Mouse oocytes connect with granulosa cells by fusing with cell membranes and form a large complex during follicle development

Abstract

Proper development and maturation of oocytes requires interaction with granulosa cells. Previous reports have indicated that mammalian oocytes connect with cumulus cells through gap junctions at the tip of transzonal projections that extend from the cells. Although the gap junctions between oocytes and transzonal projections provide a pathway through which small molecules (<1 kDa) can travel, it is unclear how molecules >1 kDa are transported between the oocytes and cumulus cells. In this study, we presented new connections between oocytes and granulosa cells. The green fluorescein protein Aequorea coerulescens green fluorescein protein (AcGFP1) localizing in oocyte cell membrane, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and dextran conjugates (10,000 MW) injected into the oocytes, which were unable to pass through gap junctions, were diffused from the oocytes into the surrounding granulosa cells through these connections. These connect an oocyte to the surrounding cumulus and granulosa cells by fusing with the cell membranes and forming a large complex during follicle development. Furthermore, we show two characteristics of these connections during follicle development-the localization of growth and differentiation factor-9 within the connections and the dynamics of the connections at ovulation. This article presents for the first time that mammalian oocytes directly connect to granulosa cells by fusing with the cell membrane, similar to that in Drosophila.

Figure 1.

Localization of AcGFP1 in the follicle and the cumulus-oocyte complex. (A and B) Follicles expressing AcGFP1 in the cultured ovary of an Oog1pro3.9 mouse. (C–F) Follicles stained using anti-GFP antibody (green), phalloidin (red), and DAPI (blue). (C) Primary follicle. (D) Secondary follicle. (E) An early antral follicle. (F) Graafian follicle. Scale bars, 50 μm.

Figure 2.

Water-resistant gel paste containing DiI injected onto the oocyte in the prepared cytosection of the ovary. (A, B, E, G) 1 h after injection. (C, D, F, H) 10 h after injection. (E–H) Enlarged images between the oocyte and granulosa cells in (D), (E) and (F), (G) and (H) are at the same location. The asterisk in (A) denotes injected water-resistant gel paste containing DiI. The arrows in (E–H) indicate CCOCs between the oocytes and granulosa cells. DiI is red in (B) and (C), and white in (D–H). Notes: o, oocyte. c, cumulus cell. Scale bar, 50 μm.

Figure 3.

Localization of AcGFP1 in CCOCs and TZPs. (A–D) Immunohistochemistry images. Anti-GFP antibody (green), phalloidin (red), and DAPI (blue). (B, C, D) Elongated images of the square area in (A). Arrows indicate AcGFP1-positive CCOCs. (E–H) Immunoelectron microscope images between an oocyte and granulosa cells. Black dots denote the signal of anti-GFP antibody. (F, G) Enlarged images of the area enclosed by a square in (E), respectively. (H) Negative control stained rabbit IgG. Notes: o, oocyte. g, granulosa cell. zp, zona pellucida. TZPs, transzonal projections. The white scale bar in (A) and (B): 20 μm; the black line in (E): 1 μm; the dashed line in (F): 200 nm.

Figure 4.

Dextran conjugates with Alexa594 injection in the oocyte of a cultured ovary. (A, B) 1 h after injection. (C, D) 46 h after injection. Dextran conjugates are red in (A) and (C) and are white in (B) and (D). Notes: o, oocyte. g, granulosa cell. Scale bar, 50 μm.

Figure 5.

Electron microscope images. (A–G) Between an oocyte and the granulosa cells in the primary follicles. (B, D, F, G) Enlarged images of the area enclosed by a square in (A), (C), and (E), respectively. Notes: o, oocyte. g, granulosa cell. zp, zona pellucida. Scale bars, 2 μm (line) and 1 μm (dashed line).

Figure 6.

The role of CCOC in follicle development. (A–C) Secondary follicle images stained with anti-GFP antibody (green), anti-GDF-9 antibody (red), and DAPI (blue). The arrows in (B) and (C) denote localization of GDF-9 in AcGFP1-positive CCOCs. (D–K) Time-lapse images of a cultured ovary in aOog1pro3.9 mouse. (D, F, H, J) Bright-field images. (E, G, I, K) AcGFP1 images. (D, E) 43 h culture. (F, G) 51 h culture. (H, I) 55 h culture. (J, K) 59 h culture. Notes: o, oocyte. g, granulosa cell. Scale bar, 100 μm.