Reproduction Abstracts

Human sperm use different behaviours for different aspects of sperm transport and fertilisation. We have investigated the effects of preparation method (density gradient method and direct swim-up) on sperm behaviour and manipulated Ca²⁺ signalling (the primary regulator of sperm motility) to induce different behaviours and assessed their effects on penetration of human sperm through viscous medium (methylcellulose).

Cells prepared by swim-up performed significantly better in penetrating methylcellulose than cells from the same sample prepared by density gradient centrifugation. CASA analysis showed that that swim-up cells had higher curvilinear and straight line velocities. In [Ca²⁺]_i imaging experiments cells prepared by density gradient included a higher proportion of dead sperm (assessed by retention of fluo4) and fewer cells that gave a [Ca²⁺]_i response to progesterone (fluo4 fluorescence) compared to cells prepared from the same sample using swim-up (P<0.01).

Treatment of cells prepared by density gradient with PGE₁ (to stimulate CatSper channels) increased curvilinear velocity (P<0.01; n=9) and enhanced performance in the viscous medium penetration assay (P<0.0002; n=10). Treatment with 4-aminopyridine, potently increased hyperactivation (assessed by CASA) and inhibited penetration of viscous medium in both density gradient and swim-up cells (n=10; P<0.001).

We conclude that i) cells prepared by density gradient centrifugation perform less well in penetrating viscous medium and are also less responsive to activation of CatSper by progesterone as measured by [Ca²⁺]_i and ii) penetration through viscous medium is inhibited by hyperactivation and enhanced by activation of CatSper (probably increased swimming velocity).