Korean J Obstet Gynecol.  2000 Feb;43(2):192-202.

Electron microscopy of the oocyte-cumulus complex and immuncytochemistry on the distribution of fibronectin, tenascin, and laminin

Abstract


OBJECTIVE
Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation 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 mechanism between the ovum and the oviduct.
METHODS
Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue.
RESULTS
The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells.
CONCLUSION
Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.

Keyword

Oocyte cumulus complex(OCC); Cumulus-corona cell; Electron microscopy; Immunocytochemistry

MeSH Terms

Actin Cytoskeleton
Adhesives
Animals
Antibodies
Antibodies, Monoclonal
Basement Membrane
Cell Membrane
Cumulus Cells
Cytoplasm
Embryonic Structures
Endoplasmic Reticulum, Smooth
Epitopes
Extracellular Matrix
Extracellular Matrix Proteins
Extracellular Space
Female
Fertilization
Fibronectins*
Fluorescein-5-isothiocyanate
Fluorescent Antibody Technique
Formaldehyde
Gap Junctions
Immunohistochemistry
Laminin*
Microscopy, Confocal
Microscopy, Electron*
Microscopy, Electron, Transmission
Microscopy, Fluorescence
Microtubules
Microvilli
Mitochondria
Oocytes
Organelles
Oviducts
Ovulation
Ovum
Sperm-Ovum Interactions
Tenascin*
Zona Pellucida
Adhesives
Antibodies
Antibodies, Monoclonal
Epitopes
Extracellular Matrix Proteins
Fibronectins
Fluorescein-5-isothiocyanate
Formaldehyde
Laminin
Tenascin
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