The extracellular matrix of porcine mature oocytes: origin, composition and presumptive roles

Abstract

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine--precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans--for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

Figure 1

TEM of the PVS of pig oocytes matured in vitro until MII. In the PVS, the ECM appears as a meshwork of fibrils (f) after tannic acid treatment (a) or as a meshwork of fibrils (f)and attached granules (g) after ruthenium red treatment (b). In the latter case, granules are also visible on the inner side of the ZP (Z). Part of the first polar body (PB) containing cortical granules is visible in (a); these granules (CG) are contrasted in both cases. bar = 0.5 μm

Figure 2

TEM autoradiography of thin sections of oocytes cultured from 0 hr post hCG until MI in presence of ³H-fucose, with (a) or without (b) cumulus cells. bar = 1 μm a : general heavy labeling of the ZP (Z) surface, of corona cell projections (P), and particularly of the PVS (*) and Golgi vesicles (G).b : low labeling in PVS (*) and ooplasm.

Figure 3

TEM of serial sections of oocytes cultured from 0 hr post hCG until MI in presence of ³H-glucosamine, with (a, b) or without (c, d) cumulus cells. bar = 2 μm a, b : heavy labeling of corona cells (C) and their projections (P) in the ZP, of the PVS (*) and of the ooplasm.c, d : light ooplasm and PVS (*) labeling.

Figure 4

Light microscope autoradiography of oocytes incubated after GVBD for 3 hrs in the presence of ³H-glucosamine and chased until MII after initiation of maturation in vivo (a, b, c) or in vitro (d). bar = 5 μm. a and b : successive semi-thin sections show similar label accumulation in the PVS (*) around the first polar body (PB).c : accumulation in the PVS (*) away from the polar body.d : after complete in vitro maturation, the PVS (*) is also heavily labeled. Some grains are found around remaining cumulus cells (C).

Figure 5

Effect of Tunicamycin and hyaluronidase on continuous incorporation of ³H-glucosamine from GVBD stage until MII after initiation of maturation in vivo (a, b, c, d) or in vitro (e, f, g, h). bar = 10 μm a : labeling of corona cells (C), of the surface of the ZP (Z), of the PVS (*) (accumulation) and of the ooplasm in a control oocyte.b : hyaluronidase treatment removes most of the labeling.c : after Tunicamycin treatment, labeling is accumulated in the PVS (*), around the polar body (PB) and on the surface of the ZP (Z).d : after Tunicamycin and hyaluronidase treatment, labeling is reduced.e : labeling of control oocyte, similar to : a.f : after hyaluronidase treatment, labeling in PVS (*) is reduced.g : heavy labeling specially of corona cells (C), ZP surface (Z), PVS (*) after Tunicamycin treatment.h : after Tunicamycin and hyaluronidase treatment, labeling of PVS (*) and ZP (Z) surface is reduced.

Figure 6

Labeling of fertilized eggs incubated in presence of ³H-glucosamine during the whole in vitro maturation and collected 20 hr after transfer in vivo in an inseminated gilt. bar = 10 μm a : heavy PVS (*) labeling still visible.b : ooplasm labeling around a pronucleus (N).c : similar labeling in an oocyte treated with Tunicamycin during maturation.