Maturation, fertilization, and the structure and function of the endoplasmic reticulum in cryopreserved mouse oocytes

Abstract

Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase II-stage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GV-stage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development. We examined the endoplasmic reticulum (ER) as one indicator of cytoplasmic structure, as well as the ability of oocytes to develop Ca(2+) release mechanisms following vitrification and in vitro maturation. Vitrified GV-stage oocytes matured in culture to metaphase II at a rate comparable to that of controls. These oocytes had the capacity to release Ca(2+) following injection of inositol 1,4,5-trisphosphate, demonstrating that Ca(2+) release mechanisms developed during meiotic maturation. The ER remained intact during the vitrification procedure as assessed using the lipophilic fluorescent dye DiI. However, the reorganization of the ER that occurs during in vivo maturation was impaired in oocytes that were vitrified before oocyte maturation. These results show that vitrification of GV-stage oocytes does not affect nuclear maturation or the continuity of the ER, but normal cytoplasmic maturation as assessed by the reorganization of the ER is disrupted. Deficiencies in factors that are responsible for proper ER reorganization during oocyte maturation could contribute to the low developmental potential previously reported in vitrified in vitro-matured oocytes.

FIG. 1.

Vitrified GV-stage oocytes are morphologically indistinguishable from fresh oocytes and mature to the MII stage in culture. A) Fresh oocyte. B) Vitrified oocyte. C) Vitrified MII-stage oocyte following IVM. Note GVBD and first polar body formation. Representative photographs from 265 vitrified oocytes are shown in B and C. Original magnification ×120.

FIG. 2.

Time course of GVBD following vitrification and in vitro maturation. Fresh (n = 400) or frozen (n = 89) oocytes were incubated in medium containing dbcAMP, and the time to GVBD was noted after washing the dbcAMP out of the culture medium. Black squares, control oocytes; black circles, vitrified oocytes.

FIG. 3.

In vitro-matured oocytes form morphologically normal MII spindles. Left: in vivo-matured MII-stage oocyte. Right: vitrified in vitro-matured MII-stage oocyte. Green, tubulin; blue, chromosomes. Original magnification ×120.

FIG. 4.

Vitrified oocytes matured in vitro develop the ability to release Ca²⁺ in response to IP_3. Oocytes were injected with the Ca²⁺ indicator dye calcium green 10-kDa dextran. The fluorescence intensity showing the relative Ca²⁺ level in the oocyte cytoplasm was measured during a subsequent injection of IP₃ (100 nM total in the oocyte). A and B) Representative tracings from control freshly ovulated MII-stage oocytes (n = 8). C) Representative tracing from vitrified oocytes matured in vitro (n = 5).

FIG. 5.

The ER structure in GV-stage oocytes. The ER was labeled using the lipophilic fluorescent dye DiI. A and B) Representative photograph from 25 fresh oocytes showing a continuous ER but the absence of cortical ER clusters that are characteristic of MII-stage oocytes [40, 48] in the cortex (A) and equator (B). C and D) Representative photograph from 19 vitrified oocytes showing the absence of cortical clusters in sections of the cortex (C) and equator (D). Bar = 5 μm (A and C) and 10 μm for (B and D).

FIG. 6.

The structure of the ER following in vitro maturation in fresh oocytes. A) Distinct ER clusters in an in vivo-matured oocyte are shown for reference. B and C) Distinct ER clusters were found in the majority of oocytes matured in CZB medium (B) but were sometimes absent (C). Bar = 5 μm.