Abstract
Spermatogenesis starts within the seminiferous tubules of the testis by mitotic division of spermatogonia that produces spermatocytes. Meiotic division of these spermatocytes produces haploid spermatids that differentiate into spermatozoa. In this study, we examined the expression of ENaC and CFTR (a Cl− channel) in rat testicular sections using confocal microscopic immunofluorescence. The structural integrity of the seminiferous tubule sections was verified by precise phalloidin staining of the actin fibers located abundantly at both basal and adluminal tight junctions. The acrosome forming regions in the round spermatids were stained using an FITC coupled lectin (wheat germ agglutinin). In all phases of the germ cells (spermatogonia, spermatocytes, and spermatids) ENaC was localized in cytoplasmic pools. Prior to spermiation, ENaC immunofluorescence appeared along the tails of the spermatids. In spermatozoa isolated from the epididymis, ENaC was localized at the acrosome and a central region of the sperm flagellum. The mature sperm are transcriptionally silent. Hence, we suggest that ENaC subunits in cytoplasmic pools in germ cells serve as the source of ENaC subunits located along the tail of spermatozoa. The locations of ENaC is compatible with a possible role in the acrosomal reaction and sperm mobility. In contrast to ENaC, CFTR immunofluorescence was most strongly observed specifically within the Sertoli cell nuclei. Based on the nuclear localization of CFTR we suggest that, in addition to its role as an ion channel, CFTR may have an independent role in gene regulation within the nuclei.
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This research was funded in part by a grant from the United States-Israel Bi-national Science Foundation (BSF Grant Number 2011370).
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Supplementary Fig. 1
A cross-section of testis was reacted with DAPI and secondary antisera alone. The blue-colored DAPI staining of cell nuclei was visible but red immunofluorescence did not appear in such slides. Scale bar 20 μm (PNG 1496 KB)
Supplementary Fig. 2
Confocal microscopic imaging of rat sperm reacted with: (a) DAPI (blue); (b) anti-α-tubulin antisera (green); (c) goat anti-rabbit Alexa Fluor 555 IgG (secondary antibody used for ENaC antisera); and (d) merged image of (a), (b), and (c). Note that the sperm nuclei stained with DAPI have the typical hooked shape for rat sperm, and the anti-tubulin shows very specific staining along the whole length of the sperm tails. The secondary antibody used for ENaC immunofluorescence did not stain sperm (c). Scale bar 10 μm (PNG 1229 KB)
Supplementary Fig. 3
A tile-scan overview image showing the expression of CFTR in many seminiferous tubules of the rat testis. The pattern of CFTR immunofluorescence observed in all the seminiferous tubules was in the consistency with previous CFTR images, confirming that CFTR is uniformly localized in the nuclei of the Sertoli cells. Scale bar 100 μm (PNG 124 KB)
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Sharma, S., Hanukoglu, A. & Hanukoglu, I. Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa. J Mol Hist 49, 195–208 (2018). https://doi.org/10.1007/s10735-018-9759-2
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DOI: https://doi.org/10.1007/s10735-018-9759-2