Deleterious genetic changes in AGTPBP1 result in teratozoospermia with sperm head and flagella defects

Abstract Approximately 10%–15% of couples worldwide are infertile, and male factors account for approximately half of these cases. Teratozoospermia is a major cause of male infertility. Although various mutations have been identified in teratozoospermia, these can vary among ethnic groups. In this study, we performed whole‐exome sequencing to identify genetic changes potentially causative of teratozoospermia. Out of seven genes identified, one, ATP/GTP Binding Protein 1 (AGTPBP1), was characterized, and three missense changes were identified in two patients (Affected A: p.Glu423Asp and p.Pro631Leu; Affected B: p.Arg811His). In those two cases, severe sperm head and tail defects were observed. Moreover, AGTPBP1 localization showed a fragmented pattern compared to control participants, with specific localization in the neck and annulus regions. Using murine models, we found that AGTPBP1 is localized in the manchette structure, which is essential for sperm structure formation. Additionally, in Agtpbp1‐null mice, we observed sperm head and tail defects similar to those in sperm from AGTPBP1‐mutated cases, along with abnormal polyglutamylation tubulin and decreasing △−2 tubulin levels. In this study, we established a link between genetic changes in AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation.


| INTRODUC TI ON
Infertility has been recognized as a global public health concern by the World Health Organization (WHO), and affects at least 8%-12% of couples worldwide 1,2 and approximately 7% of all men. 3,4ratozoospermia is the leading clinical manifestation of male sterility, and is characterized by sperm head defects accompanied by sperm DNA damage. 5[9] In the past decade, the identification of genetic variations (e.g., copy number variation, deletion, insertion, and nucleotide alterations) associated with male infertility using next-generation sequencing (NGS) has increased. 8However, research on the aetiology of male infertility typically focuses on specific patient subgroups, such as men with teratozoospermia.NGS has been used to examine various subtypes of teratozoospermia, such as sperms with multiple flagellar morphological abnormalities, 10 cases of primary ciliary dyskinesia-associated male infertility, 11 and globozoospermia. 12However, many plausible causes of male infertility have yet to be replicated and the aetiology may vary between populations. 85][16] Conversely, tubulin tyrosine ligase-like family proteins (TTLL) add polyglutamate. 17,18AGTPBP1 mutations can cause childhood-onset neurodegeneration with cerebellar atrophy (CONDCA) by decreasing deglutamylase activity and the protein amount of △−2 tubulin. 19Furthermore, deletion of the Agtpbp1 allele in spontaneous strain mice not only causes neuronal degeneration but also results in male infertility. 20,21TPBP1 regulates the post-translational modifications of tubulin, such as polyglutamation, and generates △−2 tubulin.The development of male germ cells is highly reliant on the dynamics of microtubule structures, such as sperm head shaping and tail formation. 22,23We suggest that mutated AGTPBP1 may be involved in human teratozoospermia.
In this study, we screened patients with teratozoospermia in Taiwan for genetic alterations using whole-exon sequencing (WES) and identified three genetic mutations in AGTPBP1.Notably, the sperm head and tail phenotypes observed in Agtpbp1-defective mice resembled those observed in patients carrying AGTPBP1 missense mutations, providing strong evidence that AGTPBP1 is a human male infertility gene.

| Case enrolment
This study was approved by the Ethics Committee of Cathay General Hospital (IRB approval no: CGH-P102031; CGHFJU-105006).
Informed consent was obtained from all recruited patients.Semen samples were obtained by masturbation after 3-5 days of sexual abstinence.After liquefying the semen, routine semen analysis was performed according to the WHO 2010 criteria. 24The results for the selected cases are listed in Table S1.Sperm morphology was evaluated using Kruger's criteria.

| Genomic DNA extraction, WES and bioinformatic analysis
Genomic DNA was extracted from sperm using a QIAamp DNA Micro Kit according to the manufacturer's instructions and stored at −80°C until the experiment.DNA libraries were prepared using the Illumina TruSeq Exome Library Prep Kit and sequenced using the Illumina NextSeq platform.The average coverage depth was approximately 50×.Sequenced reads were mapped using the Burrows-Wheeler Aligner.WES reads were aligned to the human reference genome (Genome Reference Consortium Human Build 37).Small-copy number variants, insertions, deletions, and variants were identified in individual cases.In collaboration with Professor Donald F. Conrad, the WES outputs were filtered and prioritized using the population sampling probability (PSAP) test, a statistical framework for assessing the significance of variants from single cases of rare genetic diseases. 25The WES data were filtered according to the following rules: (1) read depth > 10, (2)   percentage of alternated sites of the reads/all reads >20%, and (3) PSAP <0.001.The selected variants were annotated using data from the Exome Aggregation Consortium (ExAC) genome database, and pathogenic effects were predicted using SIFT and PolyPhen-2 softwares.Finally, the mutated sites of the candidate AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation.

K E Y W O R D S
AGTPBP1, genetic changes, male infertility, teratozoospermia, whole-exome sequencing | 3 of 13 genes were confirmed by Sanger sequencing after polymerase chain reaction (PCR) using the reference transcript of AGTPBP1 (NM_001286717.1).The following primes were used for the genomic typing of AGTPBP1: forward (F′: GGCTT CTG AGG GTT AATGG AG) and reverse (R′: TGGGG CTG AAG TAG GGT CTAA).
To determine the effects of mutations within the protein structure, three-dimensional (3D) models of mutated-AGTPBP1 were predicted using ColabFold 26 and the carboxypeptidase domain as templates.The results were visualized using PyMOL software (PyMol Molecular Graphics System, Version 2.5.4).
For TEM, spermatozoa were washed in 0.1 M phosphate buffer (pH 7.2), fixed and processed according to the protocol described in our previous study. 28The final sections were counterstained with lead citrate and uranyl acetate and observed using a JOEL 1200 TEM. 29

| Preparation of the murine testicular germ cell populations
All animal studies were approved by the Institutional Animal Care and Use Committee (No: A10871, date of approval: 04/10/2020; No: A10979, date of approval: 03/18/2021) of Fu Jen Catholic University.Murine male germ cells were isolated using a centrifugal system according to the density of different types of germ cells, as described previously. 28Briefly, testes were decapsulated and the seminiferous tubules were enzymatically digested, after which the germ cell suspensions were filtered through 35-μM nylon filters (Falcon).The suspension of single cells was centrifuged at different gravity levels on a Kubota centrifuge 3330.Germ cells were collected at different developmental stages.Mature spermatozoa were collected from the cauda epididymides of adult male mice.Finally, suspended male germ cells were spread on a slide and airdried for further analysis.

| Generation of Agtpbp1 knockout mice using CRISPR/Cas9
sgRNA was designed by the Gene Knockout Mouse Core Laboratory of the National Taiwan University Center of Genomic Medicine to delete the critical functional carboxypeptidase domain of the Agtpbp1 allele.The generated sgRNAs and Cas9 targeted the genomic Agtpbp1 allele in C57BL/6 mouse embryonic stem cells (MESCs) to create a deletion (del) region (Figure 4A).After replacing the wild-type allele in MESCs, clones bearing the targeted allele were confirmed using PCR and sequencing.The confirmed clones were injected into C57BL/6J blastocysts.Blastocysts were transferred to pseudopregnant female mice.Male chimeras were mated with wild-type females to generate Agtpbp1 +/del mice.The reproductive ability of each group was compared among pups from the same pregnancy.

| Sperm quality analysis
Spermatozoa collected from enrolled patients or the vas deferens of wild-type (n = 5), Agtpbp /+/del (n = 5), and Agtpbp del/del (n = 6) adult male mice was suspended in human tubal fluid (HTF) medium (Irvine Scientific).To determine sperm counts, sperm were immobilized by dilution in water and counted using a haemocytometer in duplicate.To evaluate sperm morphology, the sperm medium was diluted to 10 6 /mL with HTF and spotted onto a glass slide.
A total of 200 sperms (both motile and immotile) were counted under a microscope in duplicate to obtain the average percentage of motility.

| Identification of the novel genetic alterations in patients with teratozoospermia
To explore the genetic causes of teratozoospermia in Taiwan, we enrolled 254 males with infertility, defined as those with one or more abnormal semen parameters.Twelve patients with severe morphological sperm defects were selected (Table S1) and WES was performed.The identified genetic variants were evaluated using the PSAP test, allele frequencies from ExAC, and the pathogenicity predictors PolyPhen and SIFT (Figure 1A).Finally, heterozygous variants in seven teratozoospermia-related genes were predicted to be potentially deleterious (Figure 1A damaged the protein (Figure 1A).Furthermore, the changed site of p.Arg811His localized within the critical carboxypeptidase A domain of AGTPBP1 (Figures 1C,D).The predicted 3D protein structure revealed that the substitution of Arg811 with His would likely disrupt the oxygen-hydrogen bonding between nearby amino acids (p.Gln793, Leu1117, and Ile1130), potentially affecting structural stability and destabilizing the overall functional structure (Figure 1E).In summary, based on WES and bioinformatics tests, several genetic alterations were identified in patients with teratozoospermia, and AGTPBBP1 genetic variations appeared to be associated with human teratozoospermia.

| Dynamic patterns of AGTPBP1 during murine spermiogenesis
To determine the precise localization and possible reproductive role of AGTPBP1 during murine spermiogenesis, testicular germ cell populations were separated and subjected to immunostaining.
In steps 1-7, round spermatids containing AGTPBP1 were distributed around the whole cells (Figure 3A).During shaping of the sperm head in steps 8-10, AGTPBP1 moved towards the post-acrosomal region and was then recruited to the manchette structure in steps 11-13 (Figure 3B).Subsequently, AGTPBP1 was concentrated at the sperm neck (Step 14), followed by the removal of the middle piece at steps 15-16 (Figure 3C).In mature caudal epididymal spermatozoa, AGTPBP1 was localized in the midpiece region (Figure 3C).These results suggest that AGTPBP1 is highly expressed during spermiogenesis and is likely involved in sperm head and tail formation.

| Loss of Agtpbp1 in mice causes severe morphological defects in the sperm
To further test the causal relationship between genetic changes in AGTPBP1 and male sterility, we generated an Agtpbp1 del mouse line.(Figure 4A).AGTPBP1-deficient mice were confirmed by genotyping and immunoblotting (Figure 4B,C).Agtpbp1 del/del mice were completely sterile when paired with controls (wild-type male mice: 8 ± 0.82 pups per litter, n = 5 vs. Agtpbp1 del/del male mice: 0 pups per litter, n = 6).Sperm isolated from the vas deferens and epididymis of Agtpbp1 del/del male mice revealed severe abnormalities in sperm morphology and motility (wild-type: n = 5; Agtpbp1 +/del : n = 5; Agtpbp1 del/del : n = 6) (Figure 4 D-G).Notably, sperm morphology in Agtpbp1 null mice displayed severe defects, such as immature sperm (no tail development) and tail defects (Figure 4D,F), which was similar to the results observed in AGTPBP1-mutated patients (Figure 2B,C).These results revealed that AGTPBP1 is required for normal sperm development and male fertility (Figure 4).

| Agtpbp1 deficiency affects the de-polyglutamylation of tubulin during sperm development
The precise regulation and maintenance of tubulin stability are vital for the morphological formation of the sperm head and elongation of the tail, which are dependent on the stabilization of the microtubule structure. 22Precise regulation of the balance between polyglutamylation (poly-E) and de-polyglutamylation in the C-terminal region of tubulin is mediated by TTLL and AGTPBP1 16,31 (Figure 5A).

Therefore, we hypothesized that the loss of AGTPBP1 in mice results
in an overabundance of polyglutamylated microtubules in developing germ cells and sperm.To test this hypothesis, we performed immunoblotting and immunostaining of Agtpbp1-deficient testicular tissues to evaluate whether the loss of Agtpbp1 affected the stability of poly-E tubulin.Compared to wild-type mice, the testicular tissues of Agtpbp1-deficient mice showed abnormal poly-E tubulin lengths (Figure 5B).Within germ cells from wild-type mice, poly-E tubulin was organized as a filamentous manchette structure in elongating spermatids and elongated spermatids during sperm head formation (Figure 5C-a,b) Furthermore, poly-E tubulin was present in the tails of mature sperm (Figure 5C-c).In contrast, the loss of Agtpbp1 in mice resulted in disorganized poly-E tubulin in sperm with no tail development (Figure 5D-a,b) or a defective tail (bent and curled) (Figure 5Dc,d).Thus, the loss of Agtpbp1 disrupts the stability of poly-E-tubulin, which is critical for sperm head and tail formation.

| Loss of AGTPBP1 decreases the amount and disturbs the localization of △−2 tubulin during sperm head and tail formation
The deglutamylation activity of AGTPBP1 is a critical step for △−2 tubulin generation (Figure 6A).Therefore, we investigated whether the disrupted Agtpbp1 allele affected the generation of △−2 tubulin.
Figure 6B shows a significant decrease in the amount of △−2 tubulin in the Agtpbp1 del/del testis, compared with that in wild-type mice.
In wild-type mice, △−2 tubulin is a major component of the manchette (Figure 6C-a,b) and sperm tail (Figure 6C-c) at various developmental stages: elongating spermatids, elongated spermatids, and mature sperm.In contrast, the loss of AGTPBP1 function resulted in a disrupted △−2 tubulin structure within the sperm with no tail development (Figure 6D-a,b) and morphologically defective sperm (Figure 6D-c,d).Collectively, these data indicate that AGTPBP1 plays an essential role in the generation of △−2 tubulin in developing male germ cells (Figure 6).

| DISCUSS ION
In this study, three sporadic genetic alterations in AGTPBP1 in Taiwanese patients with teratozoospermia were identified using WES.Sperm from affected patients showed severe structural head and tail defects.AGTPBP1 was mainly observed during murine sperm head formation and tail elongation during spermiogenesis.Consistent with these findings, sperm from a Agtpbp1 deletion mouse model exhibited morphological defects in the head and tail.
At the molecular level, the loss of Agtpbp1 resulted in an abnormal ploy-E tubulin length and decreased △−2 tubulin generation in vivo.To the best of our knowledge, this is the first study to link genetic changes in AGTPBP1 with teratozoospermia from a clinical perspective.

| Identification of male sterility-related genetic changes in Taiwan from WES
Several studies investigating sterility-related genetic alterations have been conducted; however, these genetic alterations differ among populations.In this study, deleterious genetic changes were identified in seven genes using WES and data obtained from PSAP, ExAC, PolyPhen, and SIFT analyses.Five of these genes were characterized as male infertility-related genes, including Polo-like kinase 4 (PLK4), AGTPBP1, KISS1 receptor, Meiosis Expressed Gene 1 (MEIG1), and Piwi Like RNA-Mediated Gene Silencing 2 (PIWIL2) (Figure 1).PLK4, which belongs to the polo protein family of serine/threonine protein kinases, 32,33 is located in centrioles, which are microtubulebased structures within centrosomes, and is involved in centriole formation. 33In clinical observations, a heterozygous 13-bp deletion in the serine/threonine kinase domain of PLK4 was identified in azoospermia cases with Sertoli cell-only syndrome (SCOS), 34 and a heterozygous PLK4 mutation (p.Ile242Asn) in mice caused male germ cell loss in the testes, 35 similar to that in human SCOS.Another gene, MEIG1, is involved in meiosis. 36Meig1 knockout in male mice results in sterility because of the arrest of spermiogenesis before the completion of spermatid elongation. 37TEM revealed that the manchette structure was disrupted in spermatids of Meig1-deficient mice.Based on previous reports, the mutated genes identified using WES in this study appear to be potential causative candidates for male infertility in Taiwan.

| AGTPBP1 mutations cause CONDCA
CONDCA has recently been identified as a rare and severe autosomal recessive disorder that affects neurodevelopment in the central and peripheral nervous systems. 19 using WES in 13 cases from 10 families. 19Furthermore, different CONDCA-related mutations (p.R374*, p.R759L, p.T784C, p.R799L, and p.R1000*) and alteration in the splicing site (c.2342 + 2 T > G) of AGTPBP1 have been characterized in different populations. 32,38,39 these studies, mutations affecting the amount of protein or the molecular functions of AGTPBP1 were also analysed.The fibroblast cells collected from CONDCA cases with the mutants were found to have decreased AGTPBP1 levels. 19Muscle tissues from patients with p.Q856* showed an accumulation of abnormally sized poly-E tubulin.Moreover, HEK293 muscle cells transfected with the mutant AGTPBP1 exhibited impaired deglutamylase activity, which decreased the abundance of the stable form of tubulin, △−2 tubulin.However, because CONDCA is a severe childhood-onset neurodegenerative disease, its reproductive phenotype has not been evaluated.In the present study, we identified three genetic alterations in AGTPBP1 in patients with teratozoospermia (affected individual A: p.Glu423Asp and p.Pro653Leu; affected individual B: p.Arg811His) (Figure 1).Spermatozoa from patients with AGTPBP1 mutations exhibited disrupted patterns and decreased signals of AGTPBP1 as well as head and tail defects (Figure 2); however, neither patient showed neurodegenerative phenotypes (Figure 1).This suggests that different mutation sites in AGTPBP1 result in distinct genetic outcomes in neurons and male germ cells.Further investigation is required to understand the genetic changes in AGTPBP1 that lead to CONDCA and teratozoospermia.

| Loss of the Agtpbp1 allele in mice also causes male sterility
The recessive and spontaneous mouse mutation Purkinje cell degeneration (pcd) causes early onset degeneration of the cerebellar Purkinje cells, retinal photoreceptor cells, and olfactory bulb and thalamic neurons. 40,41In addition to neuronal degeneration, pcd mutant male mice also exhibit male sterility, with decreased sperm counts and few mature sperm. 32After genetic mapping, the mutated gene of the pcd strain mice was mapped to chromosome 13, and mutations, deletions, and insertions of genomic fragments within the AGTPBP1 gene were identified in pcd 2J , pcd 3J , and pcd 5J strains, 20,42 respectively.Kim et al. (2011) first detected AGTPBP1 in spermatocytes and found it to be primarily expressed in round spermatids in testicular sections. 21Loss of the AGTPBP1 allele in pcd 3J mice decreased testicular weight, increased apoptotic cell death in the testes, and produced abnormally shaped spermatozoa from the cauda epididymis. 37However, the detailed dynamic expression patterns and in vivo functions of AGTPBP1 during sperm morphological development remain unclear.In this study, we found that AGTPBP1 was specifically localized around the manchette structure of the sperm head and elongating tail during murine spermiogenesis (Figure 3).Significantly reduced sperm count and motility and an increased number of immature (no tail development) and tail-defective sperm were observed in our Agtpbp1-knockout mice (Figure 4).Immature sperm are released from the seminiferous tubules owing to disrupted sperm development.These findings indicate that AGTPBP1 is critical for sperm head and tail formation during murine spermatogenesis.

| Deglutamylation activity of AGTPBP1 is critical for the formation of sperm head and tail in mice
The balance between polyglutamylation and deglutamylation in the C-terminus of tubulin is critical for the stability of the microtubule structure during neuronal development. 43Polyglutamylases and deglutamylation modifications are catalysed by tubulin tyrosine ligase-like proteins, TTLL family proteins, and cytosolic carboxypeptidase family proteins (e.g., AGTPBP1). 44,45Using pcd strain, Shashe et al. reported that the femoral quadriceps nerve of pcd 3J mice exhibited a reduction in nerve diameter and the number of myelinated axons formed via microtubule polymerization. 19Furthermore, the cerebellum of pcd 3J mice exhibited the increased tubulin polyglutamylation and decreased △−2 tubulin generation.These results support the hypothesis that AGTPBP1 is critical for maintaining microtubule structure, the major cytoskeletal component of the axon, and preserves neuron numbers.In this study, Agtpbp1 ablation in mice revealed similar results: an increase in abnormally sized polyglutamylated tubulin and decrease in △−2 tubulin levels (Figure 5B and 6B).Compared with signals from wild-type mice, the multiplex sizes of polyglutamylated tubulin and △−2 tubulin exhibited fragmented patterns during sperm head and tail formation (Figure 5C and 6C).Therefore, we suggest that the deglutamylation activity of AGTPBP1 is critical for the formation and maintenance of neurons and male germ cells.

| Enzymatic and molecular roles of AGTPBP1 during murine spermiogenesis
Based on our results and those of previous studies, we proposed a possible molecular model of AGTPBP1 during spermiogenesis (Figure 7).AGTPBP1 was highly localized in the manchette structure in the heads of elongating spermatids and the tails of mature sperm (Figure 3).The manchette, a temporary microtubule and an actin-based structure, facilitates the transport of vesicles and proteins necessary for the formation of the sperm head and tail. 22,46n tubulin.AGTPBP1 also catalyses the generation of △2-tubulin to form stable microtubulin after the detyrosination of tubulin (Figure 7B).In AGTPBP1-defective mice, the manchette and sperm tail structures were disrupted (Figures 5 and 6) due to the impairment of the enzymatic functions of AGTPBP1.
The present study is the first to establish a connection between the genetic mutations in AGTPBP1 to teratozoospermia.Damage to AGTPBP1 in human and mouse sperm results in severe sperm tail and head defects, leading to the loss of deglutamylation function.This study identifies the role of AGTPBP1 mutations in teratozoospermia and provides potential guidance for the diagnosis and treatment of male infertility.
: PLK4: p.Pro953Leu; AGTPBP1: p.Glu423Asp, p.Pro653Leu, p.Arg811His; GRID2: p.Arg631Gln; KISS1R: p.Pro196His; P2RX2: p.Ala182Ser; MEIG1: p.Asp63Asn; PIWIL2: p.Thr937Ser).One of the candidate genes, AGTPBP1, is remarkable, as a classical spontaneous Agtpbp1 mouse mutant has been shown to exhibit abnormal sperm development in previous studies. 20,21Genetic changes in AGTPBP1 were observed in both cases.Affected individual A carried compound heterozygous mutations (NM_001286717.1:c.1336A > T [p.Glu423Asp] and c.1959C > T [p.Pro653Leu]) (Figure 1B).The other hemizygous mutation (NM_001286717.1:c.2499G > A [p. Arg811His]) was detected in affected individual B. (Figure 1C).Both PolyPhen and SIFT predicted that two amino acid changes (p.Pro653Leu and p.Arg811His) Immunostaining and TEM were used to examine genetic changes in AGTPBP1 in spermatozoa.AGTPBP1 was expressed in spermatocytes (black arrows) and was strongly expressed in spermatids (red arrows) in human testicular sections obtained from the Human Protein Atlas database (Figure2A).Immunofluorescence staining of human spermatozoa revealed that AGTPBP1 was mainly localized in the neck (white arrows) and annulus (red arrows), which are ring-like structures separating the midpiece and principal regions of the tail (Figure 2B; left panel).Spermatozoa from the affected individual A exhibited compound heterozygous mutations of AGTPBP1 (NM_001286717.1:c.1336A > T [p.Glu423Asp] and c.1959C > T [p.Pro653Leu]), while affected individual B carried a hemizygous mutation of AGTPBP1 (c.2499G > A [p. Arg811His]) that resulted in head defects (Figure 2B; middle panel) and lack of tail development (Figure 2B; left panel), respectively.Contrast immunostaining using sperm from the control, affected individual A, and affected individual B revealed mislocalized AGTPBP1 signals to the midpiece that had fragmented patterns (white arrows).Furthermore, spermatozoa from affected individual B showed significantly decreased AGTPBP1 signals (Figure 2B; Right panel).TEM revealed severe morphological and size abnormalities of the sperm head of spermatozoa from affected individual B (Figure 2C; red arrows).Most sperm showed no tail development, and a small portion of the sperm with tails showed disarranged mitochondria (Figure 2C; black arrows).These results indicated that genetically altered AGTPBP1 expression resulted in spermatozoa with severe head and tail defects.

F I G U R E 1
Identification of ATP/GTP Binding Protein 1 (AGTPBP1) genetic alterations in teratozoospermia using whole-exome sequencing (WES).(A) Screening for genetic changes in 12 teratozoospermia cases using WES and the results of bioinformatic analysis (PSAP; ExAC; PolyPhen, and SIFT).(B, C) Sanger sequencing chromatograms of AGTPBP1 variants.Chromatograms display the sequence corresponding to the control and mutated alleles (upper and lower panels, respectively).Genetic changes in AGTPBP1 in affected individual A carrying compound heterozygous mutations (NM_001286717.1:c.1336A > T [p.Glu423Asp]; c.1959C > T [p.Pro653Leu]) and affected individual B carrying a hemizygous mutation (NM_001286717.1:c.2499G > A [p. Arg811His]).Red arrows indicate the site changes compared with control cases.(D) Schematic of the AGTPBP1 gene (upper panel) and protein (lower panel).The 25 exons of the human AGTPBP1 were numbered.N = N-terminal; C = C-terminal.The near N-terminal and C-terminal regions of AGTPBP1 are encoded with the armadillo-type fold and carboxypeptidase A, respectively.The amino acid of p.Arg811His was located in exon 17 of AGTPBP1.(E) The substitution of arginine (green colour) with histidine (yellow colour) (p.Arg811His) in the predicted carboxypeptidase A domain structure disrupts the oxygenhydrogen bonding between the nearby amino acids (p.Gln793, Leu1117, and Ile1130).

F I G U R E 2
Immunostaining and electron microscopy analysis in cases with ATP/GTP Binding Protein 1 (AGTPBP1) mutations.(A) Immunohistochemical detection of AGTPBP1 signals in the human testicular sections from The HUMAN PROTEIN ATLAS.Black and red arrows indicate the signals on spermatocytes and spermatids, respectively.(B) Sperm from the control (Left), affected individual A carrying compound heterozygous mutations (NM_001286717.1:c.1336A > T [p.Glu423Asp]; c.1959C > T [p.Pro653Leu]), and affected individual B carrying a hemizygous mutation (NM_001286717.1:c.2499G > A [p. Arg811His]).AGTPBP1 is mainly located at the sperm neck (white arrows) and annulus (red arrows) in control cases.AGTPBP1 signals (white arrows) denote the disrupted patterns (fragmented) (Middle and Left) and decreased AGTPBP1 signals (Left).Sperm head stains with nuclear dye (DAPI; blue).The sperm is marked in the dotted rectangle and enlarged in the right corner.(C) Electron microscopy images of sperm from affected individual B showing severely malformed head shapes (red arrow) and sperm tail defects (black arrow).Enlarge figures have shown as the right panels.F I G U R E 3 ATP/GTP Binding Protein 1 (AGTPBP1) signals showed multiple localizations during murine spermiogenesis.From left to right: bright field, DAPI staining (blue), lectin staining (acrosome marker; red), AGTPBP1 staining (green), AGTPBP1 staining (green) combined with lectin, and AGTPBP1 signals (lectin; AGTPBP1) combined with DAPI (Merge).(A-C) AGTPBP1 signals localized at different steps of murine spermiogenesis: step 1, step 2-3, step 4-5, Step 6, step 7, step 8-9, step 9-10, step 10, step 11, step 12, step 13, step 14, step 15-16, and mature sperm (co-staining with Mito-tracker).400X magnification.

F I G U R E 4
Disruption of ATP/GTP Binding Protein 1 (Agtpbp1) allele in mice results in male sterility.(A) Schematic representation of the murine genomic structure and disrupted exon 18 of Agtpbp1 induces frameshift mutation.(B) Sanger sequencing reveals the specific deletion (del) sites within exon 18 in Agtpbp1 +/del mice, compared with wild-type mice.(C) AGTPBP1 expression was evaluated in the murine testes of Agtpbp1 del/del mice via western blotting.(D) Sperm collected from vas deferens of Agtpbp1 del/del mice shows immature sperm (roundlike; red arrows) and defective sperm tail (black arrows), compared with the wild-type mice (upper).(E-G.)Analysis of the ratio of sperm count, immature sperm (round-like), and motility in 2-month-old wild-type and Agtpbp1 del/del mice.Mice number per genotype: wild-type, n = 5; Agtpbp1 +/del , n = 5; Agtpbp1 del/del , n = 6.Sperm number > 200 per mouse.Each bar represents the mean ± standard error of the mean (SEM).*Significant differences (*p < 0.05; **p < 0.001; ****p < 0.00001, analysed using Student's t test).

Figure
Figure 7A shows that polyglutamylation and depolyglutamylation of the C-terminal sequences of tubulin are catalysed by TTLL and AGTPBP1, respectively, to balance long-chain polyglutamylation