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1 Nutrient restriction, inducer of yeast meiosis, induces meiotic initiation in mammals 2 3 Xiaoyu Zhang, Sumedha Gunewardena, Ning Wang* 4 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 5 3901 Rainbow Blvd, Kansas City, KS 66160, USA. 6 *Corresponding author. E-mail: [email protected] 7 8 ABSTRACT 9 From yeasts to mammals, the molecular machinery and chromosome structures carrying out 10 meiosis are frequently conserved. However, the signal to initiate meiosis appears divergent: 11 while nutrient restriction induces meiosis in the yeast system, retinoic acid (RA), a chordate 12 morphogen, is necessary but not sufficient to induce meiotic initiation in mammalian germ cells 13 via its target, Stra8. Here, using cultured mouse male germline stem cells without the support of 14 gonadal somatic cells, we show that nutrient restriction in combination with RA robustly induces 15 Spo11-dependent meiotic DNA double strand breaks (DSBs) and Stra8-dependent meiotic gene 16 programs recapitulating those of early meiosis in vivo. Moreover, a distinct network of 11 17 nutrient restriction-upregulated transcription factor genes was identified, whose expression does 18 not require RA and is associated with early meiosis in vivo. Thus, our study proposes a 19 conserved model, in which nutrient restriction induces meiotic initiation by upregulating 20 transcriptional factors for meiotic gene programs, and provides an in vitro platform to derive 21 haploid gametes in culture. 22 23 24 One Sentence Summary: nutrient restriction synergizes with retinoic acid to induce 25 mammalian meiotic initiation 26 27 28 29 30 31

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32 Sexual reproduction depends on the formation of haploid gametes through meiosis, which 33 exchanges replicated parental chromosomes through homologous recombination during meiotic 34 prophase, followed by two rounds of successive divisions to generate haploid gametes carrying 35 novel genetic constitution (1). Meiotic initiation requires the activation of an array of meiosis- 36 specific genes that implement the chromosomal events during meiosis prophase, including 37 homologous pairing, synapsis, and recombination (2). From yeasts to mammals, meiosis-specific 38 chromosome structures exhibit remarkable evolution conservation, including synapsis formation 39 mediated by the assembly of the synaptonemal complex (SC), homologous recombination 40 through the formation and the subsequent repair of meiotic DNA double-strand breaks (DSBs). 41 Many genes underlying these events are often evolutionarily-related or -conserved. For instances, 42 Spo11 encodes a DNA topoisomerase-like enzyme that catalyzes meiotic DSB formation. Dmc1 43 encodes a meiotic recombinase that repairs DSBs by searching for allelic DNA sequences on the 44 homologous chromatids. Hormad genes encode meiosis-specific chromosome factors (Hop1 in 45 yeasts and Hormad1/2 in mammals) that are critical for synapsis and DSB formation and repair. 46 47 Despite these evolutionary conservations in meiotic genes and structures, the overarching signal 48 to trigger meiosis appears to be divergent. In yeasts, transition from mitotic to meiotic cell cycles 49 is induced by nutrient restriction. Subsequently, nutrient-sensing pathway induces the expression 50 of inducer of meiosis 1 (IME1), which encodes a single master transcriptional activator for 51 meiotic genes, including Spo11, Dmc1, and Hop1 (3, 4). In mammalian germ cells, meiotic 52 initiation requires retinoic acid (RA), an activate metabolite of vitamin A. RA is a morphogen 53 essential for growth and development in chordate animals (5). In female oogenesis, RA is 54 synthesized primarily in the mesonephric ducts in embryonic ovaries (6). In male 55 spermatogenesis, RA is produced by meiotic and somatic cells in testes (7). RA induces meiosis 56 primarily by activating stimulated by retinoic acid gene 8 (Stra8) (8, 9). Although STRA8- 57 mediated transcriptional activation of meiotic genes by itself or with other protein has been 58 reported (10, 11), neither RA nor STRA8 is sufficient to induce meiosis, suggesting that other 59 signal(s) works with RA signaling to induce mammalian meiotic initiation. 60 61 Our recent work in Stra8-deficient mice reveals that an autophagy-inducing signal is engaged on 62 meiosis-initiating germ cells (12). Interestingly, nutrient restriction, the aforementioned inducer

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63 of yeast meiosis, is perhaps the most potent inducer of autophagy (13). Thus, we asked whether 64 nutrient restriction might have a conserved role in inducing meiotic initiation in mammalian 65 germ cells. To test this, we established primary culture for male mammalian germline stem (GS) 66 cells by using neonatal mouse testicular cells in C57BL/6 X DBA/2 F1 hybrid background (14) 67 (fig. S1A; see below for the whole transcriptome profile of the cultured GS cells). Nutrient 68 restriction was applied to cultures by adding 90% Earle's Balanced Salt Solution (EBSS) to the 69 complete GS culture medium. 70 71 Co-treatment of nutrient restriction and RA (referred to as “NRRA”) for 2 days was sufficient to 72 trigger a distinct morphology of cellular enlargement in GS cultures, suggesting cell 73 differentiation (Fig. 1A; fig. S1B). NRRA induced a significant activation of essential meiotic 74 genes, including Spo11, Dmc1, and Sycp3 (Fig. 1B). Spo11 and Dmc1 encodes enzymes that 75 specifically catalyze meiotic DNA double-strand break (DSB) formation and repair (15, 16). 76 Sycp3 encodes a lateral element of synaptonemal complex that forms between two meiotic 77 homologous chromosomes (17). Consistently, phosphorylated histone AX (γH2AX) shows that 78 DNA DSBs were most profoundly formed in NRRA-treated culture (Fig. 1C; fig. S1C). These 79 effects were not observed in cultures treated with RA or NR alone (Fig. 1A-C). Similar effects of 80 NRRA in meiotic gene activation were observed in F9 premeiotic cells (fig. S2). Importantly, the 81 meiotic origin of these DSBs was confirmed by DMC1 staining (Fig. 1D). In addition, cultured 82 GS cells generated from Spo11-deficient mice lack DMC1 foci upon NRRA treatment, 83 constituting genetic evidence that meiotic DSB formation in vitro requires Spo11 (Fig. 1D; see 84 below for the transcriptome profiles of Spo11-deficient culture). In addition, cultured GS cells 85 exhibited rapid loss of undifferentiation spermatogonia markers, Gfra1 and E-cadherin (fig. S1D). 86 87 Transcriptomic analysis shows that NRRA induced a gene expression pattern distinct from the 88 treatment of RA or NR alone (Fig. 1E; fig. S3; table S1). Four clusters of gene sets were 89 identified by unsupervised hierarchical clustering (UHC) (table S2). Notably, genes in Cluster 2, 90 which appear to be genes upregulated by RA and NRRA, are enriched with genes bearing gene 91 ontology (GO) functional terms related to meiosis (Fig. 1E and fig. S4). In contrast, Cluster 1, 3, 92 or 4 is enriched with genes for “mitochondrion organization” (Cluster 1, nutrient restriction- 93 downregulated genes), “regulation of innate immune response” (Cluster 3, nutrient restriction-

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94 upregulated genes), “ncRNA metabolic process” (Cluster 4, RA-downregulated genes) (fig. S4). 95 Consistently, nonparametric and unsupervised gene set variation analysis (GSVA) and hallmark 96 gene set analysis revealed stronger positive correlations of meiosis-related pathways (male 97 meiotic nuclear division, chromosome organization in meiotic cell cycle, synaptonemal structure) 98 in genes upregulated by NRRA than those upregulated by RA (Fig. 1F and G, fig. S5) (table 99 S3). These data together indicate that nutrient restriction is required to work with RA to induce 100 meiotic initiation in vitro. 101 102 To examine whether the effect of NRRA in inducing meiotic initiation depends on the genetic 103 background of the cultured GS cells, we established cultured GS cells using testes from neonatal 104 CD1 inbred mice. CD1 cultured GS cells exhibited comparable transcriptomic changes including 105 activation of meiotic genes in response to NRRA treatment (fig. S6), suggesting that the 106 sufficient role of NRRA in meiotic initiation is independent of genetic backgrounds. 107 108 To dissect the role of nutrient restriction in meiotic gene programs, we assembled a set of 193 109 meiosis genes by combining two collections of genes that were previously found to be associated 110 with mammalian early meiosis during mouse (2) and human meiosis (18) spermatogenesis (fig. 111 S7). Unsupervised UHC divided 165 differentially expressed genes (DEGs) into five major 112 clusters (Fig. 1H; table S4). Nutrient restriction appears to play four roles on the expression of 113 key meiotic genes by: 1) inducing a subset of meiotic gene expression that was not regulated by 114 RA, such as Rad21l; 2) initiating the activation of a subset of meiotic genes, which was further 115 enhanced by RA, such as Zfp541; 3) synergizing with RA to induce the activation of a subset of 116 meiosis gene expressions, such as Sycp1, Sycp3, Mei1, Msh5, and Stag3; and 4) augmenting the 117 expression of a subset of meiotic genes induced by RA, including Dmc1, Smc1b, Ugt8a, Stag3, 118 Hormad1, as well as Gm4969, which encodes MEIOSIN, a transcriptional cofactor for STRA8 119 required for meiotic prophase program (11). Notably, many meiotic genes whose expression 120 depends on STRA8 require nutrient restriction (Fig. 1H). Together, these data suggest crucial 121 roles of nutrient restriction by itself or by synergizing with RA in inducing meiotic gene 122 expression; in the absence of nutrient restriction, RA alone is not sufficient (Fig. 1H; see the 123 effect of in vivo RA treatment on meiotic gene expression despite Stra8 activation in fig. S) (19). 124

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125 To examine NRRA-induced meiotic initiation and its progression potential at the single cell 126 resolution, we isolated single cells from cultured GS cells treated with NRRA for 2 days to 127 induce meiotic initiation, followed by media that support progression for an additional 1 and 2 128 days . Cultured GS cells treated with normal medium for 4 days were used as control. Single cell 129 RNA-sequencing (scRNA-seq) was then performed using the 10x Genomics platform (Fig. 2A). 130 A total of 25,000 cells were sequenced from these samples. Using stringent quality control, 131 18,088 cells were selected for further analysis (fig. S9). All of the cells were pooled to perform 132 clustering analysis, which revealed four major cell clusters based on their distinct gene 133 expression patterns (Fig. 2B and C). These four cluster were subsequently annotated by using 134 known marker genes (fig. S10). Cluster 0 appears to be undifferentiated spermatogonia (e.g., 135 Gfra1, Evt5). Clusters 1, 2, and 3 appear to be differentiating spermatogonia/pre- and meiotic 136 spermatocytes at progressively advanced meiotic stages with upregulated expression of 137 spermatogonial differentiation (e.g., Sohlh1) and meiotic genes (e.g., Dmc1). Cluster 4 is mostly 138 feeder cells (mouse embryonic fibroblast or MEF) due to the expression of fibroblast genes 139 (s100a4). Progressive upregulation of meiotic genes and downregulation of undifferentiated 140 spermatogonia genes on each time points were confirmed by qRT-PCR analysis (fig. S11). 141 142 Analysis of the distribution of the different cell clusters at each time point demonstrate a 143 dramatic transition of cell populations (Fig. 2D). Analysis of GO functional terms for DEGs 144 revealed rapid changes in their cellular processes (Fig. 2E). Cluster 0 is enriched in genes 145 involved in regulation of cellular amide metabolic process (e.g., Sox4), stem cell division (e.g., 146 Zbtb16, Etv5), etc (fig. S12A, table S5). Cluster 1 is enriched in genes involved in the regulation 147 of gene silencing (e.g., Hist1h1e), mitochondrial electron transport, NADH-ubiquinone (e.g., 148 Park7), retinoic acid and metabolic process/response (e.g., Stra8), etc (fig. S12B). Cluster 2 is 149 enriched in genes involved in reproduction in multicellular organisms (e.g., Sycp3), sperm-egg 150 recognition (e.g., Ly6k), etc (fig. S12C). Cluster 3 is enriched in genes for meiotic cell cycle (e.g., 151 Prdm9), cytoplasmic translation (e.g., Rpl18a), translation initiation (e.g., Eif1), chromosome 152 condensation (e.g., Dnajc19), etc (fig. S12D). 153 154 Importantly, cells from Cluster 1 to Cluster 3 exhibit a progressive and coordinated upregulation 155 of meiotic genes bearing GO terms for mouse DNA double-strand break formation, mouse

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156 histone variants, meiotic nuclear division, mouse synapsis (fig. S13). Notably, meiotic genes that 157 are fully (Dmc1, Hormad1, Mei1, M1ap) or partially (Smc1b, Stag3, Sycp1, Sycp2, Sycp3, Ugt8a, 158 Meioc) (Fig. 2F; fig. S14) dependent on STRA8 expression displayed progressive upregulation 159 from Cluster 1 to Cluster 3. Since activation of RA signaling with Stra8 expression is not 160 sufficient to induce their expression in vitro and in vivo (fig. S8) (19), this data suggests that 161 nutrient restriction is required to work with RA to meiotic gene programs. 162 163 To assess the relationship between NRRA-induced meiotic initiation and progression in vitro 164 with those during in vivo meiosis during spermatogenesis, we performed principal component 165 analysis (PCA) analysis with a published scRNA-seq database (20) (fig. S15), which shows that 166 the transcriptional profiles of Clusters 0 to 3 correlate with meiotic initiation (leptotene, the first 167 stage of meiotic prophase) and progression (zygotene and early pachytene) during in vivo 168 spermatogenesis (20) (Fig. 2G). Subsequently, hclust and differential gene correlation analysis 169 (DGCA) demonstrate independently that cell population in Cluster 0 from in vitro meiosis 170 correlates with undifferentiated spermatogonia during in vivo spermatogenesis, cell population in 171 Cluster 1 with differentiating spermatogonia/preleptotene spermatocytes, cell population in 172 Cluster 2 with leptotene/zygotene spermatocytes, and cell population in Cluster 3 with early 173 pachytene spermatocytes (Fig. 2H and I). A hallmark event that occurs in male germ cells 174 during pachytene stage is meiotic sex chromosome inactivation (MSCI) (21). Similarly, we 175 observed a progressive decline in sex chromosome gene transcription from Cluster 0 to Cluster 3, 176 supporting that cells in Cluster 3 have reached to a pachytene-like stage (Fig. 2J). 177 178 Moreover, past study indicates that early meiosis during in vivo spermatogenesis follows a step- 179 wise pattern without lineage branching (20) (Fig. 2K). To examine the transcriptional dynamics 180 of NRRA-induced in vitro meiosis, we performed pseudo-time analysis, which shows that the 181 projected timeline recapitulated early meiosis during in vivo spermatogenesis (Fig. 2K, S16-18). 182 The pseudo-time indicates that Cluster 0 is mainly at the start of the projected timeline trajectory, 183 that Clusters 1, followed by Cluster 2, is positioned in the middle, and that Cluster 3 is at the end 184 (Fig. 2K, S16-18). Consistently, expression of genes specific to undifferentiated spermatogonia 185 was located preferentially at the beginning of the trajectory, while expression of genes 186 functionally involved in many essential meiotic programs (e.g., cohesion, DNA DSB formation,

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187 chromosome segregation) was located preferentially towards to the end of the trajectory, which 188 further supports the validity of the analysis (fig. S17 and S18). 189 190 Stra8 is a best characterized gatekeeper of meiotic initiation in vertebrates (8). To examine 191 whether NRRA-induced meiotic initiation meets this genetic requirement, we generated primary 192 GS cell culture using Stra8-deficient mice. Consistent with the in vivo role of STRA8 in meiotic 193 DSB formation (8, 22) (fig. S19), Stra8-deficient culture did not exhibit DMC1 foci upon NRRA 194 treatment (Fig. 3A), suggesting that NRRA-induced in vitro meiotic DSB formation requires 195 Stra8 (Fig. 3A). Moreover, RNA-seq analysis shows that WT, Stra8-deficient, and Spo11- 196 deficient cultures exhibited similar transcriptome profiles in normal medium (Fig. 3B). However, 197 upon parallel NRRA treatment, Stra8-deficient culture underwent a unique transcriptomic 198 change from WT and Spo11-deficient cultures, in that Stra8-deficient culture fails to upregulate a 199 cluster of genes, which is enriched for meiosis-related GO terms (meiotic cell cycle, meiosis I, 200 synapsis) (Fig. 3B; Table S6) (2). This is consistent with the essential role of STRA8 in 201 activating the gene programs of meiotic prophase, and indicates that the lack of meiotic DSBs in 202 Stra8-deficient and Spo11-deficient cultures resulted from discrete mechanisms (SPO11 is an 203 enzyme that catalyze meiotic DSBs). GSVA analysis shows that indeed STRA8 sits at the 204 foundation of NRRA-induced meiotic initiation: in contrast to WT and Spo11-deficient cultures, 205 despite activation of retinoid acid receptor signaling pathway, gametogenesis- and meiosis- 206 related pathways were not activated in Stra8-deficient culture upon NRRA treatment (Fig. 3C). 207 Moreover, STRA8-dependent meiotic genes (Sycp3, Mei1, Dmc1, Stag3, Sycp2) were only 208 upregulated in WT, but not Stra8-deficient, culture (Fig. 3D; fig. S20; Table S7). And the genes 209 only upregulated in Stra8-deficient culture bears no meiosis-related GO terms, which is 210 consistent with the robust meiotic initiation arrest phenotype of Stra8-deficient germ cells in vivo. 211 Interestingly, our system reveals that many genes associated with undifferentiated spermatogonia 212 were not downregulated in Stra8-deficient culture (e.g., Pou5f1, Etv5), which is in line with the 213 role for STRA8 in promoting spermatogonial differentiation (7) (Fig. 3E; fig. S21; Table S7). 214 215 Our results show for the first time that Stra8-dependent and Spo11-dependent meiotic DSBs are 216 induced in vitro (Figs. 1D and 3A). To further examine the processing of meiotic DSB formed in 217 vitro, our spread analysis shows that, similar to in vivo meiosis, sing-strand DNA (ssDNA)-

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218 binding proteins, SPATA22 and MEIOB (23, 24), were progressively recruited onto the meiotic 219 chromosomes from a leptotene-like to an early pachytene-like stage, suggesting that meiotic 220 DSBs formed in vitro were resected into ssDNA before recruiting meiotic recombinases RAD51 221 and DMC1 for repair (fig. S22A). Quantification show that similar numbers of SPATA22, 222 MEIOB, RAD51, and DMC1 foci were detected on meiotic chromosomes per germ cell between 223 in vivo meiosis and in vitro meiosis (fig. S22B and C). In addition, histone methyltransferase 224 PRDM9 directs meiotic DSBs to be distributed to recombination hotspots by (25, 26). PRDM9 225 expression is induced by NRRA in cultures at both mRNA and protein levels (Fig. 2; fig. S23A). 226 Consistently, chromatin immunoprecipitation (ChIP) assay for DMC1-associated ssDNA 227 fragments, a direct method to detect recombination hotspots (27), revealed that the DSBs formed 228 in vitro upon NRRA treatment were mapped to the strong hotspots of meiotic recombination in 229 vivo (28) (fig. S23B). 230 231 To examine the mechanism of nutrient restriction-induced meiotic initiation, we identified that 232 nutrient restriction upregulated 120 transcription factor (TF) genes, whose expression does not 233 require RA (Fig. 4A and B). To identify those potentially involved in regulating the meiotic 234 gene programs, we examined the expression of these genes in the transcriptomic database for 235 mouse spermatogenesis and found that 30 of them are expressed before meiotic prophase (Fig. 236 4B and C) (20). Then, we further investigated their correlation with meiotic gene expression in 237 the Genotype-Tissue Expression database (GTEx) (29). Using Dmc1, Sycp3, Hormad1 as three 238 representatives, we found that 11 of them shows strong (Pearson correlation > 0.6) or moderate 239 correlation (Pearson correlation > 0.4) with meiotic gene expression (Fig. 4B and D, fig. S24). 240 We further confirmed that the expression of these 11 TFs in vivo does not require RA (fig. S25). 241 Notably, these 11 TFs include Sohlh1 and Sox3, two characterized TFs implicated in early 242 meiosis and spermatogenesis. Sohlh1 is basic helix-loop-helix (bHLH) transcription factor, 243 whose deletion results in many tubules lacking meiotic spermatocytes (30). Recently, Sohlh1 is 244 shown to meiotic gene expression ( Sycp1, Sycp3) by directly binding to their proximal promoters 245 (31). Sox3 is expressed exclusively in spermatogonia committed to differentiation (32), and loss 246 of Sox3 impairs early meiosis (33). Thus, these data suggest that nutrient restriction induces a 247 distinct network of RA signaling-independent TFs to activate meiotic gene program. 248

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249 Nutrient restriction is a metabolic cue. Our scRNA-seq revealed that, while genes involved in the 250 glycolytic pathways were downregulated, genes involved in mitochondrial function pathways 251 were upregulated during NRRA-induced in vitro meiotic initiation and progression (Cluster 0 to 252 Cluster 3), which mirrors the transition from glycolysis to mitochondrial biogenesis and 253 oxidative phosphorylation during in vivo spermatogonial differentiation (34) (fig. S26). Together, 254 our data at the transcriptomic, cytologic, mechanistic, and metabolic levels suggest that nutrient 255 restriction in combination with RA induces meiotic initiation that faithfully recapitulates that 256 during in vivo spermatogenesis. Interestingly, unlike the nutrient-rich basal compartment where 257 mitotic spermatogonia reside, the apical compartment of the mammalian seminiferous tubule, 258 where meiosis takes place, is a nutrient-restricted microenvironment (i.e., low in glucose and 259 most amino acids) created by the blood-testis barrier (BTB) (35). Thus, nutrient restriction as a 260 physiological stimulus for meiotic initiation that requires BTB function warrants further 261 characterization. Our study provides an in vitro platform to study meiotic initiation and to 262 facilitate production of haploid gametes in culture. 263 264 Reference: 265 1. M. A. Handel, J. C. Schimenti, Genetics of mammalian meiosis: regulation, dynamics 266 and impact on fertility. Nat Rev Genet 11, 124-136 (2010). 267 2. Y. Q. Soh et al., A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary. 268 PLoS Genet 11, e1005531 (2015). 269 3. Y. Kassir, D. Granot, G. Simchen, IME1, a positive regulator gene of meiosis in S. 270 cerevisiae. Cell 52, 853-862 (1988). 271 4. H. E. Smith, S. S. Su, L. Neigeborn, S. E. Driscoll, A. P. Mitchell, Role of IME1 272 expression in regulation of meiosis in Saccharomyces cerevisiae. Mol Cell Biol 10, 6103- 273 6113 (1990). 274 5. J. Bowles, P. Koopman, Retinoic acid, meiosis and germ cell fate in mammals. 275 Development (Cambridge, England) 134, 3401-3411 (2007). 276 6. J. Bowles et al., Retinoid signaling determines germ cell fate in mice. Science 312, 596- 277 600 (2006).

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309 21. J. M. Turner, Meiotic sex chromosome inactivation. Development 134, 1823-1831 (2007). 310 22. E. L. Anderson et al., Stra8 and its inducer, retinoic acid, regulate meiotic initiation in 311 both spermatogenesis and oogenesis in mice. Proc Natl Acad Sci U S A 105, 14976- 312 14980 (2008). 313 23. S. La Salle et al., Spata22, a novel vertebrate-specific gene, is required for meiotic 314 progress in mouse germ cells. Biol Reprod 86, 45 (2012). 315 24. M. Luo et al., MEIOB exhibits single-stranded DNA-binding and exonuclease activities 316 and is essential for meiotic recombination. Nat Commun 4, 2788 (2013). 317 25. F. Baudat et al., PRDM9 is a major determinant of meiotic recombination hotspots in 318 humans and mice. Science 327, 836-840 (2010). 319 26. E. D. Parvanov, P. M. Petkov, K. Paigen, Prdm9 controls activation of mammalian 320 recombination hotspots. Science 327, 835 (2010). 321 27. P. P. Khil, F. Smagulova, K. M. Brick, R. D. Camerini-Otero, G. V. Petukhova, Sensitive 322 mapping of recombination hotspots using sequencing-based detection of ssDNA. Genome 323 Res 22, 957-965 (2012). 324 28. F. Smagulova et al., Genome-wide analysis reveals novel molecular features of mouse 325 recombination hotspots. Nature 472, 375-378 (2011). 326 29. G. T. Consortium, The Genotype-Tissue Expression (GTEx) project. Nat Genet 45, 580- 327 585 (2013). 328 30. D. Ballow, M. L. Meistrich, M. Matzuk, A. Rajkovic, Sohlh1 is essential for 329 spermatogonial differentiation. Dev Biol 294, 161-167 (2006). 330 31. Y. Li et al., Sohlh1 is required for synaptonemal complex formation by transcriptionally 331 regulating meiotic genes during spermatogenesis in mice. Mol Reprod Dev 86, 252-264 332 (2019). 333 32. D. McAninch et al., SOX3 promotes generation of committed spermatogonia in postnatal 334 mouse testes. Sci Rep 10, 6751 (2020). 335 33. G. Raverot, J. Weiss, S. Y. Park, L. Hurley, J. L. Jameson, Sox3 expression in 336 undifferentiated spermatogonia is required for the progression of spermatogenesis. Dev 337 Biol 283, 215-225 (2005). 338 34. T. Lord, B. Nixon, Metabolic Changes Accompanying Spermatogonial Stem Cell 339 Differentiation. Dev Cell 52, 399-411 (2020).

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340 35. G. M. Waites, R. T. Gladwell, Physiological significance of fluid secretion in the testis 341 and blood-testis barrier. Physiol Rev 62, 624-671 (1982). 342 343 ACKNOWLEDGEMENTS 344 We thank L. L. Heckert for helpful input on this study, P. E. Cohen for chromosome spread 345 technique, K. E. Orwig for suggestion on primary mouse GS cell culture, and J. P. Wang for 346 MEIOB antibody and insightful discussion. 347 348 Author contributions: X.Z. performed all the experiments. S.G. assisted the analyses of RNA- 349 seq and scRNA-seq data. X.Z. and N.W. designed the experiments, analyzed and curated the data. 350 N.W. and X.Z. wrote the manuscript. Funding: This work was supported by the KUMC 351 Department of Molecular and Integrative Physiology to N.W., a KUMC BRTP postdoctoral 352 fellowship to X.Z., and a National Institutes of Health (NIH) grant (NIH R21HD-087741) to 353 N.W. Genomic Sequencing Core is supported by Kansas Intellectual and Developmental 354 Disabilities Research Center (NIH U54 HD 090216), the Molecular Regulation of Cell 355 Development and Differentiation – COBRE (P30 GM122731-03) - the NIH S10 High-End 356 Instrumentation Grant (NIH S10OD021743) and the Frontiers CTSA grant (UL1TR002366). 357 Competing interests: The authors declare no competing interests. Data and materials 358 availability: All data are available in the manuscript or the supplementary material. 359 360 Supplementary Materials: 361 This manuscript contains 26 Supplementary Figures and 7 Supplementary Tables. 362 363 Figure legend 364 Fig. 1. NR is required to induce meiotic initiation with RA in primary GS cell culture. (A) 365 Bright-field images of cultured spermatogonia with indicated treatments for 2 days. Scale bars, 366 50 µm. (B) Relative expression of Spo11, Dmc1, and Sycp3 against Gapdh analyzed by qRT- 367 PCR in cultured spermatogonia with indicated treatments for 2 days. *P < 0.05. n = 3 368 independent cultures. n.s., no significant. (C) Immunostaining for γH2AX (red) and DAPI (blue) 369 with indicated treatment for 2 days. Scale bars, 10 µm. (D) Immunostaining for DMC1 (red), 370 DDX4 (green), and DAPI (blue) in Spo11+/+ and Spo11–/– primary spermatogonia culture. Scale

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371 bars, 10 µm. (E) (Left) UHC and heatmap of gene expression in cultured primary spermatogonia 372 with indicated treatments for 2 days. (Right) Top GO enrichments with representative genes in 373 each cluster. (F) GSVA analysis for indicated treatments. In the heatmap, rows are defined by 374 the selected gene sets, and columns by consensus scores for each treatment. Group enriched gene 375 sets are highlighted by different color. (G) Bar plot shows p-value of GO functions between RA 376 and NRRA treatment. (H) UHC and heatmap for the expression of 165 early meiosis-associated 377 genes. STRA8-dependent genes characterized in a previous study in ref. (2) are shown in red. 378 379 Fig. 2. scRNA-seq analysis of primary GS cell culture during NRRA-induced meiotic initiation 380 and progression. (A) Workflow of scRNA-seq experiment. Number of cells collected: 6,884 (0 381 day), 6,936 (2 day), 6,200 (3 day), and 5,587 (4 day). (B) A t-distributed Stochastic Neighbor 382 Embedding (tSNE) plot for analyzed cells. Cluster 0 – 3 were germ cells and Cluster 4 was 383 somatic cells. Number of cells selected for analysis: Cluster 0 (6,487 cells), Cluster 1 (5,286 384 cells), Cluster 2 (3,860 cells), Cluster 3 (2,170 cells), Cluster 4 (285 cells). (C) Violin plots 385 showing the expression level of representative genes in each cluster. (D) A bar plot showing the 386 proportion of the different cell clusters at different time points. (E) A heatmap showing the 387 expression of marker genes and GO functions in each cluster. (F) Gene expression patterns of 388 indicated genes on tSNE plots and violin plots. (G) PCA analysis showing the relationship 389 between single cell clusters from NRRA-treated culture in vitro (n = 4 clusters) and early mouse 390 spermatogenesis in vivo (n = 6 clusters) based on the transcriptional profiles of commonly 391 expressed genes (n = 7,803 genes). (H) Hierarchical clustering showing the relationship between 392 single cell clusters from NRRA-treated culture in vitro and early mouse spermatogenesis in vivo. 393 (I) Scatter plots comparing marker genes expression profile between in vitro clusters and in vivo 394 clusters. (J) (Left) Heatmap of sex chromosome genes in Clusters 0 – 3. (Right). Violin plots 395 showing percentage of X and Y chromosome genes profiles in Clusters 0 – 3. The median is 396 shown by a red line. (K) Scatter plots showing cells along the projected pseudo-time in Cluster 0 397 – 3 (upper) and in indicated clusters from early mouse spermatogenesis in vivo (lower). 398 399 Fig. 3. Stra8 is required for NRRA-induced meiotic initiation. (A) Immunostaining for DMC1 400 (red), DDX4 (green), and DAPI (blue) in Stra8+/+ and Stra8–/– primary spermatogonia culture. 401 Scale bars, 10 µm. (B) (Left) UHC and heatmap of gene expression in cultured primary

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402 spermatogonia with indicated genotypes for 2 days. (Right) Top GO enrichments with 403 representative genes in each cluster. (C) GSVA analysis for indicated genotypes. In the heatmap, 404 rows are defined by the selected gene sets, and columns by consensus scores for each genotype. 405 Group enriched gene sets are highlighted by different color. (D and E) Venn plots with top GO 406 enrichments and representative genes for NRRA-upregulated (D) and -downregulated (E) genes 407 in Stra8+/+ and Stra8–/– cultures. 408 409 Fig. 4. Nutrient restriction induces a network of TF genes involved in early meiosis. (A) A 410 workflow showing filters to identify genes of interest. (B) Heatmap of 120 TF genes upregulated 411 by nutrient restriction. 11 identified TF genes involved in early meiosis are shown on the right. 412 (C) Gene expression patterns of 3 representative TF genes during early spermatogenesis from a 413 published scRNA-seq database (20). (D) GTEx database showing correlation of representative 414 TF genes (Sohlh1, Pou2f2, Bcl11a) with selected meiotic genes in 165 testis samples.

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A Normal RA NR NRRA C Normal RA NR NRRA

γ H2AX / DAPI 10 um

0% 5.7% 11% 31%

+/+ −/− B 5 D Spo11 Spo11 * Normal NRRA Normal NRRA 4 n.s. Spo11 3 n.s.

2 Dmc1 / VASA DAPI Relative levels 1 Sycp3 DMC1 0 Normal RA NR NRRA 0% 29% 0% 2% EFH

GO term -0.4 -0.2 0.20 0.4 Normal_1 Normal_2 RA_1 RA_2 NR_1 NR_2 NRRA_1 NRRA_2 Normal_1 Normal_2 RA_1 RA_2 NR_1 NR_2 NRRA_1 NRRA_2 ncRNA metabolic Pparg, Fhl4, Poln,

process: Normal RA NR NRRA Tex101, Fbxo47, Nhp2, Snd1, Rtcb... Fatty_Acid_Derivative_Biosynthetic_Process Sycp2, Pramel1, Regulation of cellular amide Cellular_Response_To_Osmotic_Stress Hormad2, Spo11, metabolic process: NR/RA- Sterol_Biosynthetic_Process 4930447C04Rik, upregulated Mov10, Eif2b5, Calr... Fatty_Acid_Biosynthetic_Process Cyld, Usp32, Tex12 regulation of protein Cluster 4 Nucleoside_Bisphosphate_Metabolic_Process catabolic process: Rsph1, Rad21l, RA Unsaturated_Fatty_Acid_Biosynthetic_Process Gna12, Cdh1, Sox9... Response_To_Osmotic_Stress Top3a, Tsc22d3 lated Fatty_Acyl_COA_Metabolic_Process

Regulation of innate Gpr19, Espl1 NR-upregu- immune response: Retinoic_Acid_Receptor_Signaling_Pathway Spata5, Ecsit, Lgals9, Nr1h3, Apoe... Specific_Granule_Membrane Hsf2bp, Zfp541, response to IFN-beta: Salivary_Gland_Development Adarb1, Phka2, Acod1, Ifnar2, Ifitm3... Exocrine_System_Development Ccnb3, Tex15, Ddb2 Male_Meiotic_Nuclear_Division Zcwpw1, Dennd4a, Chr._Org._Involved_In_Meiotic_Cell_Cycle Mitochondrion Slc25a31, Dmrtc2

Regulation_Of_Myelination NR/NRRA-upregulated organization:

NRRA Multi_Organism_Behavior Hk2, Spg7, Akt1... Figla, Mei1, Msh4, GO_Defense_Response_To_Virus Ribonucleoprotein Msh5, Smc1b, Synaptonemal_Structure complex biogenesis Stag3, Sycp1, Regulation_Of_Myotube_Differentiation Pih1d2, Rrp15, Mrpl10... Sycp3, Tex11, Nucleoside Tex16, Ccdc36, monophosphate Cluster 1 Cluster 3 Wbp2nl, Ccdc155, Hfm1, Taf9b, Taf7l G NRRA-upregulated metabolic process p-value (-log10) Cox5a, Sdhd, Atp5f1... Ugt8a, Rec8, 0123 Prdm9, Stra8, Dmc1, Hormad1, Chromosome Rad21, Cntd1, RA segregation: 1700013H16Rik, Axin2, Chmp1a... Syce1, Rad51ap2, Organelle fission: Spdya, Fmr1nb, Zfy2, Tom1l2... D1Pas1, Setdb2, NRRA Meiotic cell cycle: BC051142,Syngr4,

Tubgcp3, Clgn, Syce2, 4930524B15Rik RA/NRRA-upregulated

Prkaca, Msh4... Cluster 2 Cilium assembly: Asf1b, Fzr1, Pcnt, Ocrl, Bbs1... GO_Male_Meiotic_Nuclear_Division Psmc3ip, Rad54l, Cdc25b, Plk1, Ereg, GO_Chr._Org._Involved_in_Meiotic_Cell_Cycle Mnd1, Rbpms2 NR/RA/NRRA NR/RA/NRRA GO_Synaptonemal_Structure downregulationed 14,728 DEG genes -1.5 21.510.50-0.5-1 165 meiosis genes -1.5 21.510.50-0.5-1

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.15.074542; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

ABCD Normal NRRA Progression 40 Gfra1 Proportion of samples medium medium medium 4 per cluster 4 d 100 2 d 3 d 4 d 20 0 Cyp26a1 3 80 Cell 0 Cluster

tSNE_2 Ccnb1 60 0 -20 1 2 1 40 Prdm9 2 Proportion (%) -40 20 3 Single cell RNA-seq -40 -20 0 4020 tSNE_1 Sycp1 0 Norm. 2 3 4 Cluster 0 1 2 3 4 Clustering analysis post-NRRA treatment Pseudotime/trajectory analysis Undiff. Diff./Meiotic MEF Ddx4 (days) Spg Spc E Cluster 01234 Cluster 0 Cluster 1 Cluster 2 Cluster 3 F Dmc1 Regulation of cellular amide 10 metabolic process: Sox4, Pgam1, Rpl5... Embryonic organ development: 3

Slc9a3r1, Lhx1, Rpl38... Expression level Stem cell division: 1 Zbtb16, Etv5, Hoxb4... Cluster 01234 Regulation of gene Hormad1 silencing: Hist1h1e/Dhx9... 10 Mito. electron transport: Park7, Ndufb9, Ndufb8...

Cellular process involved in 3

reproduction in multicellular Expression level organism: Sycp3, Hormad1, 1 1700013H16Rik... Cluster 0 1 2 3 4 sperm-egg recognition: Ly6k, Vdac2, Tex101... Sycp3 100 Meiotic cell cycle: Tex12, Sycp1, Tex19.1, Pttg1... Cytoplasmic translation: Rpl18a, Rps23, Rpl30... 10 tSNE2

Translational initiation: Expression level Eif1, Npm1, Eif5, Eif1b... 1 Chr. condensation: Expression Cluster 0 1 2 3 4 Dnajc19, Ndufs7, Mrpl17... tSNE1

Low High Relative Expression Low High G H I Late Pachy. Spc R=0.76 R=0.90 Late Pachy. Spc. 10 Diplo. Spc Cluster 3 Diplo. Spc. Early.Pachy. Spc Lep./Zygo. Diff./ Prelep. 5 Spc Spc. Cluster3

Early Pachy. Spc. Log 2 [Cluster 0 expression] Log 2 [Cluster 1 expression] Diff./Prelep. Spc Cluster 2 Log2[Undiff. spg. expression] Log2[Diff./Lep. spc. expression]

PC2 0 Cluster1 Cluster 1 R=0.74 R=0.92 Lep./Zygo. Spc −5 In vivo meiosis Cluster 0 (Hermann et al 2018) Cluster2 Undiff. Spg In vitro meiosis Undiff. Spg Cluster dendrogram −10 −20 −10 0 10 Cluster 0 Log 2 [Cluster expression] Log 2 [Cluster 3 expression] PC1 Log2[Lep./Zygo. spc. expression] Log2[Early pachy. spc. expression]

J Cell Cluster ChrX group genes K Cluster 0123 0.02 0 5 1 0.01 0 2 % ChrX -5 0 Component 2 3 Cluster 01234 Identity -10 0 10 20 Component 1 0.002 Cell Cluster 15 Undiff. Spg 10 Diff./PreL Spc 0.001 5 Lep./Zygo. Spc High 0 % ChrY Early Pachy. Spc Component 2 -5 Late Pachy. Spc 0 -10 Diplo. Spc Cluster 01234 Low -20 020 40 Identity Component 1

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A B

/− _normal/− _NRRA /− _normal _NRRA− − NRRA-induced in vitro meiosis In vivo meiosis − −/−

+/+ −/− +/+ −/− WT_normalWT_NRRAStra8 Stra8 Spo11 Spo11 Stra8 Stra8 Stra8 Stra8 1212 12121212

Stem cell population maintenance: Pou5f1, Tfap2c, Eomes, Nanos2...

/ VASA DAPI Stem cell division: Etv5, Zbtb16, Notch1... DNA replication: DMC1 Rpa1, Rac1, Ccne1... 31% 1.5% ATP metabolic process: Cluster 1 Cox5a, Sdhd, Atp5f1, Hk2, Tgfb1... C Meiotic cell cycle:

/− _normal/− _NRRA Syce2, Msh4, Msh5, Tex15, /− _normal/− _NRRA− − − − Tex11... WT_normalWT_NRRAStra8 Stra8 Spo11 Spo11 Meiosis I: Sycp3, Mlh3, Ddx4, Dmc1, Meiob... RETINOIC ACID RECEPTOR SIGNALING PATHWAY Synapsis: Syce2, Msh4, Msh5, Tex15, Cluster 2 RETINOIC ACID RECEPTOR BINDING Tex11, Sycp3, Mlh3, Dmc1, Meiob, Sycp1, Hormad1... OOGENESIS Glycoprotein metabolic MALE MEIOTIC NUCLEAR DIVISION process: Sdf2, Lmf1, Ncstn... SPERMATOGENESIS 0.6 Extracellular structure organization: MEIOSIS I CELL CYCLE PROCESS Fap, Egfl6, Loxl3, Mmp14... 0 Canonical Wnt signaling pathway: Cluster 3 SPERMATID DIFFERENTIATION Wnt9a, Ift20, Col1a1, Klf4, Dvl3, Sulf2, Ctnnb1... SYNAPTONEMAL STRUCTURE −0.6

DE Genes upregulated by NRRA (FC>2) Genes downregulated by NRRA (FC< -2) Synapse organization: Positive regulation Morphogenesis Fc receptor Grm5, Nrxn3, Zfp804a, WT of cytokine WT signaling Ntrk3, Pclo, Atp2b2... −/− production: of a branching −/− Stra8 structure: Stra8 pathway: Learning or memory: Mbp, Cd300c2, Nr4a3, Myo1g, Grm5, Pak7, Lhx8, Afap1l2, Irf8, Ltb, Col13a1, Rspo2, Mycn, Etv5, Fcgr2b... Ppp1r1b, Slc6a1, Rasgrp1... Epidermis Cntnap2... Leukocyte mediated Foxf1, Sall1... Regulation of development: Homologous 731 706 408 cytotoxicity: Lelp1, Sox18, Flg, chromosome (39.6%) (38.3%) (22.1%) Coro1a, Pik3r6, Wnt signaling 506 402 185 pathway: (46.3%) (36.8%) (16.9%) Apcdd1... segregation: Rasgrp1, H2-M2, cellular Fmn2, Sycp3, Mei1, Fcgr1, Vav1... Egr1, Slc9a3r1, Wnt3, Pou5f1, potassium ion Meioc, Ago4, Dmc1, Regulation of ion transport: Mei4, Meiob, Stag3... transmembrane Fgf9... Cell fate Kcnj8, Kcna3, Meiotic nuclear transport: Kcnk9, Kcnn4... division: Ntsr1, Coro1a, commitment: Pax7, Esrp1, Negative Camk2b, Prdm9, Kcns2, Gjc2, Clic6, regulation of Wnt Fbxo43, Sycp2, Zfy2, Nos1, Casq1... Foxg1, Fgf10, Notch1... signaling Top2b, Plcb1, Stag3... Reproductive pathway: structure Apcdd1, Notum, Response to interferon-beta: development: Response to wounding: Nkd2, Ptpro... Tgtp1, Tgtp2, F830016B08Rik, Ifi205, Gm4951, Gbp6... Sox8, Nanog, Wnt2, Foxa2, Entpd1, Serpinb2, F2rl1, Cd34, Gp5, Nefl... Extracellular structure organization: Umodl1, Esrrb, Bik, Positive regulation of peptidyl-tyrosine phosphorylation: Fap, Dpp4, Tgfb2, Ptx3, Pmp22, Ntn4, Col14a1, Abi3bp... Sohlh1... Cck, Il11, Areg, Ereg, Grem1, Tslp, Csf3, Csf2, Lif...

Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.15.074542; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

AB C

Deifferentially expressed TF genes ( 1283 genes) Hermann et al, 2018 RA_1 RA_2 Normal_1 Normal_2 NR_1 NR_2 NRRA_1 NRRA_2 Mga high Arid4b low NR-induced TF genes Sohlh1 expression (120 genes) Bbx

Expressed in meiosis in Zfp157 Ascl2 spermatogenesis high (30 genes) Bcl11a Pou2f2 Pou2f2 low expression Pax9 Atoh8

Positive correlation with 1.5 meiotic genes in GTEx Sox3 0 (11 TF genes) Sohlh1 −1.5 high

Bcl11a low C expression n = 165, r = 0.68(pearson), p.value= 0 n = 165, r = 0.76(pearson), p.value= 0 n = 165, r = 0.56(pearson), p.value= 0 5.0 5.0 5.0 2.5 2.5 2.5

0.0 0.0 0.0

Expression of DMC1 (log2(TPM))

−2.5 Expression of DMC1 (log2(TPM)) −2.5 Expression of DMC1 (log2(TPM)) −2.5

−30 3 01234 −3 −2 −10123 Expression of SOHLH1 (log2(TPM)) Expression of POU2F2 (log2(TPM)) Expression of BCL11A (log2(TPM))

n = 165, r = 0.92(pearson), p.value= 0 n = 165, r = 0.84(pearson), p.value= 0 n = 165, r = 0.68(pearson), p.value= 0 8 5 4 5

0 0 0 Expression of SYCP3 (log2(TPM)) Expression of SYCP3 (log2(TPM)) −4 Expression of SYCP3 (log2(TPM)) −30 3 01234 −3 −2 −10123 Expression of SOHLH1 (log2(TPM)) Expression of POU2F2 (log2(TPM)) Expression of BCL11A (log2(TPM))

n = 165, r = 0.88(pearson), p.value= 0 n = 165, r = 0.83(pearson), p.value= 0 n = 165, r = 0.68(pearson), p.value= 0

7.5 5.0 5 5 2.5

0.0 0 0

−2.5

Expression of HORMAD1 (log2(TPM)) −5.0 Expression of HORMAD1 (log2(TPM)) Expression of HORMAD1 (log2(TPM)) −30 3 01234 −3 −2 −10123 Expression of SOHLH1 (log2(TPM)) Expression of POU2F2 (log2(TPM)) Expression of BCL11A (log2(TPM))

Figure 4