Morphodynamics of oocyte follicle cells association during the development of human ovarian follicles were studied by transmission electron microscopy and high resolution scanning electron microscopy including the ODO method. For this study primordial, primary, growing preantral and antral follicles were systematically analysed in a total of 20 adult and fetal (3-8 months and at term) ovaries. In early stages of follicle development (primordial and primary stages) the flattened and/or polyhedral cells, closely associated with the growing oocyte, project an increasing number of microvillous processes. These are in apposition with the oolemma, and form bulbous terminals presenting attachment zones such as zonula adherens, desmosomes and communicating junctions (gap junctions). ''Focal contacts'' between oolemma, and lateral microvillous extensions of follicle cells were also present. Unusual forms of contact between follicle cell microvilli and oocytes in the early stages of growing primordial and primary follicles were also observed. These consist of long, thin extensions penetrating into the oocyte through deep invaginations of the oolemma. The aid of high resolution SEM of specimens subjected to the ODO method clearly reveals their 3-D arrangement within the ooplasm. They appear as long tortuous microvilli coming very close to the nucleus, and in their course are closely associated with a variety of organelles such as Golgi vesicles, endoplasmic reticulum membranes and nascent forms of smooth endoplasmic reticulum. Using integrated observations by TEM and SEM, there may be as many as 3-5 ''intraooplasmic processes'' even in only one plane of fracture of an oocyte. Therefore, if the total volume of the oocyte and associated cells is considered, their amounts appear to be higher than previously reported. Thus, they have to be considered as normal devices of deep contact between the ooplasm and associated follicle cell extensions. The presence of such structures within the ooplasm in early developing follicles well coincides with the great increase in volume of the oocyte. Although it is commonly believed that the activation of the growing oocyte may depend on the numerous contacts between the oolemma and follicle cells (mostly via gap junctions), the finding of these additional intraoocytic extensions suggests that they may in someway contribute to the initiation of growth in the human. In fact, these microvilli penetrate deep into the ooplasm, much like a sword in its sheath. After contacting numerous oocytes' organelles, they come close to the nucleus, where they might transfer, more extensively and easily, a variety of ions or molecules (as signals), including nutrients. In turn, they may mediate or integrate a parallel activation on specific oocyte organelles and their cohort of enzymes. In later stages of follicle development (pluristratified and antral follicles provided with a thick and complete zona pellucida), these intraooplasmic microvilli were very rarely observed. In large antral follicles close to ovulation, the bulbous processes of follicle cells contacting the oolemma were noted, and the attachement zones (zonula adherens, desmosome) and gap junctions appeared very numerous. All these junctions disappear by being disrupted, at the time of ovulation, by active retraction of follicle cell extensions. The long exposure of specimens to the ODO maceration method allowed full observation of the real 3-D surface pattern of follicular cells and their extensions, including those of the so-called corona radiata. In fact, through the chemical dissolution of liquor folliculi and zona pellucida, these cells, which were mainly pear shaped, showed a characteristic apical polarization of their numerous microvilli toward the oocyte. As a rule, these unusual microvilli measured 7-10 mu m in length and the apical surface of a single corona cell toward the zone/ oocyte formed up to 70 long microvilli. Similar extensions were rarely observed over the remaining surfaces of the corona cells facing the antrum, or over the surface of associated cumulus and parietal follicular cells. Single short cilia were also frequently noted in these cells. As fully revealed by SEM following the ODO method, these microvilli contribute to a tremendous increase in the surface area of corona cells containing the oocyte. In fact, anchored as bridges to the oolemma, they provide a sort of cytoplasmic skeleton supporting the zona pellucida. As revealed by their 3-D pattern, additional functions for these structures may be suggested: 1) transfer of substances to build up the zona pellucida; 2) release of nutrients into the zona and from there to the oocyte, and vice versa; and 3) removal of catabolites from the zona, as well as from the oocyte. Thus the zona, which closely resembles a thick basal lamina, can be continuosly regenerated, acting as an efficient filter for the oocyte. A final consideration that these 3-D images suggest is that in vivo the corona cell extensions are included in the zona pellucida. Therefore, narrow spaces separate the dense channels sculptured in the zona from the surface of cytoplasmic processes of corona cells contained therein. As a consequence, a kind of microlabyrinthine system of microfissures (microtunnels) arises, which may serve as areas of exchange of minute materials to/from the follicle cell, the oocyte and zona pellucida. Further considering that follicular cells and their extensions are highly pulsatile in nature because of the high number of contractile filaments, the obvious deduction is that in vivo, the follicular cell extensions may elongate and retract continuously within the microtunnels of the solidified zona pellucida. Therefore, the numerous contacts that they have with the oocyte may be not permanent but dynamic, with high functional advantages. In particular, it is possible to hypothesize that a variety of dynamic contacts are created between follicular cells and oocytes and among microvilli of corona cells. These in turn may serve to actively modulate (inhibiting or stimulating) the oocyte up until the time of ovulation through a coordination with the entire follicle oocyte complex.
