Deficiency of X-linked TENT5D causes male infertility by disrupting the mRNA stability during spermatogenesis

Dear Editor, Infertility has become a worldwide health problem affecting ~10% of couples. However, genetic causes of male infertility remain largely elusive and unexplained. In addition, advances in assisted reproduction technologies (ART) to obtain biological children are in increasing demand. Oligoasthenoteratozoospermia (OAT) is a common type of male infertility with genetic heterogeneity, manifesting low sperm concentrations, reduced motility and various malformations. A group of OAT-affected men fail to father a biological child via conventional ART, such as intracytoplasmic sperm injection (ICSI), probably due to severe defects in spermatogenesis. Modeling human genetic variants in mice has been shown to be efficient in establishing gene–disease relationships for male infertility. In addition, mouse models have superiority for the exploration and optimization of ART approaches. Herein, by taking advantage of the availability of OAT patients and a gene-edited mouse model, we investigated a novel genetic cause of male infertility and tested a “FACS+ ROSI” (fluorescence activated cell sorting+ round spermatid injection) strategy for a potential compensatory ART approach. First, whole-exome sequencing (WES) and bioinformatic analyses were performed to analyze our cohort of 186 unrelated Han Chinese men with OAT. A hemizygous stop-gain variant (c.637G > T [p.Glu213*]) of X-linked TENT5D (NCBI GenBank: NM_001170574.2) was identified in the proband (II-1 in Fig. 1a) from nonconsanguineous family HX001. Sanger sequencing showed that the mother (I-2 in Fig. 1a) is a heterozygous carrier. The variant p.Glu213* with glutamicacid that located at a conserved position of TENT5D changed to a stop codon was predicted to be deleterious by MutationTaster and CADD tools (Fig. 1a; Supplementary Table S1). This TENT5D variant is novel and absent in human population genome databases, including the 1000 Genomes Project and gnomAD (Supplementary Table S1). In the OAT patient harboring a hemizygous TENT5D variant, the sperm DNA fragmentation index and high DNA stainability were dramatically increased (Supplementary Table S2). The spermatozoa from the hemizygous TENT5Dmutated patient displayed multiple heads and/or multiple flagella upon hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM) detection (Fig. 1b; Supplementary Fig. S1 and Table S2). Transmission electron microscopy (TEM) also revealed multiple heads and/ or multiple flagellar ultrastructural abnormalities (Fig. 1c). These findings suggest that TENT5D deficiency may cause human male infertility with OAT. Considering that TENT5D is highly conserved between humans and mice (83.8% similarity in consensus positions), we used a mouse model to investigate whether TENT5D deficiency plays a causative role in male infertility. Murine ortholog X-linked Tent5d is preferentially expressed in the testis and epididymis (Fig. 1d). Furthermore, Tent5d was highly expressed at 3 to 4 weeks, the

software. We then used the ANNOVAR software for functional annotation with a variety of genome datasets and bioinformatic tools, including OMIM, Gene Ontology, SIFT, PolyPhen-2, MutationTaster, 1000 Genomes Project and gnomAD [4][5][6][7][8] . Sanger sequencing was conducted to validate the candidate pathogenic variants using the PCR primers listed in Supplementary Table S7.

Semen parameters analysis
Semen samples of human individuals were collected through masturbation after 2 -7 days of sexual abstinence and examined in the source laboratories during routine biological examination according the World Health Organization (WHO) guidelines. Sperm morphology was assessed by hematoxylin and eosin (H&E) staining and electron microscopy assays. We examined at least 200 spermatozoa to evaluate the percentages of morphologically abnormal spermatozoa.
For mouse semen samples, we collected them from the cauda epididymides. Then we diluted these samples in 1 mL of human tubal fluid (HTF; Millipore, Cat. # MR-070-D) before incubation at 37℃ for 15 minutes. Sperm counts and motility were analyzed by a computer-assisted semen analysis system.

Sperm chromatin structure assay
DNA fragmentation index (DFI) and high DNA stainability (HDS) were measured by flow cytometry using sperm DNA fragmentation staining kit (ANKEBIO). At least 5000 sperm cells were measured and repeated twice for each subject. The acridine orange staining solution was added to stain the sites of single-strand DNA breaks red and the double-strand DNA breaks green. Then the red and green fluorescence signals were collected by flow cytometry, and the DFI and HDS values were calculated using the Flowjo software.

Mouse model
Tent5d-mutated mice were generated using CRISPR-Cas9 technology. Cas9 and sgRNA were prepared as described previously 9 . Founder mice were crossed to WT C57BL/6 mice to establish the mutant mouse line 1,2,10 . The founder mice and their offspring were identified by Sanger sequencing with the primers listed in Supplementary Table S7. Young mice (4-week-old) and adult mice (8-week-old) were used in this study. All animal experiments were carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The study was approved by the animal ethics committee at the School of Life Sciences of Fudan University.

Reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR)
For RT-PCR, total RNAs were extracted from mouse various tissues using RNeasy Mini Kit (Qiagen). Approximately 1 μg of RNA was reversely transcribed to cDNA using SuperScript Ⅲ Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. RT-PCR was performed with cDNA, and Gapdh was used as an internal control (Supplementary Table S7).
For RT-qPCR, total RNAs were extracted from human spermatozoa and mouse testes and reversely transcribed as described above. The cDNAs were diluted 5-fold to be used as templates for RT-qPCR with AceQ qPCR SYBR Green Master Mix (Vazyme). The results were analyzed and shown as relative mRNA levels of the CT (cycle threshold) values, which were then converted as fold changes. GAPDH/Gapdh were used as internal controls, and primers for RT-qPCR are presented in Supplementary Table S7.

Electron microscopy assays
For scanning electron microscopy (SEM), human sperm specimens were subjected to fixation in 2.5% glutaraldehyde, washed with 0.1 mol/L phosphate buffer for 30 minutes and postfixed in 1% osmic acid. Next, the samples were progressive dehydration with ethanol and dried with a CO2 critical-point dryer (Eiko HCO-2, Hitachi). Subsequently, the samples were mounted on aluminum stubs, sputter-coated by an ionic sprayer meter (Eiko E-1020, Hitachi) and analyzed via SEM (Stereoscan 260) under an accelerating voltage of 20 kV.
For transmission electron microscopy (TEM), human semen and mouse testis/epididymis were fixed in 2.5% glutaraldehyde. The samples were progressively dehydrated with ethanol gradient (50%, 70%, 90%, and 100%) and 100% acetone. Next, the samples were embedded in Epon 812, sliced by an ultra-microtome and stained with uranyl acetate and lead citrate. Finally, the samples were observed and photographed by TEM (TECNAI-10, Philips) with an accelerating voltage of 80 kV.

Histological analysis of mouse tissues
Fresh mouse testis and epididymis tissues were fixed in Bouin's solution and 4% paraformaldehyde, respectively. After 24 hours at 4℃, the samples were embedded in paraffin and sectioned at 5 μm intervals by a microtome. The sections were routinely stained with H&E and periodic acid-Schiff (PAS) reagent, respectively.

Immunohistochemistry
Fresh mouse testis tissues were fixed in 4% paraformaldehyde at 4℃ for 24 hours and embedded in paraffin. The sections samples were performed by a microtome at 5 μm. Then, the samples were incubated with anti-TEX14 (18351-1-AP, Proteintech, 1:100).

TdT-mediated dUTP nick-end labeling
Fresh mouse testis tissues were fixed in 4% paraformaldehyde at 4℃ for 24 hours. We then embedded the samples in optimal cutting temperature compound. Tissue sections were prepared and mounted on glass slides. Apoptotic analysis was performed using the TMR Tunel Cell Apoptosis Detection Kit (G1502, Servicebio).

PCR-based poly(A) test assay
PAT assays were performed as described previously with specific primers (Supplementary Table S8) 11 . In brief, total RNAs extracted from testis were incubated with p[dT]12-18, oligo[dT]-anchor, and T4 DNA ligase before reverse transcription reaction for generating cDNA. The polyadenylation state of specific mRNAs was analyzed by PCR using a mRNA gene-specific primer (F), oligo(dT)-anchor (R). The specific mRNAs were analyzed by PCR using a mRNA gene-specific primer (F), mRNA gene-specific poly(A) start primer (R). The PCR reaction consisted of initial denaturation of 93℃ for 5 minutes; then 30 cycles of 93℃ for 30 seconds, 57℃ for 1 minute, and 72℃ for 1 minute; followed by a final extension of 72℃ for 7 minutes. The amplified DNAs were separated on 5% polyacrylamide TBE gel, and stained with ethidium bromide.

Isolation of spermatogenic cells from mouse testes
Mouse spermatocytes and round spermatids were isolated from mouse testes as previously described 12 . In brief, total spermatogenic cells were extracted from the seminiferous tubules of male mice, stained by Hoechst 33342 (Acros Organics), and then separated through fluorescence activated cell sorting (FACS). The isolated spermatogenic cells were further confirmed by their distinct nuclear morphology (Hoechst 33342 staining of nuclei).

Differential expression analysis
R software packages were used for differentially expressed genes (DEGs) analysis with the counting matrix. The differentially expressed mRNA levels were analyzed using DESeq2 19 .
The shrinkage approach of DESeq2 was used to implement a regularized logarithm (rlog) transformation for PCA analysis (prcomp function). The median-of-ratios method of DESeq2 was used to calculate the normalization counts. Only the transcripts with |FC| ≥ 2 and padj (P.adjust) < 0.05 were considered as DEGs. Clustering analysis of DEGs was conducted with numeric matrix transformed from normalization counts using the ComplexHeatmap package 20 . Volcano plots were assembled using the DESeq2 transformed output (log2FC and -log10padj) and plotted using the ggplot2 package. The target gene enrichment test of downregulated genes was annotated based on biological processes Gene Ontology terms using clusterProfiler package 21 .

Intracytoplasmic sperm injection and intracytoplasmic round spermatid injection in mice
Superovulation and intracytoplasmic sperm injection in mice were performed as previously The corresponding mutated nucleotides were shown in red. c The frameshift mutation was predicted to cause premature translational termination (p.Tyr208Glyfs*7) of mouse Tent5d.
The frameshift-mutated amino acid sequences were shown in red.

Supplementary Fig. S4 Expression analysis of Tent5d mRNA and protein in the
Tent5d-mutated male mice. a RT-qPCR assay showed that the level of Tent5d mRNA was almost undetectable in the testis from Tent5d-mutated (Tent5d − /Y) male mice. The data represent the means ± standard deviations (SDs) of three independent experiments. Two-tailed Student's paired or unpaired t tests were used as appropriate (***p < 0.001). b Expression and location of TENT5D (red) protein in mouse testis revealed by immunofluorescence staining assay. DNA was counterstained with DAPI as a nuclei marker. Scale bars: 40 μm. In vitro tailing assay shows that TENT5D extends 5 [ 32 P]-labeled A15 substrate only in the presence of ATP.

Supplementary Fig. S11 Quality control and sequencing analysis of isolated germ cells.
a Morphological confirmation of spermatocytes (SC) and round spermatids (RS) isolated from the testes of wild-type (WT, Tent5d + /Y) and Tent5d-mutated (MU, Tent5d − /Y) male mice. Scale bars: 50 μm. b Transcriptome analysis of representative genes of SC and RS isolated from the testes of Tent5d + /Y and Tent5d − /Y male mice. c Hierarchical clustering of significantly differently expressed mRNAs in the spermatocytes and round spermatids between WT and MU male mice. The expression levels of mRNAs were represented by a color scale. "Blue" represents the low relative expression level, and "red" represents the high relative expression level. Each column represents a biological repeated sample, and each row represents a single mRNA. d The numbers of differentially expressed genes between Tent5d-mutated groups and WT controls (|FC|>2, P.adjust<0.05) in SC and RS.

Supplementary Fig. S14 Intracytoplasmic sperm injection (ICSI) is inefficient for overcoming sterility of Tent5d-mutated (Tent5d − /Y) male mice.
Representative blastocysts from ICSI in mice. The rate of blastocysts was dramatically lower in the ICSI group using the spermatozoa from Tent5d − /Y male mice than that in the control ICSI group using the spermatozoa from wild-type (Tent5d + /Y) male mice. Scale bars: 25 μm.  Supplementary Table S1