Spermatozoa are transcriptionally and translationally silent cells. The development from immature to mature cells capable of fertilizing an oocyte depends on post-translational modification of pre-existing proteins. These modifications occur via interactions with proteins secreted by the epididymal epithelium while sperm traverse the epididymis. We characterized Spag11a in the mouse epididymis at both the RNA and protein levels to obtain data on the putative role of this gene in sperm maturation.
SPAG11A is a member of the beta defensin family and contains a signal peptide sequence. This protein family has antimicrobial activity and is involved in host defense . The defensin gene family has evolved by repeated gene duplication and divergence, including functional diversification . Reproductive functions are suggested by the surface localization of several defensins, including SPAG11 , DEFB118  and DEFB126 , on sperm. SPAG11E, also known as Bin1b, is known to promote motility in immature spermatozoa in the caput epididymis via a calcium uptake-dependent mechanism . Alternative spliced transcript have produced multiple Spag11 isoforms in epididymal epithelial cells. Although many members of the beta defensin family have been characterized, the exact role of Spag11a in the mouse epididymis is unknown. The presence of a signal peptide sequence suggested that mouse SPAG11A is a secretory protein that may be involved in sperm maturation. Moreover, we discovered that SPAG11A contains phosphorylation sites such that upon binding to sperm, the cell can be modified by protein kinases. This corresponded with a finding that during transit through the epididymis, spermatozoa undergo changes in tyrosine phosphorylation . Concerning binding to sperm, we identified a myristoylation site in SPAG11A. The presence of this potential N-myristoylation site suggested that the protein may covalently bind to the plasma membrane of sperm [32, 33].
Our data demonstrated that mouse Spag11a was expressed exclusively in the epididymis, not in other tissues such as the testis, vas deferens, intestine, kidney, liver, heart, muscle and brain. Moreover, mouse Spag11a exhibited a region-specific expression pattern and was mainly present in the caput region. This is similar to several genes that are important for sperm maturation such as Rnase10[34, 35], Crisp4[36, 37], and Crisp1, which are exclusively expressed in the initial segment, caput and corpus/cauda, respectively. This region-specific expression is important to create specific environments for sperm maturation. Although the highest expression was detected in the caput, low expression levels were detected in corpus epididymis, muscle and liver (Figure 2). This is possible because the beta defensin family has diverse members, and some of which function in the muscle . A study by Yamaguchi et al. also showed multiple epididymis-specific beta defensin isoforms in human and mice, including mouse EP2e, which is a synonym of Spag11a, with expression in caput, corpus and cauda . Our study is first to show Spag11a expression specificity in the caput region both at the transcript and protein levels.
Sperm maturation in the epididymis is androgen-dependent. Our data demonstrated that Spag11a was slightly up-regulated 6 hours to 1 day after castration/gonadectomy before being dramatically down-regulated 3 days after castration. The androgen dependence was confirmed with a rescue experiment in which exogenous testosterone was injected daily into castrated mice. The results indicated that exogenous testosterone could maintain Spag11a expression at nearly normal levels on days 3 and 5 after castration. This indicated that Spag11a was primarily regulated by circulating androgen. Moreover, our study in the regulation of Spag11a by androgen was also confirmed by immunohistochemistry which demonstrated a total lost of staining in the caput principal cells after 3 d castration. This is similar to the regulation of several genes involved in sperm maturation, such as Eppin (epididymal protease inhibitor) , Pate (cysteine rich prostate and testis expressed protein)  and the serine protease inhibitor HongrES1. EPPIN is an antimicrobial cysteine-rich protein that contains both Kunitz and whey acidic protein (WAP) domains and is a target for male contraception because of its critical role in sperm motility .
In addition to androgen, epididymal genes are also regulated by testicular factors. Because the observed recovery of Spag11a expression after gonadectomy/castration with testosterone replacement was slightly lower than that in the control non-gonadectomy group, we tested the possibility that testicular factors were involved in Spag11a regulation. By using efferent duct ligation (EDL), we blocked testicular fluid (lumicrine factor) from entering the epididymis while preserving testosterone supply from both testis. The result from the EDL experiment showed that the lack of testicular fluid did not affect Spag11a expression at 6 hours to 1 day after the ligation. Interestingly, Spag11a was transiently up-regulated at 3 days after EDL before down-regulated back to the level of control at 5 days after EDL (Figure 4). The reason for the transient increase is not known, but we hypothesize that it may be caused by initiation or the onset of apoptosis within the cell which somehow stimulates a temporary up-regulation of Spag11a in response to the process. This notion is based on previous studies that orchidectomy and efferent duct ligation induces apoptotic cell death in the caput epididymis that reach maximum at day 3 [44, 45]. Several epididymal genes are regulated by testicular factors, including gamma-glutamyltransferase 1 (Ggt1, regulated by fibroblast growth factor (FGF)) , 5-alpha reductase (regulated by androgen binding protein (ABP))  and proenkephalin (Penk, regulated by sperm-associated factors) . The primary regulation of caput-specific Spag11a by androgen confirmed our previous report that epididymal genes enriched in the initial segment are more dependent on testicular factors whereas androgen regulates most of the caput-enriched genes .
We also analyzed whether the expression of Spag11a mRNA was consistent with the protein expression. We performed western immunoblotting using protein extracts from four different regions of the mouse epididymis. SPAG11A protein was detected with a rabbit anti-human SPAG11A polyclonal antibody. The results demonstrated that SPAG11A was only present in the caput region (Figure 5) which confirmed the antibody specificity. Because the protein was present in the caput epididymis, we performed immunohistochemistry to localize SPAG11A at the subcellular level. SPAG11A was localized in the nucleus and cytoplasm of the principal cells in the caput region, whereas only cytoplasmic staining was detected in the corpus and cauda. This is in agreement with the tissue distribution analyses using qRT-PCR in which the highest expression was in caput whereas very weak expression was detected in the corpus and cauda (Figure 2). The cell-specific expression of SPAG11A confirmed its putative role as a secretory protein that creates a microenvironment suitable for sperm maturation. Principal cells contain secretory apparatuses, such as endoplasmic reticulum, Golgi and secretory granules, and endocytic apparatuses, including coated pits, endosomes, multivesicular bodies and lysosomes. Therefore, the primary functions of principal cells are to synthesize and secrete proteins and to perform endocytosis . This is particularly interesting because a signal peptide sequence was identified in the first twenty amino acids of the N-terminus of SPAG11A (Figure 1), which is characteristic of secretory proteins. An epididymal gene known to have a cell type-specific expression pattern is cystic fibrosis transmembrane conductance regulator (CFTR) which is also expressed specifically in the principal cell to release ATP into epididymal lumen .
It is intriguing to observe that SPAG11A, with a signal peptide indicating a secretory protein was localized in the nucleus. Although this is an unusual phenomenon, examples of similar protein behaviour do exist. A study of ADAMTS13, a secreted zinc metalloprotease involved in an array of processes including development and angiogenesis, detected the protein in the nucleus of liver cells . Other metalloproteinases, MMP-2  and MMP3 , which are involved in extracellular matrix remodeling, were detected in the nucleus of cardiac myocytes and chondrocytic cells, respectively. This suggests intracellular role for these secreted proteins. MMP3, for instance, behaves as a proteinase that degrades matrix components following its secretion, while behaving as a transcription factor when present within the nucleus. It is also possible that SPAG11A has another intracellular role and it shuttles between nucleus and cytoplasm through nuclear pore complex. This is particularly interesting if we correlate it with transient up-regulation of Spag11a at 3 d following efferent duct ligation (EDL) and returned to the normal level at 5 d post EDL. That coincided with a major change in the caput epididymal cell at day 3 following orchidectomy or EDL, particularly the onset of apoptosis [44, 45].
Our study is the first to show that SPAG11A is a secretory protein that is present in the epididymal fluid and spermatozoa taken from the cauda epididymis and vas deferens. SPAG11A is secreted mainly by principal cells of the caput epididymis and the protein was detected in epididymal fluid but minimum amount of protein was detected in the vas deferens fluid. The reduced amount of protein in the vas deferens luminal fluid indicating that most of the protein may have bound to the sperm cell. Secreted from the caput and to some extent from corpus and cauda region, the protein subsequently bind to the spermatozoa. An increasing amount of SPAG11A was detected in the protein extracted from cauda and vas deferens sperm compared to the caput sperm (Figure 8A). Furthermore, by using immunocytochemistry, we also showed more intense SPAG11A staining in the sperm cells taken from vas deferens compared to the epididymal sperm, confirming more SPAG11A protein deposited to the sperm upon exit from the epididymis. We believe that data from this study is important for a further study to determine the role of SPAG11A during epididymal sperm maturation and fertilization.