Cumulus cell contact during oocyte maturation in mice regulates meiotic spindle positioning and enhances developmental competence

Abstract

Purpose: To investigate the role of cumulus cell contact during oocyte maturation on meiotic spindle assembly and the acquisition of developmental competence.

Methods: Cumulus oocyte complexes isolated from mouse ovaries subjected to in vitro or in vivo maturation were analyzed by confocal microscopy with respect to oocyte somatic cell contacts and for their ability to develop after parthenogenic activation during embryo culture.

Results: Cell contact is maintained during maturation in vivo, predisposing oocytes to cortical meiotic spindle assembly and developmental competence acquisition. In contrast, oocytes matured in vitro lose cell contact coincident with central meiotic spindle assembly that results in cleavage delays upon egg activation and failure to form blastocysts. Experimental disruption of cell contact by the actin-depolymerizing agent latrunculin B results in the formation of enlarged meiotic spindles with dispersed chromosomes unlike the compact ordering of chromosomes observed on spindles formed after in vivo maturation, suggesting a link between cell contact and the acquisition of developmental competence.

Conclusions: Somatic cell contact optimizes oocyte quality during meiotic maturation by regulating the spatial organization and function of the meiotic spindle through actin-dependent mechanisms that enhance development.

Fig. 1

Comparison of TZP organization in living or fixed oocytes. A Confocal image of vitally stained cumulus oocyte interface [DiO16(3)}shows TZPs extending horizontally(green) or vertically (blue) TZPs relative to the surface of the zona pellucida; projections were pseudocolored using image processing. In contrast, confocal images of fixed oocytes B stained with rhodamine phalloidin (red) shows predominantly vertically-oriented TZPs at the oocyte-cumulus cell interface. Arrows depict individual TZPs. Scale bar = 10 μm

Fig. 2

Persistence of TZPs during oocyte maturation in vivo. COCs were isolated at T = 0, 2, 4 and 6 h following hCG and stained for detection of f-actin (red), microtubules (green) and DNA (blue) as described in text. Channel specific linescans below each image correspond to intrazonal optical density profile for TZPs and solid bar denotes position of GV or spindle. Note actin TZP density remains elevated and concentrated near the GV (0,2, and 4 h) and by 4 h post microtubule-rich TZPs become apparent. Insets show enlarged area of zona containing TZPs. Scalebar = 10 μm

Fig. 3

TZP retraction and GV centration occur during in vitro maturation. Confocal projections of COCs at 2(A and D), 4(B and E) and 6(C and F) hours of in vitro maturation in IVMb (A–C) or IVM + medium (D–F). Linescans below each image denote position of GV or spindle with a horizontal bar. In IVMb medium, GVs are centrally located (n = 30) and low levels of actin TZPs are evident (A–C; and compare to Fig. 2). In contrast, oocytes matured in IVM + medium display cortical GVs or spindles (n = 30) and maintenance of actin TZPs (D–E). Insets demonstrates TZPs. Scalebar = 10 μm

Fig. 4

GV positioning during in vitro maturation in living oocytes. DIC time-lapse imaging was used to map GV position during in vitro maturation in either IVMb or IVM + media. Left panel (A and C) shows GV position (black circles) at the start of imaging (n = 20–24 oocytes in each treatment group) and GV position is shown just prior to GVBD on the right for the same oocyte. Central (A,B) or cortical (C,D) patterns were observed in IVMb or IVM + respectively. Scale bar (A and B) represents 30 μm. Scale bar (C and D) represents 20 μm

Fig. 5

GV enlargement accompanies centration during IVM. Mean GV diameters were calculated for 64 COCs during IVM and are plotted as a function of IVM conditions (IVMb, IVM+) and as to their original GV location (stayed in cortex; migrated from cortex to center; or stayed in center. Note that a fixed cortical position occurs only under IVM + conditions and GVs remain smaller in diameter whereas in all other cases, GV centration results in larger GVs. Asterisks (*) denote statistical significance between treatment groups at P < 0.01

Fig. 6

Latrunculin B severs TZPs but does not impair meiotic progression. COCs incubated in control (IVM+) medium (A,B) or medium supplemented with Latrunculin B for 6 h were fixed at either 6 (A,C) or 16 (B,D) hours and analyzed by confocal microscopy. Representaive images are shown from 10 oocytes per treatment group and time point and in D, the effects of washing out Latrunculin show that the ability to extrude the fist polar body has not been impaired due to prior drug exposure. Scalebar = 25 μm and magnification is same for A/C and B/D

Fig. 7

Cumulus cell contact and actin integrity influences inter-chromosomal spacing in MI. 10–15 Distances between adjacent chromosomes were calculated from confocal z-stacks of MI oocytes matured under IVO or IVM conditions as described before. Furthermore, oocytes were subdivided a prioi with respect to whether cumulus cells were retained (C+) or not (C-) At least 10 oocytes from each group were measured ^-10 chromosomes/group) and inter-chromosome spacing is plotted in microns. Note that Coocytes exhibit a 2–3 fold increase in chromosome spacing and that latrunculin B treatment caused an increase independent of maturation conditions or cumulus cell attachment. *, Denotes statistical significance compared to all groups, P < 0.01, **, Denotes statistical significance compared to matched LatB group, P < 0.01