Pietro M. Motta

Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice

OBJECTIVE To compare histologic and ultrastructural characteristics of fresh and frozen-thawed human ovarian cortical tissue grafted into nude mice. DESIGN Experimental prospective study. SETTING An academic research environment. PATIENT(S) Ovarian biopsy specimens were obtained from 13 women undergoing laparoscopy for tubal ligation or infertility. ANIMAL(S) Forty nude mice. INTERVENTION(S) A minilaparotomy was performed to place fresh and frozen-thawed ovarian grafts subcutaneously (sc) or intraperitoneally (ip). Removal of the ovarian grafts was performed at 24 days. MAIN OUTCOME MEASURE(S) [1] the follicular population, [2] fibrosis, [3] vascularization of the grafted tissue, and [4] ultrastructural evaluation. RESULT(S) A greater fibrosis relative surface area was noted in frozen-thawed transplanted tissue than in fresh transplants. Regardless of this fibrosis, a similar follicular density was observed in fresh and frozen-thawed ovarian tissue 24 days after transplantation. Active angiogenesis was proved by both immunohistochemical study of the vascular endothelial growth factor and morphometric study of the vascular network. Normal ultrastructural characteristics were noted in frozen-thawed ovarian biopsies. CONCLUSION(S) Angiogenesis allows implantation of the graft even if it has been cryopreserved and thawed similarly to implantation of fresh tissue. The greater fibrosis observed in grafts after cryopreservation and implantation does not seem to affect the primordial and primary ovocyte population and their ultrastructural characteristics, but further studies must be conducted to prove that after cryopreservation and transplantation, ovocytes may achieve full maturation and fertilization.

Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy

SummaryThe inner surface of sinusoids and adjacent hepatocytes have been examined by scanning electron microscopy. The endothelial cells lining the sinusoids show large numbers of fenestrations which vary greatly in size and arrangement. Some are very small (0.1 μm) and arranged in clusters; others that are much larger (∼1.0 μm) are subdivided by slender strands of cytoplasm. At sites where the larger fenestrae are present it is evident that the endothelial lining of the sinusoid is double. This may represent a kind of structural assurance against complete breakdown of what seems to be a very thin and fragile endothelial wall. Junctions between adjacent endothelial cells have not been found in these preparations.The open continuity of the sinusoid is occasionally interrupted by slender extensions of cells morphologically distinct from the thin fenestrated endothelial cells. These possess a characteristically textured surface and are thought to represent stellate Kupffer cells.The SEM images describe the subendothelial Spaces of Disse as being larger and as having more extensive ramifications than is generally evident from transmission micrographs. The space, limited on one side by the hepatocyte with numerous microvilli and on the other by endothelial cells, appears actually to be only part of an extensive labyrinth of intercellular channels. These connect the more discrete Spaces of Disse and extend into the narrower spaces between the hepatocytes. The total effect of this system is to expose the greater part of the liver cell surface to the blood filtrate. Microvilli populate the hepatocyte surfaces except for narrow margins which border the bile canaliculi. Whether their presence coincides with the adsorbing surfaces and their absence with secreting surfaces can be decided best by experimental studies.

Functional and ultrastructural evidence of myocardial stunning after acute carbon monoxide poisoning

OBJECTIVE To study human myocardial ultrastructural changes after carbon monoxide (CO) poisoning inducing reversible cardiac failure. DESIGN CASE REPORT clinical, functional and morphologic findings. SETTINGS Public university-affiliated hospital and electron microscopy laboratory. PATIENT A 25-yr-old woman with functional evidence of cardiac failure after acute CO poisoning. INTERVENTIONS Hyperbaric and intensive care treatment over 10 days. Scintigraphic and cardiac angiography with endomyocardial biopsy. MEASUREMENTS AND MAIN RESULTS Scintigraphy with 99mTc hexakis 2-methoxy-2-isobutyl isonitrile (sestaMIBI) showed an uptake defect in the left anterior descending artery territory. The cardiac angiography demonstrated a slight hypokinesis of the superior two thirds of the anterior wall and of the septal region with completely normal coronary angiograms. Electron microscopy of left ventricular biopsies showed slight ultrastructural changes in the myocytes. In addition, large glycogen deposits were mostly associated with swollen mitochondria. The patient was discharged in good clinical condition on day 10. CONCLUSIONS Presence of glycogen deposits associated with abnormal mitochondria may be signs of the incapability of myocardial cells in utilizing energy substrata. In the presence of normal myocardial perfusion, our findings are consistent with the presence of a stunned myocardium-like syndrome. Early recognition and treatment of this clinical syndrome allow the prevention of myocardial infarction.

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.

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.

A scanning electron microscopic study of the rat liver sinusoid

SummaryThe surface ultrastructure of Kupffer cells in the rat liver has been studied by scanning electron microscopy (SEM). The results demonstrate that Kupffer cells are both significantly different and clearly distinct from endothelial cells. Kupffer cells have neither pores (and/or “sieve plates”) nor fenestrations, all of which are present in endothelial cells. They possess a stellate shape, and only indirectly, with slender and irregular evaginations, contribute to the lining of the sinusoidal wall. Furthermore, the luminal surface in some areas contains a large population of short microvilli, microplicae and invaginations. These elements form a kind of microlabyrinth which may correspond to the “worm-like” structures described by transmission electron microscopy (TEM).In the present study, transition forms between endothelial and Kupffer cells were never found. On the contrary, considering the highly fenestrated nature of the endothelial cells, the Kupffer cells may, by ameboid movements, easily cross the overlapping barrier of the sinusoid and protrude into the lumen. Thus, acting as activated macrophages, the Kupffer cells might function to prevent the entrance of foreign material into the tissues of the liver through the fragile and highly fenestrated endothelium.Finally, the topographical reconstruction of the sinusoid by correlated SEM and TEM studies demonstrates that Kupffer cells, with their protruding cytoplasm and ability to extend into the lumen of the sinusoid, may actually change the caliber of the vessel, and thus function as a “sphincter” which causes a temporary arrest of the blood flow when the diameter of the sinusoidal lumen is reduced.

Development of the ovarian surface and associated germ cells in the human fetus

SummaryThe ovarian surface and associated germ cells have been studied in human fetuses from 12 weeks of age until near term, using light, TEM and SEM techniques. The surface epithelium and related cords proliferate extensively, especially at midterm. The cords in the ovarian cortex appear to be linked with ingrowths from the surface epithelium, and both structures have a common basal lamina. Germ cells are always interspersed among the somatic cells of the surface epithelium and associated cords. These results indicate that both the proliferating cords and surface epithelium may contribute to the formation of early follicles. Furthermore, the occurrence, of elements having some of the features of primitive steroidogenic cells in the regions of cordsurface epithelium continuity, suggests that both structures (surface epithelium and cords) contribute somatic cells, which in addition to becoming granulosa cells, might also contribute to the provision of primitive interstitial cells.Gonocytes tend to migrate through the developing ovarian tissue towards the surface where they become extruded into the peritoneal cavity. This phenomenon might contribute to the reduction in the number of germ cells at birth and parallels the atretic processes within the ovary.