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SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.

Evans EA, Zhang H, Martin-DeLeon PA - Reprod. Biol. Endocrinol. (2003)

Bottom Line: SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA. eric.evans@stanford.edu

ABSTRACT

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.

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In situ hybridization localization of SPAM1 transcripts in human corpus epididymal epithelium. Histological sections were hybridized with either an anti-sense (A) or sense (B) SPAM1 RNA probe. SPAM1 transcripts localized by immunolabeling appear as a purple stain in the epithelium of (A). This stain is absent from the negative control (B) which shows only the neutral red counterstaining and which was incubated for the same period of time as A) in the developing solution.
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Figure 3: In situ hybridization localization of SPAM1 transcripts in human corpus epididymal epithelium. Histological sections were hybridized with either an anti-sense (A) or sense (B) SPAM1 RNA probe. SPAM1 transcripts localized by immunolabeling appear as a purple stain in the epithelium of (A). This stain is absent from the negative control (B) which shows only the neutral red counterstaining and which was incubated for the same period of time as A) in the developing solution.

Mentions: In situ transcript hybridization was performed on human caput/corpus epididymis (Subject #2; Fig. 3) to confirm the presence of SPAM1 transcript detected by LM/RT-PCR. The analysis indicates that the transcript is present in the epithelial cells where the purple label can be seen (Fig. 3A). The mRNA was located predominantly in the perinuclear region of the principal cells. Unlike test sections hybridized with the antisense probe, the control sections probed with the sense riboprobe showed only the neutral red staining (Fig. 3B). The purple label seen in the connective tissue in control and test sections may result from endogenous alkaline phosphatase activity.


SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.

Evans EA, Zhang H, Martin-DeLeon PA - Reprod. Biol. Endocrinol. (2003)

In situ hybridization localization of SPAM1 transcripts in human corpus epididymal epithelium. Histological sections were hybridized with either an anti-sense (A) or sense (B) SPAM1 RNA probe. SPAM1 transcripts localized by immunolabeling appear as a purple stain in the epithelium of (A). This stain is absent from the negative control (B) which shows only the neutral red counterstaining and which was incubated for the same period of time as A) in the developing solution.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC184449&req=5

Figure 3: In situ hybridization localization of SPAM1 transcripts in human corpus epididymal epithelium. Histological sections were hybridized with either an anti-sense (A) or sense (B) SPAM1 RNA probe. SPAM1 transcripts localized by immunolabeling appear as a purple stain in the epithelium of (A). This stain is absent from the negative control (B) which shows only the neutral red counterstaining and which was incubated for the same period of time as A) in the developing solution.
Mentions: In situ transcript hybridization was performed on human caput/corpus epididymis (Subject #2; Fig. 3) to confirm the presence of SPAM1 transcript detected by LM/RT-PCR. The analysis indicates that the transcript is present in the epithelial cells where the purple label can be seen (Fig. 3A). The mRNA was located predominantly in the perinuclear region of the principal cells. Unlike test sections hybridized with the antisense probe, the control sections probed with the sense riboprobe showed only the neutral red staining (Fig. 3B). The purple label seen in the connective tissue in control and test sections may result from endogenous alkaline phosphatase activity.

Bottom Line: SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA. eric.evans@stanford.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus