In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives.

In vitro growth culture systems of mammalian oocytes have been developed with the aims of studying regulative processes occurring during oogenesis and folliculogenesis, and of preserving fertility. Although in large mammals IVG technology does not still assure the co-ordinate development of both somatic and germinal cells and the production of high number of viable offspring, their improvement may represent an important therapeutic tool for restoring fertility in women undergoing premature menopause or cancer treatments. Morphological studies of in vitro grown follicles were not performed extensively, especially by means of scanning electron microscopy. In the present paper preliminary ultrastructural observations of in vitro cultured follicles are presented.

A micro-anatomical model of the distribution of myocardial endomysial collagen.

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.

In vitro fertilized human eggs. An electron microscopic study.

This paper describes by scanning and transmission electron microscopy the ultrastructure of the human fertilized egg and its vestments (cumulus oophorus and zona pellucida). Data are reported on the ultrastructure of a. conventional in vitro fertilized eggs (pronuclear eggs and cleaving eggs at two-to-four cell stage); b. eggs at the same developmental stage deriving fro intracytoplasmic sperm injection. The present results showed that: 1. The cumulus-enclosed fertilized egg is a highly dynamic structure in which egg vestments play a crucial role, positively affecting fertilization and healthy embryo development; 2. Intracytoplasmic sperm injection technique does not seem to significantly alter fertilized egg morphology.

Ultrastructure and distribution of interstitial glandular cells and associated elements in human fetal ovaries.

In order to understand the fine structure and distribution of the interstitial glandular cells (IGCs) and associated elements in the human fetal ovary, we studied human fetal ovaries at 16 weeks post fertilization (p. f.) by transmission electron microscopy. Semithin sections revealed voluminous typical IGCs usually grouped in clusters, located in the interstitium among the ovigerous cords. Isolated primordial follicles were seen in the cords located close to the interstitium in which IGCs were present. Besides the main ultrastructural characteristics of steroid secreting cells, the IGCs showed lipofuscin granules and stacks of annulate lamellae in their cytoplasm. Fibrocytes, macrophages and mast cells were detected close to the IGCs. In particular, the fibrocytes were located around the IGCs, with which they occasionally formed focal cell contacts. Fibrocytes issued numerous long projections, which, together with collagen fibers, surrounded the clusters of IGCs and small vessels (mainly capillaries), often extending into the intercellular spaces among IGCs. These data indicated that, already at the initiation of folliculogenesis, the IGCs are present numerously in a close association with the ovigerous cords. The morphological aspects of IGCs were comparable to that of fetal testis interstitial (Leydig) cells and hilar cells in adult ovary, and suggest that fetal IGCs may be source of adult ovary hilar cells. In addition, we have here demonstrated for the first time that IGCs are associated with stromal cells whose distribution seems to support IGCs microtopography. Fetal ovarian fibrocytes revealed a structural arrangement similar to that of the "compartmentalizing cells" previously described in the adult testis. Macrophages and mast cells presumably have a role as local modulators of steroid synthesis. Mast cells may also affect fibrocyte organization and vascular permeability. We thus suggest that IGCs and associated cells may form a glandular unit in the human fetal ovary similar to that in the adult testis, and this structure is likely involved in early steroid secretion during gonadal differentiation.

The microvasculature of the corpus luteum in pregnant rabbit. A scanning electron microscopy study of corrosion casts.

The vascular network of pregnant rabbit ovaries was studied by means of scanning electron microscopy (SEM) of corrosion casts, in order to evaluate the morphofunctional changes of the microcirculation of corpus luteum (CL). Pregnant rabbit ovary showed an overwhelming vascularization. Ovarian hilus displayed an increase in the arterial spirallisation. The arterial spiral pattern was present along the entire vessel course, up to CL tissues. The CL of pregnancy was supplied by wide vascular plexuses (2-5 plexuses were found in each pregnant ovary) whose major axis was about 2 mm. Luteal capillaries showed a tortuous course and were arranged in a three-dimensional, wide and rounded-meshed network. Postcapillary venoconstrictions were present. The venous drainage appeared more developed then the arterial supply. Tight artero-venous contacts in hilar, juxtamedullar and medullar regions of the ovary were observed. These results clearly show that the morphofunctional expression of CL of pregnancy is greatly dependent on its hemodynamic control. In particular, the increase of spirallisation exhibited by the arteries during pregnancy is likely to be considered a significant functional change. The spirallisation likely is a device for reducing the blood pressure through the CL. The artero-venous contacts, also previously described in hCG stimulated (pseudopregnant) ovaries, may support a counter-current like system that may allow a veno-arterial exchange of small molecules through the wall of the facing vessels. In addition, in 10-day pregnant rabbit CL the consolidation of a well-developed capillary network was revealed, which is a sign that the CL of pregnancy reached the full morphofunctional maturation. Furthermore, the CL of 10-day pregnant rabbit did not present significant capillary permeabilization and dilation or angiogenic processes, aspects that were previously found in stimulated periovulatory ovaries. Indeed, changes of the arterial supply and venous drainage of the CL of pregnancy were demonstrated. This suggests that the control of the blood flow through the CL of pregnancy may be transferred from the local capillary microcirculation to the regional artero/venous circulation. This may be probably related to the significant increase of the ovarian blood flow necessary for the maintenance of CL endocrine functions during pregnancy.

Egg-sperm interactions in humans: ultrastructural aspects.

A large cumulus mass usually covers the human ovulated oocyte, and voluminous clusters of cumulus cells are still seen after fertilization around the egg. Cumulus cells surround oocytes and fertilized eggs also during in vitro fertilization (IVF) procedures. This study describes, by transmission and scanning electron microscopy, the morphology and the microtopography of the cells forming the human cumulus mass surrounding IVF samples (insemined but not fertilized oocytes and fertilized eggs). Particularly emphasized is their morphodynamic role in sperm-egg interactions. A comparison with the behavior in vivo of cumulus-enclosed oocyte/fertilized eggs has been also performed. All patients have given their informed consent to participate in this protocol. An inner layer (corona radiata cells) and an outer layer (proper cumulus cells) can be microtopographically recognized in the cumulus mass. Numerous cumulus-corona cells, particularly after fertilization, showed ultrastructural characteristics typical for steroid synthetic cells, thus undergoing a sort of "luteinization" parallel to that occurring in the sister granulosa cells of the postovulatory follicle. This steroid synthetic activity, particularly enhanced in vitro but present also in vivo, may be finalized to the release of small amount of steroids (estrogens and progesterone) in the oocyte/fertilized egg milieu. Various proteins, secreted by other cell subpopulations--as revealed in other studies by our research group--, may even enrich this milieu. Lymphocytes and macrophages were often found in the cumulus mass. They may modulate the steroid secretion of the neighboring cumulus cells by production of cytokines, mimicking what occurs in the ovarian follicle and, later, in the corpus luteum. Spermatozoa, both normal (acrosome-intact or--reacted) and abnormal, were frequently seen in the cumulus mass, free in the intercellular spaces or close to the cumulus cells, that can induce sperm capacitation and acrosome reaction. Leukocytes and cumuluscorona cells appeared both capable of actively phagocytizing supernumerary and/or abnormal sperms. Such spermiophagic response is present in a lesser extent around oocytes and eggs fertilized in vivo. In vitro, instead, cumulus spermiophagy leads to the elimination of a large part of the excess spermatozoa that have reached the oocyte, thus restoring in an extracorporeal medium the spermiophagic activity physiologically exerted by leukocytes and epithelial cells in the female and male genital tracts. In conclusion, the cumulus mass surrounding oocytes and fertilized eggs appears as a highly dynamic system, in which various subpopulations of cells cooperate in order to provide a suitable and healthy microenvironment for fertilization and early embryo development.