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

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.

Effect of Human Cord Serum on the Hormone Production by Mouse Oocyte Cumulus Complexes in vitro / 대한산부인과학회잡지

In our previous study, it was shown that human cord serum stimulated cumulus expansionin vitro by cumuli oophori isolated from mice primed with pregnant mare serum gonadotropin(PMSG), and suggested that there were some gonadotropin-like components stimulating cumulusexpansion in human cord serum. In this paper we assess whether there are gonadotropinstimulating cumulus expansion in human cord serum and sex steroid hormone production fromoocyte cumulus complexes(OCCs). The contents of follicle stimulating hormone(FSH) andluteinizing hormone(LH) were measured in human cord serum: follicle stimulating hormone levelwas basal, but luteinizing hormone was as high as 142.4 mIU/ml even in inactivated serum.After short term culture(4hr), with or without OCCs, medium containing 0.4% bovine serumalbumin(BSA) as control or 10% human cord serum(HCS) was collected and analyzed for itscontent of estradiol, progesterone and testosterone. Little or no sex steroid contents were detectedin any control media with or without OCCs. In contrast, a moderate or small amount ofsex steroid contents was detected in culture medium containing cord serum. OCCs secretedminute but not significant amounts of estradiol, progesterone, and testosterone when culturedin media containing cord serum. After 4, 8, and 22hr culture with OCCs, similar patterns ofcumulus expansion were observed in media containing cord serum, human chorionic gonadotropin(HCG) instead of luteinizing hormone(LH), and HCS plus HCG. However, no cumulus expansionwas observed in any control media.It is suggested that luteinizing hormone in human cord serum induces cumulus expansionand affects the secretion of sex steroid hormones by OCCs during culture.