Although human SPAM1 mRNA expression is detected by Northern analysis only in the testis , RT-PCR assays reveal that the gene is also transcribed in somatic tissues where the message is rare. Rare SPAM1 transcripts are however not ubiquitous as revealed by their absence from human ovary, spleen, and liver  and murine skeletal muscle . Both rare and abundant SPAM1 transcripts have been found in neoplastic breast tissue  and in a number of other cancers including pharyngeal , metastatic melanomas and gliomas . In normal somatic cells rare transcripts have been found in breast tissue  and in fetal, placental, and prostate cDNA libraries .
Expression in the prostate is relevant to our findings in the present study where transcripts were detected in the human caput/corpus epididymis using LM/RT-PCR. This technique which has been effectively used to procure homogeneous populations of cells from tissue sections [26–28] revealed that SPAM1 mRNA is confined to the epididymal epithelium. In situ hybridization demonstrated that the transcript is localized in the principal cells, confirming its epithelial location, similar to that reported for the mouse [11, 12]. It must be stressed that because the probes used in the hybridization were generated from the unique 3' UTR of SPAM1 cDNA the possibility of cross-hybridization to paralogous hyaluronidases is highly unlikely. This conclusion is supported by unique BLAT assignment of the probe sequence to the human genome .
SPAM1 protein was found to be present in both the human and macaque reproductive tracts. This indicates that in the epididymis and vas deferens SPAM1 is both transcribed and translated. The results from Western analysis of protein extracts from the epididymis, which like the vas deferens were thoroughly washed to remove sperm, show conclusively that intact SPAM1 protein is synthesized in all three epididymal regions in macaques. It is noteworthy that the ~53 kDa band, representing proteolytically cleaved SPAM1 was seen only in sperm and not in the epididymis or the vas which had their own unique degradation products at ~60 kDa and <50 kDa, respectively. This suggests that the Western results may reveal an isoform that is endogenous to each tissue. It also indicates and that sperm is not the source of epididymal SPAM1. Also in macaque the intensity of the epididymal bands was equal to, or greater than, that of the testis. This also makes it highly unlikely that SPAM1 is present in the epididymis merely because it is transported there from the testis.
Western blot analysis also suggests that SPAM1 protein is expressed at higher levels in the distal regions of the epididymis, with the highest level of expression in the corpus, a region associated with a lower volume of sperm. This is supported by the Dot blot, with equal protein loading, showing the macaque corpus to have the highest amount of SPAM1. This observation not only argues against the mere transportation of SPAM1 into the epididymis, but also suggests a region-dependent expression of the gene. This finding is consistent with that reported in the mouse [11, 12] where there is differential expression of the protein in the epididymis with the highest expression in the corpus. Expression in the extratesticular pathway seems to be lowest in the vas deferens in both human and macaque. However, its presence prompted us to examine the vas in the mouse where it had not been previously studied: both the transcript and protein were found (Martin-DeLeon et al, unpublished results). Thus there seems to be a conservation of the pattern of expression of SPAM1 in the extratesticular pathways of mice and primates.
The finding from the Western blots was corroborated by those from immunohistochemistry. The latter indicates that SPAM1 is associated with the principal cells of the epithelium, consistent with the localization of the transcript and with findings in the mouse [11, 12]. The finding of human SPAM1 in the seminal vesicle, which was examined only because it was included in the tissue series on the slides from Novagen, was unexpected but not surprising. CD52, a well-known human epididymal protein, is also known to be expressed in the seminal vesicle . While the expression of human SPAM1 in the seminal vesicle remains to be confirmed, data from our lab have shown that the murine Spam1 gene is both transcribed and translated in this tissue (Martin-DeLeon, unpublished results).
HASGE shows that epididymal SPAM1 has neutral hyaluronidase activity (Fig. 5), which is typical only of sperm hyaluronidase. This further supports the identity of the protein expressed in the epididymis. In general, the expression of the enzyme activity was less robust in the epididymis compared to the testis, and inconclusive for the vas deferens. The latter is not surprising because (1) Western analysis showed SPAM1 levels to be the lowest in the vas deferens and (2) it is reasonable to expect that HASGE is less sensitive than Western analysis. More importantly, it is possible that the hyaluronidase activity in cells from non-testicular origin may be lower than that of the testis due to differences in the pattern of glycosylation. Variations in glycosylation patterns have been shown to exist among tissues  and glycosylation levels have been reported to influence hyaluronidase activity of SPAM1 in the mouse and horse [10, 32]. Note that in the mouse, tissue differences in glycosylation patterns have been reported for the testis and epididymis . It is possible that in the epididymis and vas deferens instead of hyaluronidase activity, a more important role of SPAM1 (which is a glycosidase) may be to modify surface proteins of sperm during their maturation. Taken together, the data from tissues of 5 men and 2 macaques show that SPAM1 is both transcribed and translated in the epididymal epithelium of primates.
The finding of conserved expression patterns of epididymal SPAM1 between humans and mice is supported by the findings in the promoter of the human gene. Table 1 reveals that in human SPAM1 there is a CRE transcription factor binding site, a site that was shown to be functional in the murine Spam1 promoter region  as well as putative AREs which are also present in the mouse . Of special interest are the relative locations of the AREs which are associated with genes expressed in the epididymis  and the prostate at -661 and -763; and a CRE (cyclic-AMP responsive element) which is consistent with haploid expression in the testis at -207 . The relative location of these elements are consistent with the findings of Hsia et al.  who observed that within the promoter regions of genes expressed in both the testis and the epididymis, the testis-expression elements are more proximal to the transcription start site than those mediating epididymal expression.
It should be mentioned that CRE and SRY are likely to be responsible for increasing the basal transcriptional level of SPAM1, leading to the robust expression of the mRNA that is detectable with Northern analysis only in the testis. Note that the relevant transcriptional binding factor and/or co-activator for CRE (CREM and ACT) and for SRY in the adult (SRY) are testis-specific [37, 38]. Future studies are necessary to determine if the transcriptional binding sites revealed by this study are functional in humans.
In the immunohistochemical studies the sperm concentrations in some of the lumen of the tubules in sections of the caput/corpus epididymis were low enough to allow the observation of SPAM1 localization on immature sperm. Observations of sperm from Subject #1, the 45-year-old male of known fertility with an obstruction in his corpus epididymis, as well as macaques, revealed that SPAM1 is concentrated in the posterior and deficient in the anterior head of immature primate sperm. This localization is quite different from that reported for ejaculated human sperm where SPAM1 is found uniformly over the entire head surface . Our finding suggests that SPAM1 undergoes redistribution on the sperm head during epididymal maturation in primates as it does in guinea pigs and mice [40, 10], albeit with a reverse order going from a regionalized to uniform pattern.
The finding that SPAM1 with enzymatic activity is expressed in the epididymides of three species (mice, humans and macaques) in two classes of mammals indicates that epididymal SPAM1 is functionally important. It is likely that SPAM1 may play a role in sperm maturation, since its expression is highest in the corpus where a number of maturational changes occur [41, 42]. Thus, this study increases the biomedical significance of findings regarding Spam1 in the mouse model. For example, it is reasonable to believe that like the mouse, human SPAM1 is secreted from the epididymal epithelium with an intact lipid anchor [12, 33] and may impact sperm maturation. It should also be noted that a study of the glycosylation and 2D-gel patterns of Spam1 from the epididymis and testis of mice indicates that epididymal Spam1 may be a unique isoform rather than a redundant protein . Such a unique isoform may play a specific role in sperm maturation in humans where SPAM1 is the only reproductive hyaluronidase in the cluster on 7q31. Currently studies are underway to determine the function of epididymal Spam1 in the mouse.