Ultrastructure of immature and mature human oocytes after cryotop vitrification.

In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.

Morphological expression of angiogenesis in the mammalian ovary as seen by SEM of corrosion casts.

In the mammalian ovary, follicular and corpus luteum cycle is associated with intensive microvascular remodelling. The complex angiogenic dynamics are finely tuned by numerous regulatory factors acting as activators (up-regulators) or inhibitors (down-regulators) of angiogenesis. Alterations of such a tight modulation are involved in several pathologies, including infertility, polycystic ovarian syndrome, ovarian hyperstimulation syndrome and ovarian cancer. We have demonstrated in several experimental models that ovarian function is critically and specifically dependent on angiogenesis for follicular development, ovulation, and corpus luteum growth. The aim of this review is to summarize the results we have obtained on the morphodynamic remodelling of ovarian microvascularization, in polyovulatory (rat, rabbit and pig) and monovulatory species (cow), using scanning electron microscopy of vascular corrosion casts. The knowledge of the morphological expression of the up- and down-regulation of angiogenesis occurring in mono and polyovulatory animals might provide useful information to preserve fertility and to increase of the effectiveness of reproductive management in species of domestic interest.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols.

PURPOSE: To ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed. MATERIALS AND METHODS: Cryopreservation was performed through two protocols with one-step or two-step propanediol. Fresh control oocytes were examined for comparison. Samples were processed for transmission electron microscopy analysis. RESULTS: By light microscopy, both fresh and frozen-thawed oocytes appeared regularly rounded, with intact zona pellucida, and homogeneous cytoplasm. By electron microscopy observation, organelles were abundant and uniformly dispersed. Mitochondria-smooth endoplasmic reticulum associations appeared regular. However, both the amount and density of cortical granules appeared abnormally reduced in frozen-thawed samples. Slight to moderate vacuolization was also found in the ooplasm of oocytes of both frozen groups. CONCLUSIONS: Slow cooling ensures a good overall preservation of human oocytes. However, cytoplasmic vacuolization and cortical granule loss appears associated with cryopreservation, irrespective of the protocol used.