The Motility of Mouse Spermatozoa Changes Differentially After 30-Minute Exposure Under Simulating Weightlessness and Hypergravity

Abstract

Research into the mechanisms by which gravity influences spermatozoa has implications for maintaining the species in deep space exploration and may provide new approaches to reproductive technologies on Earth. Changes in the speed of mouse spermatozoa after 30 min exposure to simulated weightlessness (by 3D-clinostat) and 2 g hypergravity (by centrifugation) were studied using inhibitory analysis. Simulated microgravity after 30 min led to an increase in the speed of spermatozoa and against the background of an increase in the relative calcium content in the cytoplasm. This effect was prevented by the introduction of 6-(dimethylamino) purine, wortmannin, and calyculin A. Hypergravity led to a decrease in the speed of spermatozoa movement, which was prevented by sodium orthovanadate and calyculin A. At the same time, under microgravity conditions, there was a redistribution of proteins forming microfilament bundles between the membrane and cytoplasmic compartments and under hypergravity conditions-proteins forming networks. The obtained results indicate that even a short exposure of spermatozoa to altered gravity leads to the launch of mechanotransduction pathways in them and a change in motility.

Keywords: cell mechanosensitivity; cytoskeleton; hypergravity; microgravity; spermatozoa.

Figure 1

Speed of mouse spermatozoa after 30 min exposure to simulated micro- and hypergravity. Study groups: C0—“zero-control”, parameters were assessed immediately after sperm collection, CS—static control—intact spermatozoa after 30 min of exposure without any impacts (dark green), CD—dynamic control—spermatozoa after 30 min of exposure on shaker with similar velocity of rotation in one plane (light green), sµg—simulated microgravity during 30 min, created by 3D-clinostat (blue), hg—hypergravity at 2 g level during 30 min, created by centrifugation (red). Values for CS and CD did not differ from each other. *—p < 0.05 in comparison to CS. (A) Exposure was carried out in a medium for spermatozoa without the addition of any agents. (B) Exposure was carried out in a medium with the addition of a broad-spectrum protein kinase inhibitor 0.5 mM 6-(dimethylamino) purine. (C) The exposure medium contained 20 μM wortmannin to inhibit phosphatidylinositol-3-kinase (PI3K). (D) The medium was supplemented with 1 μM AR-A014418, an inhibitor of glycogen synthase kinase 3 (GSK3). (E) The exposure medium contained 200 μM sodium orthovanadate to inhibit tyrosine phosphatase (Tyr PP). (F) Spermatozoa were exposed in the medium containing 20 nM calyculin A, which inhibits serine/threonine phosphatase (Ser/Thr PP). Data are presented as mean ± SD of three independent experiments. In each independent experiment, at least 50 cells were assessed for motility in each study group. Motility in each study group was compared with the corresponding CS group.

Figure 2

Relative calcium content in spermatozoa after 30 min exposure to simulated microgravity and 2 g hypergravity. Study groups—same as above, in Figure 1. Groups C0, CS, and CD did not differ from each other. *—p < 0.05 in comparison to CS. Typical images with bar 500 µm are shown on the left. Data are presented as mean ± SD of three independent experiments. In each independent experiment, at least 30 cells were assessed for fluorescence in each study group. Fluorescence in each study group was compared with CS group.

Figure 3

Relative content of the main proteins forming the sperm cytoskeleton after 30 min exposure to simulated micro- and hypergravity. Study groups—same as above, in Figure 1. M—membrane fraction, and C—cytoplasmic fraction. The protein content in groups C0, CS, and CD did not differ in either the membrane or cytoplasmic fractions. (A) Beta-actin, 42 kDa; (B) gamma-actin, 42 kDa; (C) alpha-tubulin, 50 kDa; and (D) acetylated alpha-tubulin, 55 kDa. &—p < 0.1 in comparison to CS. Typical Western blots are given under each histogram. Data are presented as mean ± SD of four biological replicas. Protein content in each study group was compared with the CS group.

Figure 4

Relative content of actin-binding proteins in spermatozoa after 30 min exposure to simulated micro- and hypergravity. As above, study groups are the same as in Figure 1. M—membrane fraction, and C—cytoplasmic fraction. The protein content in groups C0, CS, and CD did not differ between themselves in either the membrane or cytoplasmic fractions. (A) Supervillin, 201 kDa; (B) alpha-actinin1, 103 kDa; and (C) alpha-actinin4, 102 kDa. *—p < 0.05, and &—p < 0.1 in comparison to CS. Typical Western blots are shown to the right of each histogram. Data are presented as mean ± SD of four biological replicas. Protein content in each study group was compared with the CS group.

Figure 5

Possible scheme of events of formation of pattern of sperm motility after 30 min exposure to conditions of simulated weightlessness and 2 g hypergravity. Detailed description is given in the text. Proteins forming bundles of filaments—migrate into cytoplasm under simulated microgravity—are shown in blue. Proteins forming networks of filaments—migrate into cytoplasm under hypergravity—are shown in red. State under normal gravity is shown in green. Plus sign denotes activating effect, and minus sign denotes suppressive effect. Up arrows denote observed increase in parameter, and down arrows denote decrease. PIP2—phosphatidylinositol 4,5-bisphosphate, PIP3—phosphatidylinositol 1,4,5-trisphosphate, IP3—inositol 1,4,5-trisphosphate, DAG—diacylglycerol, PLC—phospholipase C, PI3K—phosphatidylinositol 3-kinase, PKC—protein kinase C, and Tyr PP—tyrosine phosphatase. Inhibitors are highlighted in yellow: 6-DMAP, 6-(dimethylamino) purine, inhibits PKC; wortmannin inhibits PI3K; and Na₃VO₄, sodium orthovanadate, inhibits Tyr PP.