X-Ray Fluorescence Microscopy Reveals the Role of Selenium in Spermatogenesis

Abstract

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis.

Mutations in the selenocysteine insertion sequence–binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

“…Consistent with the known importance of mGPx4 and TGR in latter spermatogenic stages, testicular histology in P1 showed maturation arrest, with preservation of early cell types (e.g., spermatogonia and spermatocytes) but lack of mature spermatids and spermatozoa ( Figure 2G). SEPP, acting as a source of selenium to meet tissue demand, has also been implicated in spermatogenesis, with SEPP-null mice showing abnormal sperm morphology and diminished testicular selenium content (30,31). Thus, markedly reduced circulating SEPP levels in P1 ( Figure 1B) may be an added factor contributing to his azoospermia.…”

High-Resolution Imaging of Selenium in Kidneys: A Localized Selenium Pool Associated with Glutathione Peroxidase 3

“…Specific re-expression of SelP in the liver restores Se transport and rescues phenotypes associated with SelP deficiency (25). Using X-ray fluorescence microscopy (XFM), we recently showed that SelP provides testes with Se where it is used for synthesis of the mitochondrial form of glutathione peroxidase 4 (16,20).…”

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

“…Historically, different substrates, ranging from thin polycarbonate foils/films [35][36][37] , formvar coated gold transmission electron microscopy (TEM) grids [38][39][40][41][42] and silicon nitride windows 5,17,[43][44][45][46] , have been used to grow mammalian cells and used for X-ray fluorescence imaging. In comparison among the existing and potential substrates for imaging adherent mammalian cells by XFM, silicon nitride membrane (Si 3 N 4 ) windows are most suitable due to their low fluorescence background and relatively simple elemental composition [4][5][6]47,48 . In addition Si 3 N 4 windows support robust cell attachment and proliferation.…”

“…With very high-intensity beams, even a relatively small amount of light that can be focused is sufficient to excite the endogenous metals in cells, producing signal that can be measured with currently available detector technology. Thus, studying the chemical biology of metals in the cell is one application in particular that makes use of many of the recent developments in this technique [4][5][6][7][8][9][10] .…”