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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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Immunohistochemical localization of SPAM1 in macaque epididymis and epididymal sperm using multiphoton confocal microscopy. In A and B (magnification seen in A) are shown the control and the test, respectively; after SPAM1 antibody staining in the corpus (Animal #2). As in Fig. 6, the images are taken in the red confocal channel but due to high autofluorescence the control is not as red as in Fig. 6. However sperm in the lumen serve as a positive control for the test whose epithelium and sperm are greener than the control. In C) are seen immature sperm from the corpus (magnification = ×1,000). SPAM1 is localized more heavily to the posterior region of the sperm head (as shown by the arrows) as was also seen for immature human sperm (data not shown).
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Figure 7: Immunohistochemical localization of SPAM1 in macaque epididymis and epididymal sperm using multiphoton confocal microscopy. In A and B (magnification seen in A) are shown the control and the test, respectively; after SPAM1 antibody staining in the corpus (Animal #2). As in Fig. 6, the images are taken in the red confocal channel but due to high autofluorescence the control is not as red as in Fig. 6. However sperm in the lumen serve as a positive control for the test whose epithelium and sperm are greener than the control. In C) are seen immature sperm from the corpus (magnification = ×1,000). SPAM1 is localized more heavily to the posterior region of the sperm head (as shown by the arrows) as was also seen for immature human sperm (data not shown).

Mentions: In the two animals studied the epididymal epithelia and the sperm in the lumen of test slides were consistently a brighter green (Fig. 7B) than those of control (Fig. 7A). In the latter the autofluorescence is high due to the nature of fixation. In the epididymides of both humans and macaques the protein was localized to the principal cells of the epithelium and in the heads of maturing sperm (positive control).


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)

Immunohistochemical localization of SPAM1 in macaque epididymis and epididymal sperm using multiphoton confocal microscopy. In A and B (magnification seen in A) are shown the control and the test, respectively; after SPAM1 antibody staining in the corpus (Animal #2). As in Fig. 6, the images are taken in the red confocal channel but due to high autofluorescence the control is not as red as in Fig. 6. However sperm in the lumen serve as a positive control for the test whose epithelium and sperm are greener than the control. In C) are seen immature sperm from the corpus (magnification = ×1,000). SPAM1 is localized more heavily to the posterior region of the sperm head (as shown by the arrows) as was also seen for immature human sperm (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Immunohistochemical localization of SPAM1 in macaque epididymis and epididymal sperm using multiphoton confocal microscopy. In A and B (magnification seen in A) are shown the control and the test, respectively; after SPAM1 antibody staining in the corpus (Animal #2). As in Fig. 6, the images are taken in the red confocal channel but due to high autofluorescence the control is not as red as in Fig. 6. However sperm in the lumen serve as a positive control for the test whose epithelium and sperm are greener than the control. In C) are seen immature sperm from the corpus (magnification = ×1,000). SPAM1 is localized more heavily to the posterior region of the sperm head (as shown by the arrows) as was also seen for immature human sperm (data not shown).
Mentions: In the two animals studied the epididymal epithelia and the sperm in the lumen of test slides were consistently a brighter green (Fig. 7B) than those of control (Fig. 7A). In the latter the autofluorescence is high due to the nature of fixation. In the epididymides of both humans and macaques the protein was localized to the principal cells of the epithelium and in the heads of maturing sperm (positive control).

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