Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

Abstract Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

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Faculty Opinions recommendation of BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1006519.81856 ◽

2002 ◽

Author(s):

Anjana Rao

Keyword(s):

Zinc Finger ◽

Germ Line ◽

Specific Protein ◽

Epigenetic Reprogramming ◽

Zinc Finger Domain ◽

Male Germ Line ◽

Insulator Protein

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Critically endangered fish species of Turkish Seas (Mediterranean and Aegean): Longfin gurnard, Chelidonichthys obscurus (Walbaum, 1792)

Natural and Engineering Sciences ◽

10.28978/nesciences.286039 ◽

2016 ◽

Vol 1 (1) ◽

pp. 11-15 ◽

Cited By ~ 1

Author(s):

Cemal Turan ◽

Deniz Ergüden ◽

Mevlüt Gürlek

Keyword(s):

Fish Species ◽

Critically Endangered ◽

Endangered Fish

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A Novel Chromodomain Protein, Pdd3p, Associates with Internal Eliminated Sequences during Macronuclear Development inTetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.4128-4134.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 4128-4134 ◽

Cited By ~ 47

Author(s):

Mikhail A. Nikiforov ◽

Martin A. Gorovsky ◽

C. David Allis

Keyword(s):

De Novo ◽

Germ Line ◽

Chromosome Breakage ◽

Specific Protein ◽

Developmentally Regulated ◽

Dna Elimination ◽

Internal Eliminated Sequences ◽

Subsequent Elimination ◽

Rearrangement Processes

ABSTRACT Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

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Food resource variability in an Australian dryland river: evidence from the diet of two generalist native fish species

Marine and Freshwater Research ◽

10.1071/mf07125 ◽

2008 ◽

Vol 59 (2) ◽

pp. 137 ◽

Cited By ~ 36

Author(s):

David Sternberg ◽

Stephen Balcombe ◽

Jonathan Marshall ◽

Jaye Lobegeiger

Keyword(s):

Fish Species ◽

Food Resource ◽

High Energy ◽

Filamentous Algae ◽

Boom And Bust ◽

Dryland River ◽

High Concentrations ◽

Terrestrial Insects ◽

Australian Fish ◽

Resource Variability

To examine how food resource availability links with natural variation in primary productivity in the Moonie River, south-west Queensland, the diets of two native Australian fish species (Nematalosa erebi and Macquaria ambigua) were examined from fifteen waterholes in February, May and September 2006. N. erebi diets reflected strong ‘boom and bust’ patterns of food consumption, with high concentrations of benthic (non-filamentous) algae during boom (flow) times, moving to higher concentrations of filamentous algae and detritus during bust (no flow) periods. M. ambigua diets were primarily dominated by aquatic insects in all sampling periods. Although there was no clear ‘boom to bust’ pattern in relation to flow, M. ambigua secondary prey consumption revealed a compensatory switch between high energy prey (crustaceans) during more productive periods with terrestrial insects during less productive periods. The ability of both species of fish to switch from high to low concentrations of food quality under a variable environmental background allows them to persist through both high productive and low productive periods. This interaction between native biota and variable ‘boom’ and ‘bust’ conditions, and how changes to the natural hydrology will affect it is an important consideration of any future water resource development plans.

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The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation.

Genetics ◽

10.1093/genetics/131.2.345 ◽

1992 ◽

Vol 131 (2) ◽

pp. 345-352 ◽

Cited By ~ 17

Author(s):

J C Eissenberg ◽

G D Morris ◽

G Reuter ◽

T Hartnett

Keyword(s):

Germ Line ◽

Position Effect ◽

Inducible Promoter ◽

Specific Protein ◽

P Element ◽

Position Effect Variegation ◽

Chromosomal Protein ◽

Gene Encoding ◽

Position Effects ◽

Effect Variegation

Abstract Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.

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Study of Fecundity of Ompok pabo (Hamilton, 1822) an Endangered Fish Species of Tripua, India

Journal of Fisheries & Livestock Production ◽

10.4172/2332-2608.1000153 ◽

2015 ◽

Vol 03 (04) ◽

Author(s):

Bhattacharya P ◽

Banik S

Keyword(s):

Fish Species ◽

Endangered Fish

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New directions in assisted breeding techniques for fish conservation

Reproduction Fertility and Development ◽

10.1071/rd19457 ◽

2020 ◽

Vol 32 (9) ◽

pp. 807 ◽

Cited By ~ 2

Author(s):

Nicola Rivers ◽

Jonathan Daly ◽

Peter Temple-Smith

Keyword(s):

Endangered Species ◽

Germ Cell ◽

Cell Transplantation ◽

Fish Species ◽

Captive Breeding ◽

Germ Line ◽

Management Strategies ◽

Germ Cell Transplantation ◽

Spawning Periods ◽

Breeding Techniques

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

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Freshwater Fish Species of the Oorlogskloof River, Northern Cape Province, South Africa

Our Nature ◽

10.3126/on.v10i1.7746 ◽

2013 ◽

Vol 10 (1) ◽

pp. 8-16

Author(s):

P.P. Ramollo ◽

M. Schumann ◽

W.A.J. Pretorius

Keyword(s):

South Africa ◽

Freshwater Fish ◽

Relative Abundance ◽

Fish Species ◽

Freshwater Fishes ◽

Endangered Fish ◽

Freshwater Fish Species ◽

Northern Cape

The freshwater fish of Oorlogskloof River were sampled in March 2010. The study aimed to determine the distribution and relative abundance of freshwater fish in the Oorlogskloof River. A total of 4643individuals represented by five fish species belonging to two families were sampled. The Barbus anoplus was only sampled in the upper reaches of the Oorlogskloof River gorge while endangered Labeobarbus capensis appeared downstream in the Oorlogskloof River. Barbus serra dominated the fish species in the system. The invasion of Tilapia sparmanni in this system was confirmed during the survey andthe species appeared to be widespread throughout the system. At this stage it does not appear to be posing a serious threat to the endangered fish species. The Oorlogskloof River can be considered as a potential refuge site for the conservation of some endemic and threatened freshwater fishes of South Africa.DOI: http://dx.doi.org/10.3126/on.v10i1.7746

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192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab192 ◽

2014 ◽

Vol 26 (1) ◽

pp. 210

Author(s):

M. Nowak-Imialek ◽

N. Lachmann ◽

D. Herrmann ◽

F. Jacob ◽

H. Niemann

Keyword(s):

Stem Cells ◽

Cell Population ◽

Germ Cells ◽

Germ Line ◽

Cell Mass ◽

Testicular Tissue ◽

Egfp Expression ◽

Cell Populations ◽

Marker Genes ◽

Transgenic Pigs

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

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