{"paper_id":"0fe86af1-629b-4f6a-9435-5cdd9569df06","body_text":"1 \nFull Title:  1 \nCross-Tissue Coordination between SLC Nucleoside Transporters Regulates Reproduction 2 \nin Caenorhabditis elegans 3 \n 4 \nShort Title: 5 \nMetabolite transporter coordination between tissues  6 \n 7 \nAuthor and Affiliation: 8 \n 9 \nAuthor: Youchen Guan1,2, Yong Yu3,4, Shihong M. Gao1,5, Lang Ding1,6, Qian Zhao1, and 10 \nMeng C. Wang1,4,* 11 \n 12 \n1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn VA, USA 20147 13 \n2. Molecular and Cellular Biology Graduate Program, Baylor College of Medicine, Houston TX, 14 \nUSA 77030 15 \n3. Current address: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, 16 \nFaculty of Medicine and Life Sciences, Xiamen University, Xiamen 361102, China 17 \n4. Huffington Center on Aging, Baylor College of Medicine, Houston TX, USA 77030  18 \n5. Developmental Biology Graduate Program, Baylor College of Medicine, Houston TX, USA 19 \n77030 20 \n6. Chemical, Physical & Structural Biology Graduate Program, Baylor College of Medicine, 21 \nHouston TX, USA 77030 22 \n* Correspondence and requests for materials should be addressed to Meng C. Wang 23 \n(mengwang@janelia.hhmi.org) 24 \n 25 \n 26 \n 27 \n 28 \n 29 \n 30 \n 31 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 2 \nAbstract: 32 \nMetabolism is fundamental to organism physiology and pathology. From the intricate network of 33 \nmetabolic reactions, diverse chemical molecules, collectively termed as metabolites, are 34 \nproduced. In multicellular organisms, metabolite communication between different tissues is 35 \nvital for maintaining homeostasis and adaptation. However, the molecular mechanisms 36 \nmediating these metabolite communications remain poorly understood. Here, we focus on 37 \nnucleosides and nucleotides, essential metabolites involved in multiple cellular processes, and 38 \nreport the pivotal role of the SLC29A family of transporters in mediating nucleoside 39 \ncoordination between the soma and the germline. Through genetic analysis, we discovered that 40 \ntwo Caenorhabditis elegans homologs of SLC29A transporters, Equilibrative Nucleoside 41 \nTransporter ENT-1 and ENT-2, act in the germline and the intestine, respectively, to regulate 42 \nreproduction. Their knockdown synergistically results in sterility. Further single-cell 43 \ntranscriptomic and targeted metabolomic profiling revealed that the ENT double knockdown 44 \nspecifically affects genes in the purine biosynthesis pathway and reduces the ratio of guanosine 45 \nto adenosine levels. Importantly, guanosine supplementation into the body cavity/pseudocoelom 46 \nthrough microinjection rescued the sterility caused by the ENT double knockdown, whereas 47 \nadenosine microinjection had no effect. Together, these studies support guanosine as a rate 48 \nlimiting factor in the control of reproduction, uncover the previously unknown 49 \nnucleoside/nucleotide communication between the soma and the germline essential for 50 \nreproductive success, and highlight the significance of SLC-mediated cell-nonautonomous 51 \nmetabolite coordination in regulating organism physiology. 52 \n 53 \n 54 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 3 \nAuthor Summary 55 \nMetabolism is essential for life, involving a complex network of chemical reactions that requires 56 \na well-organized system to maintain efficiency. This includes the optimal allocation of resources 57 \nand the dynamic exchange of metabolic products between various compartments within an 58 \norganism. Solute carriers (SLCs) are the largest family of transporters for metabolic products 59 \nacross the animal kingdom. In our research, we investigated how specific SLC transporters 60 \ncollaborate to move key metabolic products between different tissues. We identified two SLC 61 \ntransporters, Equilibrative Nucleoside Transporter ENT-1 and ENT-2, which are vital for 62 \ntransporting guanosine, a purine nucleoside, to support successful reproduction in the nematode 63 \nCaenorhabditis elegans. We discovered that ENT-2 acts in the gut to export guanosine to the 64 \nsurrounding body cavity, while ENT-1 functions in the germline to import guanosine from the 65 \nbody cavity. When both transporters are disrupted, the animals experience significant 66 \nreproductive defects. Our study underscores the importance of coordinated activity between SLC 67 \ntransporters in different tissues to maintain organism health. A breakdown in this communication 68 \ncan result in metabolic imbalances and physiological dysfunction.   69 \n 70 \n 71 \n 72 \n 73 \n 74 \n 75 \n 76 \n 77 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 4 \nIntroduction 78 \nIn multicellular eukaryotic organisms, a complex network of small metabolites, encompassing 79 \namino acids, glucose, lipids, and nucleotides/nucleosides, serves as the foundational framework 80 \nfor cellular homeostasis. Perturbations in this intricate metabolic network have been linked to 81 \nvarious chronic diseases such as diabetes, cancers, and neurodegenerative disorders (Diamanti et 82 \nal., 2022; Pothiwala et al., 2009; Procaccini et al., 2016). In particular, nucleosides and 83 \nnucleotides are essential for DNA replication and RNA synthesis to enable cell growth and 84 \ndivision. Imbalances in nucleotide species can disrupt genome stability, mitochondrial activity, 85 \ncell proliferation, muscle integrity, germline maintenance, and organism development (Chi et al., 86 \n2016; Diehl et al., 2022; Marsac et al., 2019; Pai and Kearsey, 2017).  87 \n 88 \nThe synthesis of DNA and RNA precursors, nucleotides, primarily involves de novo and salvage 89 \npathways. In the de novo pathway, nucleotides are synthesized from various substrates such as 90 \namino acids, PPRP (Phosphoribosyl pyrophosphate), and tetrahydrofolate (Sato et al., 2006), 91 \nthrough a series of enzymatic steps. On the other hand, the salvage pathway, known for its higher 92 \nenergy efficiency, directly produces nucleotides from free purine/pyrimidine nucleobases and 93 \nnucleosides. While nucleobases and nucleosides are preferentially salvaged within the cell, their 94 \nuptake from the extracellular space also plays crucial roles in regulating nucleotide balance, 95 \nespecially in tissues with high demand. In C. elegans, the germline, which carries cells 96 \nundergoing rapid proliferation and division, requires a large amount of nucleotide synthesis. Our 97 \nrecent findings found that the mitochondrial GTP but not ATP level in the germline regulates 98 \nreproductive activities during aging, which is coupled with bacterial inputs from the intestine 99 \n(Lee et al., 2023). 100 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 5 \n 101 \nSolute carrier (SLC) transporters are one of the two major transporter superfamilies responsible 102 \nfor the transport of a diverse range of small molecules across the plasma membrane and 103 \nsubcellular organelle membranes. These transporters are essential for cellular homeostasis, 104 \nregulating the uptake and efflux of various vital nutrients such as glucose, amino acids, fatty 105 \nacids, vitamins, and ions (Zhang et al., 2019). They also play a significant role in whole-body 106 \nphysiology, as many are expressed in a tissue-specific manner (Lin et al., 2015). Equilibrative 107 \nNucleoside Transporter (ENT) family are encoded by SLC29 family genes, and mediate the 108 \nsodium independent transportation of different nucleobases and nucleosides across membranes 109 \n(Baldwin et al., 2004; Griffith and Jarvis, 1996). Despite the relatively well-studied role of ENT 110 \ntransporters in the cell-autonomous regulation of nucleosides, understanding whether and how 111 \nthey coordinate to mediate nucleoside transport across tissues remains limited. In this work, we 112 \nfound that C. elegans ENT-1 and ENT-2 transporters control nucleoside transport between the 113 \nsoma and the germline. Specifically, ENT-2 transports nucleosides from the intestine to the body 114 \ncavity/pseudocoelom, while ENT-1 facilitates their transportation from the pseudocoelom to the 115 \ngermline. Their synergistic action is essential for reproduction. Moreover, our studies suggest the 116 \nsubstrate specificity of ENT-1/2 towards guanosine, through the integration of single-cell 117 \ntranscriptomic profiling, targeted metabolomic profiling, and chemical screening. These findings 118 \nserve as a pioneering example of SLC transporters functioning in different tissues to coordinate 119 \nthe cell non-autonomous communication of vital metabolites, thereby supporting organism 120 \nphysiology.  121 \n 122 \n 123 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 6 \nResults and Discussion 124 \nENT-1 and ENT-2 work together to regulate reproduction  125 \nIn human, four ENT members exhibit varying tissue abundance and subcellular distribution 126 \n(Baldwin et al., 2004). C. elegans possesses seven ENT family members: ENT-1 to ENT-7. 127 \nDespite significant separation in the phylogenetic tree and limited amino acid sequence similarity 128 \nbetween C. elegans ENTs and human ENTs (Fig 1A), the predicted protein structures of ceENTs 129 \nbased on AlphaFold2 are highly conserved with human ENT1 (Fig 1B) (Wright and Lee, 2019). 130 \nAmong C. elegans ENTs, ENT-6 appears relatively divergent from others, while ENT-4, ENT-5, 131 \nand ENT-7 are relatively close to each other (Fig 1A). ENT-1 and ENT-2 exhibit a notably close 132 \nrelationship (Fig 1A) and share 84% identity in exon sequences and 94% in amino acid 133 \nsequences (Appleford et al., 2004), which has been previously suggested to result from a 134 \nrelatively recent gene duplication event (Sankar et al., 2002).  135 \n 136 \nIn C. elegans adulthood, only germline cells continue to undergo proliferation and division. To 137 \ninvestigate whether successful reproduction depends on nucleoside transport from other tissues 138 \ninto the germline, we examined reproduction in worms where ENTs were knocked down by 139 \nRNA interference (RNAi). We found that RNAi knockdown of either ent-1 or ent-2 but not other 140 \nents reduces the total progeny number (Figs 1C-E). RNAi knockdown of either ent-1 or ent-2 141 \nreduced the number of progenies in the first two days of the reproductive period, but increased 142 \nthe number afterward, leading to an extension in the duration of the reproductive process 143 \n(reproductive lifespan) (Figs 1F and 1G).  144 \n 145 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 7 \nOur previous studies revealed that C. elegans exhibits different reproductive strategies when 146 \nexposed to different types of bacteria (Lee et al., 2023; Sowa et al., 2015). Specifically, wild-147 \ntype worms grown on OP50 E. coli have extended reproductive lifespan, in comparison with 148 \nthose grown on HT115 E. coli that is commonly used for RNAi. We thus examined the effect of 149 \nent-1 and ent-2 RNAi knockdown on reproduction in the background of OP50 E. coli. We 150 \nobserved a similar extension of reproductive lifespan, however the reduction in the total progeny 151 \nnumber did not reach significance (S1A and S1B Figs).  152 \n 153 \nNext, to confirm the result of RNAi inactivation, we utilized the CRISPR-Cas9 technique 154 \n(Dickinson and Goldstein, 2016) to generate knockout mutant strains for both ent-1 (ent-1KO) and 155 \nent-2 (ent-2KO) (S1C Fig). We found that in the background of either OP50 or HT115 E. coli, the 156 \nreproductive pattern of the ent-1KO mutant is indistinguishable from WT, while the ent-2KO 157 \nmutant slightly increased the progeny number on the third day of the reproductive period (S1D- 158 \nS1G Figs). Further RT-qPCR analysis revealed elevated ent-2 mRNA levels in the ent-1 KO 159 \nmutant, and increased ent-1 mRNA levels in the ent-2 KO mutant (S1H, and S1I Figs), which 160 \nsuggests that the loss of one transporter leads to the compensatory induction of the other one. In 161 \nsupporting this idea, we found that the double mutant of ent-1 KO and ent-2 KO exhibited complete 162 \nsterility with developmental delay and vulva protrusion, which is consistent with previous 163 \nobservations (Appleford et al., 2004). We also knocked down ent-2 by RNAi in the ent-1 164 \nknockout mutant (ent-1KO;ent-2KD) or knocked down ent-1 by RNAi in the ent-2 knockout 165 \nmutant (ent-1KO;ent-2KD). We found that under both conditions, the brood size is largely reduced 166 \n(Figs 1H and 1I). The phenotype associated with ent-1KO;ent-2KD is much stronger, close to 167 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 8 \nbeing sterile (Fig 1H, S2A Fig). These results suggest that ENT-1 and ENT-2 transporters 168 \nfunction together to regulate reproduction.  169 \n 170 \nGermline ENT-1 and intestinal ENT-2 coordinate to regulate reproduction 171 \nTo identify the functional tissues where these two ENTs act to regulate reproduction, we first 172 \nexamined their expression pattens. We generated two CRISPR knock-in lines in which 173 \nendogenous ENT-1 and ENT-2 is tagged with mNeonGreen and wrmscarlet at the C-terminus, 174 \nrespectively (S2B and S2C Figs). Using these lines, we revealed that ENT-1 expresses in the 175 \ngermline and intestine, and ENT-2 expresses in gonadal sheath cells and the intestine (Figs 2A, 176 \n2B and S2D, S2E Figs). Next, we restored the expression of either ent-1 or ent-2 specifically in 177 \nthe intestine of the ent-1KO or ent-2 KO mutant, and then performed RNAi knock down of either 178 \nent-2 or ent-1, respectively. We found that the intestine-specific restoration of ent-2, but not ent-179 \n1, increases the brood size in the worms with both ent-1 and ent-2 knockdown (Figs 2C, 2D). In 180 \nparallel, we restored the expression of ent-2 specifically in gonadal sheath cells of the ent-2 KO 181 \nmutant and knocked down ent-1 by RNAi. We found this restoration does not rescue the 182 \nreduction in the brood size (Fig 2E). We also attempted to restore the expression of ent-1 in the 183 \ngermline of the ent-1 KO mutant but failed to obtain a stable line.  184 \n 185 \nWe thus applied an auxin-inducible degradation (AID) system (Zhang et al., 2015) to deplete 186 \nENT-1 proteins selectively in the germline. The AID system utilized a plant-specific F-box 187 \nprotein that recognizes degron-tagged substrates upon auxin binding and induces their 188 \ndegradation by proteasome (Fig 2F). We generated a CRISPR knock-in line in which 189 \nendogenous ENT-1 is tagged with degron and V5 tag at the N-terminus (S2F Fig), and 190 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 9 \nsubsequently crossed this line with TIR1 tissue-specific expressing lines, germline (gld-191 \n1p::TIR1::mRuby) and intestine (ges-1p::TIR1::mRuby) (Fig 2F). In these crossed lines, the 192 \nauxin treatment resulted in selective degradation of ENT-1 in the germline or intestine (S2H 193 \nFig). With RNAi knockdown of ent-2, we observed that auxin-induced germline-specific 194 \ndepletion of ENT-1 resulted in a 2-fold reduction in brood size compared to the control without 195 \nthe auxin treatment (Fig 2G). In contrast, the intestine-specific depletion of ENT-1 did not 196 \nreduce the brood size compared to the control (Fig 2H), which is consistent with the result using 197 \nthe intestine-specific rescuing strain (Fig 2C). 198 \n 199 \nWe have also generated the degron-tagged ent-2 CRISPR knock-in line and crossed it with the 200 \nent-1KO mutant (Fig S2G). This strain was further crossed with the TIR1 tissue-specific 201 \nexpressing lines, gonadal sheath cell (lim-7p::TIR1::mRuby) and intestine (ges-202 \n1p::TIR1::mRuby) (Fig 2F). In these crossed lines, the treatment of auxin induces specific 203 \ndegradation of ENT-2 either in the gonadal sheath cells or intestine (Fig S2I). Consistent with 204 \nthe findings using tissue-specific rescuing strains, we found that the depletion of ent-2 205 \nspecifically in the intestine led to a largely reduced brood size (Fig 2I), while the depletion in 206 \ngonadal sheath cells does not affect brood size (Fig 2J), when compared to the controls. 207 \nTogether, these results demonstrated that ENT-1 and ENT-2 function in the germline and 208 \nintestine, respectively to regulate reproduction in a coordinated manner.  209 \n 210 \nsnRNA-seq profiling reveals the effect of ENTs on purine metabolism  211 \nFollowing our identification of the synergistic effect between ENT-1 and ENT-2 and their tissue-212 \nspecificity, we next sought to understand how they coordinate the intestine and the germline to 213 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 10 \nregulate reproduction. To this end, we conduced single-nuclei RNA sequencing (snRNA-seq) 214 \nanalysis and profiled cell type-specific transcriptomic changes in the ent-1KO, ent-2KO, and ent-215 \n1KO;ent-2KD worms. Approximately 3,000 worms were collected for each condition, and nuclei 216 \nwere isolated using fluorescence-activated cell sorting (FACS) and profiled using the 10X 217 \nGenomics platform (Gao et al., 2024). Following pre-processing, cell filtering, and quality 218 \ncontrol, we obtained 56,992 single-nucleus transcriptome profiles, which were subsequently 219 \nannotated into 14 distinct cell types (Fig 3A). Analysis of cell composition across these 220 \nconditions revealed a notable decrease in the proportion of germline cells and a higher proportion 221 \nof somatic cells collected from the ent-1KO;ent-2KD worms (Fig 3B), which is consistent with the 222 \nreduced brood size. 223 \n 224 \nWe further conducted germ cell pseudotime inference analysis (Gao et al., 2024), to examine 225 \nchanges in the germline composition. This analysis allowed us to construct a pseudotemporal 226 \norder of germ cells (S3A Fig), where the x-axis signifies pseudotime denoting the progression 227 \nfrom germline stem cells (GSCs) through mitotic cells and meiotic cells to mature oocytes. We 228 \nfound that the GSC number is increased in the ent-1KO single mutant but decreased in the ent-2KO 229 \nsingle mutant (S3A Fig), suggesting distinctive alterations in germline homeostasis despite the 230 \nabsence of brood size changes in these single mutants. Moreover, in the ent-1KO;ent-2KD worms, 231 \nwe observed an increased proportion of germ cells in proliferation stages, including GSC and 232 \nthose in the mitotic zone (Fig 3C). In contrast, the proportion of cells in the meiosis stage was 233 \ndecreased (Fig 3C), while the transition zone remained unchanged. These results suggest a 234 \ndefect in germ cell differentiation associated with the loss of both ENT-1 and ENT-2.  235 \n 236 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 11 \nWe also performed the differential expressed gene (DEG) analysis to reveal molecular changes 237 \nin the germline upon the loss of ENT-1 and ENT-2. 716 DEG genes were identified through the 238 \ncomparison between ent-1KO;ent-2KD worms and their controls, with a significance cutoff of p 239 \nadjust value < 0.05 and |log2FoldChange|>0.5. Among these genes, 302 were upregulated, while 240 \n412 were downregulated (Fig 3D). The Gene Ontology (GO) pathway analysis of the 241 \ndownregulated genes in the germline of the ent-1KO;ent-2KD worms highlighted pathways 242 \nassociated with cell cycle, chromosome organization, and mRNA processing, which is likely 243 \nresulted from the loss of differentiated germ cells in these worms (S3B Fig). When analyzing the 244 \nupregulated genes, we found that GO terms related to purine metabolism are overrepresented in 245 \nthe ent-1KO;ent-2KD germline (Fig 3E).  246 \n 247 \nA series of purine metabolic genes in both de novo and recycling pathways are conserved in C. 248 \nelegans (Marsac et al., 2019) (Fig 3F). When analyzing their expression levels using the snRNA-249 \nseq data, we found that most purine metabolic genes showed trends of up-regulation in the ent-250 \n1KO;ent-2KD germline (Fig 3G), while genes encoding enzymes involved in the de novo pathway 251 \nshowed trends of down-regulation in the intestine (Fig 3H). However, no clear trends of changes 252 \ndetected in other somatic tissues such as hypodermis and muscle (S3C and S3D Figs). 253 \nFurthermore, when analyzing conserved metabolic genes in the pyrimidine pathway (Fig 3I), we 254 \ndid not observe an obvious trend of alterations in their expression in either the germline or 255 \nintestine of the ent-1KO;ent-2KD worms (Figs 3J and 3K).  256 \n 257 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 12 \nTogether, these results suggest that the level of purine, rather than pyrimidine, is likely altered in 258 \nthe germline and intestine upon the loss of both ENT-1 and ENT-2, leading to compensatory 259 \ntranscriptional alterations of purine biosynthesis genes.  260 \n 261 \nENT-mediated guanosine transport regulates reproduction  262 \nTo directly assess whether purine and pyrimidine nucleoside levels change in association with 263 \nthe ENT deficiency, we employed targeted metabolomic analysis by liquid chromatograph 264 \ncoupled with mass spectrometry (LC/MS). We compared guanosine, adenosine, inosine, and 265 \ncytidine levels in ent-1KO, ent-2KO, and ent-1KO;ent-2KD worms with their controls. The levels of 266 \nuridine and thymidine are below the detection sensitivity. Interestingly, we observed that the 267 \nproportion of guanosine is decreased by ~20% and ~30% in the ent-1KO and ent-2KO single 268 \nmutants, respectively (Fig 4A). In the ent-1KO;ent-2KD worms, the decrease is close to be 3-fold 269 \n(Fig 4A). For the other two purine nucleosides, adenosine and inosine, their proportion exhibited 270 \nthe opposite trend (Figs 4B and 4C). On the other hand, the proportion of cytidine did not show 271 \nsignificant alterations in either ent-1KO, ent-2KO, or ent-1KO;ent-2KD worms compared to their 272 \ncontrols (Figs 4D). It is worth noting that no reduction in the brood size was observed in the ent-273 \n1KO or ent-2KO single mutant, despite the decrease in guanosine. Hence, the germline is tolerant 274 \nto a certain level of reduction in guanosine levels while sustaining normal reproduction, but 275 \nexceeding this threshold would result in reproductive defects.  276 \n 277 \nThese results support the predominant role of ENT-1/2 in regulating purine especially guanosine 278 \nhomeostasis. When integrating the tissue-specificity of ENT-1/2, the germline-specific 279 \ntranscriptional up-regulation of purine metabolic genes, and the guanosine reduction, we 280 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 13 \nhypothesized that ENT-2 functions in the intestine to export guanosine into the body 281 \ncavity/pseudocoelom, while ENT-1 is responsible for the uptake of guanosine into the germline 282 \n(Fig 4E). The loss of ENT-2 in the intestine results in reduced guanosine availability in the 283 \npseudocoelom, while the loss of ENT-1 in the germline compromises the uptake of guanosine 284 \nfrom the pseudocoelom. With the single mutation of either ent-1 or ent-2, the reduction of 285 \nguanosine in the germline does not reach the threshold for severe disruption of reproduction. 286 \nHowever, this reduction threshold will be met with the simultaneous loss of ENT-1 and ENT-2 287 \n(Fig 4F).  288 \n 289 \nIn supporting this hypothesis, we confirmed that ENT-2::wrmscarlet predominantly localizes at 290 \nthe basolateral plasma membrane of the intestine (S2E Fig), while ENT-1::mNeonGreen is 291 \npositioned at the plasma membrane in the germline (S2D Fig). We also injected guanosine or 292 \nadenosine into the pseudocoelom at the fourth larval (L4) stage to increase their levels in the 293 \npseudocoelom. We found that the microinjection of 1mM guanosine can increase the brood size 294 \nin the ent-1KO;ent-2KD worms, but adenosine microinjection showed no effect in restoring 295 \nreproduction (Fig 4G). However, feeding of either guanosine or adenosine was not able to rescue 296 \nthe brood size in the ent-1KO;ent-2KD worms (Fig 4H). We also tried directly injecting 297 \nnucleosides into the germline of the ent-1KO;ent-2KD worms; however, the severe disruption in 298 \ngermline morphology prevented this attempt. These results highlighted the importance of 299 \nguanosine levels in the pseudocoelom in the regulation of reproduction (Fig 4F).  300 \n 301 \nIn summary, our study underscores the significance of guanosine communication between the 302 \nsoma and germline in the control of reproduction, as well as the pivotal role played by specific 303 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 14 \nSLC transporters in mediating this cell non-autonomous metabolite communication. SLC 304 \ntransporters are highly conserved in humans and exhibit diverse tissue-specificity, and their 305 \nmutations have been associated with human diseases. Current research of these mammalian 306 \nhomologs focuses on identifying their substrates and understanding their cell-autonomous 307 \nregulatory functions. It would be interesting to explore how their cell non-autonomous 308 \ncoordination contributes to mammalian physiology and pathology in future studies.  309 \n 310 \nMaterials and methods 311 \nC. elegans strains and maintenance 312 \nThe strains N2, CA1352 (ieSi64 [gld-1p::TIR1::mRuby::gld-1 3 UTR + Cbr-unc-119(+)] II), 313 \nand CA1209 (ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)] II), and 314 \nMQD2383 (hqSi11 [lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-119(+)] II; daf-315 \n2(hq363[daf-2::degron::mNeonGreen]) unc-119(ed3) III), were obtained from CGC. 316 \n 317 \nKnockout mutants MCW1244 (ent-1(rax74) IV) and MCW1245 (ent-2(rax75)X) were generated 318 \nin our lab by using CRISPR/Cas9 technology, as previous outlined by Chen et al. (Chen et al., 319 \n2014), Paix et al.(Paix et al., 2015) and Arribere et al.(Arribere et al., 2014) with modifications. 320 \nBriefly, a mixture containing tracrRNA (1μg/μl), crRNAs (0.5μg/μl each for one on the 5' and 321 \none on the 3' of the target gene), dpy-10 crRNA (0.16μg/μl), and Cas9 protein (0.05μg/μl) was 322 \nmicroinjected into the gonads of N2 young adult animals. Each injected worm was then placed 323 \non individual plates, and the non-Dpy F1 progenies from the plates which contains animals with 324 \nDpy phenotypes were individualized to single plates for further analysis. After confirming the 325 \ndeletion region through PCR and Sanger sequencing of F1 animals, homozygous F2 animals 326 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 15 \ncarrying the knockout mutation were selected and individualized. The knockout deletion strains 327 \nwere backcrossed to wild-type N2 for at least four times before running reproductive 328 \nexperiments. 329 \n 330 \nStrains that carry extrachromosomal arrays: MCW1589(ent-2(rax75)X; raxEx624[lim-7p::ent-331 \n2cds::sl2RFP::tbb-2 3'UTR; lin-44p::GFP], MCW1634(ent-1(rax74) IV; raxEx628[ges-1p::ent-332 \n1cds:sl2RFP::unc-54 3'UTR; lin-44p::GFP], MCW1636(ent-2(rax75)X; raxEx630[ges-1p::ent-333 \n2cds:sl2RFP::unc-54 3'UTR; lin-44p::GFP] were generated by microinjecting DNA mixture 334 \ncontaining linearized expression construct and co-injection marker lin-44p::GFP into the 335 \ncorresponding gonad of ent-1 or ent-2 knockout young adult animals.  336 \n 337 \nPHX4218(ent-1(syb4218) ent-1::mNeonGreen IV), PHX5055(ent-2(syb5055) ent-338 \n2::wrmscarlet::3Xflag X), PHX7376(ent-1(syb7376) 3XV5::degron::ent-1 IV), PHX7457(ent-339 \n2(syb7457) 3XHA::degron::ent-2 X) were generated via CRISPR/Cas9 genome editing by 340 \nSunyBiotech (Fuzhou, China).  341 \n 342 \nMCW1638 (syb7376[3XV5::degron::ent-1]IV; ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + 343 \nCbr-unc-119(+)]II) and MCW1639 (syb7376[3XV5::degron::ent-1]IV; ieSi64 [gld-344 \n1p::TIR1::mRuby::gld-1 3'UTR + Cbr-unc-119(+)] II) were generated by crossing PHX7376 345 \nwith CA1209 or CA1352.  346 \n 347 \nMCW1640 (syb7457[3XHA::degron::ent-2]X; ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + 348 \nCbr-unc-119(+)] II and MCW1641 (syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; hqSi11 349 \nII[lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-119(+)] II) were generated by crossing 350 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 16 \nPHX7457 with CA1209 or the strain hqSi11 [lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-351 \n119(+)] II which were obtained from crossing MQD2383 and N2. 352 \n 353 \nMCW1643 (syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; ieSi61 [ges-354 \n1p::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)] II and MCW1644 355 \n(syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; hqSi11 II[lim-7p::TIR1::mRuby::unc-54 3' 356 \nUTR + Cbr-unc-119(+)] II) were generated by crossing MCW1640 or MCW1641 with 357 \nMCW1244. 358 \n 359 \nAll the C. elegans strains were grow and maintained non-starved for at least three generations at 360 \n20˚C on NGM agar plates seeded with OP50 E.coli using standard protocols (Stiernagle, 2006) 361 \nbefore experiments. The E. coli strain HT115 (DE3) and OP50 RNAi strain (OP50 bacteria 362 \n[rnc14::DTn10 laczgA::T7pol camFRT] generated by our lab (Neve et al., 2020) were used for 363 \nRNAi experiments. 364 \n 365 \nRNA interference (RNAi) experiments 366 \nRNAi libraries from Dr. Julie Ahringer's lab were utilized in the study (Kamath and Ahringer, 367 \n2003). RNAi clones for ent-1, ent-2, ent-3, ent-5, and ent-6 were obtained from the Ahringer 368 \nlibrary. RNAi clones for ent-4 and ent-7 were created in our lab using L4440 as the vector 369 \nbackbone and ent-4 or ent-7 transcript fragments as inserts. For OP50 RNAi experiments, RNAi 370 \nplasmids were transformed into the genetically modified competent OP50 bacteria 371 \n[rnc14::DTn10 laczgA::T7pol camFRT], generated by our lab (Neve et al., 2020). All RNAi 372 \ncolonies were selected for resistance to both 50 µg ml−1 carbenicillin and 50 µg ml−1 373 \ntetracycline. RNAi bacteria were cultured for 14 hours in LB with 25 µg ml−1 carbenicillin, then 374 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 17 \nseeded onto RNAi agar plates containing 1 mM IPTG and 50 µg ml−1 carbenicillin. Each RNAi 375 \nbacteria clone was allowed to dry on the plates before overnight incubation at room temperature 376 \nto induce dsRNA expression. 377 \n 378 \nMolecular cloning of expression construct 379 \nAll the expression plasmids were generated via the Gibson Assembly (NEB). The ent-1 and ent-380 \n2 coding sequences were PCR-amplified from C. elegans cDNA and then fused together with 381 \nsl2-RFP sequence by fusion PCR. The ent-1::sl2RFP and ent-2::sl2RFP fragments were then 382 \nligated into the tissue-specific promoter vectors. 383 \n 384 \nMeasure the brood size 385 \nSynchronized L1 worms obtained from egg preparation were placed onto 6cm NGM plates, each 386 \nseed with corresponding bacteria for the experimental condition, and then incubated at 20 °C. 387 \nUpon reaching the L4 stage, individual worms were transferred to new plates. Subsequently, they 388 \nwere transferred to new plates every day until reproduction ceased. Plates with progenies were 389 \nstored at 20°C until progeny reached the L4 stage for counting. Total brood size was determined 390 \nby summing viable progeny produced daily by each worm. 391 \n 392 \nStructural stimulation by AlphaFold2 393 \nAlphaFold2 predictions for the structures of ceENT-1 (UniProt: G5EDJ3) and ceENT-2 394 \n(UniProt: Q93871) were retrieved from The AlphaFold Protein Structure Database 395 \n(https://alphafold.ebi.ac.uk/) (Jumper et al., 2021; Varadi et al., 2022). Molecular graphics for 396 \nhuman ENT1, ceENT-1, and ceENT-2 were generated using UCSF Chimera (Pettersen et al., 397 \n2004). 398 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 18 \n 399 \nAuxin treatment 400 \nThe L1 worms were places on the NGM plates seeded with bacteria containing auxin 4mM 401 \nindole-3-acetic acid (IAA), following the protocol outlined by Zhang et al.(Zhang et al., 2015). A 402 \n400 mM stock solution of IAA in ethanol was prepared, filtered through a 0.22µm filter, and 403 \nstored at 4°C for up to 1 month. This stock solution was diluted into NGM plates at a ratio of 404 \n1:100. Control plates were prepared by diluting ethanol into NGM plates at the same ratio. Fresh 405 \nbacteria were then seeded onto the plates and stored at room temperature for 1 day to allow 406 \nbacterial growth. The plates containing auxin were kept in a dark place to prevent photolysis. 407 \n 408 \nRT-qPCR 409 \nTotal RNA was extracted from approximately 3000 age synchronized D1 worms using Trizol 410 \nhomogenization, chloroform phase separation, isopropanol precipitation, and subsequent 411 \nwashing with 75% ethanol.  412 \ncDNA synthesis utilized the amfiRivert Platinum cDNA Synthesis Master Mix (GenDEPOT), 413 \nfollowed by quantitative PCR with the Kapa SYBR fast qPCR kit (Kapa Biosystems) in a 96-414 \nwell Eppendorf Realplex 4 PCR machine (Eppendorf).  415 \nAll presented data are from at least six independent biological samples and were normalized to 416 \nrpl-32 as an internal control.  417 \n 418 \nFluorescent microscopy 419 \nC. elegans were immobilized in 1% sodium azide in M9 buffer and positioned on a 2% agarose 420 \npad between a glass microscopic slide and coverslip for imaging. Endogenous ENT-421 \n1::mNeonGreen and ENT-2::wrmscarlet expression patterns were visualized using a Nikon CSU-422 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 19 \nW1 spinning disk confocal microscopy system equipped with a 20x objective, oil immersion 60x  423 \nand 100x objective. Degron-mediated protein degradation was assessed using a laser scanning 424 \nconfocal FV3000 (Olympus, US) with an oil immersion 60x objective. 425 \n 426 \nInjection of nucleoside into the pseudocoelom 427 \nStock solutions of nucleosides are stored at 100mM in DMSO at -20°C for up to 1 month. Fresh 428 \n1mM nucleoside injection solutions are prepared before each injection by dissolving the 100mM 429 \nstock solution in egg buffer [118 mM NaCl, 48 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 25 430 \nmM Hepes (pH 7.3)] at a ratio of 1:100. The control injection solution consists of DMSO 431 \ndissolved at a ratio of 1:100. Mid-stage L4 animals were injected with either the nucleoside 432 \nsolution or DMSO control solution into the pseudocoelom , with the injection needle inserted 433 \ninto the pharyngeal region. Successful injection is confirmed by the observation of liquid flow in 434 \nthe pseudocoelom. 435 \n 436 \nImmunostaining 437 \nApproximately 200 Adult animals were initially transferred to unseeded NGM plates to 438 \nminimize bacterial presence. M9 solution containing 0.4μM levamisole was then added to 439 \nimmobilize animals on the plates. Subsequently, these animals, along with the M9 solution 440 \ncontaining 0.4μM levamisole, were transferred to a glass dissection plate for dissection. 441 \nDissection involved using two 25 gauge syringe needles to extrude the gonad arm and intestine 442 \ncompletely. The dissected worms were fixed with 4% PFA for 10 minutes at room temperature 443 \nin darkness, followed by washing twice with PBST and post-fixation in −20°C methanol for 1 444 \nhour. After three washes with PBST, the specimens were then blocked with 5% BSA in PBST 445 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 20 \nfor 30 minutes. Following blocking, a 200μL volume of primary antibody solution (in PBST) 446 \n[anti-HA antibody (1:200): HA-Tag (C29F4) Rabbit mAb #3724; anti-V5 antibody (1:200): V5 447 \nTag Monoclonal Antibody (SV5-Pk1) (R960-25)] was applied and incubated overnight at 4°C. 448 \nThe cut worms were then washed three times with PBST and incubated with secondary antibody 449 \n[1:500 dilution of Alexa 488 conjugated antibodies from Invitrogen] for 1 hour at room 450 \ntemperature. After three additional washes with PBST, the specimens were resuspended in a 451 \nglycerol antifade reagent and mounted on agarose pads before imaging. 452 \n 453 \nNucleoside dietary supplementation 454 \nFor dietary nucleoside supplementation, nucleoside powder was dissolved directly to a final 455 \nconcentration of 100mM in standard NGM liquid medium immediately prior to pouring into 456 \nplates. These plates were then seeded with RNAi E. coli and allowed to grow overnight before 457 \nuse. 458 \n 459 \nSingle-nucleus RNA sequencing analysis 460 \nNuclei Isolation 461 \nWe followed the nuclei isolation protocol similar to that described by Gao et al.(Gao et al., 462 \n2024). Briefly, worms were washed three times with PBS, collected in 1.5 mL tubes, and 463 \nhomogenized in 100 μl of homogenization buffer (Li et al., 2022) using a pestle motor on ice. To 464 \nprevent nuclei adhesion, all equipment was pre-coated with homogenization buffer or PBS. The 465 \nhomogenate was further processed in an autoclaved Dounce tissue grinder, with sequential 466 \nstrokes using loose and tight pestles to ensure thorough disaggregation without foaming. After 467 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 21 \nfiltering through cell strainers, the nuclei were pelleted by centrifugation, resuspended in PBS 468 \nwith additives, and prepared for sorting. 469 \nNuclei Sorting 470 \nThe nuclei were stained with Hoechst 33342 for DNA content visualization and sorted using a 471 \nSony MA800 sorter. Gating of sorting was the same as describe by Gao et al. (Gao et al., 2024). 472 \nPost-sorting, nuclei were checked for concentration and morphology before proceeding with 10X 473 \nChromium Controller. 474 \nLibrary Preparation and Sequencing 475 \nThe quality-checked nuclei were encapsulated using a 10X Chromium Controller, and libraries 476 \nwere prepared according to the 10X Chromium Single Cell 3’ v2/v3 Solution protocol, selecting 477 \nappropriate indexing options. Sequencing was performed on a NovaSeq 6000 system, using 26 478 \ncycles for Read 1, 8 cycles for the i7 index, and 98 cycles for Read 2, as recommended. 479 \nSingle-nucleus RNA-seq Data Preprocessing 480 \nRaw sequences were processed using Cell Ranger 6.0 (10x Genomics), aligned to the C. elegans 481 \ngenome (WS282), and assembled into feature-barcode matrices. Doublet exclusion was 482 \nperformed per 10X Genomics guidelines, utilizing Doubletfinder for accurate detection. 483 \nSubsequent analyses, including data integration, dimensional reduction, and clustering, were 484 \nconducted using Seurat 4.0.5 to identify and characterize cell populations. 485 \nCell Type Annotation and Analysis 486 \nFor cell type annotation, we predominantly employed a reference-based mapping approach using 487 \nthe SingleR package (Aran et al., 2019), which allowed us to automatically match the expression 488 \nprofiles of our cells to those from established reference datasets. This analysis predominantly 489 \nlinked cell clusters to specific tissues, as evidenced by previous single-nucleus RNA sequencing 490 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 22 \ndatasets produced by Gao et al. (Gao et al., 2024). We further enhanced cell type specificity by 491 \nemploying the FindMarkers function within Seurat to identify distinctive marker genes for each 492 \ncluster. These markers were then cross-referenced with microscopy-based expression profiles 493 \nand literature-reported tissue markers to confirm cell type assignments. For enrichment analysis 494 \nand further validation of our findings, we utilized tools available on Wormbase (Angeles-495 \nAlbores and Sternberg, 2018). 496 \nIn addition to broad classification, we also focused on the germline cells, isolating them into a 497 \nseparate Seurat object. This subset underwent a similar analysis pipeline, where we identified 498 \nhighly variable genes and performed CCA integration followed by dimensional reduction and 499 \nclustering to determine germline subclusters. This refined analysis enabled us to explore cell 500 \ndevelopment within the germline specifically. 501 \nGermline Trajectory Analysis 502 \nUtilizing the refined germline cluster data, we applied Slingshot for trajectory analysis, which 503 \nutilized cluster labels and UMAP embeddings to construct cell lineages and ascertain pseudotime 504 \ntrajectories. This analysis revealed the developmental progression from germline stem cells, 505 \nshowing low pseudotime values, to mature oocytes, which displayed high pseudotime values. 506 \nWe correlated these pseudotime findings with nuclei counts reported in the literature (Diag et al., 507 \n2018), allowing us to map each cluster to specific stages of germline development. This dual 508 \nanalysis of pseudotime distribution and developmental status percentage provided a 509 \ncomprehensive view of germline cell differentiation. 510 \nDifferential Expression and Gene Ontology Analysis 511 \nTo identify differentially expressed genes (DEGs) across various cell states and conditions, we 512 \nused Seurat's FindMarkers function, employing a Wilcoxon rank-sum test with thresholds set for 513 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 23 \nadjusted p-values below 0.05 and |log2 fold changes| greater than 0.5. Subsequent gene ontology 514 \nanalysis was performed using the ClusterProfiler package, which helped to understand the 515 \nbiological processes and pathways significantly associated with the identified DEGs. To visually 516 \ncompare gene expression across different samples and conditions, we used the DotPlot function 517 \nwithin Seurat, facilitating an intuitive display of data that highlights specific gene expression 518 \nchanges. 519 \n 520 \nTargeted metabolic analysis of nucleosides 521 \nSample collection: 522 \nAround 5000 C. elegans in each condition were collected in the bead beater tubes (Sarstedt Inc 523 \nScrew Cap Micro tube 2ml) with 250 µL RNAlater Stabilization Solution (Invitrogen: AM7021) 524 \nand 10µg/ml of tetrahydrouridine to prevent rapid deamination of the nucleosides (Persaud et al., 525 \n2023) and then snap freezing in liquid nitrogen which were stored at -80°C until use. 526 \nNucleosides extraction and purification 527 \nNucleosides were extracted from the samples following a previously outlined method (He et al., 528 \n2019) with some modifications. Briefly, RNAlater Stabilization Solution was removed from 529 \nthawed samples, and 250 µL LC-MS H2O with RNase Inhibitor and beads were added to the 530 \nsamples for homogenization with a bead beater. After homogenization, the sample was 531 \ntransferred to a 2 mL tube. pH adjustment to 8.5 was done using ammonium in methanol, 532 \nfollowed by addition of methanol at a ratio of 1:4 (v:v). The mixture was then centrifugated at 533 \n14,000 rpm for 20 min at 4 °C after vortexing for 3 min. The supernatant was collected and dried 534 \nusing SpeedVac. Each dried sample was dissolved then in 250 μL water. 535 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 24 \nFor nucleoside purification, each sample was loaded onto an OASIS HLB cartridge (Waters 536 \nCorp., Milford, MA, USA) activated 3 times with water and methanol. After loading, cis-diol 537 \ncompounds were eluted with 250 μL 2.8% ammonium hydroxide (NH4OH) in methanol, 538 \nrepeated 3 times. Eluates were then dried using SpeedVac 539 \nDried samples were reconstituted in 100 μL water, centrifuged at 14,000 rpm for 10 min at 4 °C, 540 \nand the clear upper solution was transferred to an LC vial for LC-MS analysis. 541 \nLC-MS/MS Analysis 542 \nThe experiments were performed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer 543 \nequipped with Ion Max API source housing with HESI-II probe and Vanquish UHPLC System 544 \n(ThermoFisher Scientific). The separation was performed on an ACQUITY UPLC HSS T3 545 \ncolumn (100 × 2.1 mm i.d., 1.8 μm) (Waters Corp., Milford, MA, USA) at a flow rate of 0.2 546 \nmL/min with column temperature of 40 °C (mobile phase A: 0.1% FA in water, mobile phase B: 547 \n0.1% FA in acetonitrile). The gradient was as follows: 0-3 min, 0-2.8% B; 3-9 min, 2.8%-10% 548 \nB; 9-9.5 min, 10-30% B; 9.5-9.8 min, 30-60% B; 9.8-9.9, 60% B; 9.9-10 min, 60-0% B (He et 549 \nal., 2019). The targeted mode (parallel reaction monitor, PRM) was applied with Orbitrap MS 550 \nacquired a full-scan survey in positive mode (m/z: 140-1300, automatic gain control target: 551 \nstandard, maximum injection time mode: auto, resolution at m/z 200: 120,000, the default charge 552 \nstate: 1, followed by tMS2 with precursor ions list of nitrogenous bases (mass range: normal, 553 \nautomatic gain control target: standard, maximum injection time mode: auto, Orbitrap with 554 \nresolution at m/z 200: 60,000, collision-induced dissociation (CID) collision energy (%): 30 with 555 \nactivation time: 10 ms. Maximum injection time: 118 ms). 556 \nLC-MS/MS Data analysis 557 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 25 \nThe raw files were analyzed by Skyline (Adams et al., 2020; Henderson et al., 2018; MacLean et 558 \nal., 2010). The transition list was generated, applied and peak area was extracted and calculated 559 \nby Skyline. All the samples were normalized based on protein concentrations. The sample 560 \nconcentration was calculated based on the standard curve. 561 \n 562 \nQuantification and statistical analysis 563 \nData were expressed as mean ± standard error of the mean (s.e.m.) and analyzed using GraphPad 564 \nPRISM. Student’s t-test (unpaired) compared the means of two groups. One-way ANOVA or 565 \ntwo-way ANOVA followed by Holm–Sidak’s or Holm-Bonferroni’s corrections, as indicated in 566 \nthe figure legends, were applied. Statistical significance in figure legends is denoted by asterisks: 567 \nns (not significant, p > 0.05), *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001. Detailed 568 \ninformation on sample size, biological replicates, and statistical analysis for each experiment is 569 \nprovided in the figure legends. Figures and graphs were generated using BioRender, GraphPad 570 \nPrism 10 (GraphPad Software), and Illustrator (CC 2019; Adobe). The researchers were not 571 \nblinded during experiments or outcome assessment. 572 \n 573 \nAcknowledgements 574 \nWe thank A. Dervisefendic and P. Svay for their assistance with worm strain maintenance. We 575 \nthank I. Neve for the great help in generating CRISPR knockout strains. Several strains were 576 \nobtained from the Caenorhabditis Genetics Center (CGC), which is supported by the NIH Office 577 \nof Research Infrastructure Programs (P40 OD010440). M.C.W. receives current support from 578 \nHoward Hughes Medical Institute. 579 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 26 \n 580 \nAuthor Contributions  581 \nConceptualization: Youchen Guan and Meng C. Wang  582 \nData Curation: Youchen Guan 583 \nFormal analysis: Youchen Guan 584 \nFunding acquisition: Meng C. Wang 585 \nInvestigation: Youchen Guan, Yong Yu, Shihong M. Gao, Lang Ding Qian Zhao, and Meng C. 586 \nWang 587 \nMethodology: Youchen Guan and Meng C. Wang  588 \nProject administration: Meng C. Wang  589 \nSupervision: Meng C. Wang  590 \nValidation: Youchen Guan 591 \nWriting – original draft: Youchen Guan 592 \nWriting – review & editing: Meng C. Wang  593 \n 594 \n  595 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 27 \nFigure Legends 596 \nFig 1. Specific ENT transporters regulate reproduction 597 \n(A) Phylogenetic analyses of human and C. elegans ENTs using protein sequences obtained from 598 \nUniprot. 599 \n(B) Comparison between human ENT1 structure (Wright and Lee, 2019), and C. elegans ENT-1 600 \n(ceENT-1) and ENT-2 (ceENT-1) structures predicted by AlphaFold2. 601 \n(C) RNA inference (RNAi) knockdown of ent-1 reduces brood size compared to the empty 602 \nvector (EV) control. n = 22 (EV), n = 26 (ent-1 RNAi).  603 \n(D)  RNAi knockdown of ent-2 reduces brood size compared to the EV control. n = 28 (EV), n = 604 \n31(ent-2 RNAi).  605 \n(E) RNAi knockdown of ent-3, ent-4, ent-5, ent-6, or ent-7 does not affect brood size compared 606 \nto the EV control. n = 17 (EV), n = 17 (ent-3 RNAi), n = 15 (ent-4 RNAi), n = 16 (ent-5 607 \nRNAi), n = 15 (ent-6 RNAi), n = 16 (ent-7 RNAi).  608 \n(F) RNAi knockdown of ent-1 alters the daily progeny number compared to the EV control. n = 609 \n22 (EV), n = 26 (ent-1 RNAi). 610 \n(G)  RNAi knockdown of ent-2 alters the daily progeny number compared to the EV control. n = 611 \n28 (EV), n = 31(ent-2 RNAi). 612 \n(H)  The ent-1 knockout (ent-1KO) mutant with ent-2 RNAi knockdown (ent-2KD) largely reduces 613 \nthe brood size and daily progeny number, compared to the control. n = 20 (ctrl), n = 22 (ent-614 \n1KO; ent-2KD). 615 \n(I) The ent-2 knockout (ent-2KO) mutant with ent-1 RNAi knockdown (ent-1KD) reduces the 616 \nbrood size and daily progeny number, compared to the control. n = 11 (ctrl), n = 10 (ent-2KO; 617 \nent1KD). 618 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 28 \nRNAi condition: HT115 E. coli (C-G); Statistics: *p<0.05; **p<0.01, ***p<0.001, 619 \n****p<0.0001, ns: p>0.05. Student’s t-test (unpaired, two-tailed) was applied for C-E, and the 620 \nbrood size graphs in H, I.  Two-way ANOVA with Bonferroni’s post hoc test for F-G, and the 621 \ndaily progeny graphs H, I. Data shown as mean ± S.E.M. 622 \n 623 \nFig 2. Germline ENT-1 and intestinal ENT-2 cooperate in regulating reproduction 624 \n(A) (B) The expression patterns of endogenously labeled ENT-1::mNeonGreen and ENT-625 \n2::wrmscarlet visualized in their CRISPR knock-in strains. Scale bar: 100 µm. White 626 \narrowheads indicate intestine; hollow white arrowhead indicates germline, and white arrows 627 \ndesignate gonadal sheath cells.  628 \n(C) Intestine-specific restoration of ent-1 expression does not rescue the reduced brood size in 629 \nthe ent-1KO mutant with ent-2 RNAi knockdown.   630 \n(D) Intestine-specific restoration of ent-2 expression rescues the reduced brood size in the ent-631 \n2KO mutant with ent-1 RNAi knockdown.   632 \n(E) Restoration of ent-2 expression in somatic gonadal sheath cells does not rescue the reduced 633 \nbrood size in the ent-2KO mutant with ent-1 RNAi knockdown.   634 \n(F)  Illustration of auxin-induced degradation of endogenous ENT-1 tagged with 3XV5 tag and 635 \ndegron and ENT-2 tagged with 3XHA tag and degron in the intestine (ges-1 promoter), in 636 \nthe germline (gld-1 promoter) and gonadal sheath cell (lim-7 promoter).  637 \n(G) Auxin-induced germline-specific degradation of ENT-1 together with ent-2 RNAi 638 \nknockdown reduces the brood size. n = 23 (-Auxin), n = 23 (+Auxin).  639 \n(H) Auxin-induced intestine-specific degradation of ENT-1 together with ent-2 RNAi 640 \nknockdown does not reduce the brood size. n = 23 (-Auxin), n = 23 (+Auxin).  641 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 29 \n(I) Auxin-induced intestine-specific degradation of ENT-2 in the ent-1KO mutant decreases the 642 \nbrood size. n = 30 (-Auxin), n = 30 (+Auxin).  643 \n(J) Auxin-induced intestine-specific degradation of ENT-2 in the ent-1KO mutant does not affect 644 \nthe brood size. n = 30 (-Auxin), n = 30 (+Auxin).  645 \nRNAi condition: HT115 E. coli (C, D, E, G, H). Statistic: *p<0.05; **p<0.01, ***p<0.001, 646 \n****p<0.0001, ns p>0.05. Student’s t-test (unpaired, two-tailed) between control group and 647 \nexperimental group was applied for the brood size graphs shown in C, D, E, G, H, I, J. Data 648 \nwere shown as mean ± S.E.M. 649 \n 650 \n 651 \nFig 3. SnRNA-seq profiling reveals the regulation of purine metabolism by ENT-1/2  652 \n(A) The Uniform Manifold Approximation and Projection (UMAP) plot displays fourteen cell 653 \ntypes in ctrl, ent-1KO, ent-2KO, and ent-1KO; ent-2KD worms.  654 \n(B) The percentage of different cell types in total captured cells shows decreased germline nuclei 655 \nin ent-1KO; ent-2KD worms.  656 \n(C) The proportion of nuclei within various regions of the germline among total germline nuclei 657 \nis compared between control and ent-1KO; ent-2KD worms 658 \n(D) The Volcano plot shows differentially expressed genes in the germline when comparing ent-659 \n1KO; ent-2KD worms vs. their controls. 660 \n(E)  Gene ontology (GO) enrichment analysis of the up-regulated genes in the germline of ent-661 \n1KO; ent-2KD worms compared to their controls. The red box highlights GO terms involved in 662 \npurine metabolism.  663 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 30 \n(F) Schematic illustration of C. elegans homologous genes involved in purine metabolism, both 664 \nde novo and salvage pathways, based on the previous study (Marsac et al., 2019). 665 \n(G, H) Dot plots show the relative expression levels of genes associated with purine metabolism 666 \nin the germline (G) and intestine (H). The size of dots indicates the percentage of nuclei 667 \nexpressing the gene, and the color indicates the average expression level.  668 \n(I) Schematic illustration of C. elegans homologous genes involved in pyrimidine metabolism 669 \npathways. 670 \n(J, K) Dot plots show the relative expression levels of genes associated with pyrimidine 671 \nmetabolism in the germline (J) and intestine (K).  672 \n 673 \n 674 \nFig 4. Guanosine specifically contributes to the regulation of reproduction by ENT-1/2 675 \n(A-D) The percentages of guanosine (A), adenosine (B), inosine (C), and cytidine (D) among all 676 \nnucleosides measured by LC/MS are compared among control, ent-1KO, ent-2KO, and ent-677 \n1KO;ent-2KD worms. n = 4 (ctrl), n = 5 (ent-1KO), n = 5 (ent-2KO), n = 4 (ent-1KO;ent-2KD). 678 \n(E)   A summary model illustrating the coordinated regulation of the reproductive process by 679 \nENT-2 mediated export of nucleosides from the intestine to the pseudocoelom and ENT-1 680 \nmediated uptake of nucleosides from the pseudocoelom to the germline. 681 \n(F) Illustration represents possible changes in guanosine levels in the intestine, pseudocoelom, 682 \nand germline under different conditions, including ctrl, ent-1KO, ent-2KO, ent-1KO;ent-2KD, 683 \nand ent-1KO;ent-2KD with pseudocoelom guanosine microinjection.  684 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 31 \n(G) Microinjection of guanosine but not adenosine partially rescues the reduced brood sizes of 685 \nent-1KO;ent-2KD worms.  n = 22 (DMSO vehicle), n = 34 (Guanosine), n = 22 (1mM 686 \nAdenosine). 687 \n(H)  Oral supplementation of neither guanosine nor adenosine rescues the reduced brood sizes of 688 \nent-1KO;ent-2KD worms.  n = 20 (DMSO vehicle), n = 20 (Guanosine), n = 20 (Adenosine). 689 \n 690 \nStatistic: *p<0.05, ***p<0.001, ****p<0.0001, ns p>0.05. One-way ANOVA with Holm–Sidak 691 \ncorrection (A-D). Student’s t-test (unpaired, two-tailed) between control group and experimental 692 \ngroup (G-H).  Data shown as mean ± S.E.M.   693 \n 694 \nSupporting information 695 \nS1 Fig. ENT-1 and ENT-2 regulate reproduction independent of bacterial inputs. 696 \n(A) RNAi knockdown of ent-1 on OP50 bacteria alters the daily progeny number compared to 697 \nthe EV control. n = 17(EV), n = 16(ent-1 RNAi). 698 \n(B) RNAi knockdown of ent-2 on OP50 bacteria alters the daily progeny number compared to 699 \nthe EV control. n = 17(EV), n = 16(ent-2 RNAi). 700 \n(C) Genome illustrations of ent-1KO and ent-2KO mutants. 701 \n(D) ent-1KO mutants on OP50 bacteria show no significant alteration of daily progeny number or 702 \nbrood size compared to wild type (WT) worms. n = 23(N2), n = 22(ent-1KO). 703 \n(E) ent-2KO mutants on OP50 bacteria show no significant alteration of daily progeny number or 704 \nbrood size compared to WT. n = 18(N2), n = 18(ent-2KO). 705 \n(F) ent-1KO mutants on HT115 bacteria show no significant alteration of daily progeny number or 706 \nbrood size compared to WT. n = 28(N2), n = 27(ent-1KO). 707 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 32 \n(G) ent-2KO mutants on HT115 bacteria show no significant alteration of daily progeny number or 708 \nbrood size compared to WT. n = 22(N2), n = 22(ent-2KO). 709 \n(H) (I)RT-qPCR analysis shows that the ent-2 mRNA level is upregulated in the ent-1KO mutant 710 \n(H), and the ent-1 mRNA level is elevated in ent-2KO (I). n = 6 biologically independent 711 \nsamples in each condition. 712 \nStatistic: **p<0.01, ***p<0.001, ****p<0.0001, ns p>0.05. Student’s t-test (unpaired, two-713 \ntailed) was applied for the brood size graphs in A, B, D, E, F and G, and RT-qPCR results in H 714 \nand I. Two-way ANOVA with Bonferroni’s post hoc test for the daily progeny graphs in A, B, 715 \nD, E, F and G. 716 \nData shown as mean ± S.E.M. 717 \n 718 \nS2 Fig. Germline ENT-1 and intestinal ENT-2 cooperate in regulating reproduction 719 \n(A) Bright-field images show sterile ent-1KO; ent-2KD mutants and their controls. Scale bar: 100 720 \nµm. 721 \n(B, C) Diagrams showing CRISPR knock-in strains: ent-1::mNeonGreen (B), ent-2::wrmscarlet 722 \n(C). 723 \n(D, E)  Endogenous localization of ENT-1::mNeonGreen in the germline and intestine (D) and 724 \nENT-2::wrmscarlet in gonadal sheath cells and the intestine (E). Scale bar: 50 µm. 725 \n(F, G) Diagrams showing CRISPR knock-in strains: degron::ent-1 (F) and degron::ent-2 (G). 726 \n(H)(I) Immunostaining results show the efficiency of tissue-specific degron-mediated protein 727 \ndegradation induced by the Auxin treatment in the degron::ent-1 strain (H) and in the 728 \ndegron::ent-2 strain (I). Scale bar: 50 µm. 729 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n 33 \nS3 Fig. Changes in the germline and other tissues in association with the loss of ENT-1 and 730 \nENT-2 731 \n(A) Pseudotime density plots illustrate the distribution of germ cell nuclei across different 732 \npseudotime points for control, ent-1KO, ent-2KO, and ent-1KO; ent-2KD worms.  733 \n(B)  Gene ontology enrichment analysis of biological processed was performed on the down-734 \nregulated expressed genes in the germline of ent-1KO; ent-2KD worms compared to their 735 \ncontrols, revealing pathways related to cell cycle and division are overrepresent. 736 \n(C) (D) Dotplots show the relative expression levels of genes that are associated with purine 737 \nmetabolism of control, ent-1KO, ent-2KO, and ent-1KO; ent-2KD animals in the hypodermis (C) 738 \nand muscle (D).  739 \n 740 \n 741 \n 742 \n 743 \nAdams, K.J., B. 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J Mol Cell Biol. 11:1-13. 875 \n 876 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}