Alpha4 Na,K-ATPase Localization and Expression Are Dynamic Aspects of Spermatogenesis and in Sperm Incubated Under Capacitating Conditions

Abstract

Utilizing high-resolution microscopy in conjunction with a new antibody highly specific for rat alpha4 Na,K-ATPase, we describe changes in alpha4 expression during spermatogenesis and in sperm incubated under capacitating and noncapacitating conditions. Immunohistochemical analyses showed alpha4 expression at low levels in spermatogonia and in pachytene spermatocytes. Alpha4 then becomes highly expressed on round spermatids and the midpiece of elongated spermatozoa within the seminiferous tubules. In noncapacitating conditions, alpha4 was confined mainly to the flagellum of mature sperm; however, under capacitating conditions, sperm acquired intense alpha4 staining along the acrosomal region of the sperm head. To visualize the precise localization of alpha4 in the sperm head, we performed an ultrastructural analysis using immuno-scanning electron microscopy. Under capacitating conditions, sperm exhibited alpha4 staining along the dorsal surface of the sperm head associated with the acrosome. In addition, after 4 h of incubation in motility buffer, we observed an increase in alpha4 protein in sperm that could be blocked with chloramphenicol, a mitochondrial-type ribosome inhibitor. These findings demonstrate that both the localization and expression level of alpha4 Na,K-ATPase are dynamic aspects of sperm maturation and suggest that sperm motility and capacitation may be supported by these changes to the location and amount of this protein.

Keywords: ATP1A4; Na,K-ATPase alpha4; Sertoli cell; capacitation; spermatozoa; testis.

Figure 1

Characterization of a new polyclonal anti-alpha4 Na,K-ATPase antibody. Total protein was extracted from various rat tissues and separated by SDS-PAGE. The blot was probed with our newly generated anti-alpha4 antibody, labeled with an HRP-conjugated goat anti-rabbit secondary antibody, and exposed on autoradiographic film. A single, testis-specific band was produced corresponding to the alpha4 Na,K-ATPase.

Figure 2

Immunohistochemistry of alpha4 Na,K-ATPase and the acrosomal compartment during spermatogenesis. Paraffin sections of rat testis were probed for alpha4 and the acrosomal compartment using anti-alpha4 primary/Alexa 594 secondary antibodies and PNA-FITC, respectively. All sections were counterstained with DAPI. (A) Low (left) and high magnification (right) images for alpha4 expression and acrosome staining in the seminiferous tubules. The arrows in the top right panel in (A) indicate alpha4 staining in presumptive Sertoli cells. For controls, (B) the anti-alpha4 antibody was preabsorbed with the immunizing alpha4 peptide and subsequently used for immunohistochemistry, or (C) the primary antibody was omitted entirely. Mag. bar = 50 µm.

Figure 3

Immunocytochemical localization of the alpha4 Na,K-ATPase in noncapacitated and capacitated sperm. Sperm were isolated from rat caudal epididymes, fixed, and probed for alpha4 (A) immediately after isolation (top row) or after being incubated for 4 h in noncapacitating (middle row) or capacitating media (bottom row). The white arrowheads in (A) (bottom panel) indicate alpha4 staining in the acrosomal region of the sperm head. (B) The primary antibody was omitted to determine the specificity of our secondary antibody. Mag. bar = 20 µm.

Figure 4

Ultrastructural localization of the alpha4 Na,K-ATPase in the sperm head. Sperm were fixed, mounted onto glass coverslips, incubated (A) with or (B) without the anti-alpha4 antibody, and labeled with 18 nm colloid gold. Following immunolabeling, sperm were postfixed, dehydrated, carbon coated, and visualized in a scanning electron microscope. Sperm were imaged using a Zeiss Supra 35 VP FEG SEM using a 12 kV accelerating voltage, 7 mm working distance, and 30 µm NA. Nuclei are outlined in the inset images. Mag. bar = 1 µm.

Figure 5

Sperm synthesize new alpha4 peptides from mitochondrial ribosomes. Sperm total protein was extracted from freshly isolated caudal sperm (0 h) or sperm incubated for 4 h in noncapacitating buffer, capacitating buffer, or capacitating buffer supplemented with chloramphenicol and were subjected to western blotting. (A) A representative western blot of alpha4 total protein levels. β-tubulin was included as a loading control. (B) Alpha4 protein levels were normalized to β-tubulin and expressed as a fold change with respect to alpha4 protein levels at 0 h. Bar chart represents the mean ± SEM fold change of three blots for three biological replicates (9 blots total). Statistical analyses were performed using a Welch’s t-test (* p < 0.05; ** p < 0.01) and unpaired student’s t-test (^### p < 0.001).