Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

Research of interrenal and chromaffin glands in barfin plaice Liopsetta pinnifasciata from Amursky Bay (Peter the Great Bay,Sea of Japan)

Studies of the barfin plaice Liopsetta pinnifasciata inhabiting Amursky Bay (Peter the Great Bay, Sea of Japan) performed in 1999–2005 showed a twofold reduction in the area of cell nuclei of the interrenal and chromaffin glands after 2001, which indicates a significant weakening of the damaging effect of the environment on fish. It is assumed that one of the main causes of the hypertrophy of both glands in barfin plaices before 2001 was the phenolic pollution of the environment. The study points to the sensitivity of the adrenal system of the barfin plaice to changes in the quality of environment.     

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 7M), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 7M). In a sauvagine radioimmunoassay, interrenal extracts displaced ¹²⁵I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.     

The Hemodynamically-Regulated Vascular Microenvironment Promotes Migration of the Steroidogenic Tissue during Its Interaction with Chromaffin Cells in the Zebrafish Embryo

Background

While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role.

Methodology and Principal Findings

In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms.

Conclusions and Significance

These results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.     

Asymmetry of the interrenal gland in two flounder species in the spawning period

Significant differences in the area of cell nuclei of the left and right lobes of the interrenal glad have been revealed within the spawning period in two flounder species—the the barfin plaice Liopsetta pinnifasciata and the Korean flounder Glyptocephalus stelleri, which testifies to the different functional activities of these lobes and, accordingly, about the functional asymmetry of the interrenal glad. Individuals with a functionally more active right lobe of the gland prevail for the barfin plaice, whereas with the Korean flounder the number of individuals with a more active left lobe is equal to the number of individuals with a more active right lobe.     

A fish with no sex: gonadal and adrenal functions partition between zebrafish NR5A1 co-orthologs

People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal–gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.     

A light- and electron-microscopic investigation of gametogenesis in Typosyllis pulchra (Berkeley and Berkeley) (Polychaeta: Syllidae)

Summary The ultrastructure of the interrenal (adrenocortical) cells of trout (Salmo fario L.) was studied after dexamethasone treatment. A procedure for identifying and isolating interrenal tissue fragments from the surrounding head kidney tissue prior to their preparation for electron microscopy is described. The peripheral plasma cortisol concentrations were measured in order to evaluate the steroidogenic activity of this tissue.The interrenal cells of control animals contain numerous mitochondria with tubular cristae, and a well developed and highly organized smooth endoplasmic reticulum (SER). The scarcity, or absence, of lipid droplets contrasts markedly with the abundance of SER.Treatment with dexamethasone results in a decrease steroidogenic activity of the interrenal cells, as indicated by the fall in plasma cortisol concentrations. The interrenal cells are small, but still contain numerous mitochondria. The SER is poorly developed, but masses of densely intermeshed smooth cisternae subsist. Lipid droplets do not accumulate in these cells; this peculiarity is discussed in connection with the virtual absence of liposomes in teleost interrenal cells.     

Morphofunctional changes of the interrenal gland and of ultrastructure of chloride cells in intact and hypophysectomized juveniles of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis> (Acipenseridae) in the course of adaptation to sea water

The impact of hypophysectomy on the state of the interrenal gland and ultrastructure of chloride cells of gills is investigated in 18-month old juveniles of starred sturgeon Acipenser stellatus in the process of its adaptation to artificial sea water (14.6‰). Hypophysectomized juveniles, similarly to intact juveniles, are able to support a relative stability of osmolarity of blood serum in the course of adaptation to sea water by transition from hyperosmotic to hypoosmotic type of osmoregulation. Changes in the investigated parameters of cells of the interrenal gland (volume of nuclei, areas of cells and of lipophilic vacuoles) occurring in the hypophysectomized and in intact specimens in the process of adaptation to sea water are generally similar, but have different dynamics. In contrast to many teleostean species, in acipenserids the hypophyectomy does not cause atrophy of the interrenal gland. The latter is incorporated in the process of regulation in the course of adaptation of fish to sea water. Hypophyecotmy results in partial destruction of organoids in some chloride cells of gills. However, when the fish are transferred to sea water, the structural changes occur in chloride cells characteristic of their transition to the excretory state. This may happen only at activation of the transport enzyme Na⁺/K⁺-ATPase of these cells by cortisol produced by the interrenal gland. In the absence of hypophysis, the functional connection of organs of the axis hypothalamus (ACTH-immunopositive cells of tuberal nucleus) → the interrenal gland → chloride cells is realized in the fish.     

Characterization of cDNAs encoding cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase in the freshwater stingray Potamotrygon motoro

The interrenal gland (adrenocortical homolog) of elasmobranchs produces a unique steroid, 1α-hydroxycorticosterone (1α-B). The synthesis of this and most other steroids requires both cholesterol side chain cleavage (CYP11A) and 3β-hydroxysteroid dehydrogenase (HSD3). To facilitate the study of elasmobranch steroidogenesis, we isolated complementary DNAs encoding CYP11A and HSD3 from the freshwater stingray Potamotrygon motoro. The P. motoro CYP11A (2182 bp total length) and HSD3 (2248 bp total length) cDNAs harbor open reading frames that encode proteins of 542 and 376 amino acids (respectively) that are similar (CYP11A: 39–61% identical; HSD3: 36–53% identical) to their homologs from other vertebrates. In molecular phylogenetic analysis, P. motoro CYP11A segregates with CYP11A proteins (and not with related CYP11B proteins) and P. motoro HSD3 segregates with steroidogenic HSD3 proteins from other fishes. CYP11A and HSD3 mRNA is found only in interrenal and gonadal tissues, indicating de novo steroidogenesis is restricted to these tissues. Because 1α-B is thought to act in the elasmobranch response to hydromineral disturbances, we examined the effect of adapting P. motoro to 10 ppt seawater on mRNAs encoding steroidogenic genes. The P. motoro response to this salinity challenge does not include interrenal hypertrophy or an increase in the levels of interrenal CYP11A, HSD3 or steroidogenic acute regulatory protein (StAR) mRNA. This study is the first to isolate full length cDNAs encoding elasmobranch CYP11A and HSD3 and the first to examine the regulation of steroidogenic genes in elasmobranch interrenal cells.     

Annual cycle of morphological changes in the interrenal gland of the flounder <Emphasis Type="Italic">Liopsetta pinnifasciata</Emphasis> from the Amur Gulf (Peter the Great Bay of the Sea of Japan)

The annual cycle of morphological changes in the interrenal gland of the flounder Liopsetta pinnifasciata, a species which had been exposed over several decades to polluted waters of the Amur Gulf (Peter the Great bay of the Sea of Japan), is investigated. The curve of annual dynamics of the volume of nuclei of interrenal cells is U-form. The maximum size of nuclei is recorded in the period of perivitellogenous growth of oocytes and the minimum size is recorded at early stages of vitellogenesis. The area of epithelium of the interrenal gland in flounders is the lowest also in the beginning of the vitellogenous growth of oocytes. It is assumed that the uncommon annual cycles of changes in the interrenal gland of this flounder living in the Amur Gulf is an index of its stressed condition.     

Interrenal of a female gymnophione amphibian,Ichthyophis beddomei,during the annual reproductive cycle

The interrenal (adrenal) of Ichthyophis beddomei lies on the ventral side of the kidney, distributed in four zones. It is separated from the renal tissue by a thin layer of connective tissue and contains both adrenocortical and chromaffin cells. Adrenocortical tissue constitutes a major portion of the interrenal islets; the chromaffin tissue consists of a few cells located at the peripheries of the interrenal islets. Histochemical studies demonstrate the presence of Δ⁵3β-hydroxysteroid dehydrogenase, 17 β-hydroxysteroid dehydrogenase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, and sudanophilic lipids in the adrenocortical tissue, suggesting its steroidogenic potential. Annual histometric and histochemical studies show two peaks of interrenal activity: (1) during the breeding phase of the reproductive cycle (January and February) and (2) during the season of heavy monsoon rains (June and July) in the postbreeding phase.     

Intracranial administration of corticotropin-like peptides increases incidence of amphibian reproductive behavior

Male rough-skinned newts (Taricha granulosa) exhibit an increase in sexual behavior (amplectic clasping) following intracerebroventricular (ICV) injection of adrenocorticotropin (ACTH 1–39), ACTH 4–10, or melanocyte-stimulating hormone (αMSH). In contrast, intraperitoneal (IP) administration of ACTH 1–39 or corticosterone significantly decreases the incidence of sexual behavior in male newts. These results suggest that a corticotropin-like peptide acts centrally to enhance sexual behavior and that systemic ACTH acts on the interrenal tissue to inhibit sexual behavior by stimulating the release of corticosterone.     

Morphology and distribution of chromaffin cells in the adrenal gland of cordylidae (Reptilia,Sauria): A comparative study

The distribution of the adrenaline and noradrenaline chromaffin cells in the adrenal glands of 10 members of the family Cordylidae have been examined. In the genus Gerrhosaurus, all the catecholamine cells lie on the surface of the adrenal gland, forming a continuous envelope of one or two layers of cells that mainly contain noradrenaline (NA). In the genus Platysaurus, the chromaffin envelope is intermittent. There are relatively large tracts of interspersed interrenal tissue containing some adrenaline cells (A). Islets of chromaffin cells are scattered between these interrenal tracts. In the genus Pseudocordylus and the genus Cordylus, the superficial chromaffin cells tend to gather into a multilayered dorsal mass, containing mainly NA cells. Inside the interrenal parenchyma, there are always numerous chromaffin islets, containing mainly A cells.     

Periodic defaunation and recovery in a subtropical epibenthic community,in relation to organic pollution

A monthly survey was carried out for two years on the epifaunal community in Tolo Harbour and Tolo Channel, Hong Kong, a subtropical embayment subjected to a gradient of organic pollution. The number of animals and species, biomass and species diversity (Shannon's function H′ and evenness J), were higher at less polluted stations in the outer Harbour and Channel than at the polluted stations in the inner Harbour. The Channel community was dominated by the crab Portunus hastatoides Fabricius and the gastropod Nassarius crematus (Hinds). Along an increasing gradient of organic pollution, N. crematus was gradually replaced by the crab Charybdis vadorum (Alcock), and a community dominated by Portunus hastatoides and Charybdis vadorum was found in the polluted inner Harbour. The abundance and dominance of predatory gastropods also showed a decrease from the Channel to the inner Harbour, reflecting changes in the trophic structure of the community in relation to pollution. No significant change in the percentage of deposit-feeders was found along the pollution gradient. Summer mortality of benthos appeared to occur regularly in inner Tolo Harbour, and was attributed to summer oxygen depletion resulting from eutrophication. The benthic community was, however, soon restored to its original state by rapid winter recolonization. It is postulated that the cyclic phenomenon of summer mortality followed by winter recovery may be a common characteristic in subtropical benthic communities subjected to a high level of organic pollution.     

The mechanism of action of adrenaline in the induction of sexual reproduction (encystation) in Opalina sudafricana parasitic in Bufo regularis

El Mofty M. M. and Sadek I. A. 1973. The mechanism of action of adrenaline in the induction of sexual reproduction (encystation) in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology3: 425–431. Adrenaline injections induced encystation in the opalinids of normal, hypophysectomized, and gonadectomized toads in the pre-breeding and post-breeding seasons. In normal and gonadectomized male and female toads, adrenaline injections may stimulate the release of ACTH of the anterior pituitary which in turn induces the secretion of androgenic steroids from the interrenal tissue. It is speculated that the breakdown products of these androgenic steroids cause the encystation of the parasites. In hypophysectomized male and female hosts, the injected adrenaline may stimulate the interstitial tissue of the testes and ovaries to secrete androgens, the breakdown products of which induce encystation of the opalinids. Adrenaline or ACTH gave negative results in vitro. Urine of male or female toad injected with adrenaline, ACTH, or testosterone propionate induced encystation in Opalina sudafricana m vitro.     

Alteration in antioxidant genes expression in some fish caught from Jeddah and Yanbu coast as a bio-indicator of oil hydrocarbons pollution

The mRNA expression profile of some antioxidant genes in skin, gills, livers, and muscles of Siganus canaliculatus and Epinephelus morio was used as an indicator of petroleum hydrocarbons pollution in six areas at Jeddah and Yanbu coasts in KSA. Total petroleum hydrocarbons (TPHs) were determined in both sea water and sediments collected from the studied areas. The mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were determined. The highest level of total petroleum hydrocarbons was observed in front of the petromine refinery at Jeddah and in S. canaliculatus when compared to E. morio. There was a significant high expression level of studied antioxidant enzymes genes in the polluted areas and the level of the expression profile tended to correlate with the degree of pollution and fish species feed habit. This was confirmed by the level of MDA which in the same way increased with an increase in the level of total hydrocarbons. In conclusion; the expression profile of antioxidant enzymes of S. canaliculatus and E. morio tissues can be used as a strong bio-indicator of total hydrocarbons pollution especially in S. canaliculatus.     

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”     

Soil TPH Concentration Estimation Using Vegetation Indices in an Oil Polluted Area of Eastern China

Assessing oil pollution using traditional field-based methods over large areas is difficult and expensive. Remote sensing technologies with good spatial and temporal coverage might provide an alternative for monitoring oil pollution by recording the spectral signals of plants growing in polluted soils. Total petroleum hydrocarbon concentrations of soils and the hyperspectral canopy reflectance were measured in wetlands dominated by reeds (Phragmites australis) around oil wells that have been producing oil for approximately 10 years in the Yellow River Delta, eastern China to evaluate the potential of vegetation indices and red edge parameters to estimate soil oil pollution. The detrimental effect of oil pollution on reed communities was confirmed by the evidence that the aboveground biomass decreased from 1076.5 g m⁻² to 5.3 g m⁻² with increasing total petroleum hydrocarbon concentrations ranging from 9.45 mg kg⁻¹ to 652 mg kg⁻¹. The modified chlorophyll absorption ratio index (MCARI) best estimated soil TPH concentration among 20 vegetation indices. The linear model involving MCARI had the highest coefficient of determination (R ² = 0.73) and accuracy of prediction (RMSE = 104.2 mg kg⁻¹). For other vegetation indices and red edge parameters, the R² and RMSE values ranged from 0.64 to 0.71 and from 120.2 mg kg⁻¹ to 106.8 mg kg⁻¹ respectively. The traditional broadband normalized difference vegetation index (NDVI), one of the broadband multispectral vegetation indices (BMVIs), produced a prediction (R ² = 0.70 and RMSE = 110.1 mg kg⁻¹) similar to that of MCARI. These results corroborated the potential of remote sensing for assessing soil oil pollution in large areas. Traditional BMVIs are still of great value in monitoring soil oil pollution when hyperspectral data are unavailable.     

Changes in the Pituitary-interrenal Axis after Gonadectomy in the Catfish,Clarias batrachus (Linn.)

The effects of gonadectomy on the cytological structure of the hypophysis and interrenal tissue of Clarias batrachus have been investigated. Forty days after gonadectomy remarkable hypertrophy of certain cyanophils, identified later as gonadotrops, has been observed followed by hyperplasia. The somatotrops also seem to undergo similar changes, and as a result the proximal pars distalis (PPD) increases enormously and encroaches upon the pars intermedia (PI). The gonadotrops undergo a gradual degranulation and become devoid of stainable cytoplasm by 90 days. No signet ring cells have been noticed at any stage. The cyanophils in the ventral region of the PPD, identified as thyrotrops, also show degranulation, but to a comparatively limited degree. The ACTH cells undergo gradual hypertrophy and degranulation. A progressive increase is noticed in the nuclear diameter. The interrenal cells are activated as indicated by the enhanced nuclear indices. It is suggested that a fall in gonadal steroids may result in ACTH release which in turn activates the interrenal cells.     

Increase of steroid-producing cells in interrenal tissue and masculinization of gonads after long-term treatment of juvenile rainbow trout with cyanoketone

Summary Cyanoketone administered via the food (0.1, 0.2 and 2 mg/g) for 8 weeks from the first feeding (day 46 after fertilization) or via the aquarium water (3 and 30 mg/100 l) for 4 weeks from day 41 does not influence the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the interstitial cells of the gonads or interrenal cells of juvenile trout in vivo. However, the number of 3-HSD-positive interrenal cells was strongly increased by administration of the highest dose of cyanoketone via both routes. These high doses furthermore affect the sex ratio in favor of males. It is concluded that interrenal tissue is responsible for the masculinizing effect of cyanoketone via increased production of androgens and/or corticosteroids. Cyanoketone at concentrations of 0.01 to 100 g/ml causes a dose-response inhibition of 3-HSD activity in the interrenal cells, when the substance is administered to an incubation medium for demonstration of this enzyme in tissue sections. The controversial in-vivo and in-vitro effects of cyanoketone on 3-HSD activity are discussed.