Galactose and its Metabolites Deteriorate Metaphase II Mouse Oocyte Quality and Subsequent Embryo Development by Disrupting the Spindle Structure

Abstract

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n = 240), with and without cumulus cells (CCs), were exposed for 4 hours to D-galactose (2 μM), galactitol (11 μM) and galactose 1-phosphate (0.1 mM), (corresponding to plasma concentrations in patients on galactose-restricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients.

Figure 1

The Leloir pathway of galactose metabolism. GALT, Galactose 1-phosphate uridyltransferase; GALE, UDP-galactose 4′-epimerase; GALK, Galactokinase; UDPGal, UDP-galactose; UDPG, UDP-glucose; Gal-1-P, Galactose 1-phosphate; Glucose-1-P, Glucose-1-phosphate; Glucose-6-P, Glucose-6-phosphate.

Figure 2

Time dependent effect of galactose 1-phosphate on oocyte quality. Percentage of oocytes with poor outcomes in MT structure and CH alignment at different incubation times (1, 2, 4 and 6 hours) when the oocytes without cumulus cells (n = 10 for each concentration) were incubated with fixed concentration of galactose 1-phosphate (0.1 mM) compared to control oocytes receiving no treatment and incubated for 6 hours (0 point on the Y axis) followed by indirect immunofluorescence staining. The table at the bottom presents the mean of percentage of oocytes with poor scores for each exposure time with the standard error of mean.

Figure 3

The effect of galactose and its metabolites on Metaphase II mouse oocyte spindles and chromosomes. (A) Representative confocal images of non-cumulus and cumulus metaphase II mouse spindles stained with β-tubulin antibody to visualize the microtubules (MT) (green) and counterstained with DAPI to visualize chromosomes (CH) (blue). After 4 hours of incubation, various abnormal configurations of spindles were observed when oocytes were exposed to D-galactose (B,F), galactitol (C,G) or Gal 1-P (galactose 1-phosphate) (D,H) compared to normal spindle shapes in untreated group (A,E) (n = 30/group). Scale bars: 1 pixel, 3 mm. Images shown are from a typical triplicated experiment. (B) The percentage of oocytes with poor scores in MT structure (upper panel) and CH alignment (lower panel) (120 cumulus and 120 without cumulus) in untreated oocytes compared to oocytes treated with galactose, galactitol and Gal 1-P (galactose 1-phosphate). Poor scores were significantly increased in oocytes exposed to galactose and its metabolites compared with controls in both MT and CH, indicated by *for oocytes without cumulus cells (p = 0.032 for MT and p = 0.05 for CH) and **for oocytes with cumulus cells (p = 0.04 for MT and p = 0.029 for CH). The experiment was conducted in triplicate.

Figure 4

Concentration dependent effect of galactose 1-phosphate on oocyte quality. Percentage of oocytes with poor outcomes in MT structure and CH alignment, at 4 hours of incubation, when the oocytes without cumulus cells were incubated with increasing concentrations of galactose 1-phosphate (0.025 mM to 1 mM) {n = 10 for each concentration}, followed by indirect immunofluorescence staining. The table at the bottom presents the mean of percentage of oocytes with poor scores for each concentration with the standard error of mean.

Figure 5

The effect of galactose and its metabolites on developmental competence of embryos generated by in-vitro fertilization. Images (A–L) represent images of embryo morphology at 24 hours (2-cell stage), 48 hours (8 cell stage) and 96 hours (blastocyst stage) after exposure to D-galactose (Images D–F), galactitol (Images G–I), and Gal 1-P (galactose 1-phosphate) (Images J–L), and untreated control (Images A–C). (M) The trends of changes in percentages of developed embryos at each stage compared to untreated controls.

Figure 6

Evaluation of ROS generation. Images (A–L) represent images of intracellular ROS generation, 4′,6-diamidino-2-phenylindole (DAPI) fluorescence and merged images of ROS generation and DAPI of cumulus oocyte complex exposed to D-galactose (Images D–F), galactitol (Images G–I), and Gal 1-P (galactose 1-phosphate) (Images J–L), and untreated control (Images A–C). Scale bars: 100 μm. Images shown are from a typical experiment performed at least three times.

Figure 7

Detection of apoptosis by TUNEL in the cumulus-oocyte complex. Images (A–L) represent images of TUNEL assay (dUTP) (green), DAPI fluorescence (blue) and merged images of dUTP and DAPI in oocyte cumulus complex exposed to galactose (Images D–F), galactitol (Images G–I), and galactose 1-phosphate (Images J–L), compared to untreated control (Images A–C). Scale bars: 100 μm. Images shown are from a typical experiment performed at least three times. The number of TUNEL-positive cumulus cells were increased by D-galactose, galactitol and galactose 1-phosphate compared to no-treatment control. TUNEL Index was determined as the ratio of TUNEL-positive cells to total counted cell nuclei and presented as mean ± SEM (standard error of mean). *p < 0.05 compared to control.