Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Oct;20(10):929-37.
doi: 10.1093/molehr/gau052. Epub 2014 Jul 11.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

Affiliations

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

Teodor G Păunescu et al. Mol Hum Reprod. 2014 Oct.

Abstract

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

Keywords: epididymis; epididymosomes; helium ion microscopy; principal and clear cells; spermatozoa.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Vibratome sections of the cauda epididymidis of B1-EGFP mouse, expressing EGFP specifically in clear cells, visualized by light (A) and epifluorescence (B) microscopy. The cauda tubule was cut longitudinally to expose the apical surface of the epithelium. Several EGFP-positive clear cells are seen. (CE) Higher magnification of the area shown in the box (A and B). Scale bars: A, B: 100 µm; C, D, E: 50 µm.
Figure 2
Figure 2
Low-magnification HIM image of a control rat cauda epididymidis. Epididymal spermatozoa were removed by perfusing the lumen with a physiological solution and the apical surface of the epithelium was exposed. Clear cells of various sizes are visible as darker spots (arrows) interspersed among principal cells that appear brighter due to their stereocilia. The inset shows the apical surface of a rat cauda epididymidis immunolabeled for the V-ATPase in red. Several clear cells expressing the V-ATPase are detected. The arrow indicates a large clear cell. Scale bar: 50 µm, Inset: 15 µm.
Figure 3
Figure 3
Characteristic high-magnification images of rat epididymal clear cells under control (A and C) and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP)-activated conditions (B and D). Long stereocilia of principal cells surrounding the clear cell are evident, making the two cell types clearly distinguishable. As also emphasized by the higher magnification image shown in C, the apical membrane of control clear cells contains microvilli and relatively short microplicae, while a large part of the flatter apical membrane domain is devoid of such formations and, thus, remains visible. Conversely, the flat membrane region of the activated clear cell (D) is no longer visible due to the increased density and size of microplicae. Scale bars: A, B: 4 µm; C, D: 1 µm.
Figure 4
Figure 4
HIM imaging of the luminal surface of the epididymal epithelium from a mouse cauda epididymidis perfused with an activating solution. The clear cells (CC) can be detected in rows and clusters, and the borders between neighboring clear cells appear dark by HIM. Their apical membranes show various levels of activation (increased size and density of microplicae and microvilli) in response to cAMP and alkaline pH. Some structures compatible with vesicles in the process of budding off the plasma membrane are also detected (arrows). PC: principal cells. Scale bar: 2 µm.
Figure 5
Figure 5
Sperm cells detected in the lumen of a mouse cauda epididymidis have their heads embedded within the layer of principal cell stereocilia (arrows), suggesting that they physically interact with the epididymal epithelium. These spermatozoa do not have a discernable cytoplasmic droplet. Scale bar: 5 µm.
Figure 6
Figure 6
(A) A low-magnification HIM image showing a mouse cauda spermatozoon, whose head is embedded within the epididymal epithelium. This spermatozoon has a prominent cytoplasmic droplet located at the mid-principal piece junction (arrow). (B) An intermediate magnification image of the same sperm cell reveals concentric rings on its midpiece (arrows) and vesicle-like structures on the surface of the cytoplasmic droplet. (C and D) High-magnification HIM imaging of the cytoplasmic droplet shows these vesicle-like membranous structures more clearly, alongside a number of surface depressions. Some vesicles have a rough appearance (arrowheads), while other have a smooth surface (arrows). A more complex structure (double arrow) compatible with a membrane protrusion that is budding from the cytoplasmic droplet is also visible. Scale bars: A: 5 µm; B:1 µm; C: 200 nm; D: 100 nm.

Similar articles

Cited by

References

    1. Aire TA, Soley JT. The surface features of the epithelial lining of the ducts of the epididymis of the ostrich (Struthio camelus) Anat Histol Embryol. 2000;29:119–126. - PubMed
    1. Anberg A. The ultrastructure of the human spermatozoon; an electronmicroscopic study of spermatozoa from sperm samples and the epididymis including some observations of the spermatid. Acta Obstet Gynecol Scand Suppl. 1957;36(Suppl 2):1–133. - PubMed
    1. Axner E. Sperm maturation in the domestic cat. Theriogenology. 2006;66:14–24. - PubMed
    1. Bazou D, Behan G, Reid C, Boland JJ, Zhang HZ. Imaging of human colon cancer cells using He-Ion scanning microscopy. J Microsc. 2011a;242:290–294. - PubMed
    1. Bazou D, Santos-Martinez MJ, Medina C, Radomski MW. Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap. Br J Pharmacol. 2011b;162:1577–1589. - PMC - PubMed

Publication types

Substances