Discovering genetic determinants for cell-to-cell adhesion in two prevalent conjugative lactococcal plasmids.

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.

Plasmid-mediated horizontal gene mobilisation: Insights from two lactococcal conjugative plasmids.

The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40- and pUC11B-mediated, high-frequency mobilisation of other, non-conjugative plasmids. The mobilisation frequencies of the smaller, non-conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non-conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40- and pUC11B-carrying host strains, was observed to confer a high-frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non-conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.

Transcriptional control of two distinct lactococcal plasmid-encoded conjugation systems.

Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry.

Delineation of a lactococcal conjugation system reveals a restriction-modification evasion system.

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Bacteriophages in the Dairy Industry: A Problem Solved?

Bacteriophages (or phages) represent one of the most persistent threats to food fermentations, particularly large-scale commercial dairy fermentations. Phages infecting lactic acid bacteria (LAB) that are used as starter cultures in dairy fermentations are well studied, and in recent years there have been significant advances in defining the driving forces of LAB-phage coevolution. The means by which different starter bacterial species defend themselves against phage predation and the chromosomal or plasmid location of the genes encoding these defense mechanisms have dictated the technological approaches for the development of robust starter cultures. In this review, we highlight recent advances in defining phage-host interactions and how phage resistance occurs in different bacterial species. Furthermore, we discuss how these insights continue to transform the dairy fermentation industry and how they also are anticipated to guide food fermentations involving plant-based alternatives in the future.

Natural Transformation in Gram-Positive Bacteria and Its Biotechnological Relevance to Lactic Acid Bacteria.

Competence refers to the specialized physiological state in which bacteria undergo transformation through the internalization of exogenous DNA in a controlled and genetically encoded process that leads to genotypic and, in many cases, phenotypic changes. Natural transformation was first described in Streptococcus pneumoniae and has since been demonstrated in numerous species, including Bacillus subtilis and Neisseria gonorrhoeae. Homologs of the genes encoding the DNA uptake machinery for natural transformation have been reported to be present in several lactic acid bacteria, including Lactobacillus spp., Streptococcus thermophilus, and Lactococcus spp. In this review, we collate current knowledge of the phenomenon of natural transformation in Gram-positive bacteria. Furthermore, we describe the mechanism of competence development and its regulation in model bacterial species. We highlight the importance and opportunities for the application of these findings in the context of bacterial starter cultures associated with food fermentations as well as current limitations in this area of research.

Biodiversity of Phages Infecting the Dairy Bacterium Streptococcus thermophilus.

Streptococcus thermophilus-infecting phages represent a major problem in the dairy fermentation industry, particularly in relation to thermophilic production systems. Consequently, numerous studies have been performed relating to the biodiversity of such phages in global dairy operations. In the current review, we provide an overview of the genetic and morphological diversity of these phages and highlight the source and extent of genetic mosaicism among phages infecting this species through comparative proteome analysis of the replication and morphogenesis modules of representative phages. The phylogeny of selected phage-encoded receptor binding proteins (RBPs) was assessed, indicating that in certain cases RBP-encoding genes have been acquired separately to the morphogenesis modules, thus highlighting the adaptability of these phages. This review further highlights the significant advances that have been made in defining emergent genetically diverse groups of these phages, while it additionally summarizes remaining knowledge gaps in this research area.

Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis.

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain.

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance.

BACKGROUND: Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. RESULTS: We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. CONCLUSION: This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.

Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain.

Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

Saccharomyces cerevisiae strains for second-generation ethanol production: from academic exploration to industrial implementation.

The recent start-up of several full-scale 'second generation' ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.

The amino-terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid.

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d-glucose and 4% d-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d-xylose over d-glucose with high d-xylose transport rates. This mutant supported efficient sugar fermentation of both d-glucose and d-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937-1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae.

BACKGROUND: Engineering of the yeast Saccharomyces cerevisiae for improved utilization of pentose sugars is vital for cost-efficient cellulosic bioethanol production. Although endogenous hexose transporters (Hxt) can be engineered into specific pentose transporters, they remain subjected to glucose-regulated protein degradation. Therefore, in the absence of glucose or when the glucose is exhausted from the medium, some Hxt proteins with high xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely xylose. RESULTS: The low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant), and Hxt5 are subjected to catabolite degradation as evidenced by a loss of GFP fused hexose transporters from the membrane upon glucose depletion. Catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. Therefore, N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination, were replaced for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely xylose concomitantly with markedly stimulated growth on xylose. The mutagenesis also improved the late stages of sugar fermentation when cells are grown on both glucose and xylose. CONCLUSIONS: Substitution of N-terminal lysine residues in the endogenous hexose transporters Hxt1 and Hxt36 that are subjected to catabolite degradation results in improved retention at the cytoplasmic membrane in the absence of glucose and causes improved xylose fermentation upon the depletion of glucose and when cells are grown in d-xylose alone.

An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

BACKGROUND: The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks. RESULTS: Evolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1-7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1-7 and GAL2 genes. CONCLUSIONS: Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.

Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

BACKGROUND: Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized. RESULTS: We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose. CONCLUSIONS: Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.

A progestin (17α,20ß-dihydroxy-4-pregnen-3-one) stimulates early stages of spermatogenesis in zebrafish.

Recently, evidence has been provided for multiple regulatory functions of progestins during the late mitotic and meiotic phases of spermatogenesis in teleost fish. For example, our previous studies suggested that 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), potentially via Sertoli cells that express the progesterone receptor (pgr) gene, can contribute to the regulation of zebrafish spermatogenesis. To further our understanding of the function of DHP at early spermatogenetic stages, we investigated in the present study the expression of genes reflecting Sertoli cell function and spermatogenic development in adult zebrafish testis after DHP treatment in tissue culture. Moreover, using an in vivo model of estrogen-mediated down-regulation of androgen production to interrupt adult spermatogenesis, we studied the effects of DHP on estrogen-interrupted spermatogenesis. In this model, DHP treatment doubled the testis weight, and all differentiating germ cell types, such as type B spermatogonia and primary spermatocytes, were abundantly present and incorporated the DNA-synthesis marker (BrdU). Accordingly, transcript levels of germ cell marker genes were up-regulated. Moreover, transcripts of two Sertoli cell-derived genes anti-müllerian hormone (amh) and gonadal soma-derived growth factor (gsdf) were up-regulated, as were three genes of the insulin-like growth factor signaling system, insulin-like growth factor 2b (igf2b), insulin-like growth factor 3 (igf3) and insulin-like growth factor 1b receptor (igf1rb). We further analyzed the relationship between these genes and DHP treatment using a primary zebrafish testis tissue culture system. In the presence of DHP, only igf1rb mRNA levels showed a significant increase among the somatic genes tested, and germ cell marker transcripts were again up-regulated. Taken together, our results show that DHP treatment induced the proliferation of early spermatogonia, their differentiation into late spermatogonia and spermatocytes as well as expression of marker genes for these germ cell stages. DHP-mediated stimulation of spermatogenesis and hence growth of spermatogenic cysts and the associated increase in Sertoli cell number may in part explain the elevated expression of Sertoli cell genes, but our data also suggest an up-regulation of the activity of the Igf signaling system.