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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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HASGE analysis at pH 7.0 of protein extracts from testis, epididymis, vas deferens and sperm of human and macaque. Equal amounts (60 μg) of protein were loaded in each lane. The large arrow shows a non-staining clear band at ~64 kDa, representing the digested substrate. A narrow band of a much higher MW (approximately 114 kDa) was seen in human caput, human sperm, and macaque corpus, as indicated by the smaller arrow.
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Figure 5: HASGE analysis at pH 7.0 of protein extracts from testis, epididymis, vas deferens and sperm of human and macaque. Equal amounts (60 μg) of protein were loaded in each lane. The large arrow shows a non-staining clear band at ~64 kDa, representing the digested substrate. A narrow band of a much higher MW (approximately 114 kDa) was seen in human caput, human sperm, and macaque corpus, as indicated by the smaller arrow.

Mentions: Using HASGE, neutral hyaluronidase activity (pH 7.0) was demonstrated in protein extracts from the epididymis, with testis and sperm serving as positive controls. Hyaluronidase activity was demonstrated by a non-staining area corresponding to the size of the intact protein at ~64 kDa (Fig. 5). In samples from the human caput, human sperm and macaque corpus there was also a narrow band with a higher MW (>114 kDa, as indicated by the smaller arrow) which may represent dimerization of the molecule. This experiment was repeated multiple times, and the results seen in Figure 5 are a concatenation of bands from several HASGE gels.


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)

HASGE analysis at pH 7.0 of protein extracts from testis, epididymis, vas deferens and sperm of human and macaque. Equal amounts (60 μg) of protein were loaded in each lane. The large arrow shows a non-staining clear band at ~64 kDa, representing the digested substrate. A narrow band of a much higher MW (approximately 114 kDa) was seen in human caput, human sperm, and macaque corpus, as indicated by the smaller arrow.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: HASGE analysis at pH 7.0 of protein extracts from testis, epididymis, vas deferens and sperm of human and macaque. Equal amounts (60 μg) of protein were loaded in each lane. The large arrow shows a non-staining clear band at ~64 kDa, representing the digested substrate. A narrow band of a much higher MW (approximately 114 kDa) was seen in human caput, human sperm, and macaque corpus, as indicated by the smaller arrow.
Mentions: Using HASGE, neutral hyaluronidase activity (pH 7.0) was demonstrated in protein extracts from the epididymis, with testis and sperm serving as positive controls. Hyaluronidase activity was demonstrated by a non-staining area corresponding to the size of the intact protein at ~64 kDa (Fig. 5). In samples from the human caput, human sperm and macaque corpus there was also a narrow band with a higher MW (>114 kDa, as indicated by the smaller arrow) which may represent dimerization of the molecule. This experiment was repeated multiple times, and the results seen in Figure 5 are a concatenation of bands from several HASGE gels.

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