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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 the human epididymis using multiphoton confocal microscopy. The magnification of tissue sections is the same as is shown in A. Images are taken in the red confocal channel to help eliminate the effect of autofluorescence which occurred in varying degrees depending on the laboratory in which the slides were fixed before sectioning. Preimmune serum was used for negative controls (A, D, and F). The epithelium (E) in A which is from the human proximal corpus (Subject #4) appears red, while SPAM1 is indicated by the yellow-green color in the epithelial lining of the test sections, B and C, also from the corpus of the same male. In C the nuclei are stained with propidium iodide to enhance the visualization of the FITC green color of the signal. Note that the green color in the elastic fibers in the stroma underlying the epithelium in test and control is due to strong autofluorescence and not the primary antibody. D and E, and F and G are from Subject #5, these slides as well as those from the macaques had very high levels of autofluorescence, however the sperm in the lumen (L) serve as a positive control, varying in staining intensity between F and G. Although no sperm are present in the caput test section, the epithelial lining for control (D) and test (E) are quite different in color.
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Figure 6: Immunohistochemical localization of SPAM1 in the human epididymis using multiphoton confocal microscopy. The magnification of tissue sections is the same as is shown in A. Images are taken in the red confocal channel to help eliminate the effect of autofluorescence which occurred in varying degrees depending on the laboratory in which the slides were fixed before sectioning. Preimmune serum was used for negative controls (A, D, and F). The epithelium (E) in A which is from the human proximal corpus (Subject #4) appears red, while SPAM1 is indicated by the yellow-green color in the epithelial lining of the test sections, B and C, also from the corpus of the same male. In C the nuclei are stained with propidium iodide to enhance the visualization of the FITC green color of the signal. Note that the green color in the elastic fibers in the stroma underlying the epithelium in test and control is due to strong autofluorescence and not the primary antibody. D and E, and F and G are from Subject #5, these slides as well as those from the macaques had very high levels of autofluorescence, however the sperm in the lumen (L) serve as a positive control, varying in staining intensity between F and G. Although no sperm are present in the caput test section, the epithelial lining for control (D) and test (E) are quite different in color.

Mentions: SPAM1 protein was localized within the human corpus (Subject #4, Fig. 6) caput and cauda epididymis (Subject #5, Fig. 6), using immunohistochemistry. The images were taken in the red confocal channel to visualize the morphology of the tissues while eliminating the effects of autofluorescence. The control slides (A, D, and F) contain no (or little) green staining, the SPAM1 signal, in the epithelium. For the corpus, epithelial staining is seen in the test samples (B and C). In C the slide is stained with propidium iodide to dye the nuclei red and therefore enhance the visualization of the yellow-green signal. Note that the yellow-green signal is of a lighter hue and is specific for the epithelium compared with the darker green autofluorescence seen in the stroma. Testis sections on the corpus slides were used as positive controls and showed the same color pattern as the epididymis for the presence and absence of the signal (data not shown).


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 the human epididymis using multiphoton confocal microscopy. The magnification of tissue sections is the same as is shown in A. Images are taken in the red confocal channel to help eliminate the effect of autofluorescence which occurred in varying degrees depending on the laboratory in which the slides were fixed before sectioning. Preimmune serum was used for negative controls (A, D, and F). The epithelium (E) in A which is from the human proximal corpus (Subject #4) appears red, while SPAM1 is indicated by the yellow-green color in the epithelial lining of the test sections, B and C, also from the corpus of the same male. In C the nuclei are stained with propidium iodide to enhance the visualization of the FITC green color of the signal. Note that the green color in the elastic fibers in the stroma underlying the epithelium in test and control is due to strong autofluorescence and not the primary antibody. D and E, and F and G are from Subject #5, these slides as well as those from the macaques had very high levels of autofluorescence, however the sperm in the lumen (L) serve as a positive control, varying in staining intensity between F and G. Although no sperm are present in the caput test section, the epithelial lining for control (D) and test (E) are quite different in color.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184449&req=5

Figure 6: Immunohistochemical localization of SPAM1 in the human epididymis using multiphoton confocal microscopy. The magnification of tissue sections is the same as is shown in A. Images are taken in the red confocal channel to help eliminate the effect of autofluorescence which occurred in varying degrees depending on the laboratory in which the slides were fixed before sectioning. Preimmune serum was used for negative controls (A, D, and F). The epithelium (E) in A which is from the human proximal corpus (Subject #4) appears red, while SPAM1 is indicated by the yellow-green color in the epithelial lining of the test sections, B and C, also from the corpus of the same male. In C the nuclei are stained with propidium iodide to enhance the visualization of the FITC green color of the signal. Note that the green color in the elastic fibers in the stroma underlying the epithelium in test and control is due to strong autofluorescence and not the primary antibody. D and E, and F and G are from Subject #5, these slides as well as those from the macaques had very high levels of autofluorescence, however the sperm in the lumen (L) serve as a positive control, varying in staining intensity between F and G. Although no sperm are present in the caput test section, the epithelial lining for control (D) and test (E) are quite different in color.
Mentions: SPAM1 protein was localized within the human corpus (Subject #4, Fig. 6) caput and cauda epididymis (Subject #5, Fig. 6), using immunohistochemistry. The images were taken in the red confocal channel to visualize the morphology of the tissues while eliminating the effects of autofluorescence. The control slides (A, D, and F) contain no (or little) green staining, the SPAM1 signal, in the epithelium. For the corpus, epithelial staining is seen in the test samples (B and C). In C the slide is stained with propidium iodide to dye the nuclei red and therefore enhance the visualization of the yellow-green signal. Note that the yellow-green signal is of a lighter hue and is specific for the epithelium compared with the darker green autofluorescence seen in the stroma. Testis sections on the corpus slides were used as positive controls and showed the same color pattern as the epididymis for the presence and absence of the signal (data not shown).

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