PG-BGA Campus de Botucatu Instituto de Biociências “Identification of germ cell-intrinsic players (GDNF and long non-coding RNAs) involved in the spermatogonial stem cell fate of adult zebrafish” LUCAS BENITES DORETTO TESE APRESENTADA AO INSTITUTO DE BIOCIÊNCIAS, CAMPUS DE BOTUCATU, UNESP, PARA OBTENÇÃO DO TÍTULO DE DOUTOR NO PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA GERAL E APLICADA, ÁREA DE CONCENTRAÇÃO BIOLOGIA CELULAR ESTRUTURAL E FUNCIONAL. PROF. DR. RAFAEL HENRIQUE NÓBREGA BOTUCATU – SP 2023 PG-BGA 1 Campus de Botucatu Instituto de Biociências UNIVERSIDADE ESTADUAL PAULISTA “Julio de Mesquita Filho” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU “Identification of germ cell-intrinsic players (GDNF and long non-coding RNAs) involved in the spermatogonial stem cell fate of adult zebrafish” LUCAS BENITES DORETTO TESE APRESENTADA AO INSTITUTO DE BIOCIÊNCIAS, CAMPUS DE BOTUCATU, UNESP, PARA OBTENÇÃO DO TÍTULO DE DOUTOR NO PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA GERAL E APLICADA, ÁREA DE CONCENTRAÇÃO BIOLOGIA CELULAR ESTRUTURAL E FUNCIONAL. PROF. DR. RAFAEL HENRIQUE NÓBREGA PG-BGA 2 Campus de Botucatu Instituto de Biociências Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências, Botucatu. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. D695i Doretto, Lucas Benites Identification of germ cell-intrinsic players (gdnf and long non-coding rnas) involved in the spermatogonial stem cell fate of adult zebrafish / Lucas Benites Doretto. -- , 2023 92 p. : il., tabs. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências, Botucatu, Orientador: Rafael Henrique Nóbrega 1. Biologia Geral. 2. Biologia Celular e Estrutural. 3. Espermatogênese. 4. Células Tronco Espermatogoniais. 5. Zebrafish. I. Título. PG-BGA 3 Campus de Botucatu Instituto de Biociências "Trabalhadores do Mundo" de Bertolt Brecht: Trabalhadores do Mundo, uni-vos! Lutai por um mundo novo e melhor! A razão e a ciência iluminarão o caminho, Se seguirdes a bandeira vermelha da revolução. Trabalhadores do Mundo, uni-vos! Lutai por um mundo novo e melhor! Sem vós não há pão, não há vida, Não há amor, nem liberdade. Trabalhadores do Mundo, uni-vos! Lutai por um mundo novo e melhor! A morte há de vir um dia, E nossas cinzas hão de unir-se às estrelas. Mas o triunfo da nossa causa Sobreviverá à própria morte. Trabalhadores do Mundo, uni-vos! Lutai por um mundo novo e melhor! PG-BGA 4 Campus de Botucatu Instituto de Biociências AGRADECIMENTOS Como estudante de pós-graduação, sou plenamente consciente das inúmeras dificuldades que enfrentamos na vida acadêmica. Nossas jornadas são marcadas por longas horas de trabalho, bolsas de estudo escassas, baixa remuneração, falta de reconhecimento, desafios relacionados à saúde mental e a escassez de oportunidades no mercado de trabalho. No entanto, é crucial ressaltar que cada um de vocês desempenha um papel fundamental no avanço da ciência, na transformação da sociedade e no fortalecimento de nossa soberania nacional. Neste momento, gostaria de expressar minha sincera gratidão a todos os meus colegas de trabalho, que têm sido uma fonte constante de apoio, colaboração e inspiração. Suas contribuições e dedicação são inestimáveis, e tenho a sorte de poder compartilhar essa jornada com pessoas tão talentosas e comprometidas. Gostaria de fazer um agradecimento especial à minha amada esposa, que tem sido meu pilar de força e apoio incansável durante todo esse percurso. Seu amor, paciência e encorajamento têm sido fundamentais para minha motivação e bem-estar emocional. Você é verdadeiramente uma fonte de inspiração para mim, e sou grato por ter você ao meu lado. Além disso, expresso minha profunda gratidão ao meu orientador, cuja orientação sábia e experiente tem sido essencial para o meu crescimento acadêmico e profissional. Sua expertise, incentivo e disponibilidade para compartilhar conhecimento têm sido inestimáveis para minha formação como cientista. Sou grato pela confiança que você depositou em mim e pelo apoio constante ao longo dessa jornada. Gostaríamos de expressar nosso sincero agradecimento à Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo apoio financeiro concedido no âmbito do processo 2019/05643-3. Em resumo, agradeço a todos os meus colegas de trabalho, minha esposa e meu orientador por estarem presentes e por seu comprometimento em superar os desafios enfrentados na vida acadêmica. Juntos, estamos construindo um futuro melhor através da ciência e da educação. Seu apoio e dedicação são inestimáveis, e sou verdadeiramente grato por tê-los em minha vida. Atenciosamente, Um colega cientista. PG-BGA 5 Campus de Botucatu Instituto de Biociências Summary 1. General introduction …………………………………………………………………. 06 1.1 Spermatogenesis and Spermatogonial Stem cells …………………………………. 06 1.2 Endocrine and Paracrine Regulation of SSCs ………………………………… 07 1.3 lncRNAs as non-coding regulators of adult stem cells ………………………… 08 2. General Objective ……………………………………………………………………... 13 3. Chapter 1-Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners... 14 4. Chapter 2 - Identification of long non-coding RNAs (lncRNAs) in germ and somatic cells of the zebrafish testis (Danio rerio) ……………………………………………… 54 5. Conclusion .................................................................................................................... 82 PG-BGA Campus de Botucatu Instituto de Biociências Apoio financeiro Processo nº 2019/05643-3, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). O presente trabalho também foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de Financiamento 001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PG-BGA 7 Campus de Botucatu Instituto de Biociências 15 16 Resumo 17 A espermatogênese é um processo controlado por células-tronco no qual uma única célula-18 tronco espermatogonial (SSC) se diferencia em muitos espermatozoides haploides. A divisão de 19 uma SSC pode resultar em duas células tronco (divisão simétrica), ou em uma célula tronco e outra 20 diferenciada (divisão assimétrica). Independentemente do tipo de divisão celular, o processo de 21 auto-renovação e diferenciação é controlado por numerosos sinais provenientes do tecido somático 22 circundante, assim como também de fatores intrínsecos da própria SSC. Nesse sentido, estudos 23 recentes mostraram que RNAs não codificantes (microRNAs, cirRNAs, lncRNAs) podem 24 desempenhar um papel fundamental na reprodução, em particular na regulação da determinação 25 sexual, diferenciação sexual e gametogênese. Entre os RNAs não codificantes, os lncRNAs se 26 destacam por desempenhar papel regulatório na transcrição de "genes mestres" envolvidos em 27 vários processos biológicos, incluindo a diferenciação e manutenção da pluripotência de células-28 tronco. Considerando esse contexto, este estudo teve como objetivo entender a sinalização Gdnf-29 Gfrα1 em testículos de zebrafish combinando abordagens in vivo, in silico e ex vivo, além dos 30 efeitos biológicos do Fsh em lncRNAs testiculares. Nossos dados revelaram que o Gdnf, fator 31 derivado de célula germinativa, está envolvido na manutenção da pluripotência das células 32 germinativas por meio da criação de nichos espermatogoniais, dando suporte ao desenvolvimento 33 de cistos espermatogônicos e inibindo a diferenciação tardia de espermatogônias de maneira 34 autócrina e parácrina. Além disso, neste estudo, identificamos 5161 novos lncRNAs dos quais 76 35 foram diferencialmente regulados pelo rzfFsh. Além disso, análises de enriquecimento das DEGs 36 demonstrou que esses transcritos estão amplamente relacionados à sinalização e sistemas 37 orgânicos, como o sistema endócrino. Quando focamos nos mRNAs, encontramos 270 DEGs, 38 sendo 174 “up-regulated” 96 “down-regulated”. Interessantemente, as vias mais enriquecidas 39 estavam relacionadas à sinalização do hormônio da tireoide e esteroidogênese. Por fim, predições 40 de interação entre lncRNAs-mRNA-proteínas mostraram que alguns lncRNAs podem interagir, e 41 consequentemente, modular a expressão de genes de pluripotência de SSCs, como o pou5f3, 42 nanos3 e nanog. Em conjunto, nossos dados indicam uma provável atividade do Fsh em criar um 43 ambiente mais favorável para a diferenciação de espermatogônias tronco. 44 PG-BGA 8 Campus de Botucatu Instituto de Biociências Abstract 45 Spermatogenesis is a process controlled by stem cells in which a single spermatogonial 46 stem cell (SSC) differentiates into many haploid spermatozoa. The division of SSC follows either 47 the asymmetric model, where one stem cell produces a differentiated cell and a stem cell (self-48 renewal), or the symmetric model, where one stem cell produces two differentiated cells or two 49 stem cells. Regardless of the model, the process of self-renewal and differentiation is regulated by 50 numerous signals from the surrounding somatic tissue (the stem cell niche) and the germline stem 51 cells themselves. Additionally, recent studies have shown that non-coding RNAs (microRNAs, 52 circRNAs, lncRNAs) from the germline niche itself can play a crucial role in reproduction, 53 particularly in the regulation of sexual determination, sexual differentiation, and gametogenesis. 54 Among the non-coding RNAs, lncRNAs stand out for their regulatory role in the transcription of 55 "master genes" involved in various biological processes, including the differentiation and 56 maintenance of stem cell pluripotency. In this context, this study aimed to understand the Gdnf-57 Gfrα1 signaling in zebrafish testes by combining in vivo, in silico, and ex vivo approaches, as well 58 as the biological effects of Fsh on testicular lncRNAs. Our data revealed that Gdnf, now a germ 59 cell-derived factor, is involved in maintaining germline stem cell pluripotency through niche 60 creation, supporting the development of spermatogonial cysts and inhibiting late spermatogonial 61 differentiation in an autocrine and paracrine manner. Furthermore, in this study, we identified 5161 62 novel lncRNAs and 76 DEGs under the influence of rzfFsh, of which 46 were upregulated and 30 63 were downregulated. Additionally, enrichment analyses of the DEGs demonstrated that these 64 transcripts are broadly related to signaling and organ systems, such as the endocrine system. When 65 focusing on mRNAs, we found 270 DEGs, with 174 upregulated and 96 downregulated. 66 Interestingly, the most enriched pathways were related to thyroid hormone signaling and 67 steroidogenesis. Finally, predictions of interaction between lncRNAs-mRNAs-proteins showed 68 that some lncRNAs can interact, and consequently, modulate the expression of pluripotency genes 69 in SSCs, such as pou5f3, nanos3, and nanog. Collectively, our data indicate a likely activity of Fsh 70 in creating a more favorable environment for spermatogonial stem cell differentiation. 71 72 73 74 75 PG-BGA 9 Campus de Botucatu Instituto de Biociências 1. General Introduction 76 1.1 Spermatogenesis and Spermatogonial Stem cells 77 78 Spermatogenesis is a stem cell-driven process by which a single spermatogonial stem cell 79 (SSC) differentiates into many haploid spermatozoa (Nóbrega et al., 2009; Schulz et al., 2010). 80 This process occurs in three phases: the mitotic or spermatogonial phase with successive rounds 81 of mitotic duplication of the spermatogonia; the meiotic or spermatocytary phase, in which 82 spermatocytes undergo the two meiotic divisions; and the third phase, spermiogenesis, where the 83 haploid spermatids emerging from meiosis differentiate into flagellated spermatozoa (Schulz et 84 al., 2010). Although many features are conserved among vertebrates, fish spermatogenesis (the 85 focus of this project) takes place in the so-called cyst or spermatocyst (Figure 1B). The cyst is 86 formed when a group of somatic Sertoli cells envelop a single SSC (Callard, 1996). As 87 spermatogonia divide, the derived daughter cells remain interconnected by cytoplasmic bridges 88 (Grier, 1993; Schulz et al., 2010; França et al., 2015). Another interesting feature of cystic 89 spermatogenesis is that the Sertoli cell supports a single germ cell clone at once, whereas in 90 amniotes (reptiles, birds, and mammals), depending on species, at least five germ cell clones at 91 different stages of development are supported by a single Sertoli cell (Figure 1) (Schulz et al., 92 2010; França et al., 2015). Another difference is that fish Sertoli cells can continuously proliferate 93 even after the onset of puberty (França et al., 2015). 94 PG-BGA 10 Campus de Botucatu Instituto de Biociências 95 Figure 1. Comparison of mammalian (A, mouse) 96 and fish (B, zebrafish) testis. Segments of 97 spermatogenic tubules are shown to illustrate the 98 differences in Sertoli/germ cell relation between cystic 99 (B) and non-cystic (A) spermatogenesis. The germinal 100 epithelium contains Sertoli (SE) and germ cells, 101 delineated by a basal lamina (BL) and peritubular 102 myoid cells (MY). The interstitial Leydig cells (LE) 103 and blood vessels (BV) are shown. A: spermatogonia 104 (SG); spermatocyte (SC); round spermatid (RST); and 105 elongated spermatid (EST). B: type A undifferentiated 106 spermatogonia (Aund); type A differentiated 107 spermatogonia (Adiff); type B spermatogonia [B 108 (early-late)]; leptotene/zygotenic primary 109 spermatocytes (L/Z); pachytene primary 110 spermatocytes (P); diplotene spermatocytes/metaphase 111 I (D/MI); secondary spermatocytes/metaphase II 112 (S/MII); early (E1), intermediate (E2) and final 113 spermatids (E3); spermatozoa (SZ). From: Schulz et al. 114 (2010). 115 116 SSCs, a small population among type 117 A undifferentiated spermatogonia, can either 118 self-renew to produce more stem cells or 119 differentiate into daughter cells dedicated to spermatogenesis (Schulz et al., 2010; Xie et al., 2020). 120 The balance between SSC self-renewal and differentiation should be properly regulated to avoid 121 stem cell tumors or spermatogenesis depletion (De Rooij, 2001). This regulation is also crucial for 122 puberty and adult gonadal maturation in seasonal breeders. For example, SSC differentiation does 123 not occur in juveniles but is activated during puberty or the beginning of a reproductive season. 124 This switch in stem cell activity, from self-renewal to differentiation, is extremely relevant for 125 aquaculture. In aquaculture, precocious puberty is considered one of the major economic problems 126 limiting the further development of aquaculture species such as salmon, European sea bass, 127 Atlantic halibut, and Chinese tongue sole among others (reviewed by Taranger et al., 2010; Carillo 128 et al., 2015; Hagen et al., 2006; Ji et al., 2011). For example, in the European sea bass aquaculture, 129 the high incidence (20-30%) of precocious sexual maturation in males during the first year of life 130 (Begtashi et al., 2004) resulted in important economic losses (Taranger et al., 2010; Carillo et al., 131 2015). This is because precocious maturation results in non-marketable fish because growth and 132 feed utilization are negatively affected by the energy invested in the production of gametes (Felip 133 et al., 2006; 2008; Taranger et al., 2010; Carillo et al., 2015). Therefore, investigations to 134 understand the physiological and molecular mechanisms controlling the switch in SSC activity are 135 the basis for developing approaches to delay the start of pubertal testis maturation. 136 PG-BGA 11 Campus de Botucatu Instituto de Biociências 1.2 Endocrine and Paracrine Regulation of SSCs 137 SSCs are maintained in a specialized microenvironment in the testis known as the testicular 138 niche (Nóbrega et al., 2010; Lacerda et al., 2012; De Siqueira-Silva et al., 2019). In a model 139 laboratory species, zebrafish (Danio rerio), this microenvironment is composed of Sertoli cells, 140 peritubular myoid cells, Leydig cells, and endothelial cells that are all near to SSCs and contribute 141 to the niche (Nóbrega et al., 2010). The niche provides growth factors and cell-to-cell interactions 142 that regulate SSC activity in the testis (Nóbrega et al., 2010). The production and release of these 143 factors are modulated by reproductive hormones, such as Fsh (follicle-stimulating hormone) and 144 androgens (Nóbrega et al., 2015; Safian et al., 2019). Information on the identity of these endocrine 145 and paracrine factors and their mode of action is largely missing in vertebrates. 146 Gdnf (Glial cell line-derived neurotrophic factor) is a closely related member of the TGF-147 β superfamily which belongs to the Gdnf family of ligands (GFLs). This family of ligands consists 148 of Gdnf, neurturin, artemin, and persephin (Airaksinen and Saarma, 2002). Gdnf exerts its 149 biological roles by activating the Ret transmembrane receptor tyrosine kinase (RET) through the 150 co-receptor Gfrα1 (Gdnf family receptor α1) (Airaksinen and Saarma, 2002). The importance of 151 GDNF for SSC maintenance was unveiled by Meng and collaborators (2000) who showed that 152 mice with impaired GDNF signaling exhibited a progressive loss of SSCs (Meng et al., 2000), 153 while its pan-ectopic overexpression promoted germ cell hyperplasia, and ultimately tumors 154 (Meng et al., 2001). Several studies have reported the presence of Gdnf and Gfrα1 homologs in 155 different species of fish (Bosseboeuf et al., 2013; Gautier et al., 2014; Bellaiche et al., 2014; 156 Nakajima et al., 2014; Wei et al., 2017; Zhao et al., 2018). Gdnf and Gfrα1 are expressed in type 157 A undifferentiated and progressively decreased their expression during germ cell development as 158 has been demonstrated in dogfish (Scyliorhinus canicula) (Gautier et al., 2014), rainbow trout 159 (Oncorhynchus mykiss) (Nakajima et al., 2014; Bellaiche et al., 2014) and medaka (Oryzias 160 latipes) (Zhao et al., 2018). Moreover, in vitro experiments have demonstrated that recombinant 161 human GDNF promoted the proliferation and long-term maintenance of dogfish spermatogonia 162 with stem characteristics (Gautier et al., 2014). Similar findings were found by Wei and 163 collaborators (2017) who showed that two medaka Gdnf homologs, named Gdnfa and Gdnfb, can 164 stimulate the proliferation of SG3, a spermatogonial cell line derived from adult medaka. On the 165 other hand, expression analysis in rainbow trout revealed that gdnfb increased during the arrest of 166 the spermatogenic cycle (end of germ cell proliferation and differentiation), suggesting that Gdnfb 167 is likely involved in the repression of SSC differentiation rather than proliferation (Bellaiche et al., 168 2014). 169 PG-BGA 12 Campus de Botucatu Instituto de Biociências 1.3 lncRNAs (long non-coding RNAs) 170 RNA is a macromolecule responsible for transmitting and processing genetic information 171 during transcription and translation (Zaha et al., 2014). While viral RNAs differ, all other RNAs 172 are transcribed from genomic DNA. Over the years, a large number of RNAs that do not code for 173 peptides or proteins have been identified, including non-mRNAs and non-coding RNAs 174 (ncRNAs). Non-mRNAs are primarily found in bacteria, while ncRNAs are present in eukaryotic 175 organisms (Wassarman et al., 1999). These ncRNAs are located in intergenic regions of the 176 genome, which do not participate in protein coding. Initially, ncRNAs were considered junk RNAs 177 due to their lack of a described function. However, identification of thousands of ncRNAs in 178 humans and experimental models have demonstrated their roles in regulating various biological 179 processes, including tumorigenesis and stem cell differentiation (Arrigo and Pulliero, 2015). There 180 are two categories of ncRNAs based on their functions. Structural ncRNAs, including tRNAs, 181 rRNAs, snRNAs, and snoR-RNAs, exert structural roles (Statello et al., 2021). Regulatory 182 ncRNAs, such as siRNAs, miRNAs, piwi-RNAs, lncRNAs, and long intergenic ncRNAs, have 183 regulatory roles on gene expression (Qu and Adelson, 2012) at various levels (Table 1). 184 PG-BGA 13 Campus de Botucatu Instituto de Biociências 185 Table 1. Functions of different classes of RNAs. Note the great diversity of ncRNA molecules in eukaryotes. Different 186 types of RNAs indicated, mRNA (messenger RNA), tRNA (transfer RNA), snoRNA (RNA nucleolar), snRNA 187 (nuclear), sRNA (small RNA), CRISPR (CRISPR RNA), miRNA (micro RNA), siRNA (interference RNA), piRNA 188 (RNA that binds to the PIWI protein) and lncRNA (long non-coding RNA). Adapted fom Costa, 2017. 189 Analytical technologies such as CAGE, Chip-chip, Chip-seq, and RNA deep sequencing 190 have allowed a better understanding of the complexity of the eukaryotic genome (Carninci, 2006; 191 Gustincich et al., 2006). In mice, about one-third of transcripts are non-coding, and recently, 192 studies have focused on long non-coding RNAs (lncRNAs). lncRNAs are longer than 200 193 nucleotides and have little or no ability to encode proteins (Cech and Steitz, 2014; Arrigo and 194 Pulliero, 2015). While this limit may seem arbitrary, it distinguishes lncRNAs from other RNA 195 categories. Recent studies have shown that lncRNAs regulate various cellular processes through 196 their direct action on gene transcription (Hezroni et al., 2015). They can act in the promoter region 197 or in other transcriptional regulatory regions, such as enhancers or locus control regions (Hezroni 198 et al., 2015). 199 PG-BGA 14 Campus de Botucatu Instituto de Biociências lncRNAs can be divided into three groups according to their action on gene expression 200 (Figure 2). In the first group, known as "transcription only", only the transcription of the molecule 201 is important, and the RNA produced does not present a characteristic function (Figure 2A). The 202 only characteristic observed for this lncRNA group is the fact that they participate in the chromatin 203 modification of the the locus that they are associated. Its sequence and structure itself are not 204 important (Perry and Ulitsky, 2016) (Figure 2A). The second group, known as cis-acting RNAs, 205 comprises lncRNAs whose function is related to the recruitment of transcripts that will act in 206 genomic or spatially close regions (Figure 2B). Finally, the third group known as trans-acting 207 RNAs that act independently of the site of their transcription, regulating the expression of other 208 loci in the nucleus or acting on transcripts in the cell cytoplasm (Figure 2C). Many lncRNAs have 209 functions strictly related to the nucleus, such as Xist, Neat1, and Malat1, however, most lncRNAs 210 act in both nucleus and the 211 cytoplasm, or only in the 212 cytoplasm (Cabili et al., 2015; 213 Derrien et al., 2012; Ulitsky e 214 Bartel, 2013). 215 216 217 Figure 2. lncRNA modes of action. (A). 218 For some lncRNA loci, the act of 219 transcription itself plays a role in 220 mediating lncRNA function, for example 221 by affecting the underlying chromatin 222 structure of the locus. In this context, the 223 RNA product itself and its sequence are 224 irrelevant. (B). In contrast, other 225 lncRNAs act close to their transcription 226 site, recruiting or repelling specific 227 factors, which may or may not recognize 228 the RNA in a sequence-specific manner. 229 (W). Other lncRNAs leave their 230 transcription site and act elsewhere, 231 usually in sequence-specific or structure-dependent ways, via interactions with proteins and other RNA factors. Figure 232 is taken from Perry and Ulitsky (2016). 233 234 PG-BGA 15 Campus de Botucatu Instituto de Biociências Long non-coding RNAs (lncRNAs) have been increasingly associated with biological 235 processes related to embryonic development and cell differentiation in adult mammals (Perry and 236 Ulistsk, 2016). Although the molecular mechanisms that regulate these processes are still largely 237 unknown, it is known that lncRNAs can control the transcription of "master" regulatory genes that 238 play a crucial role in these processes (Perry and Ulitsky, 2016). Several mechanisms have been 239 proposed for the regulation of lncRNAs in stem cell differentiation, as illustrated in Figure 3. 240 LncRNAs can repress the early differentiation of stem cells by shaping the chromatin and creating 241 a repressive environment (Figure 3A). Alternatively, lncRNAs can facilitate the binding of 242 transcription factors, thereby promoting the 243 process of differentiation (Figure 3B). In cases 244 where the lncRNA target gene is also a 245 transcription factor, lncRNA activity can be 246 enhanced by the expression of the target gene, 247 resulting in a positive feedback loop (Figure 248 3C). Furthermore, during the process of cell 249 differentiation, lncRNAs can repress molecules 250 necessary for the establishment of different cell 251 lineages (Figure 3D). 252 Figure 3. Main functions of lncRNAs in the regulation 253 of stem cells. lncRNAs can play several roles during cell 254 differentiation. Figure taken and adapted from Perry and 255 Ulitsky (2016). 256 257 While lncRNAs are mainly involved in 258 mechanisms of differentiation, several studies have suggested that they may also be associated 259 with the maintenance of stem cells and the promotion of their undifferentiated state in various 260 mammalian cell types, such as neuronal trunk cells (Ng et al., 2013; Aprea et al., 2013; Lin et al., 261 2014), epidermal cells (Kretz et al., 2012), cardiac cells (Klattenhoff et al., 2013), endodermal 262 cells (Jiang et al., 2015; Kurian et al., 2015), endothelial cells (Boulberdaa et al., 2016), adipocytes 263 (Sun et al., 2013), and hematopoietic cells (Deng et al., 2016). 264 PG-BGA 16 Campus de Botucatu Instituto de Biociências Long non-coding RNAs (lncRNAs) have been identified as playing a key role in the 265 spermatogenic process, and dysregulation of lncRNAs may be associated with male infertility 266 (Wichman et al., 2017). Regulatory elements such as enhancers and regulatory ncRNAs specific 267 for spermatogonia, spermatocytes and spermatozoa have been identified in mammals, indicating 268 the significant impact of lncRNAs in spermatogenic regulation and maintenance of fertility 269 (Choudhury et al., 2010; Wang et al., 2001; McCarrey et al., 1992; Penkner et al., 2005; Kuramochi 270 et al., 2004; Hotta et al., 1995; Shima et al., 2004). Wichman and collaborators (2017) have 271 identified lncRNAs expressed exclusively in different types of germ cell populations in mice, with 272 differentially expressed lncRNAs found in spermatogonia and pachytenic spermatocytes. In this 273 study, knockout of testis-specific lncRNA 1 resulted in reduced fertility in male mice. Another 274 lncRNA, Tsx is important in the meiosis of germ cells of male mice (Anguera et al., 2011). 275 lncRNAs have also been shown to participate in signaling 276 pathways, such as Wnt, which is important for the maintenance 277 of the spermatogenic process (Arun et al., 2012). In fish, a 278 recent study demonstrated in Nile tilapia that the knockdown 279 of the igf3 gene results in the alteration of the expression of 280 more than 124 different types of lncRNAs, indicating their 281 fundamental role in the regulation of the spermatogenic process 282 (Song et al., 2019). 283 Wichman et al. (2017) performed the knockout of the 284 lncRNA “TSLrn1”, or called “testis-specific lncRNA 1”, found 285 mainly in spermatogonia and in mice meiotic cells. The result 286 is that all males had their fertility reduced. Anguera et al. (2011) 287 also showed the importance of lncRNA Tsx in the meiosis of 288 germ cells of male mice. Other studies described the 289 participation of lncRNAs in signaling pathways, such as Wnt 290 (Arun et al., 2012), important in the maintenance of the 291 spermatogenic process (Takase et al., 2016; Sreenivasan et al., 2014). 292 Figure 4. Heatmap of differentially expressed lncRNAs among spermatogonia, meiotic (pachytene), and post-meiotic 293 (spermatid) cells of mice. Adapted from Wichman et al. (2017). 294 295 PG-BGA 17 Campus de Botucatu Instituto de Biociências Information about the performance of lncRNAs in fish testes are still unknown. More 296 recent, studies with common carp, Cyprinus carpio, demonstrated that the knockdown of igf3 297 (insulin-like growth factor 3), a gene responsible for controlling the axes of reproduction and 298 growth in fish (Reinecke., 2010) and stimulates spermatogonial differentiation in zebrafish 299 (Nóbrega et al., 2015), resulted in alteration of the expression of more than 124 different types of 300 lncRNAs (Song et al., 2019). Similar to mammals, these results suggest that lncRNAs may also 301 play a role in the regulation of the spermatogenic process in fish. 302 303 304 2. General Objective 305 The objective of this thesis is to better understand the regulatory mechanisms zebrafish 306 spermatogonial niche, focusing on germ cell-derived factors (Chapter 1) and lncRNAs (Chapter 307 2). 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 PG-BGA 18 Campus de Botucatu Instituto de Biociências Chapter 1 327 328 Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem 329 Cell Niche in Autocrine- and Paracrine-Dependent Manners 330 331 Lucas B Doretto, Arno J Butzge, Rafael T Nakajima, Emanuel R M Martinez, Beatriz Marques 332 de Souza, Maira da Silva Rodrigues, Ivana F Rosa, Juliana M B Ricci, Aldo Tovo-Neto, Daniel 333 F Costa, Guilherme Malafaia , Changwei Shao, Rafael H Nóbrega 334 335 336 337 Abstract 338 Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-339 GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in 340 mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their 341 functions remain poorly investigated in the testes. Considering this background, this study aimed 342 to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in 343 vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for 344 Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-345 specific third round of whole genome duplication. Expression analysis further revealed that both 346 ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that 347 gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia 348 (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf 349 promoted the creation of new available niches by stimulating the proliferation of both type Aund 350 spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. 351 Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in 352 type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data 353 revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the 354 creation of new available niches, supporting the development of spermatogonial cysts and 355 inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners. 356 357 358 359 360 https://pubmed.ncbi.nlm.nih.gov/?term=Doretto+LB&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Butzge+AJ&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Nakajima+RT&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Martinez+ERM&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=de+Souza+BM&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=de+Souza+BM&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Rodrigues+MDS&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Rosa+IF&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Ricci+JMB&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Tovo-Neto+A&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Costa+DF&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Costa+DF&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Malafaia+G&cauthor_id=35455974 https://pubmed.ncbi.nlm.nih.gov/?term=Shao+C&cauthor_id=35455974 PG-BGA 19 Campus de Botucatu Instituto de Biociências 1. Introduction 361 GDNF (Glial cell line-derived neurotrophic factor) is a closely related member of the TGF-362 β superfamily which belongs to the GDNF family of ligands (GFLs). This family of ligands 363 consists of Gdnf, neurturin, artemin and persephin [1]. The importance of GDNF for 364 spermatogonial stem cell (SSC) maintenance was unveiled by Meng et al. [2], who demonstrated 365 that mice with impaired GDNF signaling exhibited a progressive loss of SSCs, whereas GDNF 366 overexpression promoted germ cell hyperplasia and ultimately tumors [2]. Further studies showed 367 that GDNF promoted in vitro expansion of mouse germline stem cells [3,4], this being considered 368 an indispensable factor for long-term culture of SSCs for several species of rodents [3,5,6]. More 369 recently, experiments using mice that ectopically expressed stage-specific GDNF in Sertoli cells 370 revealed that GDNF increased SSC self-renewal by blocking differentiation rather than actively 371 stimulating their proliferation [4]. Altogether, these studies in mammals demonstrated that GDNF 372 is an important factor for SSC self-renewal, proliferation of the stem cell direct progenitors and 373 maintenance of the SSC undifferentiated state (see the review in Parekh et al. [7]; see also Mäkelä 374 and Hobbs [8]). 375 GDNF signaling occurs through binding the non-signaling co-receptor of the GDNF 376 Family Receptor α1 (GFRα1), which is attached to the cell membrane by 377 glycosylphosphatidylinositol-anchors [1]. The complex GDNF-GFRα1 associates with a single 378 transmembrane RET receptor tyrosine kinase, leading to the activation of RET’s intracellular 379 kinase domain and the subsequent stimulation of different downstream cellular pathways [1]. In 380 mammalian testes, GDNF is produced by testicular somatic cells, including Sertoli cells [2,9,10], 381 peritubular myoid cells under the influence of androgens [11,12] and testicular endothelial cells, 382 which seem to be the major GDNF-producing sources in mouse testes [13]. In rodents, GFRα1 is 383 present in a subpopulation of single type A spermatogonia (As), which also expresses the inhibitor 384 of DNA binding 4 (ID4) [14,15]. This subpopulation is considered the purest functional SSC 385 population [14,15]. However, several other studies have demonstrated that GFRα1 is not 386 exclusively detected in SSCs but is also expressed in types A paired (Apr) and aligned (Aal) 387 spermatogonia [16,17,18,19,20]. Similar expression patterns have been reported in other 388 mammalian species, such as hamsters [21], pigs [22], collared peccaries [23,24], buffaloes [25], 389 different equine species [26] and primates, including humans [27,28]. 390 In nonmammalian species, particularly in fish, Gdnf/Gfrα1 homologs have been found in 391 a limited number of species, such as dogfish (Scyliorhinus canicula) [29], rainbow trout 392 (Oncorhynchus mykiss) [30,31,32] and medaka (Oryzias latipes) [33]. In these species, Gdnf and 393 https://www.mdpi.com/2073-4409/11/8/1295#B1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B2-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B2-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B3-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B4-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B3-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B5-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B6-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B4-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B7-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B8-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B2-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B9-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B10-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B11-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B12-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B13-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B14-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B15-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B14-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B15-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B16-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B17-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B18-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B19-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B20-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B21-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B22-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B23-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B24-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B25-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B26-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B27-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B28-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B29-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B30-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B31-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B32-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B33-cells-11-01295 PG-BGA 20 Campus de Botucatu Instituto de Biociências Gfrα1 are co-expressed in type A undifferentiated spermatogonia, suggesting an autocrine 394 mechanism for Gdnf-mediated functions in fish testes [30]. The physiological relevance of Gdnf 395 for type A undifferentiated spermatogonia has been further demonstrated by in vitro studies 396 showing that recombinant human GDNF (rh GDNF) promoted the proliferation and long-term 397 maintenance of dogfish spermatogonia with stem characteristics [29]. Similar findings were 398 reported by Wei et al. [34], who showed that two Gdnf homologs in medaka, named Gdnfa and 399 Gdnfb, stimulated proliferation of SG3, a medaka spermatogonial stem cell line. On the other hand, 400 studies in rainbow trout revealed that gdnfb mRNA levels increased during the arrest of the 401 spermatogenic cycle (end of germ cell proliferation and differentiation), suggesting that Gdnfb is 402 likely involved in the repression of SSC differentiation rather than proliferation [31]. 403 Considering this background and the lack of knowledge about Gdnf-Gfrα1 signaling in 404 fish, this study aimed to unravel the autocrine/paracrine roles of Gdnf on the zebrafish germ stem 405 cell niche and to expand our knowledge about the critical factors involved in SSC activity as well 406 as improve our abilities to predict the consequences of changes involved in the physiological 407 mechanisms related to Gdnf. To these ends, we initially performed phylogenetic and synteny 408 analyses for Gfrα1 and then investigated the testicular expression profiling of gdnf (gdnfa and 409 gdnfb) and gfrα1 (gfrα1a and gfrα1b) transcripts in zebrafish testes. Subsequently, we identified 410 the cellular types expressing Gdnf and Gfrα1 and assessed the biological effects of Gdnf through 411 an ex vivo testis culture system. According to Oatley and Brinster [35], the impairment of SSC 412 function disrupts spermatogenesis and causes subfertility or infertility in males; therefore, knowing 413 the mechanisms that regulate SSC homeostasis is imperative for the conservation of species or for 414 their use as experimental models in studies focusing on the treatment of pathological conditions 415 affecting the reproductive organs in humans. 416 417 2. Material and Methods 418 2.1. Zebrafish Stocks 419 Sexually mature zebrafish (Danio rerio, outbred) (4–5 months old) were kept in 6 L water 420 tanks in a recirculating system with controlled photothermal conditions (27 °C and 14 h of light 421 and 10 h dark). Parameters such as salinity, pH, dissolved oxygen and ammonia were monitored 422 daily in all tanks. Fish were fed twice a day using commercial food (Zeigler®, Gardners, PA, USA). 423 Handling and experimentation were in accordance with Brazilian legislation regulated by the 424 National Council for the Control of Animal Experimental (CONCEA) and the Ethical Principles 425 in Animal Research of São Paulo State University (protocol no. 666-CEUA). Zebrafish is a tropical 426 https://www.mdpi.com/2073-4409/11/8/1295#B30-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B29-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B34-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B31-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B35-cells-11-01295 PG-BGA 21 Campus de Botucatu Instituto de Biociências freshwater fish species natural to rivers in Southern Asia [36,37,38] and has been considered a 427 versatile model for reproductive biology [39], besides being used as a model for translational 428 research in human health and disease [40]. Therefore, these aspects justify the choice of this 429 species in our study. 430 431 2.2. Sequence Analysis 432 The predicted amino acid sequences for Gfrα1a and Gfrα1b of D. rerio (Q98TT9 and 433 Q98TT8, respectively), GFRA1 of Homo sapiens (P56159), Rattus norvegicus (Q62997) and Mus 434 musculus (P97785) were obtained from the Universal Protein Resource (UniProt, accessed 435 09/12/2019) and aligned using the MEGA algorithm allocated in Geneious Pro 4.8.5 software [41]. 436 For the phylogenetic analysis, we retrieved the protein sequences for GFRα1 (Gfrα1a and Gfrα1b) 437 from the Universal Protein Resource (UniProt, accessed on 25 February 2020), the National Center 438 for Biotechnology Information (NCBI, 25 February 2020) and Ensembl (accessed on 25 February 439 2020 [42]). For this analysis, we retrieved vertebrate sequences for GFRα1 and Growth arrest-440 specific protein 1 (GAS1) from humans (GAS1 as an outgroup). The predicted amino acid 441 sequences were aligned using the Muscle algorithm [43] allocated in Geneious Pro 4.8.5 software 442 [41]. The choice of the best fitting model of evolution was performed with SMS [44]. Phylogenetic 443 reconstruction was determined by Bayesian methods implemented in Beast v1.7.0 software [45]. 444 This step was carried out according to Geraldo et al. [46], with adaptations. Branch values were 445 supported by posterior probabilities obtained by Bayesian analysis. For Bayesian methods, the 446 burn-in was obtained through Tracer [45] using log likelihood scores, and data were compiled in 447 TreeAnnotator [45] after trees that were out of the convergence area had been discarded. The 448 visualization and the final tree edition were generated using FigTree v1.3.1 [45]. In the 449 phylogenetic analyses, the proportion of invariable sites and γ-distributed rate variation across sites 450 were estimated, and the substitution of rate categories set in four categories. The parameter settings 451 used to reconstruct the phylogeny are shown in Table S2. To construct the synteny regions of 452 GFRA1 (human), Gfrα1 (rat and mouse), gfrα1a and gfrα1b (zebrafish), we used the GenBank 453 database, available at the National Center for Biotechnology Information (NCBI; 454 http://www.ncbi.nlm.nih.gov/) (accessed on 25 February 2020) and Ensembl [42]. 455 456 https://www.mdpi.com/2073-4409/11/8/1295#B36-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B37-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B38-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B39-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B40-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B41-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B42-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B43-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B41-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B44-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B45-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B46-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B45-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B45-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B45-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 http://www.ncbi.nlm.nih.gov/ https://www.mdpi.com/2073-4409/11/8/1295#B42-cells-11-01295 PG-BGA 22 Campus de Botucatu Instituto de Biociências 2.3. Expression Profiling of Gdnf (gdnfa and gdnfb) and Gfrα1 (gfrα1a and 457 gfrα1b) Transcripts in Zebrafish Testes 458 To investigate the expression of gdnfa (glial cell-derived neurotrophic factor a), gdnfb 459 (glial cell-derived neurotrophic factor b), gfrα1a (gdnf family receptor alpha 1a) and gfrα1b (gdnf 460 family receptor alpha 1b) in zebrafish testes, total RNA from testes (n = 4 males) was extracted 461 using an RNAqueous®-Micro kit (Ambion, Austin, TX, USA), following the manufacturer’s 462 instructions. cDNA synthesis and quantitative reverse transcription polymerase chain reaction 463 (RT-qPCR) were performed as previously described [47]. The number of amplification cycles (Ct-464 cycle threshold) for gdnfa, gdnfb, gfrα1a and gfrα1b were determined through a StepOnePlus™ 465 Real-Time PCR System (Thermo fisher, Waltham, MA, USA, EUA). Primers (Table 1) were 466 designed based on zebrafish sequences available from the Genbank database. 467 468 Target Genes Primer Sequences (5′–3′) References ef1α GCCGTCCCACCGACAAG (Fw) Morais et al. [48] CCACACGACCCACAGGTACAG (Rv) b-actin AGACATCAGGGAGTGATGGT (Fw) Tovo-Neto et al. [49] CAATACCGTGCTCAATGGGG (Rv) gdnfa GAAGCTCCGGTCTGTATGGA (Fw) This paper GGAGCTCAGGAGCAACAAAC (Rv) gdnfb AGGAGTAAATCAGTGGGCCAAA (Fw) This paper AGTAGCTGAATATGAGCTCCTCC (Rv) gfrα1a TCGACTGGCTCCCATCTATTC (Fw) This paper AGGTGTCATTCAGGTTGCAGG (Rv) gfrα1b CCTGTGCTTGATTTAGTGCA (Fw) This paper GCATCCGTACTTTCCCAAAC (Rv) igf3 TGTGCGGAGACAGAGGCTTT (Fw) Morais et al. [48] CGCCGCACTTTCTTGGATT (Rv) amh CTCTGACCTTGATGAGCCTCATTT (Fw) García-Lopez et al. [50] GGATGTCCCTTAAGAACTTTTGCA (Rv) fshr GAGGATTCCCAGTAATGCTTTCCT (Fw) García-Lopez et al. [50] TCTATCTCACGAATCCCGTTCTTC (Rv) pou5f3 GAGAGATGTAGTGCGTGTAT (Fw) Tovo-Neto et al. [49] https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#table_body_display_cells-11-01295-t001 https://www.mdpi.com/2073-4409/11/8/1295#B48-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B49-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B48-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B50-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B50-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B49-cells-11-01295 PG-BGA 23 Campus de Botucatu Instituto de Biociências Target Genes Primer Sequences (5′–3′) References GCTCGTAATACTGTGCTTCA (Rv) dazl AGTGCAGACTTTGCTAACCCTTATGTA (Fw) Morais et al. [49] GTCCACTGCTCCAAGTTGCTCT (Rv) sycp3l AGAAGCTGACCCAAGATCATTCC (Fw) García-Lopez et al. [50] AGCTTCAGTTGCTGGCGAAA (Rv) gdnfa-ish T7Rpps-CCGCAGTGAGAGCCCCG (Fw) This paper T3Rpps-TCCCGTTAGGTCATATTGTTCCTC (Rv) Fw, forward; Rv, reverse; T7Rpps–T7 RNA polymerase promoter sequence at its 5′-end (5′ 469 CCGGGGGGTGTAATACGACTCACTATAGGG-3′), T3Rpps–T3 RNA polymerase promoter sequence at its 5′-470 end (T3′GGGCGGGTGTTATTAACCCTCACTAAAGGG-3′). 471 Table 1. Primers used for gene expression analysis (RT-qPCR) and to generate DNA templates for digoxigenin (DIG)-472 labeled cRNA probe synthesis for in situ hybridization (ISH) (Supplementary Materials). 473 474 2.4. Differential Plating Method 475 To obtain testicular cellular fractions (germ or somatic cell-enriched fractions), a 476 differential plating method was carried out as previously described by Hinfray et al. [51]. To this 477 end, testes (n = 20 males) were digested with 0.2% collagenase (Sigma Aldrich, San Luis, MI, 478 USA) and 0.12% dispase (Sigma Aldrich, San Luis, MI, USA) [47]. Total cell suspension was 479 submitted to a differential plating method, in which somatic cells adhere to the bottom of the plate, 480 whereas germ cells either remain in suspension or only weakly associate with adhering somatic 481 cells [51]. By using this approach, germ and somatic cell-enriched fractions can be obtained [51]. 482 RNA from cell suspensions (total, germ and somatic cell-enriched fractions) was obtained using a 483 PureLink® RNA Mini Kit (Ambion, Austin, TX, USA), following the manufacturer’s instructions. 484 cDNA synthesis was conducted using a SuperScript® II Reverse Transcriptase kit (Invitrogen, 485 Carlsbad, CA, USA) and random hexamers. The relative mRNA levels of pou5f3 (POU domain, 486 class 5, transcription factor 3) (spermatogonia marker), vasa (spermatogonia marker), gdnfa, igf3 487 (insulin-like growth factor 3) (Sertoli cell marker), gfrα1a and gfrα1b were determined by qRT-488 PCR. β-actin and ef1 were used as housekeeping genes. The quantification cycle (Cq) values were 489 determined in a StepOne system (Life Technologies, Carlsbad, CA, USA) using SYBR Green 490 (Invitrogen, Carlsbad, CA, USA) and specific primers (Table 1), as described in section 2.3. 491 492 https://www.mdpi.com/2073-4409/11/8/1295#B49-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B50-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B51-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B51-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B51-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#table_body_display_cells-11-01295-t001 https://www.mdpi.com/2073-4409/11/8/1295#sec2dot3-cells-11-01295 PG-BGA 24 Campus de Botucatu Instituto de Biociências 2.5. Immunofluorescence and Western Blot 493 Testes (n = 10 males) were fixed with 4% paraformaldehyde in PBS (Phosphate Buffered Saline) 494 (1X, pH 7.4) for 1 h, embedded in paraplast (Sigma Aldrich, San Luis, MI, USA) and sectioned at 495 5 μm thickness. After deparaffinization and rehydration, sections were submitted to antigen 496 retrieval by heating slides in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 497 6.0) until temperatures reached 95–100 °C in a microwave. To reduce background fluorescence, 498 slides were incubated with NaBH4 (sodium borohydride—0.01g dissolved in 1 mL of distilled 499 water) (Sigma Aldrich, San Luis, MI, USA) for 3 min. Subsequently, slides were rinsed with 1X 500 PBS (pH 7.4) and incubated with the biotinylated primary antibody rabbit anti-zebrafish Gfrα1a 501 (1:300, 1X PBS pH 7.4) at 4 °C overnight. Zebrafish polyclonal biotinylated antibody anti-Gfrα1a 502 was synthesized by Rheabiotech (Campinas, SP, Brazil) using the specific antigen sequence 503 ‘RLDCVKANELCLKEPGCSSK’ located at the N-terminus of zebrafish Gfrα1a (Figure 1). This 504 antibody is also potentially able to recognize other Gfrα1 isoforms, such as GFRA1 in humans and 505 rodents and Gfrα1b in zebrafish (Figure 1). After rising, the slides were incubated with Dylight 506 488 Streptavidin (BioLegend®, San Diego, CA, USA) (1:400) or Alexa Fluor 594 Streptavidin 507 (BioLegend®, San Diego, CA, USA) (1:400) in 1X PBS (pH 7.4) for 60 min at room temperature. 508 Subsequently, sections were counterstained with Hoechst (1:2000, 1X PBS pH 7.4) (Invitrogen, 509 Carlsbad, CA, USA) or Propidium iodide (PI) (BioLegend®, San Diego, CA, USA) (1 mg/mL 510 dissolved in distilled water) and mounted with ProLong Gold Antifade (Thermo Fisher Scientific, 511 Waltham, MA, USA). Control sections were prepared by preadsorbing the zebrafish Gfrα1a 512 antibody with the corresponding peptide (10 μg/1 µL of antibody, Rheabiotech, Campinas, SP, 513 Brazil) or by omitting the primary antibody. Slides were photographed using a Leica SP5 laser 514 scanning confocal microscope (Leica, Wetzlar, Hessen, Germany) from the Electron Microscopy 515 Center, Institute of Biosciences, São Paulo State University (Botucatu, Brazil), and germ cells 516 were classified according to Leal et al. [52]. 517 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#B52-cells-11-01295 PG-BGA 25 Campus de Botucatu Instituto de Biociências 518 Figure 1. GFRα1 predicted amino acid sequence alignment. Numbers at the top left of the sequences indicate amino 519 acid positions, dashes indicate deletions and black boxes indicate shared sequences. The three cysteine-rich domains 520 (D1–D3) (orange lines), 28 cysteine residues (*) (plus 2 in the terminal region) and two triplets (MLF and RRR) 521 (green boxes) are highly conserved among humans, rodents and zebrafish. At the end of the alignment are the 522 percentage identity values of zebrafish Gfrα1a and Gfrα1b in relation to the other corresponding sequences. The blue 523 line indicates the amino acid sequence recognized by the zebrafish Gfrα1a antibody used in this study; the purple line 524 indicates the putative motifs critical for binding to GDNF. 525 526 527 For the Western blot analysis, testes (n = 10 males) were homogenized in an extraction 528 TBST buffer (10 mM Tris–HCl, pH 7.5; 150 mM NaCl; 0.1% Tween 20) containing a cocktail of 529 protease inhibitors (Roche Applied Science, Mannheim, Germany). Subsequently, the homogenate 530 was incubated on ice for 15–20 min before sonication (3 × 1 min on ice) and centrifuged at 4000 531 rpm at 4 °C for 20 min in order to determine the total protein concentration by means of a NanoVue 532 spectrophotometer (GE Healthcare, Chicago, IL, USA). A total of 40 µg protein was analyzed by 533 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein extracts were 534 blotted onto a nitrocellulose membrane (Amersham, Little Chalfont, UK) blocked with 3% non-535 fat milk diluted in 1X Tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-HCl, pH 7.6.) for 1 536 h, and incubated with the primary antibody rabbit anti-zebrafish Gfrα1a (1:500, Rheabiotech, 537 Campinas, SP, Brazil) at 4 °C overnight. The membrane was washed with TBS and incubated with 538 horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Santa Cruz Biotechnologies, Dallas, 539 TX, EUA) for 2 h. After washing, blots were developed with a chemiluminescence substrate kit 540 (Pierce ECL Western Blotting Substrate, GE Healthcare, Chicago, IL, USA) and the signal was 541 captured by a CCD camera (ImageQuant LAS 4000 mini®, GE Healthcare, Chicago, IL, USA). 542 PG-BGA 26 Campus de Botucatu Instituto de Biociências As controls, some membranes were alternatively incubated with primary antibodies that had been 543 preadsorbed with the respective peptides. 544 545 2.6. Recombinant Human GDNF 546 To evaluate the effects of Gdnf on zebrafish spermatogenesis (see below), a rhGDNF 547 purchased from PeproTech® (London, UK) (reference no. 450-10; 548 https://www.peprotech.com/en/recombinant-human-gdnf#productreviews)(accessed on 20 549 February 2020) was used. We used a recombinant human hormone because the recombinant 550 zebrafish Gdnf is not commercially available. In addition, rhGDNF has previously been used in 551 fish [53]. The rhGDNF was dissolved in sterile Lebovitz medium (L-15) (Sigma-Aldrich, St. 552 Louis, MO, USA) at a concentration of 100 µg/mL and subsequently aliquoted and stored at 553 −20 °C until use. After identifying the binding sites between rhGDNF and human GFRA1A, a 3D 554 structure model was built to predict the interaction sites between rhGDNF and zebrafish Gfrα1a 555 (Q98TT9). The 3D protein structure used was obtained through SWISS-MODEL 556 (swissmodel.expasy.org), with multiple target sequences representing different subunits of a 557 hetero-oligomer (hetero-2-2-mer), and the quality of the modeling was analyzed by means of a 558 Ramachandran plot generated with Rampage software [50]. The template (4ux8.1) and the final 559 model were viewed in the software Pymol (the PyMOL Molecular Graphics System, Version 1.8 560 Schrödinger, LLC). 561 562 2.7. Testis Tissue Culture 563 The effects of rhGDNF on zebrafish spermatogenesis were investigated using a previously 564 established ex vivo culture system [52]. In this system, one testis (left) was incubated in the 565 presence of rhGDNF (100 ng/mL, based on Gautier et al. [53]) and its contra-lateral (right) in the 566 basal culture medium (L-15). The culture medium was changed every 3 days, and after 7 days, 567 testes were collected for histomorphometrical analysis via a BrdU (bromodeoxyuridine) (Sigma 568 Aldrich, San Luis, MI, USA) incorporation assay and gene expression (RT-qPCR) (see below). 569 Additional cultures were carried out to assess the interaction of Gdnf with Fsh-mediated effects 570 on the zebrafish spermatogonial phase [54]. To this end, zebrafish testes (n = 10 males) were 571 incubated with recombinant zebrafish Fsh (rzfFsh) (100 ng/mL [55]) (U-Protein Express B.V; 572 Utrecht, the Netherlands) in the presence or absence of rhGDNF (100 ng/mL) for 7 days. After the 573 culture period, testes were collected for RT-qPCR analysis. For histomorphometry, zebrafish 574 testicular explants (n = 10) were fixed in 4% buffered glutaraldehyde at 4 °C overnight, 575 https://www.peprotech.com/en/recombinant-human-gdnf#productreviews https://www.mdpi.com/2073-4409/11/8/1295#B53-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B50-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B52-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B53-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B54-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B55-cells-11-01295 PG-BGA 27 Campus de Botucatu Instituto de Biociências dehydrated, embedded in historesin Technovit 7100 (Wehrheim, Germany), sectioned at 4µm 576 thickness and stained with 0.1% toluidine blue to estimate the frequency of the different germ cell 577 cysts using a high-resolution light microscope (Leica DM6000 BD, Leica Microsystems, Wetzlar, 578 Germany). In this analysis, five histological fields for each animal were randomly selected for 579 counting the frequency of germ cell cysts (type A undifferentiated spermatogonia (Aund), type A 580 differentiated spermatogonia (Adiff), type B spermatogonia (SPG B), spermatocytes (SPCs) and 581 spermatids (SPTs)), as previously described [47,52]. 582 To investigate the effects of rhGDNF on germ cell proliferation, BrdU (100 µg/mL) was 583 added during the final 6 h of incubation. After incubation, zebrafish testes (n = 10) were fixed at 584 4 °C overnight in freshly prepared methacarn (60% (v/v) absolute ethanol, 30% chloroform and 585 10% acetic acid) for 4 h. Subsequently, testes were dehydrated, embedded in Technovit 7100 586 (Wehrheim, Germany), sectioned at 4 µm thickness and used for BrdU immunodetection, as 587 previously described [47,55]. The mitotic index or BrdU incorporation ratio of types Aund, Adiff 588 and Sertoli cells was determined by counting the BrdU-positive and BrdU-negative cells in a total 589 of 100 cells for the same cellular type, as described previously [47,48,55]. 590 For RT-qPCR, total RNA from testicular explants (n = 20 males) was extracted using the 591 same method described in section 2.3. The relative mRNA levels of gdnfa, gfrα1a, gfrα1b, amh 592 (anti-Müllerian hormone), igf3, fshr (follicle stimulating hormone receptor), pou5f3, dazl (deleted 593 in azoospermia-like) and sycp3l (synaptonemal complex protein 3) were evaluated. The mRNA 594 levels of the targets (Cts) were normalized by β-actin levels, expressed as relative values of basal 595 expression levels, according to the 2−(ΔΔCT) method. Primer sequences are indicated in table 1. 596 597 2.8. In Silico Analysis of Putative Regulatory Sequences Upstream Human 598 GDNF, Mouse Gdnf and Zebrafish Gdnfa 599 To retrieve the putative regulatory sequences of upstream human GDNF (NM_000514.4), 600 mouse Gdnf (NM_010275.3) and zebrafish gdnfa (NM_131732.2), the transcription start site 601 (TSS) was found in the Eukaryotic Promoter Database (EPD), and the promoter regulatory regions 602 (3’ to 5’) were prospected by means of the flanking regions (2000 bp) extracted from NCBI. The 603 cAMP response elements (CRE, four different sequences), the androgen receptor binding site (AR, 604 full and half sequences), several NF-kB-binding sites, N-Box, E-Box, TATA-Box and GC-Box 605 (Table S3) were prospected using sequences described in the literature [7,56,57,58,59,60,61]. 606 607 https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B52-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B55-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B48-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B55-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#sec2dot3-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#table_body_display_cells-11-01295-t001 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B7-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B56-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B57-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B58-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B59-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B60-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B61-cells-11-01295 PG-BGA 28 Campus de Botucatu Instituto de Biociências 2.9. Statistical Analyses 608 Data were initially checked for deviations from variance normality and homogeneity 609 through the Shapiro–Wilk and Bartlett’s tests, respectively. Significant differences between two 610 groups were identified using a paired Student’s t-test, at 5% probability. Comparisons of more 611 than two groups were performed with one-way ANOVA followed by Student–Newman–Keuls 612 test, at 5% probability. Graphpad Prism 7.0 (Graphpad Software, Inc., San Diego, CA, USA) was 613 used for the statistical analysis. 614 3. Results 615 3.1. Sequence Analyses, Phylogenetic Tree and Genomic Organization of 616 Zebrafish Gfrα1a and Gfrα1b 617 618 Sequence analysis revealed that both predicted zebrafish Gfrα1a and Gfrα1b have sequence 619 characteristics of Gfrα family members, such as the three cysteine-rich domains (D1-3), 28 620 cysteine residues (plus 2 in the terminal region), and two triplets (MLF and RRR) in the domain 621 D2 (Figure 1). Sequence alignment of zebrafish Gfrα1a and 1b with different GFRA1s (human 622 and rodent) revealed that the three cysteine-rich domains (D1, D2, D3) are highly conserved 623 among the species, highlighting, in particular, the conserved residues and motifs in the domain D2 624 critical for binding to GDNF and eliciting downstream cellular pathways (Figure 1). Sequence 625 analyses also demonstrated that zebrafish Gfrα1a and 1b have 67.1% identity to each other, and 626 zebrafish Gfrα1a showed a higher identity with mammalian GFRA1 (61.7%, 61.1% and 60.9% 627 similarity to human, rat and mouse GFRA1, respectively) than zebrafish Gfrα1b (57.4%, 57.2% 628 and 57% identity to human, rat and mouse GFRA1, respectively) (Figure 1). 629 Phylogenetic analysis further confirmed that both zebrafish Gfrα1a and Gfrα1b are related 630 to other fish Gfrα1a and Gfrα1b predicted sequences, respectively, and that these isoforms diverge 631 and form two separate fish-specific subclades (estimated posterior probability = 1) (Figure 2A). 632 On the other hand, the GFRA1 sequences from other vertebrates (mammals, birds, reptiles, 633 amphibians and Chondrichthyes) are clustered and form a separate clade to the fish Gfrα1 634 (estimated posterior probability = 0.851) (Figure 2A). 635 636 637 638 639 640 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f002 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f002 PG-BGA 29 Campus de Botucatu Instituto de Biociências 641 PG-BGA 30 Campus de Botucatu Instituto de Biociências Figure 2. (A) Phylogenetic analysis of GFRα1 predicted amino acid sequences across vertebrates. Zebrafish Gfrα1a 642 and 1b (both underlined) are clustered with other fish-specific Gfrα1a (yellow box) and Gfrα1b (green box) sequences, 643 respectively, forming two separate subclades. Note that the GFRA1 sequences from other vertebrates (mammals, 644 birds, reptiles, amphibians and Chondrichthyes) formed a separate clade (brown box). Branch values represent 645 posterior probabilities obtained by Bayesian analysis (see Table S1). (B, C) Genomic organization and synteny 646 comparisons among human GFRA1, rodents Gfrα1 and zebrafish gfrα1b (B) or zebrafish gfrα1a (C). The syntenic 647 regions were analyzed according to the alignment of the target genes and genomic annotation available in the GenBank 648 database (National Center for Biotechnology Information and Ensembl). 649 650 A cross-species comparison of chromosome neighboring genes revealed that both the 651 zebrafish gfrα1a- and gfrα1b- containing regions are syntenic to human GFRA1- and rodent 652 Gfrα1-containing regions (Figure 2B). This analysis also showed that the zebrafish gfrα1b gene 653 (chromosome 12, NC_007123.7) showed a larger group of syntenic genes (8 out of 14 genes 654 analyzed) when compared with zebrafish gfrα1a (chromosome 13, NC_007124.7) (2 out of 14 655 genes analyzed) (Figure 2C). 656 657 3.2. Expression Profiling in Zebrafish Testes and Identification of Gdnfa-, 658 Gfrα1a- and Gfrα1b-Expressing Cells 659 RT-qPCR analyses revealed that both ligands (gdnfa and gdnfb) and receptors (gfrα1a and gfrα1b) 660 were expressed in zebrafish testes, although with different numbers of amplification cycles (i.e., 661 values of cycle threshold (Ct)) (Figure 3). As the Ct for gdnfb is greater than 30, this value indicates 662 lower amounts for this target nucleic acid in zebrafish testes (Figure 3). 663 664 Figure 3. Number of amplification 665 cycles (cycle threshold (Ct)) for both 666 ligands (gdnfa and gdnfb) and 667 receptors (grfα1a and grfα1b) in 668 zebrafish testes. Bars represent the 669 mean ± SEM (n = 4) for each 670 transcript. 671 672 673 674 675 676 677 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f002 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f002 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f003 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f003 PG-BGA 31 Campus de Botucatu Instituto de Biociências Considering the lower amounts of gdnfb transcripts in zebrafish testes, we focused our 678 analysis on gdnfa. We tried to identify the cellular types expressing gdnfa mRNA in zebrafish 679 testes by employing in situ hybridization with a specific antisense cRNA probe (Table 1, Figure 680 S1) and RT-qPCR using RNA from isolated testicular cell populations (germ and somatic cell-681 enriched populations) (Figure 4). The first approach showed that gdnfa is expressed in germ cells 682 (Figure S1). Nevertheless, due to limited resolution, it was not possible to unravel whether the 683 signal was present or not in the Sertoli cells (Figure S1). This was attributed to the fact that 684 cytoplasmic extensions of Sertoli cells protrude towards the lumen of a cyst in between the germ 685 cells, making it difficult to accurately locate the signal. The precise identification of gdnfa 686 expression sites was then accomplished through RT-qPCR using testicular cell populations 687 obtained after the differential plating method (Figure 4A–E). In this approach, expression analysis 688 showed higher transcript levels for gdnfa in the germ cell-enriched population when compared to 689 the levels found in the total testicular cell suspension (Figure 4D,E). When analyzing the testicular 690 somatic cell population, we found that gdnfa mRNA levels decreased significantly as compared to 691 the levels observed in the germ cell fraction (Figure 4D,E). To confirm this result, we performed 692 proper controls using specific markers for germ (vasa and pou5f3) and Sertoli cells (igf3). For the 693 germ cells, we used vasa, which is a germ cell marker mostly expressed in early germ cells, 694 including types Aund, Adiff and B spermatogonia [47]. We showed that vasa was expressed in the 695 germ cell-enriched population, although with levels not significantly higher as compared to the 696 total cell suspension (Figure 4D,E). On the other hand, vasa was not expressed in the testicular 697 somatic cell fraction (Figure 4D,E). For pou5f3, a marker of types Aund, Adiff and B spermatogonia 698 (Souza, Doretto and Nóbrega (unpublished data)), we showed higher mRNA levels in the germ 699 cell-enriched fraction, but no expression in the somatic cell population (Figure 4D,E). For the 700 Sertoli cells, we used igf3, which is a growth factor produced by Sertoli cells [54]. igf3 was not 701 expressed in the germ cell population but it was detected in the somatic cell fraction with levels 702 comparable to those found in the total cell suspension (Figure 4D,E). 703 704 705 706 707 708 709 710 https://www.mdpi.com/2073-4409/11/8/1295#table_body_display_cells-11-01295-t001 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#B47-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#B54-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 PG-BGA 32 Campus de Botucatu Instituto de Biociências 711 712 PG-BGA 33 Campus de Botucatu Instituto de Biociências Figure 4. Differential plating method and expression analysis of the cellular enriched fractions. (A) Scheme showing 713 the steps of the differential plating method, according to Hinfray et al. [51]. Briefly, a total testicular cell suspension 714 was harvested (step A) in L-15 culture medium, and after 2 days of culture, only somatic cells (Sertoli cells, brown 715 triangular shapes; Leydig cells, yellow oval shapes) adhere to the bottom of the plate (step B), while germ cells (blue 716 shapes) remain floating or loosely attached to the bottom of the plate (step C). After washing steps, germ cells (floating 717 and weakly attached to the somatic cells) can be removed, leaving the adherent somatic cells at the bottom of the plate. 718 The firmly attached somatic cells can be obtained after extensive washing with trypsin. (B) Total testicular cell 719 suspension after 2 days of culture. Note the somatic adherent cells (SCs) with cytoplasm extensions towards different 720 germ cells (GC). (C) After washing, note that only somatic adherent cells (SCs) remain attached to the bottom of the 721 plate. Scale bars: 20 µm. (D,E) Gene expression analysis of isolated zebrafish testicular cell populations: total cell 722 suspension (black bar), germ cell-enriched population (white bar) and testicular somatic cells (hatched bar). Cells 723 were obtained from three independent experiments. Bars represent relative mRNA levels of target genes expressed as 724 mean ± SEM; different letters indicate significant differences between the cell populations (one-way ANOVA 725 followed by the Student–Newman–Keuls test). (E) Heat map illustrating the relative mRNA levels of pou5f3, vasa, 726 igf3 gdnfa, gfrα1a and gfrα1b according to different cell populations. Data shown are log2 values (relative 727 quantification) relative to the average expression. Each colored cell in the heat map represents the standardized relative 728 gene expression value for each sample. Genes (rows) are hierarchically clustered using Pearson correlation and the 729 distance metric. The higher expression values are displayed in blue, moderate expression values in shades of white 730 (light blue and light red) and lower expression values in red. 731 732 We also expressed our data in a heat map and genes were hierarchically clustered using 733 Pearson correlation and the distance metric (Figure 4E). We showed through this analysis that 734 genes such as vasa, pou5f3 and gdnfa were hierarchically clustered in the germ cell fraction and 735 separated from igf3 and gfrα1b, which were clustered in the somatic cell fraction (Figure 4E). 736 gfrα1a was expressed in both germ and somatic cell fractions (Figure 4D,E). 737 738 3.3. Localization of Gfrα1a Protein in Zebrafish Testis 739 Gfrα1a was detected in all generations of zebrafish spermatogonia, although the staining pattern 740 varied among them according to the developmental stage (Figure 5A,C–E). The Gfrα1a signal was 741 finely dispersed in the cell surface and cytoplasm of type Aund spermatogonia (Figure 5C) and later 742 became more aggregated, forming intensely stained spots in type Adiff spermatogonia (Figure 5D). 743 In type B spermatogonia, the Gfrα1a signal became finely dispersed again (Figure 5E) and 744 gradually decreased as the number of spermatogonia B increased within the cyst until it became 745 undetectable in the meiotic and post-meiotic cysts (Figure 5A). Furthermore, Gfrα1a was also 746 found in Sertoli cells contacting germ cells at different stages of development (Figure 5A,B (inset) 747 and Figure S1F,G). This result was also confirmed by the expression of both gfrα1a and gfrα1b in 748 https://www.mdpi.com/2073-4409/11/8/1295#B51-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 PG-BGA 34 Campus de Botucatu Instituto de Biociências the somatic cell-enriched population (Figure 4D). Altogether, these two bodies of evidence support 749 the presence of Gfrα1a and 1b in zebrafish Sertoli cells. The specificity of the antibody (anti-750 zebrafish Gfrα1a) was confirmed by immunoblots (Figure 5F) and control sections either by using 751 a preadsorbed antibody with the corresponding peptide or omitting the primary antibody (Figure 752 S2). It is important to mention that the immunofluorescence signal should not be limited to Gfrα1a, 753 since the antibody could potentially recognize part of zebrafish Gfrα1b (see the blue line in Figure 754 1). 755 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f004 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f005 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f001 PG-BGA 35 Campus de Botucatu Instituto de Biociências Figure 5. Cellular localization of Gfrα1a in zebrafish testis. (A–E) Immunofluorescence for Gfrα1a (green—A; red—756 B–E) in testis sections of sexually mature zebrafish. The spermatogonial generations, including type A 757 undifferentiated spermatogonia (Aund), type A differentiated spermatogonia (Adiff) and type B spermatogonia (SPG 758 B), were immunoreactive to Gfra1a, although staining patterns among them varied according to developmental stage. 759 The signal was not found in spermatocytes (SPCs), spermatids (SPTs) and spermatozoa (SPZ). Note that Sertoli cells 760 (SCs) contacting germ cells at different stages of development were also immunoreactive to Gfrα1a. Cell nuclei were 761 counterstained with propidium iodide (A) or Hoechst (B–E). Scale bars: 15 µm. (F) Gfrα1a (approximately 52 kDa 762 PG-BGA 36 Campus de Botucatu Instituto de Biociências (kilodaltons)) immunoblots of whole testes with (+) or without (−) preadsorbed antibodies, confirming the presence 763 of the protein in the zebrafish testes and antibody specificity. 764 765 3.4. Three-Dimensional Model for Predicting the Interaction between rhGDNF 766 and Zebrafish Gfrα1a 767 768 In this study, we used a recombinant human hormone because the recombinant zebrafish 769 Gdnf is not commercially available. Therefore, to investigate whether rhGDNF could have effects 770 on zebrafish spermatogenesis, we first generated a 3D structure model to predict the possible 771 interaction sites between human GDNF and zebrafish Gfrα1a (Figure 6A, box 2, box 3). The 3D 772 structure (hetero-2-2-mer) was built according to the homology of the 4ux8.1 template and showed 773 a GMQE value of 0.63 with 74% of identity and a resolution of 24Å (method: Electron 774 Microscopy) when compared to human GDNF-GFRA1 interaction (merged in the 3D structure) 775 (Figure 6A, box 2, box 3). Moreover, the predictive model demonstrated that 89.8% of the amino 776 acid residues were in the most favorable regions, 7% of residues were situated in allowed regions 777 (~2% expected) and 3.1% in the outlier regions according to Ramachandran plots. The 3D 778 structures of the hetero-2-2-mer (GDNF-zebrafish Gfrα1a) were based on the homology modeling 779 templates and are shown in Figure 6A (box 2, box 3). More detailed information regarding the 780 predictive interaction model between GDNF and zebrafish Gfrα1a can be found in the 781 Supplementary Materials (Figure S3, Video S1). In agreement with the 3D model, the alignment 782 of zebrafish Gdnfa with rhGDNF showed conserved regions, particularly in the binding sites to 783 human GFRA1 or zebrafish Gfrα1a (Figure 6B). 784 785 786 787 788 789 790 791 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f006 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f006 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f006 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f006 PG-BGA 37 Campus de Botucatu Instituto de Biociências 792 Figure 6. A 3D model to predict the interaction between rhGDNF and zebrafish Gfrα1a. (A) Box 1 depicts the 793 molecular components of the complex GDNF-GFRα1-RET. Boxes 2 and 3 show the predictive 3D model (template 794 4ux8.1) in which the structural similarities between zebrafish Gfrα1a and human GFRA1 are represented by orange 795 and purple coloring and the identity of the structure formed at the binding sites is indicated in red. In box 2, green is 796 used to indicate the conserved amino acid sequences between zebrafish Gfrα1a and human GFRA1 and blue indicates 797 the GNDF protein. In box 3, we highlighted the interaction sites between human GDNF and zebrafish Gfrα1a/human 798 GFRA1. (B) Alignment of zebrafish Gdnfa with rhGDNF. The blue lines indicate the conserved binding sites to 799 zebrafish Gfrα1a or human GFRA1. 800 PG-BGA 38 Campus de Botucatu Instituto de Biociências 3.5. Biological Effects of rhGDNF 801 802 To investigate the roles of Gdnf in zebrafish spermatogenesis, we first examined whether 803 rhGDNF could affect germ cell composition and cellular proliferation, using a previously 804 established primary testis tissue culture system (Figure 7A–D). The results showed that rhGDNF 805 (100 ng/mL) increased the abundance of types Aund and Adiff spermatogonia as compared to basal 806 conditions (Figure 7C). These data are also consistent with the proliferation activity of these cells, 807 showing that treatment with rhGDNF (100 ng/mL) augmented the mitotic index of both types of 808 spermatogonia (Aund and Adiff) as compared to their basal mitotic index (approximately 1,5-fold 809 increase for Aund and Adiff, with p < 0.001 and p < 0.01, respectively) (Figure 7A,B,D). Moreover, 810 histomorphometrical analysis showed that rhGDNF decreased the frequency of type B 811 spermatogonia, whereas no effects were observed for meiotic and post-meiotic germ cells (Figure 812 7C). In this study, we also quantified Sertoli cell proliferation (Figure 7E), reasoning that change 813 in the proliferation of Sertoli cells associated with types Aund or Adiff spermatogonia would indicate 814 the creation of new niche space or support the development of differentiating spermatogonial cysts, 815 respectively [62]. Our results then demonstrated that treatment with rhGDNF stimulated Sertoli 816 cell proliferation (1,5-fold increase, p < 0.050), particularly if the Sertoli cells associated with 817 proliferating types Aund and Adiff spermatogonia (Figure 7E). 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 https://www.mdpi.com/2073-4409/11/8/1295#B62-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f007 PG-BGA 39 Campus de Botucatu Instituto de Biociências 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 Figure 7. Effects of Gdnf on germ cell composition and cellular proliferation, using a previously established primary 862 testis tissue culture system. (A, B) BrdU immunodetection from zebrafish testicular explants incubated for 7 days in 863 the absence (Basal) or presence of rhGDNF (100 ng/mL), demonstrating a higher proliferation activity for type A 864 undifferentiated spermatogonia (Aund) and type A differentiated spermatogonia (Adiff) in the presence of rhGDNF. (C) 865 Frequency of different germ cell cysts after 7 days of incubation in the absence (Basal) or presence of rhGDNF (100 866 ng/mL). Types Aund, Adiff and B spermatogonia (SPG B), spermatocytes (SPCs) and spermatids (SPTs) were identified 867 according to morphological characteristics, as described by Leal and collaborators [55]. (D) Mitotic indices of type 868 https://www.mdpi.com/2073-4409/11/8/1295#B55-cells-11-01295 PG-BGA 40 Campus de Botucatu Instituto de Biociências Aund and Adiff spermatogonia after incubation in the absence (Basal) or presence of rhGDNF (100 ng/mL) for 7 days. 869 (E) Mitotic indices of Sertoli cells in association with BrdU-negative or BrdU-positive type Aund and Adiff 870 spermatogonia in the absence (Basal) or presence of rhGDNF (100 ng/mL) for 7 days. Sertoli cells were identified 871 according to morphological characteristics, as described previously [55]. In fish, Sertoli cells (SCs) have a triangular 872 nuclear shape, dark chromatin and usually they appear surrounding spermatogenic cysts, as shown in Figure S1. Bars 873 represent the mean ± SEM (n = 10). Paired t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 15 µm. 874 875 In order to elucidate the molecular mechanisms mediated by rhGDNF on basal or Fsh-876 induced spermatogenesis, we performed gene expression analyses of selected genes related to 877 Gdnf signaling (gdnfa, gfrα1a and gfrα1b), Sertoli cell growth factors (igf3 and amh), Fsh 878 signaling (fshr) and germ cell markers (undifferentiated spermatogonia—pou5f3; differentiated 879 spermatogonia and preleptotene spermatocytes—dazl; and primary spermatocytes—scyp3l) 880 (Figure 8). 881 882 https://www.mdpi.com/2073-4409/11/8/1295#B55-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#app1-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f008 PG-BGA 41 Campus de Botucatu Instituto de Biociências Figure 8. Relative mRNA levels of genes related to Gdnf signaling (gdnfa, gfrα1a and gfrα1b) (A–C), Sertoli cell 883 growth factors (igf3 and amh) (D–E), Fsh signaling (fshr) (F) and germ cell markers (undifferentiated 884 spermatogonia—pou5f3 (G); differentiated spermatogonia and preleptotene spermatocytes—dazl (H); and primary 885 spermatocytes—scyp3l (I)). Testicular explants were cultivated for 7 days with rhGDNF, rzfFsh or both (rhGDNF + 886 rzfFsh). The relative mRNA levels were normalized with the β-actin levels. Bars represent the mean ± SEM (n = 20). 887 One-way ANOVA followed by the Student–Newman–Keuls test, in which different letters denote significant 888 differences (p < 0.05) among treatment conditions. 889 890 RT-qPCR analysis revealed that rhGDNF increased the transcript levels of gdnfa and 891 gfrα1a, whereas gfrα1b mRNA levels remained unaltered when compared with basal condition 892 levels (Figure 8A–C). The transcript abundance for the other genes (Sertoli cell growth factors, 893 Fsh signaling and germ cell markers) did not change following rhGDNF treatment (Figure 8D–I). 894 We further investigated whether rhGDNF could affect the Fsh-induced changes in testicular gene 895 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f008 https://www.mdpi.com/2073-4409/11/8/1295#fig_body_display_cells-11-01295-f008 PG-BGA 42 Campus de Botucatu Instituto de Biociências expression, since Fsh is considered the major endocrine player regulating the zebrafish 896 spermatogonial phase [48,54,63]. We first showed that Fsh did not modulate the transcript levels 897 of gdnfa, gfrα1a or gfrα1b in the zebrafish testes (Figure 8A–C). However, Fsh was able to nullify 898 the rhGDNF-increased gdnfa and gfrα1a mRNA levels following co-treatment (Figure 8A, B). 899 With respect to Sertoli cell growth factors, we demonstrated that rhGDNF did not change Fsh-900 mediated expression on igf3 (Figure 8D) or amh mRNA levels (Figure 8E). As expected, and in 901 agreement with previous studies [54,64], Fsh increased igf3 mRNA levels (Figure 8D) and down-902 regulated amh transcription (Figure 8E). The other evaluated genes were not responsive to Fsh or 903 co-treatment (Figure 8F–I). Nevertheless, it is worth mentioning that transcript levels of fshr, 904 pou5f3 and dazl were significantly higher following rhGDNF treatment than in the co-treatment 905 with Fsh (Figure 8F–H). 906 907 3.6. In Silico Analysis of Putative Regulatory Sequences Upstream of Human 908 GDNF, Mouse Gdnf and Zebrafish Gdnfa 909 To support our expression analysis, we investigated the putative regulatory sequences 910 upstream of the transcriptional start site (TSS) of human GDNF (NM_000514.4), mouse Gdnf 911 (NM_010275.3) and zebrafish gdnfa (NM_131732.2) (Figure 9). The in silico analysis showed 912 three different types of cAMP response elements (CRE), several N-box and E-box motifs, one NF-913 kB binding site and a TATA-Box within the 2000 bp upstream of human GDNF (Figure 9, Table 914 S2). The upstream sequence of the Gdnf mouse gene showed similar regulatory binding sites to 915 the human GDNF (Figure 9, Table S2). For zebrafish, we predicted a non-canonical TATA-Box, 916 one CRE close to a GC-Box, one N-Box, four E-Boxes and two androgen receptor (AR) half 917 binding sites within the 2000 bp upstream of gdnfa (Figure 9, Table S2). 918 https://www.mdpi.com/2073-4409/11/8/1295#B48-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B54-cells-11-01295 https://www.mdpi.com/2073-4409/11/8/1295#B63-cells-11-01295 https://www.mdpi.com/20