Stefania A. Nottola

Morphodynamics of the follicular-luteal complex during early ovarian development and reproductive life.

Female reproductive activity depends upon cyclic morphofunctional changes of the ovarian tissue during the females fertile period, but the primum movens of an active gonadal rearrangement can be found from early phases of embryo development. To offer a basic account of the main steps of ovarian dynamics, we review the morphofunctional behavior of the follicular-luteal complex in an integrated study using light microscopy and transmission and scanning electron microscopy as well as through the use of numerous drawings. Particular emphasis is given to some reproductive aspects including (1) germ-somatic cell relationships and onset of folliculogenesis during early gonadal development; (2) follicular development and oocyte-follicle cell associations through adult folliculogenesis, finally leading to ovulation; (3) morphodynamics of corpus luteum formation, development, and regression, and (4) degenerative processes involving germ and somatic cells. The results reported, many of which originated in our laboratory, arise from some experiments on laboratory mammals but mostly from a large selection of human specimens. The data obtained are integrated and correlated with classic reports as well as with current views. Crucial biochemical, histophysiological, and clinical aspects are also emphasized.

Is the sperm-binding capability of the zona pellucida linked to its surface structure? A scanning electron microscopic study of human in vitro fertilization

The structure of the zona pellucida and the early interactions between human oocytes and spermatozoa were investigated in an in vitro fertilization program. Thirty-five mature (preovulatory) oocytes, 10 immature oocytes lacking a germinal vesicle, and 11 atretic oocytes which had not undergone fertilization at 10–20 hr after insemination were studied by light and scanning electron microscopy. Observed through employment of these techniques, the zona pellucida showed two basically different patterns: a mesh-like, spongy structure having wide and/or close meshes; and a compact, smooth surface. The smooth-surfaced zona was most commonly seen in the cultured oocytes belonging to the immature and atretic groups. These observations seem to show that the spongy appearance of the zona pellucida is related mainly to oocyte development and maturity. In this study, greater numbers of penetrating spermatozoa were noted on oocytes showing the mesh-like zona, in contrast to the presence of a few sperm flattened against its surface or the frank absence of sperm associated with oocytes having the more compact, smooth zona. It is likely that the condensation of the outer aspect of the zona pellucida causes a disorientation of sperm-binding sites, which would probably result in markedly reduced binding and penetration capacity with spermatozoa. These changes might ultimately lead to impairment of in vitro oocyte fertilizability.

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification.

Histology of the exocrine pancreas.

The morphology of the exocrine secretory unit of the pancreas, i.e. the pancreatic acinus, is reviewed. The histological features of the acini and their relation with the duct system are described. The acinar three‐dimensional architecture was studied by means of different ultrastructural techniques, some of which are complementary. The fine structure and morphodynamics of the acinar cells are also described. In addition, the location of the organelles in specific cytoplasmic domains and their close morphofunctional relationship with the sequential stages of secretion of the digestive enzymes are specially emphasized. Finally, morphological approaches are suggested to achieve a better comprehension of the physiological and pathological pancreatic activities whose morphodynamics need to be further elucidated or are almost totally unknown. Microsc. Res. Tech. 37:384–398, 1997.

Natural history of the female germ cell from its origin to full maturation through prenatal ovarian development

This paper contains a number of sketches concerning the main morphological ultrastructural features of the human female germ cell during the prenatal period. The morphodynamic outline of primordial germ cells has been traced, both in their extraembryonic site of origin and during their migration towards the developing ovary. After gonadal settlement, the intraovarian differentiation of the germ cells into primary oocytes through the stage of oogonia, as well as the dramatic fall in the number of germ cells before birth, is described. The presence of morphofunctionally relevant interactions between the differentiating female gamete and the surrounding somatic microenvironment has also been evaluated and discussed.

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Preservation of fertility in young cancer patients: contribution of transmission electron microscopy

During the last decade, new technologies in reproductive medicine have emerged to preserve the fertility of women whose gonadal function is threatened by premature menopause or gonadotoxic treatments. To offer an individualized approach to these patients, different experimental procedures are under investigation, including oocyte cryopreservation and cryopreservation and transplantation of ovarian tissue in the form of cortical fragments, whole ovary or isolated follicles. This review shows that transmission electron microscopy (TEM), combined with other in-vivo and in-vitro analysis techniques, is a valuable tool in the establishment of new experimental protocols to preserve female fertility. Ultrastructural studies allow in-depth evaluation of the oocytes unique morpho-functional characteristics, which explain its low cryotolerance, and provide essential information on follicular, stromal and endothelial cell integrity, as well as cellular interactions crucial for normal folliculogenesis. In order to be able to offer appropriate and efficient options in every clinical situation, oocyte in-vitro maturation and ovarian tissue transplantation need to be optimized. Further development of new approaches, such as follicular isolation and whole ovary transplantation, should be encouraged. Fine ultrastructural details highlighted by TEM studies will be useful for the further optimization of these emerging technologies.

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

PurposeTo ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed.Materials and methodsCryopreservation 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.ResultsBy 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.ConclusionsSlow 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.

Heterogenous distribution of fibronectin, tenascin-c, and laminin immunoreactive material in the cumulus-coronal cells surrounding mature human oocytes from IVF-ET protocols—Evidence that they are composed of different subpopulations: An immunohistochemical study using scanning confocal laser and fluorescence microscopy

Monoclonal antibodies and immunofluorescence microscopy, including laser confocal microscopy, were used in this study to point out the production of fibronectin, tenascin‐c, and laminin in the cumulus‐corona (CC) cells surrounding mature human oocytes from IVF‐ET protocols in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm‐egg interaction, and the “complex biochemical dialogue” between the gamete and the oviduct through the tubal luminal environment.

The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes

OBJECTIVE To describe the possible effects of cryotop vitrification on maturation rate and ultrastructural morphology of human in vitro matured germinal vesicle (GV) oocytes. STUDY DESIGN A total of 301surplus immature GV oocytes obtained from infertile patients were allocated into two groups: (i) GV oocytes (n=150) matured in vitro (fIVM), and (ii) GV oocytes (n=151) that were first vitrified, then matured in vitro (vIVM). Supernumerary fresh in vivo matured oocytes (n=10) were used as controls. The maturation media was Hams F10 supplemented with FSH+LH and human follicular fluid. After 36h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM). RESULTS Oocyte maturation rates were reduced (P<0.001) in vIVM (45.92%) in comparison with fIVM oocytes (75.33%). The rate of degeneration was also significantly higher in vIVM than in the fIVM group (44.4% vs. 6.0%). Large and numerous mitochondria and minute vesicles of smooth endoplasmic reticulum (SER) complexes (MV complexes) were observed in both fIVM and vIVM groups. In addition, TEM revealed a drastic reduction in amount of cortical granules (CGs) at the cortex of vitrified-warmed GV oocytes, as well as appearance of vacuoles and small mitochondria-SER aggregates in the ooplasm. CONCLUSION The vitrification procedure is associated with ultrastructural alterations in specific oocyte microdomains, presumably related to the reduced competence of cryopreserved oocytes for maturation. This information emphasizes the need for further work on advancing the cryotechnology of human oocytes.