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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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A). Western blot analysis of protein extracts from testis, epididymis, vas deferens and sperm of human (Subject #2) and macaque #1. Equal amounts (40 μg) of protein were loaded in each lane. The arrow points to the ~64 kDa SPAM1 band in all samples. This experiment was repeated three times. A 53 kDa band is also seen for human sperm, while the epididymis shows weaker bands (partially degraded products) at ~60 kDa. In both species the vas deferens show a weak <50 kDa band. B) Dot blot analysis of protein extracts from macaque testis, caput, corpus, and cauda. Bovine serum albumin (BSA) was used as a negative control. Equal volumes with approximately equal amounts of protein (40 μg) were blotted for each tissue.
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Figure 4: A). Western blot analysis of protein extracts from testis, epididymis, vas deferens and sperm of human (Subject #2) and macaque #1. Equal amounts (40 μg) of protein were loaded in each lane. The arrow points to the ~64 kDa SPAM1 band in all samples. This experiment was repeated three times. A 53 kDa band is also seen for human sperm, while the epididymis shows weaker bands (partially degraded products) at ~60 kDa. In both species the vas deferens show a weak <50 kDa band. B) Dot blot analysis of protein extracts from macaque testis, caput, corpus, and cauda. Bovine serum albumin (BSA) was used as a negative control. Equal volumes with approximately equal amounts of protein (40 μg) were blotted for each tissue.

Mentions: SPAM1 protein was detected by Western blot analysis in extracts from testis, epididymis, vas deferens and sperm of Subject #2 and of macaque (Fig. 4A). The intact protein (~64 kDa) was found in each sample studied. Partial degradation products (~60 kDa) appear in epididymal sections. Proteolytically cleaved SPAM1 (~53 kDa) was seen in human sperm samples. Also, a low molecular weight band (<50 kDa) was seen in vas deferens in both species. In macaque where all three regions of the epididymis were studied, the levels of the protein in the epididymis were approximately equal to that seen in the testis, and the distal regions tended to have stronger bands than the caput. The Dot blot which showed that the corpus has the highest level of expression (Fig. 4B) confirmed this tendency. Due to the limited availability of fresh human tissue, only the caput epididymis was studied. The vas deferens of both species seemed to have lower amounts of SPAM1 than the epididymis.


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)

A). Western blot analysis of protein extracts from testis, epididymis, vas deferens and sperm of human (Subject #2) and macaque #1. Equal amounts (40 μg) of protein were loaded in each lane. The arrow points to the ~64 kDa SPAM1 band in all samples. This experiment was repeated three times. A 53 kDa band is also seen for human sperm, while the epididymis shows weaker bands (partially degraded products) at ~60 kDa. In both species the vas deferens show a weak <50 kDa band. B) Dot blot analysis of protein extracts from macaque testis, caput, corpus, and cauda. Bovine serum albumin (BSA) was used as a negative control. Equal volumes with approximately equal amounts of protein (40 μg) were blotted for each tissue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184449&req=5

Figure 4: A). Western blot analysis of protein extracts from testis, epididymis, vas deferens and sperm of human (Subject #2) and macaque #1. Equal amounts (40 μg) of protein were loaded in each lane. The arrow points to the ~64 kDa SPAM1 band in all samples. This experiment was repeated three times. A 53 kDa band is also seen for human sperm, while the epididymis shows weaker bands (partially degraded products) at ~60 kDa. In both species the vas deferens show a weak <50 kDa band. B) Dot blot analysis of protein extracts from macaque testis, caput, corpus, and cauda. Bovine serum albumin (BSA) was used as a negative control. Equal volumes with approximately equal amounts of protein (40 μg) were blotted for each tissue.
Mentions: SPAM1 protein was detected by Western blot analysis in extracts from testis, epididymis, vas deferens and sperm of Subject #2 and of macaque (Fig. 4A). The intact protein (~64 kDa) was found in each sample studied. Partial degradation products (~60 kDa) appear in epididymal sections. Proteolytically cleaved SPAM1 (~53 kDa) was seen in human sperm samples. Also, a low molecular weight band (<50 kDa) was seen in vas deferens in both species. In macaque where all three regions of the epididymis were studied, the levels of the protein in the epididymis were approximately equal to that seen in the testis, and the distal regions tended to have stronger bands than the caput. The Dot blot which showed that the corpus has the highest level of expression (Fig. 4B) confirmed this tendency. Due to the limited availability of fresh human tissue, only the caput epididymis was studied. The vas deferens of both species seemed to have lower amounts of SPAM1 than the epididymis.

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