Structural and functional differentiation of follicular and oviductal mouse oocytes visualised with FITC-protein conjugates.
The fluorescence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida, perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage (GV) ovarian oocytes exhibited a bright, amorphous, mesh-like staining pattern (immature type). In contrast, mature polar body stage (PB) oocytes, either ovarian or oviductal, displayed faint, spotty fluorescence labelling of the ZP (mature type). The perivitelline space (PVS) of mature ovarian oocytes (12 h post-hCG) failed to label, whereas approximately 50% of oviductal oocytes showed PVS labelling. The incidence of PVS staining increased with postovulatory age, possibly as a result of the accumulation of materials secreted by the oviduct. Following in vivo or in vitro fertilisation of oocytes, a characteristic pattern of plasma membrane (PM) labelling was observed. Similar patterns of PM labelling were seen in oocytes parthenogenetically activated with ethanol or ionophore (A23187) but not in control oocytes. The pattern of PM labelling observed with FITC-protein conjugates was strikingly similar to that observed with FITC-labelled lectins, which are thought to interact with glycoconjugates released from cortical granules. Immature type of ZP staining also occurred when GV oocytes were treated with FITC alone or with a variety of FITC-protein conjugates. Thus, protein may not be required for labelling of the ZP by FITC-protein conjugates as previously thought. FITC-conjugated proteins including casein, bovine serum albumin, peroxidase and non-immune immunoglobulin G (IgG), all labelled the PM of activated oocytes; however, FITC-IgG failed to label the PVS. Results demonstrate for the first time that various components of viable mouse oocytes exhibit and undergo characteristic structural and functional changes during periovulatory differentiation as evidenced by their interaction with one or more FITC-protein conjugates and/or FITC. On the basis of these results the intrafollicular and oviductal mechanisms mediating these changes are discussed as is the possibility that the fluorescent molecule attached to conjugates may play a role in oocyte labelling.
Kim
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Johns Hopkins University, School of Hygiene and Public Health, Department of Population Dynamics, Baltimore, Maryland 21205.
Schuetz
A W
AW
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D016650:Fluorescein-5-isothiocyanate; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D006080:Ovarian Follicle / Q000166:cytology; D010312:Parthenogenesis / Q000502:physiology; D011485:Protein Binding; D014817:Vitelline Membrane / Q000378:metabolism; D015044:Zona Pellucida / Q000378:metabolism
D002364:Caseins; D016650:Fluorescein-5-isothiocyanate