Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione

Abstract

Background: In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide.

Methodology/principal findings: The proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6+/-2.4% versus 27.3%+/-2.8% in wells alone). On the assumption that the soluble factor could be H(2)O(2), reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%+/-2.0% versus 21.2%+/-1.9%), while addition of oxidized glutathione did not (82.7%+/-6.5% with reduced glutathione; 44.5+/-8.8% with oxidized glutathione; 47.8%+/-12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.

Conclusions/significance: IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.

Figure 1. In vitro fertilization rates with cryopreserved C57BL/6J sperm in cell culture inserts and wells.

The bar patterns, in groups of 4, indicate the volume of cryoprotectant used to extend the sperm prior to freezing. From L to R: 105 µL, 180 µL, 400 µL and 800 µL. Numbers within each group of 4 bars indicate IVF Protocol: Protocol 1, 6.5 mm insert; Protocol 2, 6.5 mm well; Protocol 3, 12 mm insert; Protocol 4, 12 mm well. Data are presented as mean ± SEM, n = 4. Significance of differences is described in text.

Figure 2. In vitro fertilization rates in medium containing reduced glutathione (GSH) and in medium lacking GSH.

(A) C57BL/6J, (B) 129S1/svImJ, (C) FVB/NJ and (D) C3H/HeJ sperm, in fertilization medium containing reduced glutathione (GSH) and in medium lacking GSH. The bar patterns, in groups of 4, indicate the volume of cryoprotectant used to extend the sperm. From L to R: 105 µL, 180 µL, 400 uL and 800 µL. Numbers within each group of 4 bars indicate IVF Protocol: Protocol 1, 6.5 mm well + GSH; Protocol 2, 6.5 mm well − GSH; Protocol 3, 12 mm well + GSH; Protocol 4, 12 mm well − GSH. The fertilization medium was supplemented with 1.25 mM GSH for the C57BL/6J, 129S1, and FVB sperm and with 0.5 mM GSH for the C3H sperm. Data are presented as mean ± SEM, n = 4 and significance of differences is described in text.

Figure 3. Effect of reduced glutathione (GSH) on mouse eggs.

(A) C57BL/6J eggs incubated for 6 h in fertilization medium containing 1.25 mM GSH. The zona pellucida has expanded resulting in an increase in width of the perivitelline space, compared with eggs incubated medium lacking GSH (B). Each scale bar  = 100 µm.