Full Length ArticleEffects of short-term exposure to genistein and overfeeding diet on the neural and retinal progenitor competence of adult zebrafish (Danio rerio)
Graphical abstract
Introduction
Neurogenesis is a complex and highly regulated process resulting in the production of new neurons (Otteson and Hitchcock, 2003; Zupanc and Zupanc, 2006; Zupanc, 2006). Particularly interesting from a comparative point of view to mammals, teleost fish exhibit remarkable adult neurogenesis and neuronal regeneration capacity (Hitchcock et al., 2004; Otteson and Hitchcock, 2003; Zupanc and Zupanc, 2006; Zupanc, 2006). In fact, in contrast to mammals, the brain of adult zebrafish displays an important number of neurogenic niches throughout (Dorsemans et al., 2017; Ghaddar et al., 2020). Such neurogenic capacities in this species are related to the maintenance of several neural stem cells (radial glial cells) during adulthood and further committed progenitors (neuroblasts) (Dorsemans et al., 2017; Ghaddar et al., 2020; Schmidt et al., 2013). However, this process might be affected, in both the adult and embryonic hypothalamus, by nutritional cues and metabolic disorders such as obesity. In recent years, several publications have reported that obesity has been strongly associated, in zebrafish and other species, with an impairment of hippocampal synaptic plasticity (Lindqvist et al., 2006; Molteni et al., 2002), alterations of cognitive abilities such as learning and memory (Stranahan et al., 2008), and ocular diseases including age-related cataract and maculopathy, glaucoma, and diabetic retinopathy (Castro-Sánchez et al., 2019; Centanin et al., 2011; Chow et al., 2009; Guo and Rahmouni, 2011; Huang et al., 2012; Otteson and Hitchcock, 2003). Moreover, several studies have provided evidence that obesity negatively affects neurogenesis, decreasing the proliferation capacity of progenitor neuronal cells (Ghaddar et al., 2020; Park et al., 2010; Tozuka et al., 2009).
Taking into account this emergent issue, soy and phytoestrogens have received increasing attention due to the health benefits associated with their consumption, leading to them being considered potential therapeutic compounds for several metabolic disorders (Cederroth and Nef, 2009; Tuli et al., 2019), including obesity (Behloul and Wu, 2012; Rietjens et al., 2017; Shen et al., 2019; Velasquez and Bhathena, 2007; Xin et al., 2019). Soybeans contain isoflavones, mainly daidzein and genistein, a class of phytoestrogens structurally similar to estradiol and that mimic its effects (Cederroth and Nef, 2009; Vitale et al., 2013). As a phytoestrogen, genistein can bind to estrogen receptors and activate estrogen receptor-dependent pathways, which exert pleiotropic effects on many target organs such as the nervous system (Diotel et al., 2013). Indeed, several reports have provided evidence that soybean has a neuroprotective effect on both the brain (Park et al., 2016; Soltani et al., 2015; Uddin and Kabir, 2019) and retina (Elgayar et al., 2015; Hayashi et al., 1997; Ibrahim et al., 2010) beyond its health benefits on the prevention of obesity (Rietjens et al., 2017; Shen et al., 2019; Velasquez and Bhathena, 2007; Xin et al., 2019).
Within this conceptual framework, zebrafish (Danio rerio) emerged as an interesting and recognized model for studying overweight/obesity (Faillaci et al., 2018; Ghaddar et al., 2020; Landgraf et al., 2017; Montalbano et al., 2019; Oka et al., 2010; Tainaka et al., 2011; Zang et al., 2018) and related metabolic disorders like adult neurogenesis (Dorsemans et al., 2017; Ghaddar et al., 2020; Schmidt et al., 2013) or retinogenesis alterations (Castro-Sánchez et al., 2019; Centanin et al., 2011; Chow et al., 2009; Guo and Rahmouni, 2011; Huang et al., 2012; Otteson and Hitchcock, 2003). Aiming to improve the understanding of how obesity and genistein trigger an impact on adult zebrafish neural and retinal progenitor competence, fish were exposed to genistein (GEN - 2 μg L−1) alone or combined with two dietary groups (control and overfeed - OFD) up to 9 weeks. Zebrafish were fed once per day with Artemia sp. in the control and GEN (2% of BW, control diet), and three times per day in the OFD and OFD + GEN groups (12% BW, overfeeding diet). In this sense, gene expression analysis was performed by qRT-PCR and stereological analysis was based on immunohistochemistry. The proliferating cell nuclear antigen (PCNA) and sox transcription factor-2 (Sox-2) were evaluated in the zebrafish brain and retina to assess the impact of obesity and/or genistein on both cell proliferation and the presence of neuronal progenitor cells, respectively. On the other hand, in an attempt to explore the effects of obesity on zebrafish, biometric parameters were assessed, and the liver was used as an indicator of lipid accumulation rate, with peroxisome proliferator-activated receptor gamma (PPAR-γ), a regulator of lipid metabolism, also being analyzed through immunohistochemistry.
Section snippets
Chemicals
Genistein (GE) (≥98% purity) was purchased from Sigma (St. Louis, MO, USA) and diluted in ethanol. Working solutions were prepared in MilliQ water and all, including the control water, had an insignificant final ethanol volume (0.000002%) in the experimental system.
Zebrafish maintenance
Adult zebrafish (Danio rerio) AB strain were maintained under a controlled temperature (28 ± 1 °C) and photoperiod 14:10 h (light: dark cycle) in dechlorinated and aerated water in a recirculation system with both mechanical and
Biometric parameters
Zebrafish body weights (BW) and total lengths (TL) were assessed (Fig. 2), and body mass index (BMI) was calculated (Table 1). The one-way factorial ANOVA of BMI revealed significant differences between groups (F(3,11) = 26.681 p < 0.001). Firstly, overfeeding diet (12% BW) induced obesity in zebrafish compared to the control diet (2% BW) (Fig. 2 and Table 1). In fact, zebrafish exposed to OFD and OFD + GEN showed a significant increase in body mass index (BMI, Table 1) (p < 0.05). At week 0,
Discussion
Over the last few years, zebrafish (Danio rerio) emerged as an interesting and recognized model for studying overweight/obesity (Faillaci et al., 2018; Ghaddar et al., 2020; Landgraf et al., 2017; Montalbano et al., 2019; Oka et al., 2010; Tainaka et al., 2011; Zang et al., 2018). Additionally, while Artemia nauplii is considered an aquaculture live food with a high nutritional content, previous studies reported that zebrafish overfed with Artemia, as high-fat diet, induced obesity (Landgraf et
Conclusion
In summary, to the best of our knowledge, this is the first study to evaluate the interaction between overfeeding diet, genistein and t effects on zebrafish neural and retinal progenitor competence. The findings suggest that phytoestrogens can represent a resilience strategy, aiming to improve the use of these natural bioactive compounds as phytopharmaceuticals to counteract the negative effects of obesity. From a biological perspective, our results show that zebrafish overfed with an
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020.
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These authors contributed equally to this work.