Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10 days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30 days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

High temperature stress response is not sexually dimorphic at the whole-body level and is dependent on androgens to induce sex reversal.

The understanding of the molecular and endocrine mechanisms behind environmentally-induced sex reversal in fish is of great importance in the context of predicting the potential effects of climate change, especially increasing temperature. Here, we demonstrate the global effects of high temperature on genome-wide transcription in medaka (Oryzias latipes) during early development. Interestingly, data analysis did not show sexual dimorphic changes, demonstrating that thermal stress is not dependent on genotypic sex. Additionally, our results revealed significant changes in several pathways under high temperature, such as stress response from brain, steroid biosynthesis, epigenetic mechanisms, and thyroid hormone biosynthesis, among others. These microarray data raised the question of what the exact molecular and hormonal mechanisms of action are for female-to-male sex reversal under high temperatures in fish. Complementary gene expression analysis revealed that androgen-related genes increase in females (XX) experiencing high water temperature. To test the involvement of androgens in thermal-induced sex reversal, an androgen antagonist was used to treat XX medaka under a high-temperature setup. Data clearly demonstrated failure of female-to-male sex reversal when androgen action is inhibited, corroborating the importance of androgens in environmentally-induced sex reversal.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.