Limits...
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.

Show MeSH

Related in: MedlinePlus

The presence of steady-state SPAM1 mRNA as detected by LM/RT-PCR of the lysate recovered from human epididymal epithelial cells (H EPI, test) and stromal cells (H STR, control). RNA was subjected to first strand synthesis with (+) or without (-) reverse transcriptase (RT), followed by PCR amplification. The expected 320 bp band for the SPAM1 cDNA fragment is seen in A) for Subjects 1–3 in Lanes 2, 4, 6, for RT(+) reactions. This band was verified to be SPAM1 by sequencing. No 320 bp band is detected in Lanes 3, 5, 7, in the absence of RT showing that the SPAM1 bands are not from DNA contamination. In Lanes 8 and 9 no SPAM1 bands are detected for the stromal cells, but one is seen in Lane 10 for genomic DNA, the positive control. In Lane 11, no sperm-specific band for PRM1 cDNA could be amplified from the cDNA of epithelial cells of Subject #1, verifying the homogeneity of the cells. In Lane 12 the 110 bp band (arrowed on the right) is the PRM1 DNA fragment amplified from genomic DNA, as a positive control. A band of the same size should be generated from the cDNA in the presence of mRNA from the lysate. Note that the primer cloud for SPAM1 is not present for PRM1 in Lanes 11 and 12. In B) the 320 bp band in Lanes 2 and 4 are from the corpus of Animal #1 and the cauda of Animal #2, respectively. They were also verified to be SPAM1 by sequencing. The marker, 100 bp ladder, is shown in Lane 1 of both A) and B). The same results were obtained for replicate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC184449&req=5

Figure 2: The presence of steady-state SPAM1 mRNA as detected by LM/RT-PCR of the lysate recovered from human epididymal epithelial cells (H EPI, test) and stromal cells (H STR, control). RNA was subjected to first strand synthesis with (+) or without (-) reverse transcriptase (RT), followed by PCR amplification. The expected 320 bp band for the SPAM1 cDNA fragment is seen in A) for Subjects 1–3 in Lanes 2, 4, 6, for RT(+) reactions. This band was verified to be SPAM1 by sequencing. No 320 bp band is detected in Lanes 3, 5, 7, in the absence of RT showing that the SPAM1 bands are not from DNA contamination. In Lanes 8 and 9 no SPAM1 bands are detected for the stromal cells, but one is seen in Lane 10 for genomic DNA, the positive control. In Lane 11, no sperm-specific band for PRM1 cDNA could be amplified from the cDNA of epithelial cells of Subject #1, verifying the homogeneity of the cells. In Lane 12 the 110 bp band (arrowed on the right) is the PRM1 DNA fragment amplified from genomic DNA, as a positive control. A band of the same size should be generated from the cDNA in the presence of mRNA from the lysate. Note that the primer cloud for SPAM1 is not present for PRM1 in Lanes 11 and 12. In B) the 320 bp band in Lanes 2 and 4 are from the corpus of Animal #1 and the cauda of Animal #2, respectively. They were also verified to be SPAM1 by sequencing. The marker, 100 bp ladder, is shown in Lane 1 of both A) and B). The same results were obtained for replicate experiments.

Mentions: In all LM epididymal samples from humans (Subjects 1 and 3 corpus, Subject 2 caput/corpus) and macaques (#1, corpus and 2, cauda) the expected 320 bp SPAM1 cDNA fragment was amplified in the RT-PCR reactions. The control RT (-) reactions for the epithelium as well as the cDNA from the stromal cells of Subject #2 gave no 320 bp product, unlike the test samples and the positive control which was from human genomic DNA (Fig. 2A). The 320 bp band was verified to be a human SPAM1 fragment by sequencing. Absence of a SPAM1 cDNA band from the stromal cells demonstrates that epididymal SPAM1 mRNA is localized to the tubule epithelium.


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)

The presence of steady-state SPAM1 mRNA as detected by LM/RT-PCR of the lysate recovered from human epididymal epithelial cells (H EPI, test) and stromal cells (H STR, control). RNA was subjected to first strand synthesis with (+) or without (-) reverse transcriptase (RT), followed by PCR amplification. The expected 320 bp band for the SPAM1 cDNA fragment is seen in A) for Subjects 1–3 in Lanes 2, 4, 6, for RT(+) reactions. This band was verified to be SPAM1 by sequencing. No 320 bp band is detected in Lanes 3, 5, 7, in the absence of RT showing that the SPAM1 bands are not from DNA contamination. In Lanes 8 and 9 no SPAM1 bands are detected for the stromal cells, but one is seen in Lane 10 for genomic DNA, the positive control. In Lane 11, no sperm-specific band for PRM1 cDNA could be amplified from the cDNA of epithelial cells of Subject #1, verifying the homogeneity of the cells. In Lane 12 the 110 bp band (arrowed on the right) is the PRM1 DNA fragment amplified from genomic DNA, as a positive control. A band of the same size should be generated from the cDNA in the presence of mRNA from the lysate. Note that the primer cloud for SPAM1 is not present for PRM1 in Lanes 11 and 12. In B) the 320 bp band in Lanes 2 and 4 are from the corpus of Animal #1 and the cauda of Animal #2, respectively. They were also verified to be SPAM1 by sequencing. The marker, 100 bp ladder, is shown in Lane 1 of both A) and B). The same results were obtained for replicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The presence of steady-state SPAM1 mRNA as detected by LM/RT-PCR of the lysate recovered from human epididymal epithelial cells (H EPI, test) and stromal cells (H STR, control). RNA was subjected to first strand synthesis with (+) or without (-) reverse transcriptase (RT), followed by PCR amplification. The expected 320 bp band for the SPAM1 cDNA fragment is seen in A) for Subjects 1–3 in Lanes 2, 4, 6, for RT(+) reactions. This band was verified to be SPAM1 by sequencing. No 320 bp band is detected in Lanes 3, 5, 7, in the absence of RT showing that the SPAM1 bands are not from DNA contamination. In Lanes 8 and 9 no SPAM1 bands are detected for the stromal cells, but one is seen in Lane 10 for genomic DNA, the positive control. In Lane 11, no sperm-specific band for PRM1 cDNA could be amplified from the cDNA of epithelial cells of Subject #1, verifying the homogeneity of the cells. In Lane 12 the 110 bp band (arrowed on the right) is the PRM1 DNA fragment amplified from genomic DNA, as a positive control. A band of the same size should be generated from the cDNA in the presence of mRNA from the lysate. Note that the primer cloud for SPAM1 is not present for PRM1 in Lanes 11 and 12. In B) the 320 bp band in Lanes 2 and 4 are from the corpus of Animal #1 and the cauda of Animal #2, respectively. They were also verified to be SPAM1 by sequencing. The marker, 100 bp ladder, is shown in Lane 1 of both A) and B). The same results were obtained for replicate experiments.
Mentions: In all LM epididymal samples from humans (Subjects 1 and 3 corpus, Subject 2 caput/corpus) and macaques (#1, corpus and 2, cauda) the expected 320 bp SPAM1 cDNA fragment was amplified in the RT-PCR reactions. The control RT (-) reactions for the epithelium as well as the cDNA from the stromal cells of Subject #2 gave no 320 bp product, unlike the test samples and the positive control which was from human genomic DNA (Fig. 2A). The 320 bp band was verified to be a human SPAM1 fragment by sequencing. Absence of a SPAM1 cDNA band from the stromal cells demonstrates that epididymal SPAM1 mRNA is localized to the tubule epithelium.

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