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The expression of the new epididymal luminal protein of PDZ domain containing 1 is decreased in asthenozoospermia

Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1), also named Na+/H+ exchange regulatory cofactor 3 (NHERF3), which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.


The differentially enriched proteins between the caput and cauda
In this study, luminal proteins were extracted from mouse caput and cauda, and the absence of nuclear protein Protamine 1 (Prm1) in all samples showed that they were not contaminated by sperm (Supplementary Figure 1). In the analysis by Protein Pilot 5.0 search engine, a total of 66 794 spectra and 20 799 distinct peptides were identified. These peptides could be to 2720 proteins with a confidence ≥ 95%, and a total of 2132 nonredundant proteins with a false discovery rate < 1% were detected (Supplementary Table 1). With a criterion of 1.2-fold cutoff and P < 0.05, 128 differentially enriched proteins were identified, of which 46 were caput fluid enriched and 82 were cauda fluid enriched (Supplementary Table 2). The proteins with no difference in enrichment between the two fluids are listed in Supplementary Table 3.

The different functions of the caput and cauda epididymidis
The 128 differentially enriched proteins were subjected to bioinformatic analysis by GO annotation and enrichment to analyze the different functions of the caput and cauda regions. Biological process analysis found that processes related to small-molecule metabolism, organic acid metabolism, oxidation-reduction, organonitrogen compound metabolism, and organic substance catabolism existed in both the caput and cauda. However, lipid metabolism was found in the caput, while those associated with phosphorus and organophosphate metabolism were found in the cauda (Figure 1a and 1b). The results of molecular function annotation indicated that binding activity was the main function of these proteins. Identical protein binding, protein domain-specific binding, protein complex binding, and coenzyme binding activities were all identified in the caput and cauda while anion and cation binding activities were identified only in cauda (Figure 1c and 1d). These results indicate that the caput and cauda play different roles in the process of sperm maturation.

Pdzk1 is a newly identified epididymal luminal protein
GO analysis showed that Pdzk1 was found in the lipid metabolism group, as an epididymal luminal protein newly identified by iTRAQ (Supplementary Table 2). The expression and localization of Pdzk1 were studied by western blotting and immunofluorescence analyses.
Western blot analysis showed that Pdzk1 was expressed in heart, liver, kidney, epididymis, and efferent ducts, but not in the testis or ovary 14 (Figure 2a and Supplementary Figure 2a). The expression of Pdzk1 in the caput, corpus, and cauda (Figure 2b) was confirmed, and the expression level of Pdzk1 in the spermatozoa and fluid from the caput, corpus, and cauda was also analyzed (Figure 2c and 2d). Immunoblotting result confirmed that Pdzk1 was caput fluid enriched (Figure 2b-2d).
The localization of Pdzk1 was shown by immunofluorescence to have a dynamic expression pattern in the epididymis (Figure 3). In the proximal and distal caput, Pdzk1 was expressed over a wide area and had a stronger signal in the distal caput. Then, an intense signal was observed at the surface of corpus and cauda epithelia. The expression pattern of Pdzk1 in the kidney has been reported and was used here as a positive control. 15 In order to illuminate the sperm localization of Pdzk1, spermatozoa from the mouse caput, corpus, and cauda were analyzed by immunofluorescence (Figure 4). Pdzk1 is localized to the sperm tail, especially in the middle piece, where the mitochondrial sheath lies (Figure 4).

The expression of Pdzk1 in asthenozoospermia
Spermatozoa from normozoospermic men (mean age: 29 years) and asthenozoospermic patients (mean age: 30 years) were collected to investigate the role of Pdzk1 in male fertility ( Table 1). The localization of Pdzk1 in human spermatozoa was similar to that in the mouse, and the localization pattern did not differ between normozoospermia and asthenozoospermia ( Figure 5). The expression of Pdzk1 was analyzed by western blotting analysis, which showed  that Pdzk1 was decreased in the spermatozoa in asthenozoospermia (Figure 6 and Supplementary Figure 2b), suggesting that Pdzk1 may function in sperm maturation and may be associated with male infertility.

DISCUSSION
When spermatozoa are transiting through the convoluted epididymal tubule, they successively interact with the epididymal fluid to acquire progressive motility and fertilizing capacity, a process that is called epididymal sperm maturation. To reveal the mechanism of sperm maturation, transcriptomic and proteomic analyses of epididymis, [16][17][18][19] proteomic analyses of sperm, 20,21 and proteomic analyses of epididymal fluid 6,9 have been performed by other groups.
Proteomic analyses of luminal proteins were mainly carried out by 2D-PAGE. 6,9 However, this method has drawbacks, in that it is difficult to use it to analyze low-abundance proteins, hydrophobic proteins, and membrane proteins. 22 The epididymal fluid contains many low-abundance proteins, and studies by 2D-PAGE can only identify hundreds of proteins or fewer in this fluid in various species, such as 525 proteins in human, 23 146 proteins in boars, 24 117 proteins in stallions, 25 172 proteins in bulls, 26 12 proteins in rams, 27 198 proteins in monkeys, 28 27 proteins in platypuses, 29 19 proteins in rats, 30,31 and 23 proteins in mice. 32 Thus, the epididymal luminal proteins, especially  the proteins differentially enriched between the caput and cauda, are still largely unknown.
Several methods for epididymal fluid collection have been developed. Mincing and pressing the epididymis was the earliest used method. However, samples processed by this method may be contaminated with serum, lymph, or epididymal cellular proteins. As an alternative, the microperfusion method can provide much purer epididymal fluid, but this method is mainly performed on large animals, and is not suitable for mice, especially for the collection of mouse caput fluid. 25,33 Thus, the method of making small slits in the epididymis tubules with pressing was adopted to collect mouse caput and cauda luminal fluids in the study.
In the present study, the iTRAQ method was applied to reveal the proteins differentially enriched between the caput and cauda. The results of bioinformatic analysis showed that these proteins took part in different biological processes and played different molecular functions. These results indicate that the caput and cauda have distinct functions during sperm maturation.
Pdzk1 is a newly identified epididymal luminal protein by iTRAQ, which was found in lipid metabolism. Pdzk1, also named NHERF3, belongs to the Na + /H + exchange regulatory cofactor (NHERF) family. It is a scaffold protein with four PDZ domains and participates in the regulation of cell surface protein expression, cholesterol metabolism, and carnitine transport, which are important for sperm maturation and motility. 15, [34][35][36] Pdzk1 prompts carnitine uptake by upregulating the activity of the carnitine transporter OCTN2. 15,37 It has been reported that Pdzk1 is expressed in the epididymis and efferent ducts, 14 which was confirmed by our findings. The cauda has a higher concentration of carnitine and the cauda sperm are more motile than that in the caput. 38 However, we found that Pdzk1 was enriched in the caput fluid but not in the cauda fluid. The function of Pdzk1 in the cauda fluid may be compensated by another NHERF family member, NHERF1, which interacts with another carnitine transporter, OCTN1, 15 since NHERF1 is enriched in the cauda fluid.
Pdzk1 was localized at the sperm tail, but had no expression in the testis. Pdzk1 may be secreted into the luminal fluid from the efferent ducts or epididymis and bound to the sperm surface. Since Pdzk1 has no typical signal sequence (Supplementary Figure 3), its secretion may be mediated by epididymosomes, specific exosomes in the epididymis. 5 This manner of secretion was confirmed by the finding that Pdzk1 was present in urinary exosomes. 39,40 The expression of Pdzk1 in human spermatozoa was analyzed and showed that Pdzk1 was decreased in asthenozoospermia. Mice without Pdzk1 are fertile but have increased serum cholesterol levels, which may affect sperm motility. However, this speculation requires further investigation because the sperm motility and sperm fertilizing ability in the knockout mice are unclear. 41 The results obtained here indicate that Pdzk1 may be associated with sperm maturation and male infertility, but further research is needed to reveal the mechanisms behind this involvement.

CONCLUSION
Taking the obtained findings together, this work reveals the proteins differentially enriched between the caput and cauda fluid, shedding light on the distinct functions of the caput and cauda in sperm maturation. The epididymal luminal protein Pdzk1 newly identified here by iTRAQ has been associated with asthenozoospermia. This work provides a foundation for future mechanistic research on sperm maturation and male infertility.