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GRTH KI transgenic mice were generated with the human GRTH gene containing the R²⁴²H mutation found in Japanese infertile men as described previously (Kavarthapu et al., 2019). Briefly, WT and GRTH KI transgenic mice were genotyped after confirming the presence/absence of the transgene using two sets of primers Geno F1/Geno R1 and Geno F2/Geno R2 (Supplementary Table 1). All the animals were housed in pathogen-free and temperature- and light-controlled conditions (22°C), with 14:10-h light/dark cycle. All studies were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee.

Isolation of CBs from adult mouse testes was carried out using the protocol (Meikar and Kotaja, 2014) with minor modifications. Testes from 10 WT and homozygous GRTH KI mice (45 days) (two mice for each sample, N = 5) were finely minced and digested in phosphate-buffered saline (PBS) containing 0.5 mg/ml of collagenase with 1 mg/ml glucose for 60 min at room temperature. Digested seminiferous tubules were filtered using a 100-μm cell strainer (BD Biosciences) and centrifuged for 5 min at 300 g at 4°C. After two washes in cold PBS, the cells were crosslinked with 0.1% paraformaldehyde (PFA; Thermo Scientific, MA, United States) for 20 min at 22°C. The reaction was stopped by adding 0.25 M glycine, pH 7.0 (Bostonbio, MA, United States). The cells were washed in PBS once, resuspended in RIPA buffer [(Millipore-Merck); 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1 mM DTT, 1% Non-idet P40, 0.5% sodium deoxycholate with 1:200 RNase Inhibitor (Thermo Scientific), and protease inhibitor cocktail (Thermo Scientific)], and sonicated (five cycles of 20 s with 45-s intervals). The cell lysate was centrifuged for 10 min at 500 g at 4°C. The pellet containing the CBs was suspended in RIPA buffer and sonicated (two cycles of 20 s with 45-s intervals), filtered with a 5-μm filter (Millipore-Merck), and used for CB isolation. Aliquots of germ cells, pellet (PEL), and filtered pellet (FPEL) fractions were collected to monitor the success of the procedure. Fifty microliters of Protein G Dynabeads (Thermo Scientific) was coupled with 8 μg of commercial rabbit polyclonal anti-MVH antibody (cst#8761; Cell Signaling) or control rabbit IgG for 3 h according to the manufacturer’s instructions. After two washes with citrate phosphate buffer containing 0.1% Tween, the Dynabeads–IgG complex was stored in RIPA buffer briefly. The FPEL was pre-cleared with 10 μl of washed Protein G Dynabeads (Thermo Scientific) for 30 min at 4°C and used for immunoprecipitation (IP) overnight with rotation at 4°C with the Dynabeads–IgG complex. On the next day, the Dynabeads–IgG–CB complexes were washed three times in 1 ml of RIPA buffer. Crosslinking was reversed in the same buffer (for RNA analysis) or in 1 × Laemmli/SDS sample buffer (for protein analysis) at 70°C for 45 min. Subsequently, the Dynabeads–IgG complex containing CBs was used for RNA isolation.

To monitor the success of the CB isolation procedure, the CB samples (5 μl) were fixed using a drying-down fixing solution (0.15% TritonX-100 and 1% PFA in PBS; 10 μl/slide) on a precleaned slide and air-dried overnight in a humidified chamber at 22°C. Once the slides were completely dried, these were post-fixed using 2% PFA for 5 min and then washed and treated with 0.2% Triton-X-100 in PBS for 2 min. Then, the slides were blocked using 5% BSA for 1 h in a humidified chamber and immunoprobed using anti-MVH/DDX4 antibody (1:1,000; cst#8761; Cell Signaling) overnight at 4°C. Following incubation with Alexa Fluor 568 secondary antibody (1:500) or Alexa Fluor 488 secondary antibody (1:500) for 1 h at 22°C, the slides were washed with phosphate-buffered saline–Tween^® 20 (PBST; twice, 5 min each) and PBS (twice, 5 min each) and mounted with ProLong Diamond antifade reagent (Thermo Scientific). These slides were imaged using a Zeiss LSM 710 confocal microscope (Carl Zeiss, CA, United States) and analyzed using ZenPro software (Carl Zeiss, CA, United States). Immunofluorescent staining of drying-down slide preparations is a reliable method to visualize the CBs. Testicular sections or germ cell preparations obtained from WT and KI mice were immunoprobed using anti-MVH/DDX4 antibody (1:1,000; cst#8761; Cell Signaling) overnight at 4°C. Following incubation with Alexa Fluor 488 secondary antibody (1:500) for 1 h at 22°C, the slides were washed with PBST (twice, 5 min each) and PBS (once, 5 min each) and mounted with ProLong Diamond antifade reagent with DAPI (Thermo Scientific). These slides were imaged using a Ziess Axioplan 2 fluorescence microscope (Carl Zeiss, CA, United States) and were analyzed using ZenPro blue and Axiovision v4.5 software (Carl Zeiss, CA, United States). Electron microscopy studies were carried out as described previously (Kavarthapu et al., 2019). Briefly, testicular tissue isolated from WT and GRTH KI mice were first fixed in 2.5% glutaraldehyde buffer at 4°C overnight, and then the tissues were post-fixed with 1% osmium tetroxide and stained using 2% uranyl acetate, dehydrated, and then embedded in Spurr’s epoxy. An EM ultramicrotome (Leica, Wetzlar, Germany) was used for making ultra-thin sections and were post-stained with lead citrate and observed under a JEOL JEM-1400 transmission electron microscope (JEOL USA, Inc., Peabody, MA, United States).

CBs isolated from the testes of WT and GRTH KI mice were subjected to western blot analysis. The protein G–IgG–CB complex was washed in RIPA buffer and reverse cross-linked at 70°C for 45 min in the 1× Laemmli/SDS sample buffer containing reducing agents. The resulting protein samples were centrifuged at 1,500 g for 5 min at 22°C. The supernatants collected were run on 4–12% Bis-Tris gel (Thermo Scientific) and transferred onto a nitrocellulose membrane in an iBlot (Thermo Scientific). The membrane was blocked with 5% skimmed milk powder in tris-buffered saline and then incubated with either of the following primary antibodies: affinity-purified anti-GRTH rabbit polyclonal antibody (1:500 dilution), anti-MVH/DDX4 polyclonal (1:500; cst#8761; Cell Signaling), mouse Argonaute/PIWI family RNA binding protein (MIWI) monoclonal antibody (CB control; 1:500; cst#6915; Cell Signaling), brain and muscle ARNT-like 1 (BMAL1) rabbit monoclonal antibody (1:500 dilution; cst#14020; Cell Signaling), circadian locomotor output cycles protein kaput (CLOCK) rabbit monoclonal antibody (1:500 dilution; cst#5157; Cell Signaling), Ybx3 mouse monoclonal antibody (1:500 dilution; LS-C105064; LS Bio), PRM1 rabbit polyclonal antibody (1:500 dilution; HPA055150; Millipore Sigma, St. Louis, MO, United States), and PRM2 rabbit monoclonal antibody (1:500 dilution; Sc-30172; Santa Cruz Biotechnology, Dallas, Texas). After the antibody incubation and washing steps, the membranes were incubated with the respective secondary antibodies conjugated with Poly-HRP (1:2,000; Thermo Scientific). Immunosignals were detected by a super-signal chemiluminescence system (Thermo Scientific).

Total RNA was extracted from reverse cross-linked Dynabeads–IgG–CB complexes using phenol/chloroform/isoamyl alcohol (25:24:1, v/v; Thermo Scientific). RNA quality and quantity were assessed using the RNA Nano 6000 Assay Kit in an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). Sequencing libraries were prepared using a TruSeq Stranded mRNA Prep Kit (Illumina), without the polyA selection step. Sequencing was performed via a paired-end 75 cycle on Illumina HiSeq 2500 (Molecular Genomics Core, NICHD). The RNA-Seq data have been submitted to the NCBI (https://www.ncbi.nlm.nih.gov/geo), with GEO accession number GSE148897.

RNA-Seq reads were trimmed and aligned using STAR to mouse mm10 reference genome sequences using Partek Flow for next-generation sequencing data (http://www.partek.com/partek-flow/). The transcript abundance was quantitated using Partek E/M (mm10-Ensembl Transcripts release 97) annotation model to obtain gene counts. Principal component analysis was done on gene counts to determine the variability in the data set (data not shown). Differences in mRNA/transcript abundance between WT and GRTH KI were generated using DESeq2 (fold change < -1.5 or > 1.5; false discovery rate/P_adj < 0.05; P < 0.05) in Partek Flow.

Gene Ontology (GO) enrichment analysis was performed to identify the functional classes using metascape software. Transcripts were annotated to three main GO categories—biological process (BP), cellular component (CC), and molecular function (MF)—and were represented separately. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on all the transcripts which show differential abundance using metascape to identify the important pathways (P < 0.05).

To better illustrate the interactions among the differentially enriched transcripts, The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to construct and visualize the PPI network using Cytoscape software (version 3.7.2), and to analyze by network analyzer plugin, cytoHubba app was used to find the top 100 hub genes based on the maximal clique centrality (MCC) algorithm, and the PPI network was constructed. Four different clusters were identified using the MCODE app in Cytoscape and were further assessed based on functional enrichment analysis.

qRT-PCR was used to validate the differentially enriched transcripts obtained from RNA-Seq transcriptome analysis. Total RNA prepared for RNA sequencing library preparation was used to perform qRT-PCR. One microgram of total RNA was used to prepare cDNA using the Iscript first-strand synthesis kit (Bio-Rad Laboratories, Hercules, CA, United States), and qRT-PCR was performed with Fast SYBR green using a set of specific gene primers (Supplementary Table 1) in a 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, United States). All reactions were performed in triplicates, and the cycle threshold (Ct) values were normalized to DDX-4/Vasa/MVH as the reference gene, and the comparative quantification of mRNA was performed using the 2^–ΔΔCt method.

The CB-enriched fraction (FPEL; 0.5 mg) isolated from the testes of WT and GRTH KI mice was reverse cross-linked and pre-cleared using 50 μl of protein A-agarose beads (Thermo Scientific) and 1 μg of rabbit IgG containing IP binding buffer (Thermo Scientific, United States) for 30 min at 4°C in a rocker. Upon spinning, the resulting pre-cleared supernatant obtained was incubated with 5 μg of anti-GRTH rabbit polyclonal antibody (Kavarthapu et al., 2019) or rabbit IgG at 4°C overnight to co-immunoprecipitate the GRTH–RNP complex. Upon overnight incubation, the GRTH–RNP complex was incubated with 50 μl of protein A-agarose beads and incubated for 2 h at 4°C. The resulting protein–RNP complex was washed with IP binding buffer (four washes), and the total RNA was isolated using the phenol/chloroform/isoamyl alcohol (25:24:1, v/v; Thermo Scientific, Waltham, MA, United States) method. The first-strand cDNA was prepared using iscript first-strand synthesis kit (Bio-Rad Laboratories, Hercules, CA, United States), and qRT-PCR was performed with Fast SYBR green using a set of gene-specific primers (Supplementary Table 1) in a 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, United States).

A germ cell suspension was obtained by squeezing the seminiferous tubules of WT and GRTH KI mice with fine forceps in 1% PFA containing 100 mM sucrose (RNase free) in a petri dish. The germ cell suspensions were spread onto slides pre-coated/cleaned with 1% PFA with 0.15% Triton X-100 and dried overnight in a humidified chamber. In situ hybridization was carried out using a slightly modified procedure (Rajakumar and Senthilkumaran, 2014). In short, the fixed cells were permeabilized with Proteinase K (in PBST) for 10 min and washed twice with PBST (5 min each). Pre-hybridization was carried out using a hybridization buffer: formamide (1:1 v/v) at 55°C for 1 h in a hybridization oven. The respective probes (100 ng) in hybridization buffer/formamide (1:1 v/v) were pre-heated at 85°C for 5 min, cooled on ice for a few minutes, and added onto the slides and incubated for 4 h in a hybridization oven. For post-hybridization washes, wash buffer containing a decreasing concentration of SSC at 60°C was used. The slides were then blocked with a blocking buffer (Thermo Scientific) containing 5% normal goat serum (Sigma) in 1× maleic acid buffer (Thermo Scientific) for 1 h at room temperature in a humidified chamber. The slides were incubated with anti-DIG Rhodamine secondary antibody (1:500) and specific anti-MVH/DDX4 rabbit polyclonal antibody (Cell Signaling, cst#8761; 1:1,000 dilution) in blocking buffer (Thermo Scientific) containing 5% normal goat serum (Sigma) in maleic acid buffer at 4°C overnight. The slides were subsequently washed with DIG wash buffer (twice for 10 min each), PBST (5 min), and PBS (5 min) and incubated with Alexa Fluor 488 for 1 h at room temperature. The slides were washed with PBST (twice, 5 min each), PBS (twice, 5 min each), and mounted with ProLong Diamond antifade reagent with DAPI (Thermo Scientific). These slides were imaged using a Ziess Axioplan 2 fluorescence microscope (Carl Zeiss, CA) and were analyzed using ZenPro blue and Axiovision v4.5 software (Carl Zeiss, CA, United States).

The significance of the differences between groups was determined by Tukey’s multiple-comparison test (one-way ANOVA analysis) using the Prism software program (GraphPad Software, Inc., San Diego, CA, United States) and Microsoft Excel (Microsoft).

The size of the testis of KI mice was significantly reduced (P < 0.05) when compared to that of WT mice (Supplementary Figure 2). The CBs were isolated successfully from WT and KI mice (Figure 1A). The KI mice have smaller CBs in the testicular sections (Figure 1B) and round spermatids (Figure 1C). The success of the CB isolation procedure was confirmed using microscopy (Supplementary Figure 3). The CBs obtained from the WT group were markedly bigger in size compared to those of GRTH KI mice (Figure 1D). Furthermore, EM studies showed smaller and condensed CBs in KI mice, which is evident by a significant reduction in their size/diameter when compared to CBs of WT mice which display an amorphous “nuage” texture (Figure 1E). Among proteins extracted from CBs of WT and KI mice, the levels of MVH/DDX4, MIWI (CB control), and non-phospho GRTH were unaltered, while pGRTH protein was completely absent in the CBs of KI mice compared to WT (Figure 1F; Table 1A; Kavarthapu et al., 2019). In addition, there was no change in the expression of BMAL1, CLOCK, and YBX3 proteins in the CB of WT and KI mice. PRM1/PRM2 expression was not detected in the CB of WT and KI mice (Supplementary Figure 4).

RNA-Seq was performed on RNA samples obtained from the CBs of WT and KI mice testes (N = 5) to analyze the transcriptomic profiles of CBs in the presence (WT mice) and in the absence of pGRTH (KI mice). Sequencing of CBs from WT and GRTH KI mice yielded 75.6 and 125.9 million reads, respectively. The average Phred score was 38 (with base calling error rate of almost 1 in 10,000), and more than 75% of reads could be mapped to the mouse reference genome. Separation of the GRTH KI and WT genotypes was evident in the hierarchically clustered heat map shown in the transcript abundance profiles (Figure 2A).

The transcript levels of several genes involved in spermatogenesis (Tnp1/2, Prm1/2, TSSK 2/3/6, etc.) were also significantly reduced in the CB of KI mice similar to that which we found in RNA-seq data from germ cells of KI mice (Kavarthapu et al., 2020; Supplementary Table 5). Differences in the transcript abundance between CBs isolated from WT and KI mice were analyzed using DESeq2. In total, 1,421 genes show differential abundance, including 474 genes that showed an increase in abundance and 947 genes with a decrease in abundance (P_adj < 0.05; Supplementary Table 6). Volcano plots (Figure 2B) were used to infer the overall distribution of differentially enriched transcripts. The GO functional enrichment analysis was classified into three categories: BP, CC, and MF (Supplementary Table 7). Only significant GO categories with P < 0.05 were chosen for analysis, and differentially abundant transcripts (top 10) of gene-enriched GO terms were identified. In the BP group, transcripts which show differential abundance were mainly enriched in spermatogenesis, spermatid differentiation, and spermatid development, motility, and fertilization (Figure 3A). In the CC group, transcripts which show differential abundance were enriched in sperm principle piece, microtubule organizing center, cation channel complex, etc., while in the MF group, transcripts which show differential abundance were enriched in protein kinase binding, voltage-gated cation channel activity, proline-rich region binding, passive transmembrane transporter activity, etc. (Figure 3A). KEGG pathway enrichment analysis revealed that all the differentially enriched transcripts (P < 0.05) were associated with either RNA transport or protein processing in the endoplasmic reticulum pathways (Figure 3B; Table 1B). We also compared the transcript abundance (fold change) of genes from CB RNA-seq (this study) and germ cells RNA-seq data (expression in fold change) that were previously reported from our laboratory (Kavarthapu et al., 2020). Among 947 genes with a decrease in abundance in CBs of KI mice, we found 272 genes that were downregulated and eight genes that were upregulated in the germ cells of KI mice. Among 474 genes with an increase in abundance in CBs of KI mice, we found 18 genes that were upregulated in the germ cells of KI mice, which were illustrated in the form of Venn diagrams (Supplementary Figure 8). The detailed list of genes with fold change between these datasets are given in Supplementary Table 5.

All 1,421 differentially enriched transcripts (P_adj < 0.05 and < −1.5/ > 1.5 fold) were analyzed using the STRING tool in Cytoscape software to visualize the PPI network. The top 100 hub genes in the PPI network were chosen based on the MCC algorithm using cytoHubba app in Cytoscape (Figure 4). A total of 99 nodes and 541 edges were mapped in the PPI network, and all these four important modules (squared) which were interconnected to each other belong to major pathways/processes, including RNA surveillance/transport, spermatogenesis, chromatin condensation/compaction, ubiquitin-proteasome pathway (UPP), centrosome organization, and organelle assembly (Figure 4).

To validate the genes that are enriched in RNA-Seq data, specific sets were selected based on GO and KEGG results, and their transcript abundance was analyzed using qRT-PCR. Overall, the qRT-PCR analysis results showed a good correlation with the RNA-Seq/transcriptome analysis results. The transcript levels of Tnp1/2, Prm1/2, Spem1, Tssk 3/6, Grth, tAce, and Upf2 involved in spermatid development, differentiation, chromatin compaction, and remodeling were significantly (P < 0.05) reduced in the CB of GRTH KI mice compared to that of WT mice (Figure 5A). Furthermore, transcripts encoding for factors involved in RNA transport, storage, transcriptional, and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were found to be increased (Figure 5B) in the CB of GRTH KI mice compared to CB of WT mice, confirming the consistency of the RNA-Seq data.

Successful GRTH-IP were confirmed using western blot (Supplementary Figure 9) prior to isolating GRTH-bound mRNAs. GRTH-IP mRNA binding experiments reveal that GRTH protein binding to specific germ cell mRNAs (Tnp1/2, Prm1/2, Grth, and Tssk6) in CBs was decreased significantly to basal IgG levels (P < 0.05) in GRTH KI mice (Figure 6). The mRNA binding function in CBs was impacted significantly in GRTH KI mice (lacks pGRTH), which resulted in impaired chromatin compaction and spermatid elongation and stalled spermiogenesis at step 8.

The ISH analysis revealed that the presence of Tnp2, Prm2, and Grth mRNAs was more abundant in CBs than in the cytoplasm of round spermatids (Figures 7A–C). In WT mouse germ cells, Tnp2, Prm2, and Grth mRNAs (red) and MVH-protein (green) were distinctively noticeable (Figures 7A–C), while in GRTH KI mouse germ cells, Tnp2, Prm2, and Grth mRNAs were drastically reduced in CBs, and the sizes of the CBs were smaller, with less distinction through the cytoplasm (Figures 7A–C). In GRTH KI mice, the mRNA storage decreases due to lack of pGRTH, which is one of the CB structural proteins. Taken together, these results indicate that loss of pGRTH impaired the storage of these specific mRNAs in the CBs of round spermatids which are later required for their translation and are essential for the progress of spermiogenesis. These findings reveal the importance of CBs in mRNA storage and the role of pGRTH in maintaining their structure during specific stages of spermiogenesis.