Giuseppe Familiari

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.

Ultrastructure of the Ovary

1. Migration of germ cells, development of the ovary, and folliculogenesis.- 2. Maturation of the human oocyte.- 3. Expansion of the cumulus-oocyte complex during the preovulatory period: Possible roles in oocyte maturation, ovulation, and fertilization.- 4. Structure and function of the zona pellucida.- 5. Quantitative analysis of the membrana granulosa in developing and ovulatory follicles.- 6. Digital image analysis studies of folliculogenesis and oogenesis in mammals.- 7. Modulation of granulosa cell ultrastructure during differentiation: The role of the cytoskeleton.- 8. Ultrastructure and functional development of the theca interna.- 9. Muscular apparatus of the ovarian follicle.- 10. Ultrastructure of the ovulatory process.- 11. Ultrastructural dynamics during corpus luteum development and growth.- 12. Relaxin and other luteal secretory peptides: Cell localization and function in the ovary.- 13. Interstitial cells.- 14. Ultrastructure and immunocytochemical aspects of steroid-synthesizing and secretory cells with special reference to the mechanism of estrogen biosynthesis.- 15. Microvasculature of the ovary.- 16. Innervation of the ovary.- 17. Follicular atresia.- 18. Biopathology of ovarian mesothelium.- 19. Culture of ovarian tissues.

The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

AIMS Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. METHODS AND RESULTS We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. CONCLUSION These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs.

Arterial Repair after Microvascular Anastomosis

In order to study the morphological aspects of endothelial regeneration and vascular wall reaction after microvascular anastomosis, rat femoral arteries were sectioned and successively sutured (end-to

Structural changes of the zona pellucida during fertilization and embryo development

The zona pellucida (ZP) is a unique extracellular coat surrounding the maturing oocyte, during ovulation, fertilization, and early embryo development. It is formed by three/four glycoproteins. Ultrastructural data obtained with transmission (TEM) and scanning electron microscopy (SEM) were compared with molecular data on the glycoproteins network from ovulation to blastocyst formation. Molecular models are quite different to the morphology obtained with TEM, which shows a microfibrillar architecture, or with SEM, which shows a spongy or smooth surface. The saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide technique allows to show the ZP real microfilamentous structure and the related functional changes. These results support an ultrastructural supramolecular model, more similar and comparable to molecular models related with the glycoprotein network. A detailed mapping of single mammalian ZP proteins and their relationship within the supramolecular architecture of the zona matrix would clearly supply insights into the molecular basis of sperm-egg recognition. Differences in ZP glycoproteins among mammals do not affect structural morphology; further studies are needed to clarify the relationships between ultrastructural and molecular organizations.

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.

Whole-Depth Change in Bovine Zona Pellucida Biomechanics after Fertilization: How Relevant in Hindering Polyspermy?

Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone.

SummaryMorphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3α- and 3β-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable.The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation.No significant differences (0.7<P<0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P<0.001). This increase was probably related to the large amounts of androgens present in the treated animals.Theca interna cells possessed small gap junctions; no significant differences (P>0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.

A Deep Look Into Erionite Fibres: an Electron Microscopy Investigation of their Self-Assembly.

The exposure of humans to erionite fibres of appropriate morphology and dimension has been unambiguously linked to the occurrence of Malignant Mesothelioma. For this reason, a detailed morpho-structural investigation through Electron Microscopy techniques has been performed on erionite samples collected at two different localities, Durkee (ED) and Rome (ER), Oregon, USA. The sample from Rome has been also investigated after a prolonged leaching with Gamble’s solution (ER4G) in order to evaluate the possible occurrence of morpho-structural modifications induced by this Simulated-Lung-Fluid (SLF). Here we report how the micrometric erionite fibres evolve in irregular ribbon- or rod-like bundles as a function of different nano-structural features. The reasons for the observed morphological variability have been explained by considering the structural defects located at ED surface fibrils (bi-dimensional ribbons) and the presence of nontronite, an iron-bearing clay mineral embedding the ER fibrils (mono-dimensional rods). ER4G shows a decrease in width of the rod-like fibres due to their partial digestion by SLF leaching, which synchronously dissolves nontronite. The reported results represent a valuable background toward the full comprehension of the morphological mechanisms responsible for potentially damage of lung tissue through the potential relocation of fibers to extrapulmonary sites, increasing the carcinogenic risk to humans.