Germ Cell Transplantation Using Sexually Competent Fish: An Approach for Rapid Propagation of Endangered and Valuable Germlines

The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25°C) treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2–13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species

Arc Statistics in Clusters: Galaxy Contribution

The frequency with which background galaxies appear as long arcs as a result of gravitational lensing by foreground clusters of galaxies has recently been found to be a very sensitive probe of cosmological models by Bartelmann et al. (1998). They have found that such arcs would be expected far less frequently than observed (by an order of magnitude) in the currently favored model for the universe, with a large cosmological constant $\Omega_\Lambda \sim 0.7$. Here we analyze whether including the effect of cluster galaxies on the likelihood of clusters to generate long-arc images of background galaxies can change the statistics. Taking into account a variety of constraints on the properties of cluster galaxies, we find that there are not enough sufficiently massive galaxies in a cluster for them to significantly enhance the cross section of clusters to generate long arcs. We find that cluster galaxies typically enhance the cross section by only $\lesssim 15$.Comment: 19 pages, 1 figure, uses aasms4.sty, submitted to Ap

Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey, a Teleost Fish with Temperature-Dependent Sex Determination?

BACKGROUND: Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals "sense" environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation. METHODOLOGY/PRINCIPAL FINDINGS: Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29 degrees C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17 degrees C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a "sexually neutral" temperature (24 degrees C) caused significant increases in the proportion of males. CONCLUSIONS/SIGNIFICANCE: Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in Medaka

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that ndrg1b is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.Fil: Arias Padilla, Luisa Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Castañeda Cortés, Diana C.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Rosa, Ivana F. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Moreno Acosta, Omar David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Hattori, Ricardo S.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Nóbrega, Rafael H.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Fernandino, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

rst Transcriptional Activity Influences kirre mRNA Concentration in the Drosophila Pupal Retina during the Final Steps of Ommatidial Patterning

Background: Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. Methodology/Principal Findings: By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. Conclusions/Significance: These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level

Chemical Tagging in the Sdss-Iii/Apogee Survey: New Identifications of Halo Stars with Globular Cluster Origins

We present new identifications of five red giant stars in the Galactic halo with chemical abundance patterns that indicate they originally formed in globular clusters. Using data from the Apache Point Observatory Galactic Evolution Experiment (APOGEE) Survey available through Sloan Digital Sky Survey (SDSS) Data Release 12, we first identify likely halo giants, and then search those for the well-known chemical tags associated with globular clusters, specifically enrichment in nitrogen and aluminum. We find that 2% of the halo giants in our sample have this chemical signature, in agreement with previous results. Following the interpretation in our previous work on this topic, this would imply that at least 13% of halo stars originally formed in globular clusters. Recent developments in the theoretical understanding of globular cluster formation raise questions about that interpretation, and we concede the possibility that these migrants represent a small fraction of the halo field. There are roughly as many stars with the chemical tags of globular clusters in the halo field as there are in globular clusters, whether or not they are accompanied by a much larger chemically untaggable population of former globular cluster stars

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Coexistence of Genotypic and Temperature-Dependent Sex Determination in Pejerrey Odontesthes bonariensis

In this study, we examined whether a homolog of the master sex-determining gene amhy of Odontesthes hatcheri is present and plays any role in testis determination of pejerrey O. bonariensis, a species otherwise known for its strong temperature-dependent sex determination (TSD). Screening of wild and laboratory-reared pejerrey for amhy revealed a high, although not complete linkage with phenotypic sex. The sex ratio in an amhy+/−/amhy−/− full sibling progeny reared during the thermolabile period of sex determination at an intermediate temperature of 25°C was 68.7% male:31.3% female; all amhy+/− fish developed as males whereas about 2/3 and 1/3 of the amhy−/− were female and male, respectively. Expression analyses revealed that amhy transcription began during embryo stage and decreased by the end of sex determination period. The autosomal amha was present in all individuals regardless of amhy genotype; its expression increased significantly from the end of the same period in the gonads of all amhy+/− but only in part of the amhy−/− animals. After histological gonadal differentiation, all gonads of amhy−/− animals with amha ISH signals were testes and those without it were ovaries. These results suggest that amhy is important for testicular differentiation in pejerrey, at least at intermediate temperatures. Thus, we hypothesize that amhy+/− animals differentiate as males by expression of either amhy alone or amhy and amha together whereas the amhy−/− probably rely solely on amha expression. These findings represent the first clear genomic evidence that genotypic and environmental sex determinants can coexist in species with marked TSD such as the pejerrey. The finding of amhy will make possible to monitor wild pejerrey populations for mismatches between genotypic and phenotypic sex and may prove instrumental for field studies addressing the effects of endocrine disruptors or abnormal temperatures on reproduction and the ecological relevance of TSD for this species

Sex determination in fish: Odontesthes spp. (Atherinopsidae) as experimental models

The sex of the offspring is the result of a unique process leading, in a binary fate, to the development of male or female characteristics. In this context, the aim of the present work is to present atherinopsid fish as excellent models to study sex determination. Here we present two atherinopsid fish from South America; one species, Odontesthes bonariensis, has a strong temperature-dependent sex determination (TSD) and the other, Odontesthes hatchery, has a genotypic sex determination (GSD). However, the results obtained in the last years from our laboratories in both species show that the boundaries between these two sex determination mechanisms within Atherinopsidae are are not as rigid as previously thought and support the notion that TSD and GSD arethe extremes of a continuum