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

The duplicated Y-specific amhy gene is conserved and linked to maleness in silversides of the genus Odontesthes

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.Fil: Hattori, Ricardo Shohei. Sao Paulo Fisheries Institue. Unidade de Pesquisa e Desenvolvimento de Campos do Jordão; BrasilFil: Somoza, Gustavo Manuel. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Fernandino, Juan Ignacio. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Colautti, Dario César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Miyoshi, Kaho. Tokyo University of Marine Science and Technology; JapónFil: Gong, Zhuang. Zhejiang Ocean University; ChinaFil: Yamamoto, Yoji. Tokyo University of Marine Science and Technology; JapónFil: Strüssmann, Carlos A.. Tokyo University of Marine Science and Technology; Japó

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

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae)

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self‐sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south‐east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark‐based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishesFil: Colautti, Dario César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Miranda, Leandro Andres. 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: González Castro, M.. Universidad Nacional de Mar del Plata; ArgentinaFil: Villanova, Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Strüssmann, Carlos A.. Tokyo University Of Marine Science And Technology; JapónFil: Mancini, Miguel Alberto. Universidad Nacional de Río Cuarto; ArgentinaFil: Maiztegui, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Berasain, Gustavo. Provincia de Buenos Aires. Ministerio de Asuntos Agrarios; ArgentinaFil: Hattori, Ricardo Shohei. Tokyo University of Marine Science and Technology; JapónFil: Grosman, Manuel Fabián. Universidad Nacional del Centro de la Provincia de Buenos Aires; ArgentinaFil: Sanzano, Pablo Miguel. Universidad Nacional del Centro de la Provincia de Buenos Aires; ArgentinaFil: Lozano, I.. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Llamazares Vegh, Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Salinas, V.. Universidad Nacional de Río Cuarto; ArgentinaFil: Del Ponti, O.. Universidad Nacional de La Pampa; ArgentinaFil: Fresno, P.. 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: Minotti, Priscilla Gail. Universidad Nacional de San Martín; ArgentinaFil: Yamamoto, Y.. Tokyo University Of Marine Science And Technology; JapónFil: Baigún, C.. Universidad Nacional de San Martín; Argentin

A Duplicated, Truncated amh Gene Is Involved in Male Sex Determination in an Old World Silverside

A master sex-determining gene, the Y chromosome-linked anti-Müllerian hormone (amhy) gene, has been described in two New World atheriniform species but little is known on the distribution, evolution, and function(s) of this gene in other Atheriniformes. Interestingly, amhy has been found to coexist with temperature-dependent sex determination (TSD), providing a unique opportunity to explore the interplay between genotypic and environmental sex determination. In this study, the search for an amhy homolog was extended to an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae (Atherinidae). The full sequences, including the coding and noncoding regions, of the autosomal amh (amha) and a putative amhy were obtained. The deduced Amha and Amhy proteins comprised 511 and 340 amino acids (aa), respectively. PCR analysis with genomic DNA from wild adults and from laboratory-reared juveniles revealed a high, but not complete association of ∼95% between amhy and maleness. The spatiotemporal expression of amhy and amha during gonadal sex differentiation was analyzed by qRT-PCR and in situ hybridization (ISH). amhy transcription (in amhy-positive larvae) started before and peaked during histological differentiation of the gonads whereas amha was negligible during the same period in both genotypes. These results demonstrate that the amhy, although with some structural differences in relation to the amhy of some New World atheriniforms, is strongly associated with maleness and probably important for testicular development in this Old World atheriniform. Thus, amhy is a candidate sex determination gene in cobaltcap silverside and it will be key to scrutinize the mechanism of sex determination in this species

Where the ends meet: An overview of sex determination in Atheriniform fishes

Atheriniform fishes have recently emerged as attractive models for evolutionary, ecological, and molecular/physiological studies on sex determination. Many species in this group have marked temperature-dependent sex determination (TSD) and yet many species also have a sex determinant gene that provides a strong drive for male differentiation. Thus, in these species the 2 forms of sex determination that were once considered to be mutually exclusive, environmental (ESD) and genotypic (GSD) sex determination, can coexist at environmentally relevant conditions. Here, we review the current knowledge on sex determination in atheriniform fishes with emphasis on the molecular and physiological mechanisms of ESD and GSD, the coexistence and cross-talk between these 2 mechanisms, the possibility of extragonadal transduction of environmental information and/or extragonadal onset of sex determination, and the results of field studies applying novel tools such as otolith increment analysis and molecular markers of genetic sex developed for selected New World and Old World atheriniform species. We also discuss the existence of molecular and histological mechanisms to prevent the discrepant differentiation in parts of the gonads because of ambiguous or conflicting environmental and genetic signals and particularly the possibility that the female is the default state in these species.Fil: Strüssmann, Carlos A.. Tokyo University Of Marine Science And Technology; JapónFil: Yamamoto, Yoji. Tokyo University Of Marine Science And Technology; JapónFil: Hattori, Ricardo Shohei. Tokyo University Of Marine Science And Technology; JapónFil: 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); ArgentinaFil: Somoza, Gustavo Manuel. 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