Amniotic Membrane Restores Chronic Wound Features to Normal in a Keratinocyte TGF-ß-Chronified Cell Model.

Unsuccessful wound closure in chronic wounds can be linked to altered keratinocyte activation and their inability to re-epithelize. Suggested mechanisms driving this impairment involve unbalanced cytokine signaling. However, the molecular events leading to these aberrant responses are poorly understood. Among cytokines affecting keratinocyte responses, Transforming Growth Factor-ß (TFG-ß) is thought to have a great impact. In this study, we have used a previously characterized skin epidermal in vitro model, HaCaT cells continuously exposed to TGF-ß1, to study the wound recovery capabilities of chronified/senescent keratinocytes. In this setting, chronified keratinocytes show decreased migration and reduced activation in response to injury. Amniotic membrane (AM) has been used successfully to manage unresponsive complicated wounds. In our in vitro setting, AM treatment of chronified keratinocytes re-enabled migration in the early stages of wound healing, also promoting proliferation at later stages. Interestingly, when checking the gene expression of markers known to be altered in TGF-ß chronified cells and involved in cell cycle regulation, early migratory responses, senescence, and chronic inflammation, we discovered that AM treatment seemed to reset back to keratinocyte status. The analysis of the evolution of both the levels of keratinocyte activation marker cytokeratin 17 and the spatial-temporal expression pattern of the proliferation marker Ki-67 in human in vivo biopsy samples suggests that responses to AM recorded in TGF-ß chronified HaCaT cells would be homologous to those of resident keratinocytes in chronic wounds. All these results provide further evidence that sustained TGF-ß might play a key role in wound chronification and postulate the validity of our TGF-ß chronified HaCaT in vitro model for the study of chronic wound physiology.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Profiling Human CD55 Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation.

Xenotransplantation of pig organs receives substantial attention for being comparable to human's. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human-CD55 have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), hCD55's product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five hCD55 transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater hCD55/DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for hCD55 transcript after challenge with human and baboon sera. Moreover, hCD55 expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the hCD55 transgene functioning and performance. Moreover, the correlations found could allow us to predict hCD55/DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation.

Chronic Wound Healing by Amniotic Membrane: TGF-ß and EGF Signaling Modulation in Re-epithelialization.

The application of amniotic membrane (AM) on chronic wounds has proven very effective at resetting wound healing, particularly in re-epithelialization. Historically, several aspects of AM effect on wound healing have been evaluated using cell models. In keratinocytes, the presence of AM induces the activation of mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) pathways, together with the high expression of c-Jun, an important transcription factor for the progression of the re-epithelialization tongue. In general, the levels of transforming growth factor (TGF)-ß present in a wound are critical for the process of wound healing; they are elevated during the inflammation phase and remain high in some chronic wounds. Interestingly, the presence of AM, through epidermal growth factor (EGF) signaling, produces a fine-tuning of the TGF-ß signaling pathway that re-conducts the stalled process of wound healing. However, the complete suppression of TGF-ß signaling has proven negative for the AM stimulation of migration, suggesting that a minimal amount of TGF-ß signaling is required for proper wound healing. Regarding migration machinery, AM contributes to the dynamics of focal adhesions, producing a high turnover and thus speeding up remodeling. This is clear because proteins, such as Paxillin, are activated upon treatment with AM. On top of this, AM also produces changes in the expression of Paxillin. Although we have made great progress in understanding the effects of AM on chronic wound healing, a long way is still ahead of us to fully comprehend its effects.

Role of TGF-ß in Skin Chronic Wounds: A Keratinocyte Perspective.

Chronic wounds are characterized for their incapacity to heal within an expected time frame. Potential mechanisms driving this impairment are poorly understood and current hypotheses point to the development of an unbalanced milieu of growth factor and cytokines. Among them, TGF-ß is considered to promote the broadest spectrum of effects. Although it is known to contribute to healthy skin homeostasis, the highly context-dependent nature of TGF-ß signaling restricts the understanding of its roles in healing and wound chronification. Historically, low TGF-ß levels have been suggested as a pattern in chronic wounds. However, a revision of the available evidence in humans indicates that this could constitute a questionable argument. Thus, in chronic wounds, divergences regarding skin tissue compartments seem to be characterized by elevated TGF-ß levels only in the epidermis. Understanding how this aspect affects keratinocyte activities and their capacity to re-epithelialize might offer an opportunity to gain comprehensive knowledge of the involvement of TGF-ß in chronic wounds. In this review, we compile existing evidence on the roles played by TGF-ß during skin wound healing, with special emphasis on keratinocyte responses. Current limitations and future perspectives of TGF-ß research in chronic wounds are discussed.

Human Skin Keratinocytes on Sustained TGF-ß Stimulation Reveal Partial EMT Features and Weaken Growth Arrest Responses.

Defects in wound closure can be related to the failure of keratinocytes to re-epithelize. Potential mechanisms driving this impairment comprise unbalanced cytokine signaling, including Transforming Growth Factor-ß (TFG-ß). Although the etiologies of chronic wound development are known, the relevant molecular events are poorly understood. This lack of insight is a consequence of ethical issues, which limit the available evidence to humans. In this work, we have used an in vitro model validated for the study of epidermal physiology and function, the HaCaT cells to provide a description of the impact of sustained exposure to TGF-ß. Long term TGF-ß1 treatment led to evident changes, HaCaT cells became spindle-shaped and increased in size. This phenotype change involved conformational re-arrangements for actin filaments and E-Cadherin cell-adhesion structures. Surprisingly, the signs of consolidated epithelial-to-mesenchymal transition were absent. At the molecular level, modified gene expression and altered protein contents were found. Non-canonical TGF-ß pathway elements did not show relevant changes. However, R-Smads experienced alterations best characterized by decreased Smad3 levels. Functionally, HaCaT cells exposed to TGF-ß1 for long periods showed cell-cycle arrest. Yet, the strength of this restraint weakens the longer the treatment, as revealed when challenged by pro-mitogenic factors. The proposed setting might offer a useful framework for future research on the mechanisms driving wound chronification.

SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression.

Breast cancer remains a significant female mortality cause. It constitutes a multifactorial disease for which research on environmental factors offers little help in predicting onset or progression. The pursuit for its foundations by analyzing hormonal changes as a motive for disease development, indicates that increased exposure to estrogens associates with increased risk. A prevalent number of breast cancer cases show dependence on the increased activity of the classic nuclear estrogen receptor (ER) for cell proliferation and survival. SIRT1 is a Type III histone deacetylase which is receiving increasing attention due to its ability to perform activities over relevant non-histone proteins and transcription factors. Interestingly, concomitant SIRT1 overexpression is commonly found in ER-positive breast cancer cases. Both proteins had been shown to directly interact, in a process related to altered intracellular signaling and aberrant transcription, then promoting tumor progression. Moreover, SIRT1 activities had been also linked to estrogenic effects through interaction with the G-protein coupled membrane bound estrogen receptor (GPER). This work aims to summarize present knowledge on the interplay between SIRT1 and ER/GPER for breast cancer onset and progression. Lastly, evidences on the ability of SIRT1 to interact with TGFß signaling, a concurrent pathway significantly involved in breast cancer progression, are reported. The potential of this research field for the development of innovative strategies in the assessment of orphan breast cancer subtypes, such as triple negative breast cancer (TNBC), is discussed.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Amniotic membrane stimulates cell migration by modulating transforming growth factor-ß signalling.

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c-Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor-ß (TGF-ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c-Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF-ß-dependent Smad-activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF-ß had a synergistic effect on the AM-induced cell migration. Moreover, antagonizing TGF-ß with specific antibodies in both cell lines or knocking out TGF-ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF-ß-Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF-ß in a process dependent on mitogen-activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation-specific inhibitors, revealing a differential influence on AM-induced migration for each Smad. Thus, AM TGF-ß-Smad signalling abating is essential for optimal cell migration and wound closure.

Sirt1 interaction with active Smad2 modulates transforming growth factor-ß regulated transcription.

BACKGROUND: The simplicity of Transforming Growth Factor ß (TGFß) signaling pathway, linear and non-amplified, hardly sustains its variety of responses. This is often justified by the complex regulation showed by Smad proteins, TGFß signaling intracellular transducers, object of post-translational modifications that modulate TGFß-dependent transcription. Protein acetylation is emerging as a compelling mechanism affecting the activities of significant transcription factors, including p53, FOXO or NF-kB. Smad proteins might be controlled by this mechanism, implying that accessory factors capable of altering Smads-transcriptional complexes acetylation status and hence regulate TGFß responses remain to be identified. Understanding this interaction may help in the assessment of TGFß signaling outcomes, extending from healthy physiology to pathological conditions and cancer. METHODS: A two-hybrid chimera interacting system allowed to identify Sirt1, a NAD+ dependent type III histone deacetylase, as a novel Smad2 interactor. Several well stablished cellular models were applied to characterize this interaction by means of co-immunoprecipitation of tagged proteins and immuno-fluorescence staining. The occurrence of the interaction at Smad2 driven transcriptomic complexes was studied by means of DNA-pull-down and chromatin immunoprecipitation (ChIP), while its effects were assessed by protein over-expression and siRNA applied into a TGFß-dependent reporter gene assay. RESULTS: The interaction was confirmed and observed to be enhanced upon Smad2 acetylation, a known feature of active and nuclear Smad2. However, Sirt1 did not play a major role in Smad2 deacetylation. Anti-Sirt1 ChIP showed increased recovery of promoter regions corresponding to Smad2-driven genes after TGFß-stimulation, while its occurrence at Smad2-dependent transcriptomic complexes on DNA was found to effectively modulate gene expression. CONCLUSIONS: Sirt1 presence on Smad2-driven TGFß-dependent regulatory elements was detected and found to increase after TGFß treatment. Moreover, Sirt1 overexpression resulted in a decrease of the activity of a Smad2-driven TGFß-dependent reporter gene, while Sirt1 interference increased its activity. This would confirm the relevance of the discovered Sirt1-Smad2 interaction for the regulation of TGFß-dependent gene transcription.

Amniotic membrane promotes focal adhesion remodeling to stimulate cell migration.

During wound healing, the migration of keratinocytes onto newly restored extracellular matrix aims to reestablish continuity of the epidermis. The application of amniotic membrane (AM) to chronic, deep traumatic, non-healing wounds has proven successful at stimulating re-epithelialization. When applied on epithelial cell cultures, AM activates extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinases 1/2 (JNK1/2), with the overexpression and phosphorylation of c-Jun along the wound edge. The effect of AM on the migration of cells was investigated by studying critical proteins involved in the focal adhesions turn-over: Focal Adhesion Kinase (FAK), Paxillin and Vinculin. In Mv1Lu and HaCaT cells, validated models for cell migration and wound healing, AM affected the expression and activation of Paxillin, but did not affect Vinculin expression, both factors which integrate into focal adhesions. Moreover, AM regulation also affected FAK activity through phosphorylation. Finally, we have determined that AM regulation of focal adhesions involves both JNK and MEK MAP kinase signaling pathways. This data provides a molecular background to understand how AM regulates critical cell and molecular aspects of cell migration, organizing and directing the movement of cells by the continuous formation, maturation, and turnover of focal adhesion structures at the migration leading edge.

Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation.

During wound healing, skin function is restored by the action of several cell types that undergo differentiation, migration, proliferation and/or apoptosis. These dynamics are tightly regulated by the evolution of the extra cellular matrix (ECM) contents along the process. Pharmacologically active flavonoids have shown to exhibit useful physiological properties interesting in pathological states. Among them, oleanolic acid (OA), a pentacyclic triterpene, shows promising properties over wound healing, as increased cell migration in vitro and improved wound resolution in vivo. In this paper, we pursued to disclose the molecular mechanisms underlying those effects, by using an in vitro scratch assay in two epithelial cell lines of different linage: non-malignant mink lung epithelial cells, Mv1Lu; and human breast cancer cells, MDA-MB-231. In every case, we observed that OA clearly enhanced cell migration for in vitro scratch closure. This correlated with the stimulation of molecular pathways related to mitogen-activated protein (MAP) kinases, as ERK1,2 and Jun N-terminal kinase (JNK) 1,2 activation and c-Jun phosphorylation. Moreover, MDA-MB-231 cells treated with OA displayed an altered gene expression profile affecting transcription factor genes (c-JUN) as well as proteins involved in migration and ECM dynamics (PAI1), in line with the development of an epithelial to mesenchymal transition (EMT) status. Strikingly, upon OA treatment, we observed changes in the epidermal growth factor receptor (EGFR) subcellular localization, while interfering with its signalling completely prevented migration effects. This data provides a physiological framework supporting the notion that lipophilic plant extracts used in traditional medicine, might modulate wound healing processes in vivo through its OA contents. The molecular implications of these observations are discussed.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish.

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity.

As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca(2+)/TGF-ß-activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response.

Estrogen receptor 2b deficiency impairs the antiviral response of zebrafish.

Although several studies have demonstrated the ability of some endocrine disruptive chemicals (EDCs) to alter the physiology of zebrafish, the immune-reproductive interaction has received little attention in this species. In this study, we used a homozygous line carrying an insertion of 8 amino acids in the ligand-binding domain of the estrogen receptor 2b gene (esr2b) to further understand the role of estrogen signaling on innate immunity. Adult mutant fish showed distorted sexual ratios related with alterations in testicular morphology and supraphysiological testosterone and 17ß-estradiol (E2) levels. Immunity-wise, although esr2b mutant fish showed unaltered antibacterial responses, they were unable to mount an effective antiviral response upon viral challenge. RT-qPCR analysis demonstrated that mutant fish were able to induce the genes encoding major antiviral molecules, including Ifnphi1, Ifnphi2, Infphi3, Mxb and Mxc, and the negative feedback regulator of cytokine signaling Socs1. Notably, although esr2b mutant larvae showed a similar resistance to SVCV infection to their wild type siblings, waterborne E2 increased their viral susceptibility. Similarly, the exposure of adult wild type zebrafish to E2 also resulted in increased susceptibility to SVCV infection. Finally, the administration of recombinant Ifnphi1 hardly reversed the higher viral susceptibility of esr2b mutant zebrafish, suggesting that elevated socs1 levels impair Ifn signaling. All together, these results uncover an important role for E2 and Esr signaling in the fine-tuning of sexual hormone balance and the antiviral response of vertebrates.

Histological effects and localization of dissolved microcystins LR and LW in the mayfly Ecdyonurus angelieri Thomas (Insecta, Ephemeroptera).

The ability of microcystins (MCs), the main group of cyanotoxins, to affect the physiological processes and tissues of insects has received little attention. Fresh water dissolved MCs represent one of the main sources of cyanotoxins. In the experiment described herein, captured wild mayfly Ecdyonurus angelieri Thomas, 1968 larvae were exposed to 5 ppb of two distinct microcystins, MC-LR and MC-LW, in separate assays. Evidence of induced mortality, MCs bioaccumulation and severe histological damage affecting fat body and alterations in the tracheal system were evident. Our results reveal the acute sensitivity of the mayfly E. angelieri to MCS, which may serve as early indicators or cyanotoxins production and the quality of freshwater streams.

17α-Ethynylestradiol alters the immune response of the teleost gilthead seabream (Sparus aurata L.) both in vivo and in vitro.

There is increasing public attention concerning the effect of endocrine disruptor chemicals (EDCs) on the immune system. One important group belonging to EDCs are the environmental estrogens. Commonly found in the effluents in wastewater treatment plants, 17α-ethynylestradiol (EE(2)) which is used in contraceptive pills, is an endocrine disruptor with strong estrogenic effects. This study aims to investigate the capacity of EE(2) to modulate in vivo and in vitro the innate immune response of the gilthead seabream (Sparus aurata L.), a teleost species of great commercial value. For this purpose, adult specimens were bath-exposed to EE(2) (0, 5 and 50 ng/L) and then immunized with hemocyanin in the presence of the adjuvant aluminum. The results indicate that, after 15 days of EE(2)-exposure, the disruptor was able to inhibit in a dose-dependent manner the induction of interleukin-1ß (IL-1ß) gene expression, but did not significantly alter the specific antibody titer. To shed light on the role played by EE(2) into seabream immune response, leukocytes were exposed in vitro to several concentrations of EE(2) (0, 0.5, 5, 50 and 500 ng/ml) for 3, 16 and 48 h and the production of reactive oxygen intermediates, the phagocytic activity and the gene expression profile of these cells were analyzed. EE(2) was seen to inhibit both cellular activities and to alter the immune gene expression profile in primary macrophages. Thus, low concentrations of EE(2) increase the mRNA levels of IL-1 ß, IL-6, tumour necrosis factor α and tumour growth factor ß in non-activated macrophages. In contrast, EE(2) treatment of activated macrophages resulted in the decreased expression of pro-inflammatory genes and the increased expression of genes encoding anti-inflammatory and tissue remodeling/repair enzymes. Taken together, our results suggest that EE(2) might alter the capacity of fish to appropriately respond to infection although it does not behave as an immunosuppressor.

Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis.

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17beta-oestradiol (E(2)) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E(2) ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.