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. 2015 Jul-Sep;16(3):138-47.

Ultrastructural and Morphalogical Changes of Mouse Ovarian Tissues Following Direct Cover Vitrification with Different Cryoprotectants

Maryam Ghavami  1 , Daryoush Mohammadnejad  2 , Rahim Beheshti  3 , Jafar Solmani-Rad  1 , Ali Abedelahi  1
Affiliations

Ultrastructural and Morphalogical Changes of Mouse Ovarian Tissues Following Direct Cover Vitrification with Different Cryoprotectants

Maryam Ghavami et al. J Reprod Infertil. 2015 Jul-Sep.

Abstract

Background: Cryopreservation of mammalian ovaries has been reported with different levels of success. Cryopreservation of ovarian tissue may be a potential alternative for treatment of infertility and many attempts have been done to improve the efficiency of ovarian cryopreservation. The objective of the present study was to compare the direct cover vitrification (DCV) with ethylene glycol (EG), dimethyl sulfoxide (DMSO) and EG plus DMSO.

Methods: Eighty five mice were sacrificed by cervical dislocation and their ovaries were cryopreserved in the presence of 5% EG or DMSO alone or as mixture, 10% EG or DMSO alone or as mixture and a group with ascending concentrations of cryoprotectants. After toxicity testing and vitrification warming, the ovaries were fixed for histological and ultrastructural studies. In addition, the viability of mechanically isolated follicles was studied by trypan blue staining. All data were compared by ANOVA (p<0.05).

Results: Ovarian tissues frozen in EG plus DMSO in ascending concentrations retained a higher percentage of morphologically normal and or viable follicles than tissues frozen in 10 M EG plus DMSO or in either concentration of EG and DMSO alone (p<0.001). Ultrastructural analysis of ovarian tissues frozen in ascending concentrations of EG plus DMSO showed that these follicles were well preserved and it was very similar to the control group.

Conclusion: Cryopreservation of ovarian tissue in EG plus DMSO is the most effective method for preserving the structural integrity of follicles within the ovary.

Keywords: Cryopreservation; Direct cover vitrification; Ovarian tissue.

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Figures

Figure 1.
Figure 1.
Trypan Blue staining for viability of follicles from vitrified ovarian tissues in different stages. A: Primordial follicles. B: Primary follicle; C: Secondary follicle. Scale bar=30 μm. In non-viable follicles, oocyte or the surrounding granulosa cells had blue coloration and viable ones were not stained
Figure 2.
Figure 2.
Morphology of the secondary follicles after direct cover vitrification; A: normal or intact follicle; B: influenced follicle whit slightly disruption of contact between innermost granulosa cells and oocyte (black arrow); C: degenerated follicle
Figure 3.
Figure 3.
Light microscopic images of mouse ovarian tissue; A: non-frozen; B: DCV1 group that vitrified alone in 5% EG, C: DCV2 group that vitrified in 10% EG; D: DCV3 group that vitrified alone in 5% DMSO, E: DCV4 group that vitrified in 10% DMSO, F; DCV5 group that vitrified in 5% EG + 5% DMSO, G: DCV6 group that vitrified in 10% EG + 10% DMSO and H: DCV7 group that equilibrated in DCV5 and then DCV6 and finally vitrified in DCV6 concentration
Figure 4.
Figure 4.
Photomicrograph of early primary follicle (intermediary follicle) in the DCV7 group showing the oocyte (O) encircled by one layer of both flattened and cuboidal granulosa cells (GC). Note the slightly centric nucleus (N), a homogeneous basal lamina (BL) and the theca cells (T) showing fibroblasts with elongated nuclei. The nucleus and the cytoplasmic organelles are well preserved in all combination cryoprotectants (EG +DMSO) groups. TEM: Ax1293, Bx 4646
Figure 5.
Figure 5.
The electron micrograph of granulosa cells of secondary follicles from control or non-frozen (Ax4795), DCV1 (Bx2784) and DCV2 (Cx3597) DCV5 (Dx3597), DCV6 (Ex2156) and DCV7 (Fx2784). The granulosa cells in single cryoprotectant appeared to have cytoplasmic retraction with nuclear shrinkage (A, B). In DCV5 and DCV6 groups were some perinuclear space (D, E). Granulosa cells were well preserved in non-frozen and DCV 7 groups (A, F). N: nucleus, PS: perinuclear space
Figure 6.
Figure 6.
The electron micrograph of mithochondria (M) arrangement of secondary follicles. A: Numerous lipid droplets (L) are found in close association with atypical or irregularly shaped mithochondria with longitudinally oriented cristae in DCV 1–4 groups, TEM:x10000. B: Numerous elongated mithochondria with tubular-vesicular cristae in DCV 5–6 groups, TEM:x10000. C: Note the presence of numerous round mithocondria with continuous membranes and normal cristae and numerous parallel stacks of RER in DCV7 group, TEM:x7750, D: The zona pellucid (ZP) was fully developed and forming a thick layer around the oocyte of secondary follicle combination cryoprotectants. TEM:x10000
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