OOCYTE FOLLICLE CELLS ASSOCIATION DURING DEVELOPMENT OF HUMAN OVARIANFOLLICLE - A STUDY BY HIGH-RESOLUTION SCANNING AND TRANSMISSION ELECTRON-MICROSCOPY

Morphodynamics of oocyte follicle cells association during the develop ment 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 adu lt and fetal (3-8 months and at term) ovaries. In early stages of foll icle 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 appositio n with the oolemma, and form bulbous terminals presenting attachment z ones such as zonula adherens, desmosomes and communicating junctions ( gap junctions). ''Focal contacts'' between oolemma, and lateral microv illous extensions of follicle cells were also present. Unusual forms o f contact between follicle cell microvilli and oocytes in the early st ages of growing primordial and primary follicles were also observed. T hese consist of long, thin extensions penetrating into the oocyte thro ugh deep invaginations of the oolemma. The aid of high resolution SEM of specimens subjected to the ODO method clearly reveals their 3-D arr angement within the ooplasm. They appear as long tortuous microvilli c oming very close to the nucleus, and in their course are closely assoc iated 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 a s 3-5 ''intraooplasmic processes'' even in only one plane of fracture of an oocyte. Therefore, if the total volume of the oocyte and associa ted cells is considered, their amounts appear to be higher than previo usly reported. Thus, they have to be considered as normal devices of d eep contact between the ooplasm and associated follicle cell extension s. The presence of such structures within the ooplasm in early develop ing follicles well coincides with the great increase in volume of the oocyte. Although it is commonly believed that the activation of the gr owing oocyte may depend on the numerous contacts between the oolemma a nd follicle cells (mostly via gap junctions), the finding of these add itional intraoocytic extensions suggests that they may in someway cont ribute to the initiation of growth in the human. In fact, these microv illi 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 va riety of ions or molecules (as signals), including nutrients. In turn, they may mediate or integrate a parallel activation on specific oocyt e organelles and their cohort of enzymes. In later stages of follicle development (pluristratified and antral follicles provided with a thic k and complete zona pellucida), these intraooplasmic microvilli were v ery 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 junct ions appeared very numerous. All these junctions disappear by being di srupted, at the time of ovulation, by active retraction of follicle ce ll extensions. The long exposure of specimens to the ODO maceration me thod allowed full observation of the real 3-D surface pattern of folli cular cells and their extensions, including those of the so-called cor ona radiata. In fact, through the chemical dissolution of liquor folli culi and zona pellucida, these cells, which were mainly pear shaped, s howed a characteristic apical polarization of their numerous microvill i 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 w ere rarely observed over the remaining surfaces of the corona cells fa cing the antrum, or over the surface of associated cumulus and parieta l follicular cells. Single short cilia were also frequently noted in t hese cells. As fully revealed by SEM following the ODO method, these m icrovilli contribute to a tremendous increase in the surface area of c orona cells containing the oocyte. In fact, anchored as bridges to the oolemma, they provide a sort of cytoplasmic skeleton supporting the z ona pellucida. As revealed by their 3-D pattern, additional functions for these structures may be suggested: 1) transfer of substances to bu ild up the zona pellucida; 2) release of nutrients into the zona and f rom 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 close ly resembles a thick basal lamina, can be continuosly regenerated, act ing as an efficient filter for the oocyte. A final consideration that these 3-D images suggest is that in vivo the corona cell extensions ar e included in the zona pellucida. Therefore, narrow spaces separate th e dense channels sculptured in the zona from the surface of cytoplasmi c processes of corona cells contained therein. As a consequence, a kin d of microlabyrinthine system of microfissures (microtunnels) arises, which may serve as areas of exchange of minute materials to/from the f ollicle cell, the oocyte and zona pellucida. Further considering that follicular cells and their extensions are highly pulsatile in nature b ecause of the high number of contractile filaments, the obvious deduct ion is that in vivo, the follicular cell extensions may elongate and r etract continuously within the microtunnels of the solidified zona pel lucida. Therefore, the numerous contacts that they have with the oocyt e may be not permanent but dynamic, with high functional advantages. I n particular, it is possible to hypothesize that a variety of dynamic contacts are created between follicular cells and oocytes and among mi crovilli 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.