SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.
BACKGROUND
The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates.


METHODS
We used laser microdissection (LM)/RT-PCR on frozen and paraffin-embedded epididymal sections of humans (n = 3) and macaques (n = 2) as well as in situ transcript hybridization to determine if transcripts are present in the epididymal epithelium. Western analysis and immunohistochemistry were used to detect and confirm the protein expression, and hyaluronic acid substrate gel electrophoresis analyzed its hyaluronidase activity. An in silico analysis of the proximal promoter of SPAM1 was also performed to identify relevant putative transcription binding sites for the androgen receptor.


RESULTS
We demonstrate that mRNA unique to SPAM1 is present in the principal cells of the epididymal epithelium in all individuals of both species studied. SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts. SPAM1 was shown to have hyaluronidase activity at pH 7.0. In the proximal promoter of SPAM1 were uncovered putative epididymal transcription factor binding sites including androgen receptor elements (AREs), consistent with epididymal expression.


CONCLUSIONS
These findings allow us to conclude that epididymal SPAM1 is conserved in at least two mammalian classes, rodents and primates. This conservation of expression suggests that the protein is likely to play an important function, possibly in sperm maturation.
Evans
Eric A
EA
Department of Biological Sciences, University of Delaware, Newark, Delaware, USA. eric.evans@stanford.edu
Zhang
Hong
H
Martin-DeLeon
Patricia A
PA
D000328:Adult; D000368:Aged; D000818:Animals; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics; D004822:Epididymis / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D008252:Macaca fascicularis / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D013079:Sperm Maturation / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014649:Vas Deferens / Q000378:metabolism / Q000648:ultrastructure
D015815:Cell Adhesion Molecules; D012333:RNA, Messenger; D014157:Transcription Factors; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20