Na+,K+-ATPase α isoforms in sperm show a highly structured and distinct pattern of distribution

Abstract

In brief: This manuscript shows that the Na+ and K+ transporter Na+,K+-ATPase α4, specific to sperm, is expressed on the surface of the sperm head and flagellum in a very structured manner. This is also true for Na+,K+-ATPase α1, the other Na+,K+-ATPase isoform present in sperm and also in all cells. However, Na+,K+-ATPase α4 distribution changes when the cells are capacitated, an event necessary for fertilization. This dynamic remodeling, along with the distinct functional properties of Na+,K+-ATPase α4 and α1, provides evidence for the refined level of specialization that sperm have developed to achieve the amazing goal of fertilizing the oocyte.

Abstract: Na+,K+-ATPase α4 is a unique Na+ and K+ transporter of the plasma membrane of spermatozoa, which is essential for male fertility. While previous studies have found Na+,K+-ATPase α4 to be mainly expressed in the sperm flagellum, less is known about its localization in the sperm head. Moreover, the spatial arrangement of Na+,K+-ATPase α4 at the subcellular level and its relationship to the functional state of the cells are unclear. We studied this using stimulated emission depletion (STED) super-resolution microscopy. We show that, under non-capacitated conditions, Na+,K+-ATPase α4 is distributed in a trilinear pattern along the midpiece and as a scattered single line along the principal piece of the sperm flagellum. Under capacitated conditions, Na+,K+-ATPase α4 pattern undergoes remodeling and its distribution shifts to a single line along the flagellum. On the other hand, Na+,K+-ATPase α1, the somatic isoform of Na+,K+-ATPase also present in sperm, exhibits a similar trilaminar localization at the flagellar midpiece but a bilinear pattern in the principal piece. This distribution, unlike that of Na+,K+-ATPase α4, does not change during sperm capacitation. We also found Na+,K+-ATPase α1 and α4 in the sperm head, where they present a complex distribution under both non-capacitated and capacitated conditions. These differences in the localization pattern and spatial dynamics of Na+,K+-ATPase isoform expression, along with their different functional properties, highlight the distinct roles that both isoforms play to support sperm function.

Keywords: Na+,K+-ATPase α4 and α1; STED microscopy; male fertility; protein localization; protein remodeling; sperm capacitation; sperm motility; sperm subcellular localization.

Figure 1:. Na⁺,K⁺-ATPase α4 organization along the flagellum of noncapacitated sperm.

(A) 3D reconstructed image of the sperm flagellum showing distribution of Na⁺,K⁺-ATPase α4 (NKAα4) along the whole length of the flagellum, including the midpiece (MP) and principal piece (PP). (B) Trilinear distribution of Na⁺,K⁺-ATPase α4 in the midpiece (MP). The scale bars represent 5 µm. (C) Sperm flagellar segment showing the mid-principal piece boundary demonstrates the three lanes merging into a single lane at the end of midpiece. (D) The depth color coded 3D image of Na⁺,K⁺-ATPase α4 is showed in the whole flagellum. (E) The y-z cross section of the image obtained from D, sectioned at the midpiece (at the site marked with an arrow in D) showing three tight clusters (1, 2 and 3) of Na⁺,K⁺-ATPase α4. (F) The y-z cross section of the image obtained from the principal piece (PP) (at the site marked with an arrow in D) showing one cluster. Colors in D, E and F represent the z positions (refer to the color scale bar G). (H) Distance between Na⁺,K⁺-ATPase α4 clusters measured from the cross section of 3D images using LAS X analysis software. D1 represents the distance between lane 1 and 3, D2 is the distance between lane 1 and 2 and D3 is the distance between lane 2 and 3. Bars represent the mean ±SEM for 8 different mice. ns indicates no significant statistical differences between values, with P < 0.05. (I) Schematic of the spatial distribution of three Na⁺,K⁺-ATPase α4 (NKAα4) clusters at the cross section of the midpiece shown in (E). The clusters or the lanes are equally distant from each other as derived from (H), forming an equilateral triangle connecting them as depicted in (I). The schematic was created with BioRender.com.

Figure 2:. Na⁺,K⁺-ATPase α4 is redistributed upon sperm capacitation.

(A) 3D reconstructed image of Na⁺,K⁺-ATPase α4 along the entire flagellum in capacitated sperm. (B) Depth coded 3D image of Na⁺,K⁺-ATPase α4. (C) The y-z cross section of the flagellum showing a compact Na⁺,K⁺-ATPase α4 cluster. The site of cross section is marked with an arrow in B. The scale bars are 5 µm. Colors in B and C represent the z positions (see color scale bar D). (E) Hyperactive motility in noncapacitated (Noncap) and capacitated (Cap) sperm evaluated with CASA. (F) A schematic showing the three lanes to be remodeled into one. The schematic was created with BioRender.com. Experiments were conducted three times, and fifteen to twenty cells were analyzed in each of the experiments. (G) The scattered plot showing a positive trend between the hyperactive motility and the redistribution of Na⁺,K⁺-ATPase α4 (NKA α4) in sperm (n=3). (H) Percentage of cells with redistributed NKAα4 at different incubation time of capacitation were determined (Pearson’s coefficient, r = 0.97; p value <0.05). Sperm collected from cauda epididymis are incubated with FertiUp medium for 20 minutes, 40 minutes and 60 minutes. The percentage of hyperactive motile cells was also determined at those time points (Pearson’s coefficient, r = 0.95; p value <0.05).

Figure 3:. Na⁺,K⁺-ATPase α1 organization in the sperm flagellum under noncapacitated conditions.

(A) 3D distribution of Na⁺,K⁺-ATPase α1 along the entire flagellum. (B) Na⁺,K⁺-ATPase α1 localization in the midpiece (MP) showing a trilinear pattern in the midpiece (MP) and a bilinear pattern at the principal piece (PP). Scale bar represents 5 µm (A) and 2 µm (B) respectively. (C) Color coded 3D image showing the position of Na⁺,K⁺-ATPase α1 along the z direction (see color bar in F for reference). (D) y-z cross section obtained from (C) shows three domains (1,2 and 3) at the midpiece (MP). (E) Cross section showing Na⁺,K⁺-ATPase α1 distributed as two lines at the principal piece of the flagellum. Arrows in C denote the sites of cross sections that result in D and E. (G) Distance between the lanes or clusters measured from the cross section of 3D images using LAS X analysis software. D1 represents the distance between lane 1 and 3, D2 is the distance between lane 1 and 2 and D3 is the distance between lane 2 and 3. Values are expressed as mean ±SEM for n=7 different mice. ns shows no significant statistical differences, with P < 0.05.

Figure 4:. The pattern of distribution of Na⁺,K⁺-ATPase α1 remains unchanged upon sperm capacitation.

(A) 3D organization of Na⁺,K⁺-ATPase α1 along the sperm flagellum, showing a trilinear pattern in the midpiece which is discontinued into two lanes in the principal piece. Scale bar represents 5 µm. (B) Expression of Na⁺,K⁺-ATPase α1 color-coded along the z axis (refer to color bar E for depth of field). (C) y-z cross section at the midpiece showing three domains (1, 2 and 3) (D) y-z cross section at the principal piece showing only two Na⁺,K⁺-ATPase α1 clusters. Arrows in B shows the sites of cross section from midpiece (C) and principal piece (D) respectively. Experiments were conducted thrice and in total 40 cells per sample were analyzed.

Figure 5:. Na⁺,K⁺-ATPase α4 and Na⁺,K⁺-ATPase α1 are also expressed in the sperm head under both noncapacitated and capacitated conditions.

(A) 3D organization of Na⁺,K⁺-ATPase α4 in sperm head under both noncapacitated and capacitated condition. (B) Expression of Na⁺,K⁺-ATPase α1 in sperm under both capacitated and noncapacitated condition. Scale bar represents 5 µm.

Figure 6:. Mutual distribution of Na⁺,K⁺-ATPase α4 and Na⁺,K⁺-ATPase α1 in sperm captured by 3D STED.

(A) The 2D projection of 3D images was obtained by summing the intensity of all slices along z-axis showing the localization of Na⁺,K⁺-ATPase α4 (NKAα4), Na⁺,K⁺-ATPase α1 (NKAα1) under noncapacitated condition. Each z stack is 0.16 µm and in total 25 slices were included. (B) shows the colocalization under capacitated condition. Scale bar represents 5 µm. (C) y-z cross section of the 3D images (obtained with 3D viewer) at the midpiece showing three domains representing localization NKAα4 (red), NKAα1 (green) and both under noncapacitated condition. (D) y-z cross section at the midpiece showing one cluster representing localization NKAα4 (red), three domains for NKAα1 (green) and both under capacitated condition.