Assessment of mouse germinal vesicle stage oocyte quality by evaluating the cumulus layer, zona pellucida, and perivitelline space

Abstract

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications.

Figure 1. GV stage oocyte classification based on thickness of the cumulus layers.

GV stage COCs were collected from ovaries from mice following administration of PMSG. The COCs were classified into three categories according to the thickness of their cumulus layers (C): COCs with C≥50 µm (upper line, left), COCs with 30 µm≤C<50 µm (upper line, middle), COCs with C<30 µm (upper line, right). For the experiment using COCs with dispelled cumulus layers, COCs with C≥50 µm (bottom line, left) were treated using gentle pipetting to generate C<30 µm (bottom line, right). Scale = 50 µm.

Figure 2. GV stage oocyte classification based on thickness of the ZP.

GV stage COCs were collected from ovaries from mice following administration of PMSG. The COCs were classified into three categories according to the thickness of their ZP (Z): COCs with Z≥8 µm (upper line, left), COCs with 5 µm≤Z<8 µm (upper line, middle), COCs with Z<5 µm (upper line, right). For the ZP digestion experiment, oocytes with Z≥8 µm (bottom line, left) were treated with tyrode solution to generate 5 µm≤Z<8 µm (bottom line, right). Scale = 50 µm.

Figure 3. GV stage oocyte classification based on width of the perivitelline space.

GV stage COCs were collected from ovaries from mice following administration of PMSG. The COCs were classified into three categories according to the width of their perivitelline space (P): COCs with P≥5 µm (upper line, left), COCs with 1 µm≤P<5 µm (upper line, middle), COCs with P<1 µm (upper line, right). For the perivitelline space enlargement experiment, oocytes with P<1 µm (bottom line, left) were treated by extracting ooplasm to generate 1 µm≤P<5 µm (bottom line, right). Scale = 50 µm.