The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

Liberase treatment allows the isolation of highly viable follicles from human ovarian tissue, with an unaltered morphology and ultrastructure. This purified endotoxin-free enzyme preparation is a promising alternative to impure collagenase preparations for the reproducible isolation of intact primordial and primary follicles for culture and grafting purposes.

Ovarian damage, with consequent permanent infertility, is one of the more common side-effects which occurs during chemotherapeutic treatment of patients affected by Hodgkin's disease. This damage is morphologically represented by a marked loss of primordial and primary follicles. The administration of contraceptive drugs before starting chemotherapy enhances survival of a greater number of ovarian follicles, as revealed by morphometric analyses, nevertheless, total ovarian protection is not assured. This study evaluated the number and the morphology of ovarian follicles, by means of transmission electron microscopy, in patients with Hodgkin's disease treated with multi-drug chemotherapeutic protocols following the administration of medroxyprogesterone acetate. Ovarian biopsies were performed prior to any pharmacological treatment, after medroxyprogesterone therapy, and after this therapy plus chemotherapy. Particular attention was given to the ultrastructure of primordial follicles. After morphometric evaluation, primordial follicles were numerous in controls and medroxyprogesterone therapy (28.55 +/- 6.59/mm3 of ovarian cortex). After chemotherapy and medroxyprogesterone acetate, the number of follicles was slightly reduced (19.37 +/- 3.41/mm3 of ovarian cortex) in contrast to the dramatic loss usually observed when protection is not given, although more follicles were atretic. Medroxyprogesterone may protect follicles only from acute, toxic effects of chemotherapy, which dramatically reduce their number and lead to sterility. Nevertheless, the quality of follicles is still impaired, and many undergo atresia, resulting in a shortened fertility period.

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This presentation, on both printed copy and CD-ROM, summarizes a series of original data on the ultrastructure of human reproduction produced by our research group. In particular, female germ cell behaviour at the time of migration and colonization of the gonad and germ-somatic cell interactions inside the developing ovary are reviewed from a morphodynamic point of view. The results mostly consist of black-and-white transmission and scanning electron microscopy (TEM and SEM) images. Artificially coloured SEM pictures, light microscopy images and drawings have also been selected for iconography to render complex microanatomical details and their morphofunctional relationships more comprehensible. In all, 35 images are presented in this article, each related to a concise text section and accompanied by a self-explaining caption. A list of pertinent references is also provided.

Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.

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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.