Global characterization of mouse testis O-glycoproteome landscape during spermatogenesis
Infertility has emerged as a global health problem and is estimated to affect 10–20% of couples of reproductive age worldwide . In China, over 40 million people suffer from infertility, and this number is still showing a rapid increasing trend . Of all the infertility cases, approximately 50% are due to males. Congenital or acquired factors that impair or affect spermatogenesis are major causes of male infertility . Spermatogenesis is a highly complex process that occurs in the seminiferous tubules of testis. The spermatogonia undergo mitosis and meiosis to form spermatocytes, round spermatids, and eventually elongated spermatids which are then released into the lumen of the seminiferous tubules . The surface of mammalian sperm is coated with a 20–60 nm thick glycocalyx containing abundant N-glycoproteins, O-glycoproteins, glycolipids and so on . It is becoming increasingly evident that sperm glycosylation plays essential roles in the entire process of spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. Over the past two decades, due to the rapid development of mass spectrometry (MS)-based technologies, considerable progress has been made in in-depth characterization of N-glycoproteins in sperm. Increasing glycomic and glycoproteomic studies have delineated a clearer picture of the composition of N-glycoproteins, N-glycosylation sites, and N-glycan structures in spermatozoa , , epididymal sperm , and seminal plasma – . These glycomic knowledge further contribute to our understanding of the roles of N-glycans in spermatogenesis. For example, MS analysis showed that the majority of N-glycan structures in seminal plasma was complex type , . Functional studies confirmed that the loss of complex N-glycans caused a block in spermatogenesis by promoting the premature upregulation of genes normally expressed during late stages of spermatogenesis . However, compared with many studies in N-glycosylation, there are still less studies on O-glycosylation in sperm. O-glycosylation is one of the most common and diverse types of glycosylation, which primarily occurs at serine (Ser) or threonine (Thr). Based on the differences in the first monosaccharide linked to amino acids, O-glycosylation can be divided into many forms. O-GalNAc glycosylation (also known as mucin type O-glycosylation) is the most abundant and diverse type of O-glycosylation, playing essential roles in many physiological and pathological processes such as cell differentiation, tissue development, immune recognition, and viral infection , . O-GalNAc glycosylation is initiated by the polypeptide N-acetyl-α-galactosaminyltransferase family (ppGalNAc-Ts) comprising 20 isoenzymes in human, which transfer GalNAc monosaccharide onto Ser/Thr residues resulting in the formation of Tn antigen (GalNAc-α1-Ser/Thr) , . The initiating Tn structure can be subsequently extended to form the most common core 1 structure (T antigen, Galβ1,3GalNAc-α1-Ser/Thr) , or other common core structures, which can be further elongated or branched in a stepwise manner (Fig. ) , . In our previous work, we developed a lectin microarray-based method to detect the glycome profile in different organs of adult mice, and surprisingly found that testis had the highest level of O-GalNAc glycosylation compared with brain, liver, spleen, and kidney . The high level of O-GalNAc glycans in spermatozoa was also verified by O-glycan antibodies . Additionally, studies on specific proteins have shown the importance of O-GalNAc glycosylation in sperm development. For example, Franke et al. found a significant difference in the O-GalNAc glycosylation pattern of mucin 1 (MUC1) between testes with normal and impaired spermatogenesis. Notably, two members of ppGalNAc-Ts family, ppGalNAc-T3 (GALNT3) and T19 (GALNTL5) were highly expressed in spermatocytes and/or spermatids in the testis, and knockout mice for both genes were sterile , . In detail, the deficiency of Galnt3 resulted in a severe reduction of mucin-type O-glycans in spermatids and caused impaired acrosome formation, leading to oligoasthenoteratozoospermia . In human semen samples, ppGalNAc-T3 and the O-glycans Tn and T were found in spermatozoa, with a positive correlation observed between the expression of ppGalNAc-T3 and classical semen parameters . The mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted the localization of ubiquitin–proteasome system, leading to asthenozoospermia . Moreover, the lectins Agaricus bisporus agglutinin (ABA) and Maclura pomifera lectin (MPL), which recognize O-GalNAc glycans, have been reported as potential biomarkers for the diagnosis of male subfertility caused by DEFB126 deficiency . Overall, these studies indicate that O-GalNAc glycosylation (hereinafter referred to as O-glycosylation) in the testis plays an essential role in spermatogenesis and is closely related to infertility. To deeply investigate the functional mechanisms involved, the first key questions to be addressed are: which proteins are O-glycosylated, what are the location and structure of O-glycans on them, and how do they change during spermatogenesis. However, the characterization of the O-glycoproteome has lagged behind due to the lack of conserved amino acid sequences at O-glycosites as well as the complexity and heterogeneity of O-glycan structures. To the best of our knowledge, no systematic O-glycoproteomics studies of the testis have been performed to date. Here, we present a large-scale site-specific O-glycoproteome study to characterize protein O-glycosylation in the testis during spermatogenesis. We first detect the modification pattern of O-glycan in the testis at different stages of spermatogenesis by lectin staining. Our findings highlight prominent O-glycan levels in round and elongated spermatids, but not in spermatogonia or spermatocytes. Subsequently, using lectin affinity enrichment coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further perform a systematic qualitative and quantitative analysis of the O-glycoproteome in 24-day-old and 12-week-old testes, elucidate the distribution properties of O-glycoproteins and O-glycosites, and discover the dynamic level of O-glycosylation with increased Tn structure and decreased T structure in mature testes. Finally, we preliminarily explore the possible functions of O-glycosylation in highly O-glycosylated proteins related to fertilization, and verify the regulatory roles of GALNT3, providing resources for future functional studies targeting O-glycosylation in spermatogenesis and fertilization. 

O-glycans are located in round and elongated spermatids In this study, we first analyzed the glycome profile data from 14 tissues in a public database and reconfirmed that the testis is an organ exhibiting a high level of O-glycans (Supplementary Fig. ). To investigate the detailed location of O-glycans in different testicular germ cells, we performed lectin staining of testicular tissues at six time points according to the developmental timeline of mouse germ cell (Fig. ) – . At 0 and 7 days after birth, only spermatogonia are present. At 16 days postpartum, spermatocytes are dominant cell type. At 24 days postpartum, elongated spermatids are not yet appeared, while round spermatids account for a higher proportion of germ cells compared to earlier stages, despite spermatocytes remaining a predominant cell type. At 8 and 12 weeks postpartum, elongated spermatids become the most abundant type of germ cells in seminiferous tubules. Two lectins, namely vicia villosa agglutinin (VVA) and peanut agglutinin (PNA), were used to detect the main types of O-glycan in the testis. The VVA lectin is a family of tetrameric proteins consisting of combinations of A and B subunits . The isolectin used in this study is primarily composed of the B subunit, which is also the predominant subtype of VVA lectin and has been widely reported to recognize terminal α-linked GalNAc residues (Tn antigen) , . In contrast, the PNA lectin has been demonstrated to exhibit the strongest specificity for the disaccharide Galβ1→3GalNAc (T antigen) (Fig. ). As shown in Fig. , both signals of PNA and VVA appeared only after 24 days postnatal, indicating that O-glycans in the testis are mainly located in round and elongated spermatids, whereas they are barely present in spermatogonia or spermatocytes. Notably, the signals of O-glycans were strongest in the acrosome of the spermatid heads. In addition, we found that spermatids have preferences for different O-glycans. That is, the signals of T structure recognized by PNA were stronger in round spermatids (hollow arrowheads), whereas the signals of Tn structure recognized by VVA were stronger in elongated spermatids (solid arrowheads), suggesting that O-glycans may be involved in the spermatozoa formation and maturation. Global characterization of O-glycoproteome in mouse testes To identify proteins undergoing O-glycosylated during spermiogenesis, we analyzed the global O-glycoproteome using testes of 24-day-old mice characterized by a higher proportion of round spermatids compared to earlier stages, and 12-week-old mice characterized by elongated spermatids being the predominant germ cells within the seminiferous tubules. As shown in Fig. , de-sialylated peptides from testes were enriched by VVA and PNA lectins respectively. O-glycopeptides were then identified by mass spectrometry using higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) strategy and analyzed with pGlyco3 and MetaMorpheus . All the spectrum were checked by HexNAcQuest to exclude O-GlcNAc glycopeptides. As a result, a total of 349 O-GalNAc glycoproteins (Supplementary Data ) with 16,227 glycopeptide-spectrum matches (GPSM) were detected. On these glycoproteins, there were 647 unique O-glycopeptides containing 799 unambiguous O-glycosites (Fig. ). Notably, more O-glycoproteins were found in the testes of 24-day-old mice (307/349), especially within the PNA-enriched group, reflecting active O-glycoprotein biosynthesis in round spermatids when the Golgi synthesizes large amounts of glycoproteins and delivers them to the acrosome . Comparison with the recently reported atlas of mouse O-glycoproteins summarized by Yang et al. , we identified an additional 185 mouse O-glycoproteins (Fig. ). In particular, when compared with the O-glycoproteins of the other 10 mouse organs in that report , we found that the testis had the most abundant and specific O-glycoproteins (Fig. ). Moreover, since O-glycoproteomic research in humans is much more advanced, we summarized 3975 published human O-glycoproteins identified by lectin weak affinity chromatography (LWAC) enrichment strategy – , chemical material enrichment strategy – , and extraction of O-linked glycopeptides (EXoO) using OpeRATOR protease , (Fig. ). We converted the mouse glycoproteins identified in this study to human homologs and discovered 85 additional glycoproteins not reported in the human O-GalNAc glycoprotein database (Fig. and Supplementary Data ). Distribution characteristics of O-glycosites in mouse testes Since the density and location of the O-glycosites on proteins are closely related to the potential functions of O-glycans , , , , we next sought to map these characteristics of the O-glycosites identified in mouse testes. First, we performed overall statistical analyses on O-glycosites. As a result, 69.6% of the O-glycopeptides and 56% of the O-glycoproteins identified in the present study carried ≤ 2 unambiguous O-glycosites, whereas 20 peptides (3.1%) and 11 proteins (3.2%) were found to be highly glycosylated, carrying ≥ 5 sites and ≥ 10 sites, respectively (Fig. ). Notably, many of these highly O-glycosylated proteins have been reported to be directly involved in spermatogenesis and fertilization, such as acrosin-binding protein (ACRBP), equatorial segment protein 1 (SPESP1), and sperm acrosome-associated protein 7 (SPACA7), which not only have a large number of glycosylation sites but also high coverage of glycopeptides ( ≥ 5) and high abundance of GPSM ( > 100) (Fig. ). Intriguingly, statistics of GPSM for the 20 O-glycopeptides with dense glycosylation ( ≥ 5 sites) showed that almost all glycopeptides specifically or highly expressed in testes had the highest GPSM counts in 12-week-old testes (Fig. ), suggesting that the degree of O-glycosylation in mature spermatozoa is higher than that in round spermatozoa. Next, we further analyzed the distribution pattern of the 799 unambiguous O-glycosites in protein structural domains annotated in the UniProt database. We found that only 7.5% (60 sites) were located within specific domains and tended to be localized on both sides rather than in the middle of the domains, which was particularly evident in domains of LDL receptor class A, Ig-like C2-type, fibronectin type III, and EF-hand (Supplementary Fig. ). In contrast, up to 19.4% (155 sites) were located within 20 amino acids of the N- or C-terminal of the domains. It was observed that many O-glycosites were distributed at the outer edges rather than the inner edges of the domains (Fig. ). Additionally, using the mouse proteome database consisting of 17,228 proteins (UniProtKB reviewed, Swiss-Prot) as a background, we found that proteins containing O-glycosites near cleavage sites, stem regions (within 50 amino acids from transmembrane domain), and ER retention motifs (KDEL and similar sequences), as well as on precursor peptides, were almost significantly enriched in the testis ( p < 0.05) (Supplementary Fig. and Supplementary Data ). This result is in accordance with previous reports using other biological models , . Notably, apart from these findings, we identified a region near the N-terminal of peptidase S1 domain which is susceptible to O-glycosylation (Fig. ). The dense O-glycosites around the peptidase S1 domain on different members of this protease family (PRSS40, 42, 43, 44, 50) suggest that O-glycosylation may play a significant role in the maturation and shearing of proteins during spermatogenesis. Overall, among the 799 unambiguous O-glycosites identified in testes, 37.5% (300 sites) were distributed in disordered regions, and 19.4% (155 sites) were located within 20 amino acids of the N- or C-terminal edges of specific protein structural domains. Additionally, 11.0% (88 sites) were localized to the stem regions of transmembrane proteins, suggesting the role of O-glycans in  maintaining structural stability . Another 10.9% (87 sites) were located in specific functional regions related to protein interactions, folding and trafficking, indicating the potential effect of site-specific O-glycosylation on protein functions. Taken together, we systematically mapped the association between identified O-glycosites and their location on proteins, providing important clues for understanding the biological functions of site-specific O-glycosylation in spermatids. Differential O-glycopeptides involve in spermatogenesis and fertilization After mapping the O-glycoproteins and O-glycosites in the testis, we performed label-free quantitative analysis of the O-glycopeptides. Correlation analysis revealed a high correlation among the four biological replicates within the same group (r 2 > 0.85), suggesting good repeatability and reliable quantification (Supplementary Fig. ). Principal component analysis (PCA) demonstrated that intact O-glycopeptides enriched at different developmental stages using distinct lectins were clearly separated (Supplementary Fig. ). Hierarchical clustering analysis further showed that overall glycopeptide intensity was higher in 12-week-old testes than in 24-day-old testes in the VVA-enriched subset, whereas it was opposite in the PNA-enriched subset (Fig. ). Moreover, it was observed that in 24-day-old testes, the H1N1 glycoform accounted for 57.2% (2668) of total GPSM, and the N1 glycoform represented 21.2% (989). In contrast, in 12-week-old samples, the proportions of H1N1 and N1 glycoform were 47.4% (2847) and 27.4% (1647), respectively, indicating an increase in Tn structure and a decrease in T structure in 12-week-old testes with more mature elongated spermatids (Fig. ). After the exclusion of glycopeptides identified in fewer than two biological replicates in at least one group, a p < 0.05 and a | fold change| > 2 were set as thresholds for differential glycopeptides. As a result, 63 up-regulated and eight down-regulated O-glycopeptides in the VVA-enriched subset, and 56 up-regulated and 126 down-regulated O-glycopeptides in the PNA-enriched subset, were identified (Fig. , and Supplementary Data ). Considering that changes in O-glycopeptide abundance might result from either altered O-glycosylation levels or altered protein expression, we analyzed reported protein expression levels from round to elongated spermatids , . However, only 74 of the 232 differential glycopeptides had corresponding protein abundance data. Among these, 59 (79%) glycopeptides exhibited glycosylation alterations that were at least two-fold greater than protein expression alterations, indicating that the observed changes in these O-glycopeptide abundance were primarily due to altered levels of O-glycans (Supplementary Fig. and Supplementary Data ). For example, the protein level of CD55 did not change significantly, but the glycoforms at all four O-glycosites on the identified glycopeptide were transitioned from T structure in 24-day-old testes to Tn structures in 12-week-old testes (Supplementary Fig. , ). Additionally, 15 (21%) glycopeptides whose changes in abundance may be due to altered protein expression, such as vitronectin (VTN), were also observed (Supplementary Fig. , ). Further Gene Ontology (GO) analysis showed that the up-regulated O-glycoproteins in the 12-week-old testes were primarily concentrated in the acrosome and spermatid tail, and mainly involved in spermatogenesis and fertilization processes, whereas the down-regulated proteins were predominantly located in membrane, lipid, and extracellular regions, and most significantly enriched in extracellular structure organization (Fig. and Supplementary Fig. ). This observation may be related to the maturation and release process of elongated spermatids. Moreover, we focused on O-glycoproteins associated with spermatogenesis and fertilization, mapping the landscape of their O-glycosites (Fig. ) and GPSM (Fig. ). Notably, ACRBP, acrosin (ACR), SPACA7, SPESP1, insulin-like peptide 6 (INSL6), and equatorin (EQTN) exhibted the highest number of O-glycosites and GPSM, implying that O-glycosylation may affect spermatogenesis and fertilization through these proteins. Different O-glycosylation patterns between round and elongated spermatids After comprehensively analyzing the O-glycoproteome in testes at developmental stages, we further conducted a preliminary exploration and validation of the O-glycosylation patterns in purified round and elongated spermatids. Using FACS-based DNA ploidy analysis and cell imaging technology, we successfully isolated round spermatids from early developmental mice (day 24, n = 20) (Fig. ) and both round and elongated spermatids from mature developmental mice (week 12, n = 10) (Fig. ). The results of VVA blot and PNA blot suggested that the O-glycosylation profiles of round spermatids from day 24 and week 12 were similar, while the O-glycosylation patterns of elongated spermatids were different from those of round spermatids (Fig. ). In detail, VVA blot results revealed that the Tn antigen signals at the 50-70 KD protein bands in 12-week-old elongated spermatids were significantly stronger than those in 24-day-old round spermatids (Fig. ). This observation partially aligns with mass spectrometry quantification finding that, compared to 24-day-old testes, many up-regulated O-glycoproteins with the highest intensity in 12-week-old testicular tissues (e.g., MENT, NUB1, ACR, and ACRBP) also exhibited molecular weights within the 50–70 kDa range (Fig. ). Similarly, reductions in T antigen signals across different molecular weight ranges were observed in both 12-week-old elongated spermatids (Fig. ) and 12-week-old testicular tissues (Fig. ). These results suggest that the O-glycosylation changes detected by mass spectrometry from 24-day and 12-week-old testicular tissues may primarily originate from the elongated spermatid population. O-glycosylation Affects Interaction between pro-ACR and ACRBP Finally, we investigated the possible functions of O-glycosylation on testicular proteins. Among the differential proteins associated with spermatogenesis and fertilization, ACR is reported as the major protease of mammalian spermatozoa and plays an indispensable role in sperm penetration of the zona pellucida, with its knockout male mice exhibiting sterile , . ACR is synthesized and stored in the acrosome as a precursor peptide (pro-ACR), and activated by sequential shearing of C-terminal fragments. ACRBP is the protein that binds to pro-ACR and prevents it from being activated prematurely before the acrosome reaction, with its knockout mice showing severely reduced fertility . It has been reported that ACRBP has two binding domains, B1 and B2, which bind to the SIII and SI domain at the C-terminus of pro-ACR, respectively . In this study, we found that both pro-ACR and ACRBP were highly O-glycosylated, with nine unambiguous O-glycosites on five O-glycopeptides of pro-ACR (Fig. ) and 16 unambiguous O-glycosites on seven O-glycopeptides of ACRBP (Fig. ). Notably, an increase of O-glycosites for both proteins was found in 12-week-old testis, many of which were located on the binding regions of pro-ACR and ACRBP (Fig. ). To further verify whether O-glycosylation affects the interaction between pro-ACR and ACRBP, we overexpressed Flag-tagged pro-ACR and His-tagged ACRBP in CHO and CHO-ldlD cells. The CHO-ldlD cell line is an O-GalNAc glycosylation-deficient cell line due to the mutation in the epimerase that converts UDP-Glc/GlcNAc to UDP-Gal/GalNAc . The results of VVA blot confirmed the absence of O-GalNAc glycosylation on both pro-ACR and ACRBP expressed in CHO-ldlD cells (Fig. ). Moreover, co-immunoprecipitation analysis showed that the interaction between ACRBP and pro-ACR was attenuated when O-glycosylation was absent (Fig. ), suggesting that O-glycosylation may regulate the functions of pro-ACR and ACRBP by influencing their interaction. To further explore what glycosyltransferases catalyze O-glycosylation on pro-ACR and ACRBP, we performed in vitro enzyme activity assays using six synthetic peptides targeting the main glycosylation regions of these two proteins (Fig. ). Using a published single-cell RNA-seq dataset of mouse testes , we found that Galntl5 and Galnt3 were the predominant members of ppGalNAc-Ts highly expressed in haploid spermatids, while Galnt1 was the most highly expressed member in Sertoli cells (Supplementary Fig. ). The results showed that GALNT3 exhibited stronger catalytic activities than GALNT1, as evidenced by its ability to glycosylate four of the six peptides within a 4 h reaction (Fig. ). Notably, two peptides localized in the region where pro-ACR interacts with ACRBP (pro-ACR ① and ③) could be glycosylated by GALNT3 on Thr382, Thr421, and Ser426 (Supplementary Fig. ). Although GALNTL5 showed remarkably higher expression in round and elongated spermatids, it has no glycosyltransferase activity toward pro-ACR or ACRBP (Fig. ), consistent with previous in vitro results using other peptides . Additionally, we tested whether Galnt7 and Galnt10 contribute to O-glycosylation of pro-ACR and ACRBP, as these two enzymes were moderately expressed in spermatids. As shown in Supplementary Fig. , neither GALNT7 nor GALNT10 exhibited enzyme activity on all naked peptides of pro-ACR and ACRBP. However, when using GALNT3-catalyzed glycopeptides as substrates, both GALNT7 and GALNT10 showed catalytic activities toward glycopeptides of pro-ACR①, pro-ACR③, and ACRBP①, consistent with previous studies indicating that GALNT7 and GALNT10 are strict glycopeptide-preferring isoenzymes . Taken together, these results suggest that GALNT3 may be the major glycosyltransferase responsible for O-glycosylation on pro-ACR and ACRBP, while GALNT7 and GALNT10 may further catalyze these proteins following initial catalysis by GALNT3. O-glycosylation functions on SPACA7, INSL6, EQTN, and SPESP1 Apart from ACR and ACRBP, we also explored the potential functions of O-glycosylation on four additional highly glycosylated proteins involved in fertilization, i.e., SPACA7, INSL6, EQTN, and SPESP1 (Fig. ). First, VVA blot results confirmed that the four proteins expressed in CHO-ldlD cells indeed lack O-GalNAc glycosylation (Fig. ). Moreover, we found that for all four proteins, a decrease in O-glycosylation corresponded to a decrease in protein stability (Fig. and Supplementary Fig. ), suggesting that maintaining protein stability may be a fundamental and universal function of O-glycosylation on testicular proteins. Furthermore, we found that O-glycosylation has a variety of other functions. In detail, INSL6 is a secreted protein that undergoes cleavage processing from preproprotein (pro-INSL6) to mature protein . In this study, all eight unambiguous O-glycosites identified were located at its precursor peptide (Supplementary Figs. and ). It was also observed that when O-glycosylation was absent, the maturation process of INSL6 was hindered in CHO-ldlD cells, as evidenced by a reduction in the secretion of cleaved peptides (Fig. ). EQTN is a type I transmembrane protein widely distributed in the acrosome membrane of mammalian spermatid , with 12 unambiguous O-glycosites identified in this study (Supplementary Figs. and ). It has been reported that EQTN could interact with SNAP25, a core component of the SNARE complex, during the acrosome reaction . The co-immunoprecipitation analysis showed that the interaction between EQTN and SNAP25 was greatly reduced when O-glycosylation was decreased (Fig. ). SPESP1 is another important equatorial protein that initially appears in the matrix of acrosomal vesicles, with its N-terminal region (amino acids 58–102) involved in oligomerization, membrane insertion, and pore formation . In this study, we found eight of the 15 unambiguous O-glycosites on SPESP1 were located at the N-terminal region (Supplementary Figs. and ). The results showed that the inhibition of O-glycosylation in CHO-ldlD cells significantly affects the dimerization of SPESP1 (Fig. ). Taken together, these results indicate that O-glycosylation on testicular proteins may ultimately affect acrosome formation and acrosome reaction by maintaining protein stability and aggregate structures, as well as promoting protein maturation and protein interactions. 

In this study, we first analyzed the glycome profile data from 14 tissues in a public database and reconfirmed that the testis is an organ exhibiting a high level of O-glycans (Supplementary Fig. ). To investigate the detailed location of O-glycans in different testicular germ cells, we performed lectin staining of testicular tissues at six time points according to the developmental timeline of mouse germ cell (Fig. ) – . At 0 and 7 days after birth, only spermatogonia are present. At 16 days postpartum, spermatocytes are dominant cell type. At 24 days postpartum, elongated spermatids are not yet appeared, while round spermatids account for a higher proportion of germ cells compared to earlier stages, despite spermatocytes remaining a predominant cell type. At 8 and 12 weeks postpartum, elongated spermatids become the most abundant type of germ cells in seminiferous tubules. Two lectins, namely vicia villosa agglutinin (VVA) and peanut agglutinin (PNA), were used to detect the main types of O-glycan in the testis. The VVA lectin is a family of tetrameric proteins consisting of combinations of A and B subunits . The isolectin used in this study is primarily composed of the B subunit, which is also the predominant subtype of VVA lectin and has been widely reported to recognize terminal α-linked GalNAc residues (Tn antigen) , . In contrast, the PNA lectin has been demonstrated to exhibit the strongest specificity for the disaccharide Galβ1→3GalNAc (T antigen) (Fig. ). As shown in Fig. , both signals of PNA and VVA appeared only after 24 days postnatal, indicating that O-glycans in the testis are mainly located in round and elongated spermatids, whereas they are barely present in spermatogonia or spermatocytes. Notably, the signals of O-glycans were strongest in the acrosome of the spermatid heads. In addition, we found that spermatids have preferences for different O-glycans. That is, the signals of T structure recognized by PNA were stronger in round spermatids (hollow arrowheads), whereas the signals of Tn structure recognized by VVA were stronger in elongated spermatids (solid arrowheads), suggesting that O-glycans may be involved in the spermatozoa formation and maturation. 

To identify proteins undergoing O-glycosylated during spermiogenesis, we analyzed the global O-glycoproteome using testes of 24-day-old mice characterized by a higher proportion of round spermatids compared to earlier stages, and 12-week-old mice characterized by elongated spermatids being the predominant germ cells within the seminiferous tubules. As shown in Fig. , de-sialylated peptides from testes were enriched by VVA and PNA lectins respectively. O-glycopeptides were then identified by mass spectrometry using higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) strategy and analyzed with pGlyco3 and MetaMorpheus . All the spectrum were checked by HexNAcQuest to exclude O-GlcNAc glycopeptides. As a result, a total of 349 O-GalNAc glycoproteins (Supplementary Data ) with 16,227 glycopeptide-spectrum matches (GPSM) were detected. On these glycoproteins, there were 647 unique O-glycopeptides containing 799 unambiguous O-glycosites (Fig. ). Notably, more O-glycoproteins were found in the testes of 24-day-old mice (307/349), especially within the PNA-enriched group, reflecting active O-glycoprotein biosynthesis in round spermatids when the Golgi synthesizes large amounts of glycoproteins and delivers them to the acrosome . Comparison with the recently reported atlas of mouse O-glycoproteins summarized by Yang et al. , we identified an additional 185 mouse O-glycoproteins (Fig. ). In particular, when compared with the O-glycoproteins of the other 10 mouse organs in that report , we found that the testis had the most abundant and specific O-glycoproteins (Fig. ). Moreover, since O-glycoproteomic research in humans is much more advanced, we summarized 3975 published human O-glycoproteins identified by lectin weak affinity chromatography (LWAC) enrichment strategy – , chemical material enrichment strategy – , and extraction of O-linked glycopeptides (EXoO) using OpeRATOR protease , (Fig. ). We converted the mouse glycoproteins identified in this study to human homologs and discovered 85 additional glycoproteins not reported in the human O-GalNAc glycoprotein database (Fig. and Supplementary Data ). 

Since the density and location of the O-glycosites on proteins are closely related to the potential functions of O-glycans , , , , we next sought to map these characteristics of the O-glycosites identified in mouse testes. First, we performed overall statistical analyses on O-glycosites. As a result, 69.6% of the O-glycopeptides and 56% of the O-glycoproteins identified in the present study carried ≤ 2 unambiguous O-glycosites, whereas 20 peptides (3.1%) and 11 proteins (3.2%) were found to be highly glycosylated, carrying ≥ 5 sites and ≥ 10 sites, respectively (Fig. ). Notably, many of these highly O-glycosylated proteins have been reported to be directly involved in spermatogenesis and fertilization, such as acrosin-binding protein (ACRBP), equatorial segment protein 1 (SPESP1), and sperm acrosome-associated protein 7 (SPACA7), which not only have a large number of glycosylation sites but also high coverage of glycopeptides ( ≥ 5) and high abundance of GPSM ( > 100) (Fig. ). Intriguingly, statistics of GPSM for the 20 O-glycopeptides with dense glycosylation ( ≥ 5 sites) showed that almost all glycopeptides specifically or highly expressed in testes had the highest GPSM counts in 12-week-old testes (Fig. ), suggesting that the degree of O-glycosylation in mature spermatozoa is higher than that in round spermatozoa. Next, we further analyzed the distribution pattern of the 799 unambiguous O-glycosites in protein structural domains annotated in the UniProt database. We found that only 7.5% (60 sites) were located within specific domains and tended to be localized on both sides rather than in the middle of the domains, which was particularly evident in domains of LDL receptor class A, Ig-like C2-type, fibronectin type III, and EF-hand (Supplementary Fig. ). In contrast, up to 19.4% (155 sites) were located within 20 amino acids of the N- or C-terminal of the domains. It was observed that many O-glycosites were distributed at the outer edges rather than the inner edges of the domains (Fig. ). Additionally, using the mouse proteome database consisting of 17,228 proteins (UniProtKB reviewed, Swiss-Prot) as a background, we found that proteins containing O-glycosites near cleavage sites, stem regions (within 50 amino acids from transmembrane domain), and ER retention motifs (KDEL and similar sequences), as well as on precursor peptides, were almost significantly enriched in the testis ( p < 0.05) (Supplementary Fig. and Supplementary Data ). This result is in accordance with previous reports using other biological models , . Notably, apart from these findings, we identified a region near the N-terminal of peptidase S1 domain which is susceptible to O-glycosylation (Fig. ). The dense O-glycosites around the peptidase S1 domain on different members of this protease family (PRSS40, 42, 43, 44, 50) suggest that O-glycosylation may play a significant role in the maturation and shearing of proteins during spermatogenesis. Overall, among the 799 unambiguous O-glycosites identified in testes, 37.5% (300 sites) were distributed in disordered regions, and 19.4% (155 sites) were located within 20 amino acids of the N- or C-terminal edges of specific protein structural domains. Additionally, 11.0% (88 sites) were localized to the stem regions of transmembrane proteins, suggesting the role of O-glycans in  maintaining structural stability . Another 10.9% (87 sites) were located in specific functional regions related to protein interactions, folding and trafficking, indicating the potential effect of site-specific O-glycosylation on protein functions. Taken together, we systematically mapped the association between identified O-glycosites and their location on proteins, providing important clues for understanding the biological functions of site-specific O-glycosylation in spermatids. 

After mapping the O-glycoproteins and O-glycosites in the testis, we performed label-free quantitative analysis of the O-glycopeptides. Correlation analysis revealed a high correlation among the four biological replicates within the same group (r 2 > 0.85), suggesting good repeatability and reliable quantification (Supplementary Fig. ). Principal component analysis (PCA) demonstrated that intact O-glycopeptides enriched at different developmental stages using distinct lectins were clearly separated (Supplementary Fig. ). Hierarchical clustering analysis further showed that overall glycopeptide intensity was higher in 12-week-old testes than in 24-day-old testes in the VVA-enriched subset, whereas it was opposite in the PNA-enriched subset (Fig. ). Moreover, it was observed that in 24-day-old testes, the H1N1 glycoform accounted for 57.2% (2668) of total GPSM, and the N1 glycoform represented 21.2% (989). In contrast, in 12-week-old samples, the proportions of H1N1 and N1 glycoform were 47.4% (2847) and 27.4% (1647), respectively, indicating an increase in Tn structure and a decrease in T structure in 12-week-old testes with more mature elongated spermatids (Fig. ). After the exclusion of glycopeptides identified in fewer than two biological replicates in at least one group, a p < 0.05 and a | fold change| > 2 were set as thresholds for differential glycopeptides. As a result, 63 up-regulated and eight down-regulated O-glycopeptides in the VVA-enriched subset, and 56 up-regulated and 126 down-regulated O-glycopeptides in the PNA-enriched subset, were identified (Fig. , and Supplementary Data ). Considering that changes in O-glycopeptide abundance might result from either altered O-glycosylation levels or altered protein expression, we analyzed reported protein expression levels from round to elongated spermatids , . However, only 74 of the 232 differential glycopeptides had corresponding protein abundance data. Among these, 59 (79%) glycopeptides exhibited glycosylation alterations that were at least two-fold greater than protein expression alterations, indicating that the observed changes in these O-glycopeptide abundance were primarily due to altered levels of O-glycans (Supplementary Fig. and Supplementary Data ). For example, the protein level of CD55 did not change significantly, but the glycoforms at all four O-glycosites on the identified glycopeptide were transitioned from T structure in 24-day-old testes to Tn structures in 12-week-old testes (Supplementary Fig. , ). Additionally, 15 (21%) glycopeptides whose changes in abundance may be due to altered protein expression, such as vitronectin (VTN), were also observed (Supplementary Fig. , ). Further Gene Ontology (GO) analysis showed that the up-regulated O-glycoproteins in the 12-week-old testes were primarily concentrated in the acrosome and spermatid tail, and mainly involved in spermatogenesis and fertilization processes, whereas the down-regulated proteins were predominantly located in membrane, lipid, and extracellular regions, and most significantly enriched in extracellular structure organization (Fig. and Supplementary Fig. ). This observation may be related to the maturation and release process of elongated spermatids. Moreover, we focused on O-glycoproteins associated with spermatogenesis and fertilization, mapping the landscape of their O-glycosites (Fig. ) and GPSM (Fig. ). Notably, ACRBP, acrosin (ACR), SPACA7, SPESP1, insulin-like peptide 6 (INSL6), and equatorin (EQTN) exhibted the highest number of O-glycosites and GPSM, implying that O-glycosylation may affect spermatogenesis and fertilization through these proteins. 

After comprehensively analyzing the O-glycoproteome in testes at developmental stages, we further conducted a preliminary exploration and validation of the O-glycosylation patterns in purified round and elongated spermatids. Using FACS-based DNA ploidy analysis and cell imaging technology, we successfully isolated round spermatids from early developmental mice (day 24, n = 20) (Fig. ) and both round and elongated spermatids from mature developmental mice (week 12, n = 10) (Fig. ). The results of VVA blot and PNA blot suggested that the O-glycosylation profiles of round spermatids from day 24 and week 12 were similar, while the O-glycosylation patterns of elongated spermatids were different from those of round spermatids (Fig. ). In detail, VVA blot results revealed that the Tn antigen signals at the 50-70 KD protein bands in 12-week-old elongated spermatids were significantly stronger than those in 24-day-old round spermatids (Fig. ). This observation partially aligns with mass spectrometry quantification finding that, compared to 24-day-old testes, many up-regulated O-glycoproteins with the highest intensity in 12-week-old testicular tissues (e.g., MENT, NUB1, ACR, and ACRBP) also exhibited molecular weights within the 50–70 kDa range (Fig. ). Similarly, reductions in T antigen signals across different molecular weight ranges were observed in both 12-week-old elongated spermatids (Fig. ) and 12-week-old testicular tissues (Fig. ). These results suggest that the O-glycosylation changes detected by mass spectrometry from 24-day and 12-week-old testicular tissues may primarily originate from the elongated spermatid population. 

Finally, we investigated the possible functions of O-glycosylation on testicular proteins. Among the differential proteins associated with spermatogenesis and fertilization, ACR is reported as the major protease of mammalian spermatozoa and plays an indispensable role in sperm penetration of the zona pellucida, with its knockout male mice exhibiting sterile , . ACR is synthesized and stored in the acrosome as a precursor peptide (pro-ACR), and activated by sequential shearing of C-terminal fragments. ACRBP is the protein that binds to pro-ACR and prevents it from being activated prematurely before the acrosome reaction, with its knockout mice showing severely reduced fertility . It has been reported that ACRBP has two binding domains, B1 and B2, which bind to the SIII and SI domain at the C-terminus of pro-ACR, respectively . In this study, we found that both pro-ACR and ACRBP were highly O-glycosylated, with nine unambiguous O-glycosites on five O-glycopeptides of pro-ACR (Fig. ) and 16 unambiguous O-glycosites on seven O-glycopeptides of ACRBP (Fig. ). Notably, an increase of O-glycosites for both proteins was found in 12-week-old testis, many of which were located on the binding regions of pro-ACR and ACRBP (Fig. ). To further verify whether O-glycosylation affects the interaction between pro-ACR and ACRBP, we overexpressed Flag-tagged pro-ACR and His-tagged ACRBP in CHO and CHO-ldlD cells. The CHO-ldlD cell line is an O-GalNAc glycosylation-deficient cell line due to the mutation in the epimerase that converts UDP-Glc/GlcNAc to UDP-Gal/GalNAc . The results of VVA blot confirmed the absence of O-GalNAc glycosylation on both pro-ACR and ACRBP expressed in CHO-ldlD cells (Fig. ). Moreover, co-immunoprecipitation analysis showed that the interaction between ACRBP and pro-ACR was attenuated when O-glycosylation was absent (Fig. ), suggesting that O-glycosylation may regulate the functions of pro-ACR and ACRBP by influencing their interaction. To further explore what glycosyltransferases catalyze O-glycosylation on pro-ACR and ACRBP, we performed in vitro enzyme activity assays using six synthetic peptides targeting the main glycosylation regions of these two proteins (Fig. ). Using a published single-cell RNA-seq dataset of mouse testes , we found that Galntl5 and Galnt3 were the predominant members of ppGalNAc-Ts highly expressed in haploid spermatids, while Galnt1 was the most highly expressed member in Sertoli cells (Supplementary Fig. ). The results showed that GALNT3 exhibited stronger catalytic activities than GALNT1, as evidenced by its ability to glycosylate four of the six peptides within a 4 h reaction (Fig. ). Notably, two peptides localized in the region where pro-ACR interacts with ACRBP (pro-ACR ① and ③) could be glycosylated by GALNT3 on Thr382, Thr421, and Ser426 (Supplementary Fig. ). Although GALNTL5 showed remarkably higher expression in round and elongated spermatids, it has no glycosyltransferase activity toward pro-ACR or ACRBP (Fig. ), consistent with previous in vitro results using other peptides . Additionally, we tested whether Galnt7 and Galnt10 contribute to O-glycosylation of pro-ACR and ACRBP, as these two enzymes were moderately expressed in spermatids. As shown in Supplementary Fig. , neither GALNT7 nor GALNT10 exhibited enzyme activity on all naked peptides of pro-ACR and ACRBP. However, when using GALNT3-catalyzed glycopeptides as substrates, both GALNT7 and GALNT10 showed catalytic activities toward glycopeptides of pro-ACR①, pro-ACR③, and ACRBP①, consistent with previous studies indicating that GALNT7 and GALNT10 are strict glycopeptide-preferring isoenzymes . Taken together, these results suggest that GALNT3 may be the major glycosyltransferase responsible for O-glycosylation on pro-ACR and ACRBP, while GALNT7 and GALNT10 may further catalyze these proteins following initial catalysis by GALNT3. 

Apart from ACR and ACRBP, we also explored the potential functions of O-glycosylation on four additional highly glycosylated proteins involved in fertilization, i.e., SPACA7, INSL6, EQTN, and SPESP1 (Fig. ). First, VVA blot results confirmed that the four proteins expressed in CHO-ldlD cells indeed lack O-GalNAc glycosylation (Fig. ). Moreover, we found that for all four proteins, a decrease in O-glycosylation corresponded to a decrease in protein stability (Fig. and Supplementary Fig. ), suggesting that maintaining protein stability may be a fundamental and universal function of O-glycosylation on testicular proteins. Furthermore, we found that O-glycosylation has a variety of other functions. In detail, INSL6 is a secreted protein that undergoes cleavage processing from preproprotein (pro-INSL6) to mature protein . In this study, all eight unambiguous O-glycosites identified were located at its precursor peptide (Supplementary Figs. and ). It was also observed that when O-glycosylation was absent, the maturation process of INSL6 was hindered in CHO-ldlD cells, as evidenced by a reduction in the secretion of cleaved peptides (Fig. ). EQTN is a type I transmembrane protein widely distributed in the acrosome membrane of mammalian spermatid , with 12 unambiguous O-glycosites identified in this study (Supplementary Figs. and ). It has been reported that EQTN could interact with SNAP25, a core component of the SNARE complex, during the acrosome reaction . The co-immunoprecipitation analysis showed that the interaction between EQTN and SNAP25 was greatly reduced when O-glycosylation was decreased (Fig. ). SPESP1 is another important equatorial protein that initially appears in the matrix of acrosomal vesicles, with its N-terminal region (amino acids 58–102) involved in oligomerization, membrane insertion, and pore formation . In this study, we found eight of the 15 unambiguous O-glycosites on SPESP1 were located at the N-terminal region (Supplementary Figs. and ). The results showed that the inhibition of O-glycosylation in CHO-ldlD cells significantly affects the dimerization of SPESP1 (Fig. ). Taken together, these results indicate that O-glycosylation on testicular proteins may ultimately affect acrosome formation and acrosome reaction by maintaining protein stability and aggregate structures, as well as promoting protein maturation and protein interactions. 

Among mammalian organs, the testis is very unique and enigmatic, with the highest number of tissue-enriched expressed proteins ( https://www.proteinatlas.org/humanproteome/tissue ) and the highest level of O-glycosylation . While the literature has documented the possible role of protein O-glycosylation in male infertility , , the spatial distribution and diversity of O-glycoproteome and temporal dynamics during spermatogenesis remain unmapped. In this study, we took advantage of advances in lectin enrichment and high-resolution MS with HCD-pd-EThcD, identifying a total of 647 unique O-glycopeptides containing 799 unambiguous O-glycosites from 349 O-glycoproteins and detected 16,227 GPSM, representing the most comprehensive O-glycoproteome map in testis to date. Recently, Yang et al. identified a total of 595 glycoproteins from nine mouse tissues (brain, lung, liver, etc.) and whole blood using EXoO combined with HCD-pd-EThcD MS strategy . Compared to these proteins, we identified 210 O-glycosylated proteins exclusively detected in the testis (Fig. ), significantly complementing and expanding the existing mouse tissue O-glycoprotein database. Comparison of identified O-glycoproteins among the 11 organs confirmed that testis is indeed an organ with higher levels of O-glycosylation. In addition, through comparative analysis with previously reported human O-glycoproteins, we were still able to identify an additional 85 O-glycoproteins, including some highly O-glycosylated proteins associated with spermatogenesis (Fig. and Supplementary Data ). These findings suggest that testicular O-glycosylation is an important, but long-neglected aspect of O-glycosylation research. Moreover, Luo et al. recently identified a total of 68 O-glycoproteins in human seminal plasma and spermatozoa by combining two fragmentation methods of EThcD-sceHCD and sceHCD , with EQTN also identified in our study. Despite differences in species, sample sources, and mass spectrometry detection methods, the O-glycosites of EQTN identified in mouse testes and human sperm were mainly located in a similar domain (from amino acids 82–122), and 5 of the 12 O-glycosites we identified were also found in human sperm. Since the positions of O-glycosites on proteins are closely related to their potential functions, global mapping of the distribution of O-glycosites has gained increasing attention and has been reported in diverse biological models, including genetic engineering cells , , hemostatic system (plasma, platelets, and endothelial cells) , and keratinocyte cell lines . In the present study, we assessed whether the O-glycosites identified in testes were enriched around specific protein structural features based on the annotation in the UniProt database. Our results showed that O-glycosites in testes tended to appear at the outer edges of specific domains, near stem regions and protein cleavage sites, as well as on precursor peptides (Figure ). This finding is similar to previous reports in the hemostatic system and keratinocytes , suggesting that this may be a universal rule of O-glycan distribution. Compared with statistical results in keratinocytes , the highest percentage (19.4%) of O-glycosites in testes was located at the domain edges (Fig. ), reinforcing the possibility that O-glycosylation may influence spermatogenesis by stabilizing domain folding or by directly participating in domain function. In addition, our results showed that in testes, LDL receptor Class A was the structural domain with the highest number of identified O-glycosites, with dense O-glycosylation distributed at both the inner and outer edges (Fig. and Supplementary Fig. ). This domain is mainly found on low-density lipoprotein receptor (LDLR) and different LDLR-related proteins (LRPs). In this study, we identified LRP1, LRP2, LRP8, LDLR, and VLDLR. Among them, LRP1 has been reported to be required for early embryonic development , while LRP8 regulates sperm maturation, and its deficient male mice are sterile . The relatively high abundance of O-glycosylation on LRP1 and LRP8 that we observed may contribute to these functions. Of note, we also identified a structural domain susceptible to O-glycosylation, i.e., the peptidase S1 domain (Fig. ). The protease members PRSS40, PRSS42, PRSS43, PRSS44, and PRSS50, which are specifically expressed in germ cells , , were all identified to have intensive O-glycosites near this domain, suggesting that O-glycosylation may modulate spermatogenesis and fertilization by regulating the substrate shearing processes of these proteases. Additionally, we found that the testicular O-glycoproteome changes dynamically during spermatogenesis. By lectin staining analysis, we found that O-glycosylation in testes was mainly localized in round and elongated spermatids. The glycoforms were predominantly composed of Tn and T antigens, with the Tn structure mainly present in elongated spermatids whereas the T structure signals were stronger in round spermatids (Fig. ). This finding is basically consistent with previous report by Mandel et al., who stained human testicular tissues and ejaculated spermatozoa using O-glycan antibodies. Their study revealed that O-glycans were not detected in spermatogonia. In contrast, the T structure was present in spermatocytes, haploid spermatids and ejaculated spermatozoa, while the Tn structure was present only in haploid spermatids and ejaculated spermatozoa . Consistent with the lectin staining results, glycan composition analysis and quantitative O-glycoproteome analysis revealed an increased Tn structure and decreased T structure level in sexually mature 12-week-old testes with more elongated spermatids. Moreover, our detection of O-glycosyltransferase suggests that the observed dynamic change in O-glycosylation are at least partially due to elevated expression of GALNT3 and reduced expression of C1GALT1C1 (Supplementary Fig. ). Intriguingly, it has been reported that GALNT3 was the only active ppGalNAc-T exclusively expressed in spermatocytes and haploid germ cells , , and the VVA signal in the acrosomal regions of spermatids in Galnt3 −/− mice was drastically reduced . Tabak et al. demonstrated that GALNT3 exhibits a substrate preference for glycine (Gly) and alanine (Ala) at the +2 position, and for proline (Pro), valine (Val), and tyrosine (Tyr) at the −1 position from glycosites . This finding partly align with the amino acid frequencies at the −1 and +2 positions across the 799 unambiguous O-glycosites identified in this study (Supplementary Fig. ). Our results, combined with these reports, support the significant role of GALNT3 in the dynamic changes of O-glycosylation during spermatogenesis. In addition, our results also suggest that GALNT7 and GALNT10 may contribute to the O-glycosylation in the testes following the initial catalysis by GALNT3. Finally, since the protein profile of the testis has become clearer only in recent years, the functions of many testicular proteins remain largely unknown. Our studies on O-glycosylation of proteins in the testis provide another window into investigating their functional mechanisms. For example, ACR is preserved in the acrosome as an enzymatically inactive zymogen (pro-ACR) by binding to ACRBP until acrosome exocytosis . This process is important for maintaining the sperm fertilizing ability, and therefore, deletion of these two genes results in infertility or reduced fertility in mice – . Previous studies have elucidated specific structural domains mediating the binding between ACRBP and pro-ACR. Our study further demonstrates that this binding is dependent on O-glycans of the proteins (Fig. ), expanding previous knowledge of the interaction of these two proteins. As another example, SPACA7 is a male germ cell-specific protein localized to the sperm acrosome. Currently, only a few studies suggest that it may promote fertilization through some unknown mechanism . Our study reveals that SPACA7 is a highly O-glycosylated protein, with a total of 346 identified GPSM from eight unique O-glycopeptides containing 12 unambiguous O-glycosites, most of which are only detected in 12-week-old testis (Supplementary Figs. and ). We also revealed the O-glycosylation of SPACA7 affects its protein stability. These results  will provide insights for investigating the functional mechanism of SPACA7. Notably, through preliminary functional studies of several proteins (Fig. ), we not only discovered the conventional roles of O-GalNAc glycosylation in maintaining protein stability and regulating protein secretion , but also found that it may maintain protein dimerization through some direct or indirect effects. This observation may be highly related to the condensation and packaging of secretory granules during acrosomal biogenesis and deserves further investigation. While this study represents a significant step forward in understanding O-glycosylation in the testis, several open questions also remain. First, we used testicular tissues from different developmental stages rather than purified cell populations for the O-glycoproteomic analysis. Due to the inherently low abundance of glycopeptides and the ion suppression effects of glycosylated sequences compared to unmodified peptides, enrichment of glycopeptides is typically required in O-glycoproteomic experiments. Therefore, the initial sample amount usually needs to be at the milligram level, making it difficult to analyze O-glycoproteome profiling of specific cell populations derived from tissue samples. Furthermore, despite our advanced mass spectrometry strategy enabling the elucidation of over 80% of GPSM for O-glycosites, challenges remain due to the presence of multiple and densely distributed Ser/Thr sites on O-glycopeptides. Consequently, many sites could not been unambiguously identified, leading to a potential underestimation of the relationship between the O-glycans positioning and their regulatory functionalities. In the future, with the development of enrichment methods, mass spectrometry instruments, and analytical software, conducting more in-depth, refined, and comprehensive O-glycoproteomic studies at the level of specific cell populations or even single cell will be the next milestone. In summary, O-glycosylation is an abundant and important modification in the testis. We established an in-depth and dynamic O-glycoproteome map of mouse testes, assessed O-glycosite preferences, and used acrosomal proteins as an example to illustrate the possible role of ppGalNAc-T-catalyzed O-glycosylation in testicular protein function. Our findings serve as a data resource and roadmap for future functional studies of site-specific O-glycosylation in sperm development and fertilization, and provide insights for investigating the function of testicular proteins that are currently uncharacterized, which will be beneficial for the development of diagnostic and therapeutic approaches for male infertility. 

Mice and testes tissue collection All experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. C57BL/6 J mice were purchased from Charles River Laboratories (Zhejiang, China) and were housed with free access to water and food in a controlled environment with 12h light-dark cycle, room temperature of 20–25 °C and humidity of 40–70%. Mating cages were monitored each morning to determine the exact date of birth, defined as day 0 postnatal. Male mice were euthanized by carbon dioxide inhalation and cervical dislocation, and testes were immediately harvested. For lectin staining, testes were dissected at different postnatal days (on 0, 7, 16, 24 days and 8, 12 weeks, n = 5 per group), then fixed with 4% paraformaldehyde. For O-glycoproteomic analysis and Western blotting, testes were collected on postnatal day 24 and week 12 ( n = 4 per group). For flow cytometry analysis, testes were collected from 24-day-old ( n = 20) and 12-week-old mice ( n = 10). Cell lines, cell culture and transfection The Chinese hamster ovary epithelial cells CHO-K1 (RRID: CVCL_0214) and its mutant cells CHO-ldlD (RRID: CVCL_1V03) were gifts from Prof. Tatsuro Irimura (The University of Tokyo and Juntendo University School of Medicine, Japan). Both cell lines were maintained in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F12) 1:1 medium containing 10% FBS and cultured in a humidified atmosphere containing 5% CO 2 at 37 °C. Plasmids containing Flag-tagged pro-ACR, Flag-EGFP-tagged SPACA7/INSL6/EQTN/ SPESP1, His-tagged ACRBP, or His-EGFP-tagged SNAP25 were transfected into CHO-K1 or CHO-ldlD cells using EZ Trans Transfection Reagent (AC04L091, Life-iLab, Shanghai, China) according to the manufacturer’s instructions. Flow cytometry sorting of spermatids The testes were surgically removed, and the tunica albuginea along with the remaining blood vessels were removed. After being washed twice in phosphate-buffered saline (PBS), the testes were minced into small fragments using surgical scissors and repeatedly pipetted for 1 min. The suspension was placed at room temperature (RT) for 15 min to enable the precipitation of the remaining large fragments of intact tubules. The supernatant was centrifuged at 700 × g for 10 min, and the pellet was resuspended in 1 mL PBS. Subsequently, the cell suspension was transferred to a FACS tube and stained with 20 μg/mL of Hoechst 33342 (364-07951; Dojindo, Tokyo, Japan) for 30 min at room temperature. The haploid spermatids were gated on Hoechst blue (UV5) and Hoechst red (UV16) channels, and the round and elongated spermatids were distinguished by two imaging parameters (eccentricity and short axis moment) using BD FACSDiscover™ S8 (BD Biosciences, San Jose, CA, USA). The sorted spermatids were visualized by BD CellView™ imaging, and data analysis was performed with BD FACSChorus™ software. H&E staining and lectin staining Formalin-fixed, paraffin-embedded (FFPE) testicular sections (5 μm thickness) were deparaffinized and dehydrated. The sections were subjected to Hematoxylin and Eosin (H&E) staining for histological examination. For lectin staining, the sections were transferred to Citrate Antigen Retrieval Solution (P0081; Beyotime, Shanghai, China) to expose antigen epitopes. After washing with PBS, the sections were incubated with Carbo-Free Blocking Solution (SP-5040; Vector Laboratories, Burlingame, CA, USA) for 30 min at RT in a humidified chamber to reduce background staining. The sections were incubated with 10 μg/mL of fluorescein isothiocyanate (FITC)-conjugated Vicia villosa agglutinin (VVA) and Peanut agglutinin (PNA) (F-4601 and F-2301; EY Laboratories, San Mateo, CA, USA) at 4 °C overnight in the dark. Finally, the sections were incubated with 20 μg/mL of Hoechst 33342 (364-07951; Dojindo) and mounted with VECTASHIELD® Mounting Medium (H-1000; Vector Laboratories). The slides were visualized on a Nikon NI-U ortho-fluorescence microscope. Immunoprecipitation and immunoblot Cells were washed with ice-cold PBS and lysed using lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 0.5% NP-40, pH 7.4) containing protease inhibitor cocktail (04693132001; Roche, Mannheim, Germany). Cell lysate (1 mg) was incubated with 30 μL anti-Flag Affinity Beads (SA042005; Smart-Lifesciences, Shanghai, China) or Ni-NTA Beads 6FF (SA005100; Smart-Lifesciences) overnight at 4 °C with gentle rotation. After washing three times with TBS, the immunoprecipitates were eluted with 20 μL TBS containing 0.2% SDS at 95 °C for 5 min and then centrifuged. The resuspended proteins were subjected to lectin blot or Western blot analysis. The cell immunoprecipitates, testicular tissue or spermatid cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the appropriate antibodies at 4 °C overnight, or with horseradish peroxidase-conjugated VVA/PNA (HRP-VVA/PNA) (1:1000, H-4601/H-2013; EY Laboratories) at room temperature for 2 h. Primary antibodies included anti-Flag antibody (1:1000, M20008L; Abmart, Shanghai, China), anti-His antibody (1:1000, M30111M; Abmart), anti-GALNT3 antibody (1:1000, A13985; ABclonal, Wuhan, China), anti-C1GALT1 antibody (1:1000, A12865; ABclonal), anti-C1GALT1C1 antibody (1:1000, A7590; ABclonal), and anti-GAPDH antibody (1:2000, 10494-1-AP; Proteintech, Rosemont, IL, USA). Immunoreactive bands were visualized using the Amersham ImageQuant 800 ECL imaging system (GE Healthcare, Buckinghamshire, UK) or Odyssey Infrared Imaging System (Li-COR, Lincoln, NE, USA). Total intensities were semi-quantified with Quantity One software (Bio-Rad, Hercules, CA, USA). HPLC-based GALNTs activity assay The enzyme activity assay used six naked peptides of pro-ACR and ACRBP (SynPeptide Co., Ltd., Nanjing, China) as substrates. The reaction mixture contained the following components in a final volume of 10 μL: 100 ng of GALNTs in Tris-HCl (25 mM, pH 7.4), 100 μM peptides, 2 mM UDP-GalNAc, and 10 mM MnCl2. The mixture was incubated at 37 °C for 4 h, and then boiled at 95 °C for 5 min to terminate the reaction. The products were separated and detected by reverse-phase HPLC (Shimadzu, Kyoto, Japan) using a C18 analytical column (COSMOSIL 5C18-AR-II, 4.6 × 250 mm) with a flow rate of 1 mL/min. Glycoproteome analysis Samples pretreatment Frozen 24-day or 12-week testes ( ~100 mg) were resuspended in 500 µL of 50 mM NH 4 NO 3 containing 0.1% RapiGest SF (186001861; Waters Corporation) and homogenized using a mortar grinder at 60 Hz for 60 s, repeated three times. The tissue lysates were centrifuged at 150,000 × g at 4 °C for 15 min, and the supernatant was collected for protein quantitation using Pierce BCA protein assay kit (23225; Thermo Fischer Scientific, Waltham, MA, USA). The extracted proteins were heated for 10 min at 80 °C, followed by reduction with 5 mM dithiothreitol (DTT, D0632; MilliporeSigma, St. Louis, MO, USA) at 60 °C for 45 min and alkylation with 10 mM iodoacetamide (IAA, V900335; MilliporeSigma) at RT for 30 min in the dark. Proteins were then digested with trypsin (P01001; Enzyme Zhiyuan Biotechnology, Beijing, China) at a 1:50 wt/wt ratio at 37 °C overnight. The enzyme reaction was terminated by adding trifluoracetic acid (TFA, 302031; MilliporeSigma) and incubating at 37 °C for 20 min. After desalting using a C18 Sep-Pak (WAT054955; Waters Corporation), the sample was treated with 100 U of neuraminidase (N2876; MilliporeSigma) at 37 °C for 2 h. O-glycopeptides enrichment Pretreated samples from 24-day or 12-week testes were dissolved in 1 mL binding buffer (VVA-binding buffer: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl 2 /MgCl 2 /MnCl 2 /ZnCl 2 , and 1 M urea; PNA-binding buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl 2 , 0.1 mM MnCl 2 , and 0.1 mM ZnCl 2 ). Then, 100 μL VVA or PNA agarose beads (AL-1233 and AL-1073; Vector Laboratories) were washed with binding buffer and added to the sample with gentle rotation at 4 °C overnight. After being centrifuged at 800 × g for 3 min, samples were resuspended in 1 mL wash buffer (VVA-washing buffer: binding buffer with 0.4 M glucose; PNA-washing buffer: same as binding buffer) at 4 °C for 5 min to remove the non-specific binding. Finally, the agarose beads were eluted with 100 μL of 0.2 M GalNAc (G0750; MilliporeSigma) for VVA-enriched glycopeptides or 0.5 M galactose (A113374; Aladdin, Shanghai, China) for PNA-enriched glycopeptides with rotation at 4 °C overnight. The eluted of O-glycopeptides were collected and desalted using C18 Spin Tips (84850; Thermo Fischer Scientific) for LC-MS/MS analysis. Each group included four biological replicates. LC-MS/MS The enriched glycopeptide fractions were analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 nanoLC system (Thermo Fisher Scientific). The nanoLC system was operated using an analytical column (Dikma Inspire C18, 3 μm, Canada, 150 mm × 75 μm, self-packed). All samples were dissolved in Buffer A (0.1% formic acid, FA) were injected onto the column and separated using a 120-min gradient from 2% to 5% Buffer B (0.1% formic acid in 100% acetonitrile) in 2 min, from 5% to 20% in 95 min, from 20% to 30% in 10 min and from 30% to 100% in 1 min followed by isocratic elution at 100% for 12 min at a flow rate of 300 nL/min. MS1 scans were acquired with the following settings: scan range, 350–1800 m/z; resolution, 120,000; AGC target, 400,000; maximum injection time, 75 ms; dynamic exclusion time, 60 s. For MS2 scans, precursors with charge states 2–7 were fragmented with higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). In HCD scans, collision energy, 28%; resolution, 30,000; maximum injection time, 100 ms; AGC target, 100,000. A subsequent ETD scan of the same precursor was triggered if two of the top 20 most abundant ions in the HCD-MS2 spectrum were included in the following m/z: 126.0550, 138.0549, 144.0655, 168.0654, 186.0760, 204.0865, 274.0921, 292.1027, 290.0870, 308.0976, 366.1395. In ETD scans, resolution, 30,000; AGC target, 100,000; maximum injection time, 150 ms; using the charge-dependent ETD parameters, with 25% supplemental activation. Glycopeptide identification and quantification Raw files of LC-MS/MS were identified and quantified using pGlyco3.0 and MetaMorpheus (v1.0.2) . The search parameters were as follows: the mass tolerance of the precursor was set to 10 ppm and that of the fragment ion was to 0.02 Da; maximum missed cleavages allowed was two with full specificity; carbamidomethylation of cysteine (Cys) residues was the fixed modification, while methionine (Met) oxidation and protein N-terminal acetylation were variable modifications; the false discovery rate (FDR) was set to 1%. A mouse-specific database (UniProt, Aug. 2023, containing 17,174 reviewed proteins) was the reference proteome. Moreover, the analysis results derived from pGlyco3.0 were used for label free quantitation by pGlycoQuant (v202302) with the same parameters. Match Between Run (MBR) function in a ± 4 min retention time window was applied and the false quantitation rate (FQR) of the quantitation results was set to 1%. Peptides that had not been quantified in duplicate were excluded. Bioinformatic annotation and analysis Protein annotations (domain, cleavage site, etc.) were retrieved from UniProt database and further data analysis was performed by Python (v3.9.13) with in-house scripts and visualized by IBS2.0 website ( https://ibs.renlab.org/ ). The Venn diagrams, upset diagrams, heatmaps and volcano plots were made by Hiplot Pro ( https://hiplot.com.cn/ ). PCA, correlation analysis, functional enrichment analysis and sankey diagram were conducted by in-house R (v4.3.1) using factoextra (v1.0.7), corrplot (v0.92), clusterProfiler (v4.8.2), sankeyD3 (v0.3.2) and ggplot2 (v3.4.2) packages. The glycoprotein interaction network was analyzed by STRING database (v11.5) ( https://cn.string-db.org/ ) and visualized by Cytoscape (v3.8.1). Interrogation of available single-cell transcriptome resources The single-cell transcriptome data of mouse testes was downloaded from GEO database (GSE121904), processed on the 10X Genomics Chromium System. Data quality control, normalization, and cell clustering were conducted by Seurat (v4.3.0.1) R package. Cell types were manually annotated by marker genes . Statistical analysis All statistical tests used biological replicates and are indicated by group size (n) in figure legends. Data were expressed as mean ± SEM (standard error of the mean). Statistical analysis was conducted using GraphPad Prism (v8.3.0). Significance was calculated using unpair two-tailed Student’s t test or one-tailed Fisher’s exact test. Reporting summary Further information on research design is available in the linked to this article. 

All experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. C57BL/6 J mice were purchased from Charles River Laboratories (Zhejiang, China) and were housed with free access to water and food in a controlled environment with 12h light-dark cycle, room temperature of 20–25 °C and humidity of 40–70%. Mating cages were monitored each morning to determine the exact date of birth, defined as day 0 postnatal. Male mice were euthanized by carbon dioxide inhalation and cervical dislocation, and testes were immediately harvested. For lectin staining, testes were dissected at different postnatal days (on 0, 7, 16, 24 days and 8, 12 weeks, n = 5 per group), then fixed with 4% paraformaldehyde. For O-glycoproteomic analysis and Western blotting, testes were collected on postnatal day 24 and week 12 ( n = 4 per group). For flow cytometry analysis, testes were collected from 24-day-old ( n = 20) and 12-week-old mice ( n = 10). 

The Chinese hamster ovary epithelial cells CHO-K1 (RRID: CVCL_0214) and its mutant cells CHO-ldlD (RRID: CVCL_1V03) were gifts from Prof. Tatsuro Irimura (The University of Tokyo and Juntendo University School of Medicine, Japan). Both cell lines were maintained in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F12) 1:1 medium containing 10% FBS and cultured in a humidified atmosphere containing 5% CO 2 at 37 °C. Plasmids containing Flag-tagged pro-ACR, Flag-EGFP-tagged SPACA7/INSL6/EQTN/ SPESP1, His-tagged ACRBP, or His-EGFP-tagged SNAP25 were transfected into CHO-K1 or CHO-ldlD cells using EZ Trans Transfection Reagent (AC04L091, Life-iLab, Shanghai, China) according to the manufacturer’s instructions. 

The testes were surgically removed, and the tunica albuginea along with the remaining blood vessels were removed. After being washed twice in phosphate-buffered saline (PBS), the testes were minced into small fragments using surgical scissors and repeatedly pipetted for 1 min. The suspension was placed at room temperature (RT) for 15 min to enable the precipitation of the remaining large fragments of intact tubules. The supernatant was centrifuged at 700 × g for 10 min, and the pellet was resuspended in 1 mL PBS. Subsequently, the cell suspension was transferred to a FACS tube and stained with 20 μg/mL of Hoechst 33342 (364-07951; Dojindo, Tokyo, Japan) for 30 min at room temperature. The haploid spermatids were gated on Hoechst blue (UV5) and Hoechst red (UV16) channels, and the round and elongated spermatids were distinguished by two imaging parameters (eccentricity and short axis moment) using BD FACSDiscover™ S8 (BD Biosciences, San Jose, CA, USA). The sorted spermatids were visualized by BD CellView™ imaging, and data analysis was performed with BD FACSChorus™ software. 

Formalin-fixed, paraffin-embedded (FFPE) testicular sections (5 μm thickness) were deparaffinized and dehydrated. The sections were subjected to Hematoxylin and Eosin (H&E) staining for histological examination. For lectin staining, the sections were transferred to Citrate Antigen Retrieval Solution (P0081; Beyotime, Shanghai, China) to expose antigen epitopes. After washing with PBS, the sections were incubated with Carbo-Free Blocking Solution (SP-5040; Vector Laboratories, Burlingame, CA, USA) for 30 min at RT in a humidified chamber to reduce background staining. The sections were incubated with 10 μg/mL of fluorescein isothiocyanate (FITC)-conjugated Vicia villosa agglutinin (VVA) and Peanut agglutinin (PNA) (F-4601 and F-2301; EY Laboratories, San Mateo, CA, USA) at 4 °C overnight in the dark. Finally, the sections were incubated with 20 μg/mL of Hoechst 33342 (364-07951; Dojindo) and mounted with VECTASHIELD® Mounting Medium (H-1000; Vector Laboratories). The slides were visualized on a Nikon NI-U ortho-fluorescence microscope. 

Cells were washed with ice-cold PBS and lysed using lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 0.5% NP-40, pH 7.4) containing protease inhibitor cocktail (04693132001; Roche, Mannheim, Germany). Cell lysate (1 mg) was incubated with 30 μL anti-Flag Affinity Beads (SA042005; Smart-Lifesciences, Shanghai, China) or Ni-NTA Beads 6FF (SA005100; Smart-Lifesciences) overnight at 4 °C with gentle rotation. After washing three times with TBS, the immunoprecipitates were eluted with 20 μL TBS containing 0.2% SDS at 95 °C for 5 min and then centrifuged. The resuspended proteins were subjected to lectin blot or Western blot analysis. The cell immunoprecipitates, testicular tissue or spermatid cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the appropriate antibodies at 4 °C overnight, or with horseradish peroxidase-conjugated VVA/PNA (HRP-VVA/PNA) (1:1000, H-4601/H-2013; EY Laboratories) at room temperature for 2 h. Primary antibodies included anti-Flag antibody (1:1000, M20008L; Abmart, Shanghai, China), anti-His antibody (1:1000, M30111M; Abmart), anti-GALNT3 antibody (1:1000, A13985; ABclonal, Wuhan, China), anti-C1GALT1 antibody (1:1000, A12865; ABclonal), anti-C1GALT1C1 antibody (1:1000, A7590; ABclonal), and anti-GAPDH antibody (1:2000, 10494-1-AP; Proteintech, Rosemont, IL, USA). Immunoreactive bands were visualized using the Amersham ImageQuant 800 ECL imaging system (GE Healthcare, Buckinghamshire, UK) or Odyssey Infrared Imaging System (Li-COR, Lincoln, NE, USA). Total intensities were semi-quantified with Quantity One software (Bio-Rad, Hercules, CA, USA). 

The enzyme activity assay used six naked peptides of pro-ACR and ACRBP (SynPeptide Co., Ltd., Nanjing, China) as substrates. The reaction mixture contained the following components in a final volume of 10 μL: 100 ng of GALNTs in Tris-HCl (25 mM, pH 7.4), 100 μM peptides, 2 mM UDP-GalNAc, and 10 mM MnCl2. The mixture was incubated at 37 °C for 4 h, and then boiled at 95 °C for 5 min to terminate the reaction. The products were separated and detected by reverse-phase HPLC (Shimadzu, Kyoto, Japan) using a C18 analytical column (COSMOSIL 5C18-AR-II, 4.6 × 250 mm) with a flow rate of 1 mL/min. 

Samples pretreatment Frozen 24-day or 12-week testes ( ~100 mg) were resuspended in 500 µL of 50 mM NH 4 NO 3 containing 0.1% RapiGest SF (186001861; Waters Corporation) and homogenized using a mortar grinder at 60 Hz for 60 s, repeated three times. The tissue lysates were centrifuged at 150,000 × g at 4 °C for 15 min, and the supernatant was collected for protein quantitation using Pierce BCA protein assay kit (23225; Thermo Fischer Scientific, Waltham, MA, USA). The extracted proteins were heated for 10 min at 80 °C, followed by reduction with 5 mM dithiothreitol (DTT, D0632; MilliporeSigma, St. Louis, MO, USA) at 60 °C for 45 min and alkylation with 10 mM iodoacetamide (IAA, V900335; MilliporeSigma) at RT for 30 min in the dark. Proteins were then digested with trypsin (P01001; Enzyme Zhiyuan Biotechnology, Beijing, China) at a 1:50 wt/wt ratio at 37 °C overnight. The enzyme reaction was terminated by adding trifluoracetic acid (TFA, 302031; MilliporeSigma) and incubating at 37 °C for 20 min. After desalting using a C18 Sep-Pak (WAT054955; Waters Corporation), the sample was treated with 100 U of neuraminidase (N2876; MilliporeSigma) at 37 °C for 2 h. O-glycopeptides enrichment Pretreated samples from 24-day or 12-week testes were dissolved in 1 mL binding buffer (VVA-binding buffer: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl 2 /MgCl 2 /MnCl 2 /ZnCl 2 , and 1 M urea; PNA-binding buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl 2 , 0.1 mM MnCl 2 , and 0.1 mM ZnCl 2 ). Then, 100 μL VVA or PNA agarose beads (AL-1233 and AL-1073; Vector Laboratories) were washed with binding buffer and added to the sample with gentle rotation at 4 °C overnight. After being centrifuged at 800 × g for 3 min, samples were resuspended in 1 mL wash buffer (VVA-washing buffer: binding buffer with 0.4 M glucose; PNA-washing buffer: same as binding buffer) at 4 °C for 5 min to remove the non-specific binding. Finally, the agarose beads were eluted with 100 μL of 0.2 M GalNAc (G0750; MilliporeSigma) for VVA-enriched glycopeptides or 0.5 M galactose (A113374; Aladdin, Shanghai, China) for PNA-enriched glycopeptides with rotation at 4 °C overnight. The eluted of O-glycopeptides were collected and desalted using C18 Spin Tips (84850; Thermo Fischer Scientific) for LC-MS/MS analysis. Each group included four biological replicates. LC-MS/MS The enriched glycopeptide fractions were analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 nanoLC system (Thermo Fisher Scientific). The nanoLC system was operated using an analytical column (Dikma Inspire C18, 3 μm, Canada, 150 mm × 75 μm, self-packed). All samples were dissolved in Buffer A (0.1% formic acid, FA) were injected onto the column and separated using a 120-min gradient from 2% to 5% Buffer B (0.1% formic acid in 100% acetonitrile) in 2 min, from 5% to 20% in 95 min, from 20% to 30% in 10 min and from 30% to 100% in 1 min followed by isocratic elution at 100% for 12 min at a flow rate of 300 nL/min. MS1 scans were acquired with the following settings: scan range, 350–1800 m/z; resolution, 120,000; AGC target, 400,000; maximum injection time, 75 ms; dynamic exclusion time, 60 s. For MS2 scans, precursors with charge states 2–7 were fragmented with higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). In HCD scans, collision energy, 28%; resolution, 30,000; maximum injection time, 100 ms; AGC target, 100,000. A subsequent ETD scan of the same precursor was triggered if two of the top 20 most abundant ions in the HCD-MS2 spectrum were included in the following m/z: 126.0550, 138.0549, 144.0655, 168.0654, 186.0760, 204.0865, 274.0921, 292.1027, 290.0870, 308.0976, 366.1395. In ETD scans, resolution, 30,000; AGC target, 100,000; maximum injection time, 150 ms; using the charge-dependent ETD parameters, with 25% supplemental activation. Glycopeptide identification and quantification Raw files of LC-MS/MS were identified and quantified using pGlyco3.0 and MetaMorpheus (v1.0.2) . The search parameters were as follows: the mass tolerance of the precursor was set to 10 ppm and that of the fragment ion was to 0.02 Da; maximum missed cleavages allowed was two with full specificity; carbamidomethylation of cysteine (Cys) residues was the fixed modification, while methionine (Met) oxidation and protein N-terminal acetylation were variable modifications; the false discovery rate (FDR) was set to 1%. A mouse-specific database (UniProt, Aug. 2023, containing 17,174 reviewed proteins) was the reference proteome. Moreover, the analysis results derived from pGlyco3.0 were used for label free quantitation by pGlycoQuant (v202302) with the same parameters. Match Between Run (MBR) function in a ± 4 min retention time window was applied and the false quantitation rate (FQR) of the quantitation results was set to 1%. Peptides that had not been quantified in duplicate were excluded. 

Frozen 24-day or 12-week testes ( ~100 mg) were resuspended in 500 µL of 50 mM NH 4 NO 3 containing 0.1% RapiGest SF (186001861; Waters Corporation) and homogenized using a mortar grinder at 60 Hz for 60 s, repeated three times. The tissue lysates were centrifuged at 150,000 × g at 4 °C for 15 min, and the supernatant was collected for protein quantitation using Pierce BCA protein assay kit (23225; Thermo Fischer Scientific, Waltham, MA, USA). The extracted proteins were heated for 10 min at 80 °C, followed by reduction with 5 mM dithiothreitol (DTT, D0632; MilliporeSigma, St. Louis, MO, USA) at 60 °C for 45 min and alkylation with 10 mM iodoacetamide (IAA, V900335; MilliporeSigma) at RT for 30 min in the dark. Proteins were then digested with trypsin (P01001; Enzyme Zhiyuan Biotechnology, Beijing, China) at a 1:50 wt/wt ratio at 37 °C overnight. The enzyme reaction was terminated by adding trifluoracetic acid (TFA, 302031; MilliporeSigma) and incubating at 37 °C for 20 min. After desalting using a C18 Sep-Pak (WAT054955; Waters Corporation), the sample was treated with 100 U of neuraminidase (N2876; MilliporeSigma) at 37 °C for 2 h. 

Pretreated samples from 24-day or 12-week testes were dissolved in 1 mL binding buffer (VVA-binding buffer: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl 2 /MgCl 2 /MnCl 2 /ZnCl 2 , and 1 M urea; PNA-binding buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl 2 , 0.1 mM MnCl 2 , and 0.1 mM ZnCl 2 ). Then, 100 μL VVA or PNA agarose beads (AL-1233 and AL-1073; Vector Laboratories) were washed with binding buffer and added to the sample with gentle rotation at 4 °C overnight. After being centrifuged at 800 × g for 3 min, samples were resuspended in 1 mL wash buffer (VVA-washing buffer: binding buffer with 0.4 M glucose; PNA-washing buffer: same as binding buffer) at 4 °C for 5 min to remove the non-specific binding. Finally, the agarose beads were eluted with 100 μL of 0.2 M GalNAc (G0750; MilliporeSigma) for VVA-enriched glycopeptides or 0.5 M galactose (A113374; Aladdin, Shanghai, China) for PNA-enriched glycopeptides with rotation at 4 °C overnight. The eluted of O-glycopeptides were collected and desalted using C18 Spin Tips (84850; Thermo Fischer Scientific) for LC-MS/MS analysis. Each group included four biological replicates. 

The enriched glycopeptide fractions were analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 nanoLC system (Thermo Fisher Scientific). The nanoLC system was operated using an analytical column (Dikma Inspire C18, 3 μm, Canada, 150 mm × 75 μm, self-packed). All samples were dissolved in Buffer A (0.1% formic acid, FA) were injected onto the column and separated using a 120-min gradient from 2% to 5% Buffer B (0.1% formic acid in 100% acetonitrile) in 2 min, from 5% to 20% in 95 min, from 20% to 30% in 10 min and from 30% to 100% in 1 min followed by isocratic elution at 100% for 12 min at a flow rate of 300 nL/min. MS1 scans were acquired with the following settings: scan range, 350–1800 m/z; resolution, 120,000; AGC target, 400,000; maximum injection time, 75 ms; dynamic exclusion time, 60 s. For MS2 scans, precursors with charge states 2–7 were fragmented with higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). In HCD scans, collision energy, 28%; resolution, 30,000; maximum injection time, 100 ms; AGC target, 100,000. A subsequent ETD scan of the same precursor was triggered if two of the top 20 most abundant ions in the HCD-MS2 spectrum were included in the following m/z: 126.0550, 138.0549, 144.0655, 168.0654, 186.0760, 204.0865, 274.0921, 292.1027, 290.0870, 308.0976, 366.1395. In ETD scans, resolution, 30,000; AGC target, 100,000; maximum injection time, 150 ms; using the charge-dependent ETD parameters, with 25% supplemental activation. 

Raw files of LC-MS/MS were identified and quantified using pGlyco3.0 and MetaMorpheus (v1.0.2) . The search parameters were as follows: the mass tolerance of the precursor was set to 10 ppm and that of the fragment ion was to 0.02 Da; maximum missed cleavages allowed was two with full specificity; carbamidomethylation of cysteine (Cys) residues was the fixed modification, while methionine (Met) oxidation and protein N-terminal acetylation were variable modifications; the false discovery rate (FDR) was set to 1%. A mouse-specific database (UniProt, Aug. 2023, containing 17,174 reviewed proteins) was the reference proteome. Moreover, the analysis results derived from pGlyco3.0 were used for label free quantitation by pGlycoQuant (v202302) with the same parameters. Match Between Run (MBR) function in a ± 4 min retention time window was applied and the false quantitation rate (FQR) of the quantitation results was set to 1%. Peptides that had not been quantified in duplicate were excluded. 

Protein annotations (domain, cleavage site, etc.) were retrieved from UniProt database and further data analysis was performed by Python (v3.9.13) with in-house scripts and visualized by IBS2.0 website ( https://ibs.renlab.org/ ). The Venn diagrams, upset diagrams, heatmaps and volcano plots were made by Hiplot Pro ( https://hiplot.com.cn/ ). PCA, correlation analysis, functional enrichment analysis and sankey diagram were conducted by in-house R (v4.3.1) using factoextra (v1.0.7), corrplot (v0.92), clusterProfiler (v4.8.2), sankeyD3 (v0.3.2) and ggplot2 (v3.4.2) packages. The glycoprotein interaction network was analyzed by STRING database (v11.5) ( https://cn.string-db.org/ ) and visualized by Cytoscape (v3.8.1). 

The single-cell transcriptome data of mouse testes was downloaded from GEO database (GSE121904), processed on the 10X Genomics Chromium System. Data quality control, normalization, and cell clustering were conducted by Seurat (v4.3.0.1) R package. Cell types were manually annotated by marker genes . 

All statistical tests used biological replicates and are indicated by group size (n) in figure legends. Data were expressed as mean ± SEM (standard error of the mean). Statistical analysis was conducted using GraphPad Prism (v8.3.0). Significance was calculated using unpair two-tailed Student’s t test or one-tailed Fisher’s exact test. 

Further information on research design is available in the linked to this article. 

Supplementary Information Peer Review file Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Reporting Summary 

Source Data 

