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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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LM-Mediated Isolation of Epithelial cells from 8 μm thick sections of the human corpus epididymis. A) shows a cross-section of a tubule prior to microdissection flanked by two regions of stromal cells (Str) that were microdissected for use as a negative control. The lumen of the tubule is marked with an (L) while the epithelium is marked with an (E). B) shows the tubule in A) after microdissection of the epithelium outlined in red and blue-dotted, and C) displays a low power view of a microdissected section on a slide showing microdissected tubules (1–5), microdissected stromal cells (Str) and an undissected tubule (E).
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Figure 1: LM-Mediated Isolation of Epithelial cells from 8 μm thick sections of the human corpus epididymis. A) shows a cross-section of a tubule prior to microdissection flanked by two regions of stromal cells (Str) that were microdissected for use as a negative control. The lumen of the tubule is marked with an (L) while the epithelium is marked with an (E). B) shows the tubule in A) after microdissection of the epithelium outlined in red and blue-dotted, and C) displays a low power view of a microdissected section on a slide showing microdissected tubules (1–5), microdissected stromal cells (Str) and an undissected tubule (E).

Mentions: To analyze the mRNA expression of SPAM1 in the epididymal epithelium, frozen sections from Subjects #1 and #2 and paraffin-embedded sections from Subject #3 were used. For macaques, frozen sections of the corpus were obtained from Animal #1 and paraffin-embedded sections of the cauda were from the Animal #2. All paraffin-embedded sections were fixed with 4% paraformaldehyde. The PALM Microbeam system (PALM Microlaser Technologies AG, Bernreid, Germany) was used for laser cutting and separation of selected tissue regions. Immediately prior to microdissection, water was removed from frozen sections by soaking the slides in 100% ethanol for 5 min followed by air-drying for 5 min. Paraffin-embedded sections were treated to remove the paraffin with xylene, prior to the ethanol treatment. Once the tissues were air-dried, the target cells, (epididymal epithelial cells) were carefully laser-microdissected according to the manufacturer's instructions with the cells being collected in mineral oil. The microdissection was performed with care to exclude sperm present in the lumen (Fig. 1). In addition to microdissection of the epithelial cells, stromal cells were also microdissected from the sections to be used as a negative control (Fig. 1). LM conditions were as follows: laser focus 67–69, laser energy 72–76 cut speed 8. These values varied slightly depending on the thickness (8–12 μm) of the sections. The total areas of epididymal cells that were microdissected ranged from approximately 350,000 to 450,000 μm2 . The samples in mineral oil were kept at -80°C until RNA extraction was performed.


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)

LM-Mediated Isolation of Epithelial cells from 8 μm thick sections of the human corpus epididymis. A) shows a cross-section of a tubule prior to microdissection flanked by two regions of stromal cells (Str) that were microdissected for use as a negative control. The lumen of the tubule is marked with an (L) while the epithelium is marked with an (E). B) shows the tubule in A) after microdissection of the epithelium outlined in red and blue-dotted, and C) displays a low power view of a microdissected section on a slide showing microdissected tubules (1–5), microdissected stromal cells (Str) and an undissected tubule (E).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: LM-Mediated Isolation of Epithelial cells from 8 μm thick sections of the human corpus epididymis. A) shows a cross-section of a tubule prior to microdissection flanked by two regions of stromal cells (Str) that were microdissected for use as a negative control. The lumen of the tubule is marked with an (L) while the epithelium is marked with an (E). B) shows the tubule in A) after microdissection of the epithelium outlined in red and blue-dotted, and C) displays a low power view of a microdissected section on a slide showing microdissected tubules (1–5), microdissected stromal cells (Str) and an undissected tubule (E).
Mentions: To analyze the mRNA expression of SPAM1 in the epididymal epithelium, frozen sections from Subjects #1 and #2 and paraffin-embedded sections from Subject #3 were used. For macaques, frozen sections of the corpus were obtained from Animal #1 and paraffin-embedded sections of the cauda were from the Animal #2. All paraffin-embedded sections were fixed with 4% paraformaldehyde. The PALM Microbeam system (PALM Microlaser Technologies AG, Bernreid, Germany) was used for laser cutting and separation of selected tissue regions. Immediately prior to microdissection, water was removed from frozen sections by soaking the slides in 100% ethanol for 5 min followed by air-drying for 5 min. Paraffin-embedded sections were treated to remove the paraffin with xylene, prior to the ethanol treatment. Once the tissues were air-dried, the target cells, (epididymal epithelial cells) were carefully laser-microdissected according to the manufacturer's instructions with the cells being collected in mineral oil. The microdissection was performed with care to exclude sperm present in the lumen (Fig. 1). In addition to microdissection of the epithelial cells, stromal cells were also microdissected from the sections to be used as a negative control (Fig. 1). LM conditions were as follows: laser focus 67–69, laser energy 72–76 cut speed 8. These values varied slightly depending on the thickness (8–12 μm) of the sections. The total areas of epididymal cells that were microdissected ranged from approximately 350,000 to 450,000 μm2 . The samples in mineral oil were kept at -80°C until RNA extraction was performed.

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