a gonadotropin-releasing hormone agonist.

The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.

Effect of 17 beta-estradiol, testosterone, and 11-ketotestosterone on 17,20 beta-dihydroxy-4-pregnen-3-one production in the rainbow trout testis.

The principal hormone related to spermiation in Oncorhynchus mykiss is 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP). In the present study we analyzed the possible paracrine/autocrine effects of three other testicular steroids (17 beta-estradiol, 11-ketotestosterone, and testosterone) on the synthesis and secretion of this progestin in male rainbow trout. Pieces of testis at various stages of spermatogenesis were incubated for 24 or 48 hr with one of these steroids (5 to 800 ng ml-1) either alone or with the gonadotropin GtH II. Following incubation, 17,20 beta OHP was measured by RIA in the culture media. In vitro, only 17 beta-estradiol (E2) decreased 17,20 beta OHP secretion repeatedly and significantly when doses higher than or equal to 50 ng ml-1 were used. This effect was observed mainly at the spermiating stage and under gonadotropic stimulation. In turn, E2 did not seem to modify the testicular capacity to convert 17-hydroxyprogesterone into 17,20 beta OHP. In vivo, the circulating levels of E2 decreased at the beginning of spermiation, concomitantly with an increase of 17,20 beta OHP in plasma. These in vitro and in vivo data suggest a possible role for E2 in the regulation of 17,20 beta OHP secretion by testes, in particular during the spermiating period.

15 beta-hydroxylated steroids may be diagnostically misleading in confirming congenital adrenal hyperplasia suspected by a newborn screening programme.

UNLABELLED: In a Swiss screening programme for detection of congenital adrenal hyperplasia (CAH), 27 of over 120,000 newborns examined from 1992 to 1994 were further studied because of persistingly high 17 alpha hydroxyprogesterone (17OHP). Out of 27, 11 were later confirmed to have CAH by specific gas chromatography of urinary steroids and ACTH test at age 3-4 months. Of 27, 11 were born at term (7 confirmed 21-hydroxylase deficiency, one 11 beta-hydroxylase deficiency). Out of 27, 16 were preterm newborns. Of them, only 2 were confirmed to have CAH (one 21-, one 11 beta-hydroxylase deficiency). In 3 cases with high 17OHP, but later not confirmed CAH, what appeared to be a pregnanetriolone peak in the gas chromatograms was shown to be 3 beta, 15 beta, 17 alpha-pregnenetriol. This compound may be misleading in confirming the diagnosis of CAH. 15 beta-Hydroxylated compounds occur in fetuses, neonates, and amniotic fluid. Since human tissues do not have 15 beta-hydroxylating capacity, their origin is unclear. However, since some bacteria (Bacillus megatherium) and mycelial fungi (fusaria) are known to hydroxylate steroids in position 15 beta, it is likely that this compound is formed by micro-organisms in the enterohepatic circulation of newborns or their mothers. CONCLUSION: For the confirmation of the diagnosis of CAH in cases suspected by screening, later ACTH stimulation and specific steroid analysis are necessary.

Steroids in human intrauterine fluids of early pregnancy.

OBJECTIVE: Little is known of the hormone environment of the developing early human embryo. We have therefore measured selected steroids in the intrauterine fluids of early pregnancy. DESIGN: Measurement of progesterone, 17 alpha-hydroxyprogesterone, oestradiol-17 beta, testosterone, androstenedione, cortisol and dehydroepiandrosterone sulphate in matched samples of coelomic fluid, amniotic fluid and maternal serum collected before pregnancy termination from 12 women between 8 and 12 weeks gestation. RESULTS: Mean concentrations of progesterone, oestradiol-17 beta and 17 alpha-hydroxyprogesterone in coelomic fluid were respectively 20, 6 and 2 times greater than in maternal serum and 8, 13 and 2.6 times those in amniotic fluid. Concentrations of testosterone and androstenedione were highest in maternal serum and lowest in amniotic fluid. Cortisol and dehydroepiandrosterone sulphate were found in intrauterine fluids only at the limit of detection but in normal concentrations in maternal serum. CONCLUSIONS: Coelomic fluid contains relatively high concentrations of progesterone, oestradiol-17 beta and 17 alpha-hydroxyprogesterone which may be synthesized locally. Amniotic fluid contains lower concentrations of steroids (other than progesterone) than are found in coelomic fluid or maternal serum. Free diffusion of steroids across the amnion appears limited. This may constitute a mechanism to protect the embryo from unwanted exposure to biologically active steroids.

Efecto de la gonadectomía prepuberal en el síndrome de resistencia parcial a los andrógenos / Effect of prepuberal gonadectomy on partial Resitance to Androgens Syndrome

El síndrome de resitencia a los andrógenos es un padecimiento dominante y recesivo ligado a X con manifestaciones clínicas heterogéneas. Se presenta el caso de un paciente de 17 años con amenorrea primaria y abigüedad genital. Tenía el antecedente de hernioplastía inguinal bilateral y probable gonadectomía a los 14 meses de edad. Su cariotipo fue masculino 46, XY y los exámenes hormonales mostraron la existencia de hipogonadismo hipergonadotrópicos. Se descartó la existencia de hiperplasia suprearrenal congénita (deficiencia de 21 hidroxilasa). En el síndrome de resistencia parcial a los andrógenos, la gonadectomía prepuberal evita la virilización progresiva de los genitales externos. Sin embargo, es necesario administrar terapia estrogénica de reemplazo