Carlos Augusto Strüssmann

A Y-linked anti-Müllerian hormone duplication takes over a critical role in sex determination

Gonadal sex determination in vertebrates generally follows a sequence of genetically programmed events. In what is seemingly becoming a pattern, all confirmed or current candidate “master” sex-determining genes reported in this group, e.g., SRY in eutherian mammals, DMY/dmrt1bY in medaka, DM-W in the African clawed frog, and DMRT1 in chicken encode transcription factors. In contrast, here we show that a male-specific, duplicated copy of the anti-Müllerian hormone (amh) is implicated in testicular development of the teleost fish Patagonian pejerrey (Odontesthes hatcheri). The gene, termed amhy because it is found in a single metacentric/submetacentric chromosome of XY individuals, is expressed much earlier than the autosomal amh (6 d after fertilization vs. 12 wk after fertilization) and is localized to presumptive Sertoli cells of XY males during testicular differentiation. Moreover, amhy knockdown in XY embryos resulted in the up-regulation of foxl2 and cyp19a1a mRNAs and the development of ovaries. These results are evidence of a functional amh duplication in vertebrates and suggest that amhy may be the master sex-determining gene in this species. If confirmed, this would be a unique instance of a hormone-related gene, a member of the TGF-β superfamily, in such a role.

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.

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17°C and 23°C. However, sex ratios become female-biased at <15°C and male-biased at 25°C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.

Effects of global warming on fish reproductive endocrine axis, with special emphasis in pejerrey Odontesthes bonariensis

The ongoing of global warming trend has led to an increase in temperature of several water bodies. Reproduction in fish, compared with other physiological processes, only occurs in a bounded temperature range; therefore, small changes in water temperature could significantly affect this process. This review provides evidence that fish reproduction may be directly affected by further global warming and that abnormal high water temperature impairs the expression of important genes throughout the brain-pituitary-gonad axis. In all fishes studied, gonads seem to be the organ more readily damaged by heat treatments through the inhibition of the gene expression and subsequent synthesis of different gonadal steroidogenic enzymes. In view of the feedback role of sex steroids upon the synthesis and release of GnRH and GtHs in fish, it is possible that the inhibition observed at brain and pituitary levels in treated fish is consequence of the sharp decrease in plasma steroids levels. Results of in vitro studies on the inhibition of pejerrey gonad aromatase expression by high temperature corroborate that ovary functions are directly disrupted by high temperature independently of the brain-pituitary axis. For the reproductive responses obtained in laboratory fish studies, it is plausible to predict changes in the timing and magnitude of reproductive activity or even the total failure of spawning season may occur in warm years, reducing annual reproductive output and affecting future populations.

Analysis of sexually dimorphic expression of genes at early gonadogenesis of pejerrey Odontesthes bonariensis using a heterologous microarray.

The process of morphological development of a differentiated gonad from an undifferentiated primordium is a very important step of gonadogenesis. Studies on sexually dimorphic gene expression are important to increase our understanding of this process and to investigate how environmental factors such as temperature can regulate gonadal development. The aim of this study was to identify putative genes involved in sex differentiation in pejerrey (Odontesthes bonariensis) reared at male- and female-producing temperatures (MPT and FPT, respectively) using a microarray heterologous from the medaka (Oryzias latipes), a closely phylogenetic species. Genes related to numerous processes presented higher expression at MPT, including those involved in muscular contraction, metabolic pathways, developmental processes, and reproduction. Genes induced by FPT were classified under the gene ontology terms of response to stimulus, transport and proteolysis. From genes selected for validation, at MPT ndrg3 expression was observed in the somatic cells, whereas pen-2 was detected in germ cells in the caudal portion of the gonads, where no apoptotic signals were observed. Finally, hsp90 was highly expressed in somatic cells of the gonads at the FPT. The results suggest that the interplay of pro-apoptotic and anti-apoptotic genes is important during the masculinization process and for the prevention of sterility following exposure to warm temperatures.

Genotypic sex determination in teleosts: Insights from the testis-determining amhy gene

The master sex-determining genes identified so far in fishes are clearly not conserved, as evidenced by several unrelated genes reported to play critical roles in sex determination. In this study, we reviewed the molecular process of sex determination in the Patagonian pejerrey Odontesthes hatcheri, an emerging model due to the recent discovery that a Y-chromosome linked, duplicated copy of the anti-Müllerian hormone gene, amhy plays a pivotal role in sex determination. A comparative analysis with other newly found sex-determining genes of teleost fish, DMY/dmrt1bY, sdY, amhr2, and gsdf(Y) is performed and alternative ideas are proposed to explain the mechanism involved in the rise of various types of non-homologous sex-determining genes.

Extrahypophyseal expression of gonadotropin subunits in pejerrey Odontesthes bonariensis and effects of high water temperatures on their expression

It has been traditionally accepted that the gonadotropins (GtHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), are synthesized and secreted only by the pituitary. However, the presence of theses hormones in extrapituitary tissues has been demonstrated in mammals, and more recently also in fish. In this study, we cloned the cDNAs and characterized the expression of FSH-β, LH-β, and glycoprotein hormone α (GPH-α) subunits from brain and gonads of male and female pejerrey Odontesthes bonariensis at different stages of gonadal maturation. In situ hybridization revealed that, in addition to their classical location in pituitary cells, the three GtH transcripts were also located in the gonads. FSH-β and GPH-α subunits were found in the cytoplasm of oogonia, previtellogenic and vitellogenic oocytes in ovaries. LH-β expression was detected in previtellogenic and vitellogenic oocytes but not in oogonia. In males, the three subunits were expressed in spermatogonia and to a lesser extent in spermatocytes. Exposure of fish to high water temperatures that impair pejerrey reproduction also induced a decrease of extrahypophyseal expression of GtH subunits.

Surrogate Production of Eggs and Sperm by Intrapapillary Transplantation of Germ Cells in Cytoablated Adult Fish

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6–39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature‐dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17°C, FPT), both sexes (25°C, MixPT), and only males (29°C, MPT). The pejerrey Fshr cDNA had 3,069u2009bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936u2009bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey. Mol. Reprod. Dev. 77: 521–532, 2010.

Gonadal differentiation and temperature effects on sex determination in the freshwater pike silverside Chirostoma estor Jordan 1880

The gonadal description of the freshwater atherinopsid pike silverside Chirostoma estor suggests that the gonads differentiate as ovaries or testes by 8xa0weeks after hatching when raised at 21xa0°C. Thermal treatments at 14xa0°C, 21xa0°C and 29xa0°C applied from fertilisation, clearly affected phenotypic sex ratios, suggesting that the thermolabile window of sex determination occurred early in development. In this study, exposure to the highest temperature led to male-biased sex ratios in this species. However, the effects of the lower and medium temperatures on the sex ratios were less clear, suggesting the presence of a mixture of genotypic and temperature-dependent sex determination (TSD) mechanisms in C. estor, similar to other atherinopsids. This work further enhances our knowledge regarding the diversity and plasticity of TSD mechanisms in atherinopsid and teleost fish.