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Abe G.,National Institute of Genetics | Suster M.L.,Sars International Center for Marine Molecular Biology | Kawakami K.,National Institute of Genetics | Kawakami K.,Graduate University for Advanced Studies
Methods in Cell Biology | Year: 2011

The Tol2 transposable element was originally found in the genome of the Japanese medaka fish (Oryzias latipes). Tol2 contains a gene encoding an active transposase that can catalyze DNA transposition in vertebrate cells. In zebrafish, Tol2 generates genomic integrations in the germ cells very efficiently. By using the Tol2 transposition system, we have developed important genetic methods including transgenesis, gene trapping, enhancer trapping, and the Gal4-UAS system in zebrafish. In this chapter, we describe how these methods can be performed. © 2011 Elsevier Inc.

Kugler J.E.,Cornell University | Kerner P.,Cornell University | Bouquet J.-M.,Sars International Center for Marine Molecular Biology | Jiang D.,Sars International Center for Marine Molecular Biology | Di Gregorio A.,Cornell University
BMC Evolutionary Biology | Year: 2011

Background: The notochord is a defining feature of the chordate clade, and invertebrate chordates, such as tunicates, are uniquely suited for studies of this structure. Here we used a well-characterized set of 50 notochord genes known to be targets of the notochord-specific Brachyury transcription factor in one tunicate, Ciona intestinalis (Class Ascidiacea), to begin determining whether the same genetic toolkit is employed to build the notochord in another tunicate, Oikopleura dioica (Class Larvacea). We identified Oikopleura orthologs of the Ciona notochord genes, as well as lineage-specific duplicates for which we determined the phylogenetic relationships with related genes from other chordates, and we analyzed their expression patterns in Oikopleura embryos. Results: Of the 50 Ciona notochord genes that were used as a reference, only 26 had clearly identifiable orthologs in Oikopleura. Two of these conserved genes appeared to have undergone Oikopleura- and/or tunicate-specific duplications, and one was present in three copies in Oikopleura, thus bringing the number of genes to test to 30. We were able to clone and test 28 of these genes. Thirteen of the 28 Oikopleura orthologs of Ciona notochord genes showed clear expression in all or in part of the Oikopleura notochord, seven were diffusely expressed throughout the tail, six were expressed in tissues other than the notochord, while two probes did not provide a detectable signal at any of the stages analyzed. One of the notochord genes identified, Oikopleura netrin, was found to be unevenly expressed in notochord cells, in a pattern reminiscent of that previously observed for one of the Oikopleura Hox genes. Conclusions: A surprisingly high number of Ciona notochord genes do not have apparent counterparts in Oikopleura, and only a fraction of the evolutionarily conserved genes show clear notochord expression. This suggests that Ciona and Oikopleura, despite the morphological similarities of their notochords, have developed rather divergent sets of notochord genes after their split from a common tunicate ancestor. This study demonstrates that comparisons between divergent tunicates can lead to insights into the basic complement of genes sufficient for notochord development, and elucidate the constraints that control its composition. © 2011 Kugler et al.

News Article | September 7, 2016
Site: www.nature.com

All zebrafish work was performed according to standard protocols approved by The University of Chicago (ACUP #72074). No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. In situ hybridization for the hox13, Cre, and1 and shha genes were performed according to standard protocols29 after fixation in 4% paraformaldehyde overnight at 4 °C. Probes for hox13 and shha were as previously described18. Primers to clone Cre and and1 into vectors can be found in Extended Data Tables 1 and 2. Specimens were visualized on a Leica M205FA microscope. In order to create a destination vector for lineage tracing, we first designed a random sequence of 298 bp that contained a SmaI site to be used in downstream cloning. This sequence was ordered as a gBlocks fragment (IDT) and ligated into the pCR8/GW/TOPO TA cloning vector (Invitrogen). We then performed a Gateway LR reaction according to the manufacturers specifications between this entry vector and pXIG–cFos–GFP, which abolished an NcoI site present in the gateway cassette and introduced a SmaI site. We then removed the GFP gene with NcoI and BglII of the destination vector and ligated in Cre with (primers in Extended Data Table 1), using the ‘pCR8GW–Cre–pA–FRT–kan–FRT’ (kind gift of M. L. Suster, Sars International Center for Marine Molecular Biology, University of Bergen, Bergen, Norway) as a template for Cre PCR and Platinum Taq DNA polymerase High Fidelity (Invitrogen). In order to add a late phase enhancer to this vector, we first ordered four identical oligos (IDT gBlocks) of the core e16 sequence from gar, each flanked by different restriction sites. Each oligo was then ligated into pCR8/GW/TOPO, and sequentially cloned via restriction sites into a single pCR8/GW/TOPO vector. This entry vector was used a template to PCR the final Lo-e16x4 sequence and ligate it into the Cre destination vector using XhoI and SmaI, creating Lo-e16x4–Cre. The early phase enhancer Dr-CNS65x3 was cloned into the destination vector using the same strategy. Final vectors were confirmed by sequencing. A full list of sequences and primers used can be found in Extended Data Table 1. *AB zebrafish embryos were collected from natural spawning and injected according to the Tol2 system as described previously21. Transposase RNA was synthesized from the pCS2-zT2TP vector using the mMessage mMachine SP6 kit (Ambion)21. All injected embryos were raised to sexual maturity according to standard protocols. Adult F0 fish were outcrossed to wild-type *AB, and the total F1 clutch was lysed and DNA isolated at 24 hpf for genotyping (see Extended Data Table 1 for primers) to confirm germline transmission of Cre plasmids in the F0 founders. Multiple founders were identified and tested for the strongest and most consistent expression via antibody staining and in situ hybridization. One founder fish was identified as best, and all subsequent experiments were performed using offspring of this individual fish. Founder Lo-e16x4–Cre and Dr-CNS65x3–Cre fish were crossed to the Tg(ubi:Switch) line (kind gift from L. I. Zon). Briefly, this line contains a construct in which a constitutively active promoter (ubiquitin) drives expression of a loxP flanked GFP protein in all cells of the fish assayed. When Cre is introduced, the GFP gene is removed and the ubiquitin promoter is exposed to mCherry, thus permanently labelling the cell. We crossed our founder Cre fish to Tg(ubi:Switch) and fixed progeny at different time points to track cell fate. In order to detect the mCherry signal, embryos or adults were fixed overnight in 4% paraformaldehyde and subsequently processed for whole-mount antibody staining according to standard protocols30 using the following antibodies and dilutions: 1st rabbit anti-mCherry/DsRed (Clontech #632496) at 1:250, 1st mouse anti-Zns-5 (Zebrafish International Resource Center, USA) at 1:200, 2nd goat anti-rabbit Alexa Fluor 546 (Invitrogen #A11071) at 1:400, 2nd goat anti-mouse Alexa 647 (Invitrogen #A21235) at 1:400. Stained zebrafish were mounted under a glass slide and visualized using an LSM 710 confocal microscope (Organismal Biology and Anatomy, the University of Chicago). Antibody stains on adult zebrafish (90 dpf) fins were imaged on a Leica SP5 II tandem scanner AOBS Laser Scanning Confocal (the University of Chicago Integrated Light Microscopy Core Facility). Two mutations were simultaneously introduced into the first exon of each hox13 gene by CRISPR/Cas9 system as previously described in Xenopus tropicalis31. Briefly, two gRNAs that match the sequence of exon 1 of each hox13 gene were designed by ZiFiT (http://zifit.partners.org/ZiFiT/). To synthesize gRNAs, forward and reverse oligonucleotides that are unique for individual target sequences were synthesized by Integrated DNA Technologies, Inc. (IDT). Each oligonucleotide sequence can be found in Extended Data Table 2. Subsequently, each forward and reverse oligonucleotide were hybridized, and double stranded products were individually amplified by PCR with primers that include a T7 RNA promoter sequence, followed by purification by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). Each gRNA was synthesized from the purified PCR products by in vitro transcription with the MEGAscript T7 Transcription kit (Ambion). Cas9 mRNA was synthesized by mMESSAGE mMACHINE SP6 Transcription Kit according to the manufacturer’s instructions (Ambion). Two gRNAs targeting exon 1 of each hox gene were injected with Cas9 mRNA into zebrafish eggs at the one-cell stage. We injected ~2 nl of the injection solution (5 μl solution containing 1,000 ng of each gRNA and 500 ng Cas9 diluted in nuclease-free water) into the single cell of the embryo. Injected embryos were raised to adulthood, and at three months were genotyped by extracting DNA from tail clips. Briefly, zebrafish were anaesthetized by Tricaine (0.004%) and tips of the tail fin (2–3 mm2) were removed and placed in an Eppendorf tube. The tissue was lysed in standard lysis buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5% SDS, 200 μg/ml proteinase K) and DNA recovered by ethanol precipitation. Approximately 800-1,100 bp of exon 1 from each gene was amplified by PCR using the primers described in Extended Data Table 2. To determine whether mutations were present, PCR products were subjected to T7E1 (T7 endonuclease1) assay as previously reported32. After identification of mutant fish by T7E1 assay, detailed analysis of mutation patterns were performed by sequencing at the Genomics Core at the University of Chicago. Identified mutant fish were outcrossed to wild type to select frameshift mutations from mosaic mutational patterns and establish single heterozygous lines. Obtained embryos were raised to adults (~3 months), then analysed by T7E1 assay and sequenced. Among a variety of mutational patterns, fish that have frameshift mutations were used for assays as single heterozygous fish. We obtained several independent heterozygous mutant lines for each hox13 gene to compare the phenotype among different frameshift mutations. To obtain hoxa13a+/−, hoxa13b+/− double heterozygous mutant fish, each single heterozygous mutant line was crossed with the other mutant line. Offspring were analysed by T7E1 assay and sequenced after three months, and double heterozygous mutant fish were selected. To generate double homozygous hoxa13 mutant embryos and adult fish (hoxa13a−/−, hoxa13b−/−), double heterozygous fish (hoxa13a+/−, hoxa13b+/−) were crossed with each other. The ratio of each genotype from crossing heterozygous fish is summarized in Extended Data Table 4. After mutant lines were established, single (hoxa13a or hoxa13b) or double (hoxa13a, hoxa13b) mutant embryos and adult fish were genotyped by PCR for each analysis. Primer sequences for PCR are listed in Extended Data Table 2. To identify an 8 bp deletion in exon 1 of hoxa13a, the PCR product was treated by Ava1 at 37 °C for 2 h, because the 8 bp deletion produces a new Ava1 site in the PCR product (‘zebra hoxa13a_8 bp del’ primers, wild type; 231 bp, mutant; 111 bp and 119 bp). Final product size was confirmed by 3% agarose gel electrophoresis. To identify a 29 bp deletion in exon 1 of hoxa13a, the PCR product was confirmed by gel electrophoresis (‘zebra hoxa13a_29 bp del’ primers, wild type; 110 bp, mutant; 81 bp). To identify a 14 bp insertion in exon 1 of hoxa13b, the PCR product was treated by Bcc1 at 37°C for 2 h, because the 14 bp insertion produces a new Bcc1 site in the PCR product (‘zebra hoxa13b_14 bp ins’ primers, wild type; 98 bp, mutant; 53 bp + 57 bp). The final product size was confirmed by 3% agarose gel electrophoresis. The details of the mutant sequence are summarized in Extended Data Table 3a–c. Two gRNAs targeting exon 1 of hoxa13b and two gRNAs targeting exon 1 of hoxd13a were injected with Cas9 mRNA into zebrafish one-cell eggs that were obtained from crossing hoxa13a+/− and hoxa13a+/−, hoxa13b+/−, hoxd13a+/− (gRNAs were same as that were used to establish single hox13 knockout fishes and found in Extended Data Table 2). Injected eggs were raised to adult fish and genotyped by extracting DNA from tail fins. PCR products of each hox13 gene were cloned into PCRIITOPO (Invitrogen) and deep sequencing was performed (Genomic Core, the University of Chicago). At four months old, skeletal staining and CT scanning were performed to analyse the effect of triple gene deletions. The knockout ratios of each hox13 allele were calculated from the results of deep sequencing. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 72 hpf or 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Pectoral fins of wild type and hoxa13a−/−, hoxa13b−/− were detached from the embryonic body and placed horizontally on glass slides. The fins were photographed with a Leica M205FA microscope, and the fin fold length along the proximodistal axis at the centre of the fin was measured using ImageJ. The resulting data were analysed by t-test comparing the means. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Wild type and hoxa13a−/−, hoxa13b−/− embryos were stained by DAPI (1:4,000 in PBS-0.1% Triton) for 3 h and washed for 3 h by PBS−0.1% Triton. Pectoral fins were detached from the embryonic body, placed on glass slides and covered by a coverslip. The DAPI signal was detected by Zeiss LSM 710 (Organismal Biology and Anatomy, the University of Chicago). Individual nuclei were manually marked using Adobe Illustrator and the number of nuclei was counted. The data were analysed by t-test comparing the means. Skeletal staining was performed as previously described33. Briefly, fish were fixed in 10% neutral-buffered formalin overnight. After washing with milli-Q water, solutions were substituted by 70% EtOH in a stepwise fashion and then by 30% acetic acid/70% EtOH. Cartilage was stained with 0.02% alcian blue in 30% acetic acid/70% EtOH overnight. After washingwith milli-Q water, the solution was changed to a 30% saturated sodium borate solution and incubated overnight. Subsequently, specimens were immersed in 1% trypsin/30% saturated sodium borate and incubated at room temperature overnight. Following a milli-Q water wash, specimens were transferred into a 1% KOH solution containing 0.005% Alzarin Red S. The next day, specimens were washed with milli-Q water and subjected to glycerol substitution. Three replicates for each genotype were investigated. After skeletal staining, girdles and pectoral fins were manually separated from the body. Girdles and fins were stained with 0.5% PMA (phosphomolybdic acid) in milli-Q water for 16 h and followed by washes with milli-Q water. Specimens were placed into 1.5 ml Eppendorf tubes with water and kept overnight to settle in the tubes. The next day, tubes containing specimens were set and scanned with the UChicago PaleoCT scanner (GE Phoenix v/tome/x 240kv/180kv scanner) (http://luo-lab.uchicago.edu/paleoCT.html), at 50 kVp, 160 μA, no filtration, 5×-averaging, exposure timing of 500 ms per image, and a resolution of 8 μm per slice (512 μm3 per voxel). Scanned images were analysed and segmented using Amira 3D Software 6.0 (FEI). Three replicates for single and double homozygotes and five for mosaic triple knockout were investigated.

Hejnol A.,Sars International Center for Marine Molecular Biology
International Journal of Developmental Biology | Year: 2011

The Saint-Petersburg Society of Naturalists awarded the 2009 "Alexander Kowalevsky Medal" to Mark Q. Martindale, Professor of Organismal Biology at the University of Hawaii and Director of the Kewalo Marine Laboratory, Honolulu. This international award inaugurated first in 1910 was re-established only in 2001. In memory of Alexander Onufrievich Kowalevsky, it is awarded to outstanding zoologists and embryologists who have made great contributions to the field of embryology and developmental biology from an evolutionary perspective. Mark Q. Martindale has worked on a wide range of animals, mostly marine species, in contrast to many evo-devo researchers who often use a single "well-established" model organism. His work demonstrates how the insights gained by studying less "popular" animal taxa not only complement, but also significantly enrich our knowledge of the evolution of metazoan body plans and of the events that have led to the current animal diversity. © 2011 UBC Press.

Church S.H.,Brown University | Ryan J.F.,Whitney Laboratory for Marine Biosciences | Ryan J.F.,Sars International Center For Marine Molecular Biology | Dunn C.W.,Brown University
Systematic Biology | Year: 2015

The Swofford-Olsen-Waddell-Hillis (SOWH) test evaluates statistical support for incongruent phylogenetic topologies. It is commonly applied to determine if the maximum likelihood tree in a phylogenetic analysis is significantly different than an alternative hypothesis. The SOWH test compares the observed difference in log-likelihood between two topologies to a null distribution of differences in log-likelihood generated by parametric resampling. The test is a well-established phylogenetic method for topology testing, but it is sensitive to model misspecification, it is computationally burdensome to perform, and its implementation requires the investigator to make several decisions that each have the potential to affect the outcome of the test. We analyzed the effects of multiple factors using seven data sets to which the SOWH test was previously applied. These factors include a number of sample replicates, likelihood software, the introduction of gaps to simulated data, the use of distinct models of evolution for data simulation and likelihood inference, and a suggested test correction wherein an unresolved "zero-constrained" tree is used to simulate sequence data. To facilitate these analyses and future applications of the SOWH test, we wrote SOWHAT, a program that automates the SOWH test. We find that inadequate bootstrap sampling can change the outcome of the SOWH test. The results also show that using a zero-constrained tree for data simulation can result in a wider null distribution and higher p-values, but does not change the outcome of the SOWH test for most of the data sets tested here. These results will help others implement and evaluate the SOWH test and allow us to provide recommendations for future applications of the SOWH test. SOWHAT is available for download from https://github.com/josephryan/SOWHAT. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

Alie A.,University Pierre and Marie Curie | Leclere L.,University Pierre and Marie Curie | Leclere L.,Sars International Center for Marine Molecular Biology | Jager M.,University Pierre and Marie Curie | And 5 more authors.
Developmental Biology | Year: 2011

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny. © 2010 Elsevier Inc.

Radax R.,University of Vienna | Hoffmann F.,University of Bergen | Hoffmann F.,Max Planck Institute for Marine Microbiology | Rapp H.T.,University of Bergen | And 4 more authors.
Environmental Microbiology | Year: 2012

The association of archaea with marine sponges was first described 15 years ago and their role in the nitrification process in Mediterranean and tropical sponges has been suggested. Here we explore the occurrence and abundance of potential ammonia-oxidizing archaea (AOA) in four morphologically different cold-water sponges (Phakellia ventilabrum, Geodia barretti, Antho dichotoma and Tentorium semisuberites) from the sublittoral and upper bathyal zone [Correction added on 30 December 2011, after first online publication on 19 December 2011: The term 'mesopelagic zone' has been replaced.] of the Norwegian coast, and relate them to nitrification rates determined in laboratory incubations. Net nitrification rates, calculated from the sum of nitrite and nitrate release during 24h, were up to 1880nmol N cm -3 day -1; i.e. comparable with those measured in Mediterranean sponges. Furthermore, a high abundance of archaeal cells was determined by fluorescence in situ hybridizations (CARD-FISH) and quantitative PCR, targeting archaeal amoA genes (encoding the alpha subunit of ammonia monooxygenase). AmoA genes as well as amoA transcripts were either exclusively detectable from archaea or were orders of magnitudes higher in abundance than their bacterial counterparts. Phylogenetic analyses of AOA and bacterial nitrite oxidizers (genus Nitrospira) confirmed the presence of specific populations of nitrifying microorganisms in the sponge mesohyl, which either were affiliated with groups detected earlier in marine sponges or were typical inhabitants of cold- and deep-water environments. Estimated cell-specific nitrification rates for P.ventilabrum were 0.6 to 6fmol N archaeal cell -1 day -1, thus comparable with planktonic organisms. Our results identify AOA as the major drivers of nitrification in four cold-water sponges, and indicate that these archaea may be considered as a relevant factor in nitrogen cycling in ocean regions with high sponge biomass. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Hejnol A.,Sars International Center for Marine Molecular Biology
Integrative and Comparative Biology | Year: 2010

Synopsis Recent progress in reconstructing animal relationships enables us to draw a better picture of the evolution of important characters such as organ systems and developmental processes. By mapping these characters onto the phylo-genetic framework, we can detect changes that have occurred in them during evolution. The spiral mode of development is a complex of characters that is present in many lineages, such as nemerteans, annelids, mollusks, and polyclad platyhelminthes. However, some of these lineages show variations of this general program in which sub-characters are modified without changing the overlying pattern. Recent molecular phylogenies suggest that spiral cleavage was lost, or at least has deviated from its original pattern, in more lineages than was previously thought (e.g., in rotifers, gastrotrichs, bryozoans, brachiopods, and phoronids). Here, I summarize recent progress in reconstructing the spiralian tree of life and discuss its significance for our understanding of the spiral-cleavage character complex. I conclude that more detailed knowledge of the development of spiralian taxa is necessary to understand the mechanisms behind these changes, and to understand the evolutionary changes and adaptations of spiralian embryos. © The Author 2010.

Vocking O.,University of Greifswald | Vocking O.,Sars International Center for Marine Molecular Biology | Uhl G.,University of Greifswald | Michalik P.,University of Greifswald
PLoS ONE | Year: 2013

Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process. © 2013 Vöcking et al.

Skaar K.S.,University of Bergen | Nobrega R.H.,University Utrecht | Magaraki A.,University Utrecht | Olsen L.C.,University of Bergen | And 3 more authors.
Endocrinology | Year: 2011

Anti-Müllerian hormone (Amh) is in mammals known as a TGFβ type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here,wereport on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis. Copyright © 2011 by The Endocrine Society.

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