Author's address (for correspondence): K Reynaud, ENVA, UMR 1198 Biologie du Développement et Reproduction, F-94700 Maisons-Alfort, France. E-mail: email@example.com
*Present address: Reproduction, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex, France.
In canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (<20%). Several IVM media have been tested without significant improvements. A critical step in the evaluation of culture conditions is the observation of the meiotic stage reached by the oocytes. The present study was designed to investigate the chromatin patterns of in vitro matured oocytes by visualizing Germinal Vesicle (GV) and Germinal Vesicle Breakdown (GVBD) structures at 72 h of IVM. Nuclear stages of 1678 oocytes were evaluated by confocal microscopy after IVM. 1204 oocytes were non-degenerated, and 94.4% were still immature and at GV stage. Five different patterns of chromatin configuration were observed. Higher percentages of oocytes with unmodified GV and with diffuse (58%; Type A) and filamentous chromatin (19%; Type B) were observed in comparison with those with modifications in the GV such as patched chromatin (12.5%; Type C), surrounded-nucleolus (3%; Type D) and in vivo type chromatin/fully grouped chromatin (2.5%; Type E). These results indicate that GVBD (absence of nucleolus, nucleus breakdown) is rarely observed in vitro. The percentage of type C-D-E GVs and MI (meiotic resumption) and of MII (completion of meiosis) can be used to evaluate meiotic resumption after IVM. Our results indicate that although a low number of in vitro matured oocytes exhibit the chromatin configurations observed in in vivo collected oocytes, chromatin changes in the GV can be induced during IVM.
In canine species, the oocyte is ovulated at an immature Germinal Vesicle (GV) stage (Reynaud et al. 2005) and requires intra-oviductal post-ovulatory maturation of 56–72 h to become a fertilizable oocyte. Moreover, meiotic maturation rates obtained after in vitro maturation (IVM) remain low (Luvoni et al. 2005).
In most mammalian species, meiotic resumption is defined as the transition from GV to Germinal Vesicle Breakdown (GVBD) stage, where condensations of chromatin, dissolution of nuclear envelop and disappearance of the nucleolus occur. As GVBD is a short phase, meiotic resumption is confirmed by the extrusion of the first polar body. In mouse and bovine oocytes, MII phase is reached in 80–90% of IVM oocytes (Sorensen and Wassarman 1976; Sirard et al. 1988).
In the bitch, the vast majority of IVM oocytes do not reach the MI or MII phases; thus, an accurate discrimination of GV/GVBD stages is of primordial importance to correctly identify meiotic resumption. The canine oocyte cytoplasm contains lipid droplets that inhibit the visualization of the nucleus by stereomicroscopy. Therefore, confocal microscopy is a relevant tool to observe the immature stages and to help minimizing the rate of undetermined stages in canine oocytes (Saint-Dizier et al. 2004).
In the present study, we performed IVM, observed oocyte nuclear stages by confocal microscopy and proposed a classification of GV stages in the bitch oocytes. These observations aimed at enriching basic knowledge of maturation in canine oocyte and serve as a tool for evaluating the maturation media.
Material and Methods
Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).
Cumulus-Oocyte-Complexes collection and IVM
Canine ovaries have been collected from mongrel and purebred bitches (mean, 3.1 ± 0.3 years) at the diestrous or anoestrous phases. Ovaries were sliced in M199/Hepes medium supplemented with 20% foetal calf serum (FCS; Invitrogen, Cergy-Pontoise, France). Oocytes with regular shape, >130 μm in diameter (zona pellucida included), with homogeneous dark ooplasm surrounded by at least two layers of cumulus cells, classified as Grade 1, were selected and cultured in groups of 20 cumulus–oocyte complexes in M199/20% FCS in 4-well dishes, under a 5% CO2 atmosphere in air, at 38.5°C.
After 72 h of IVM, oocytes were mechanically denuded of cumulus and corona cells and fixed and permeabilized as described previously (Reynaud et al. 2009).
Determination of oocyte chromatin configuration and classification of chromatin patterns
Immunocytochemistry was performed to detect DNA and tubulin, as previously described (Reynaud et al. 2009), and nuclear stages and tubulin status were evaluated by confocal laser scanning microscopy (CLSM 510 Carl Zeiss, Gottingen, Germany).
According to their chromatin aspect/condensation and to the level of tubulin polymerization, oocytes were classified into three groups: (i) GV/GVBD oocytes, (ii) MI/MII oocytes (chromosomes aligned on the metaphase plate, absence (MI) or presence (MII) of a polar body) and (iii) degenerated oocytes (absence of DNA material, abnormal DNA structure). The chromatin configurations of all GV/GVBD oocytes were then observed, and oocytes were classified into groups of homogeneous appearance.
Results are expressed as mean ± SEM.
A total of 1378 oocytes were placed in IVM, and the nuclear status of non-degenerated oocytes (1204) was examined at 72 h of IVM (Table 1). Ninety-four percentage of the analysed oocytes were immature and classified into one of the five nuclear stage types.
Table 1. Degeneration rate and nuclear stages of in vitro matured canine oocytes
GV stages (non-degenerated oocytes)
MI and MII
Type B (filamentous)
Type C (patched)
Oocyte number (n)
Type A nuclear stage, described with a diffuse or a slightly condensed chromatin, was present in 57.8% of the oocytes (Fig. 1A,B). Type B nuclear stage, where the chromatin was filamentous, in the whole nuclear area, and a red-stained nucleolus always present (Fig. 1C,D) was observed in 18.7% of the oocytes. In this group, two nuclear diameters were observed and classified as with a large (Fig. 1C) or small nucleus (Fig. 1D).
Type C nuclear stage, defined with ‘patched chromatin’ and characterized by the presence of a large nucleolus with masses of condensed chromatin throughout the nucleoplasm (Fig. 1E,F), was observed in 12.5% of the oocytes. Similarly to the type B nuclear stage, two nuclear diameters were observed and classified as large (Fig. 1E, 21.2% of the oocytes) and small nucleus (Fig. 1F, 78.8% of the oocytes).
Type D nuclear stage, observed in 3% of the oocytes, was characterized by a chromatin localization restricted around the nucleolus, which appeared unstained (Fig. 1G).
Type E nuclear stage, described as ‘in vivo type’, was observed in 2% of the oocytes analysed (Fig. 1H). Chromatin was less condensed than that of in type D nuclear stage, restricted to part of the nucleus and had similar appearance to that of in vivo collected oocytes (chromatin fully grouped around the nucleus).
In the present study, we described the oocyte GV patterns of canine oocytes after IVM to determine whether immature oocytes from diestrous and anestrous ovaries were able to mimic normal GV rearrangements observed in vivo (Reynaud et al. 2009).
The majority of oocytes analysed were at the type A nuclear stage (diffuse-dots chromatin), which is consistent with the chromatin pattern previously reported in canine oocytes after in vitro culture (Saint-Dizier et al. 2004). Similar diffuse nuclear configuration has been described in other mammalian species, in smaller oocytes collected from small follicles or younger animals (for review, Tan et al. 2009). In mouse, dispersed chromatin was described as ‘non-surrounded nucleolus’ (NSN) and was present in transcriptionally active oocytes. Because type A oocytes are similar to the unmatured oocytes, it suggests that nucleus, in almost 60% of canine oocytes, is not modified during maturation.
The type B nuclear stage (filamentous) was observed in fewer oocytes and comparable to the GV-II stage previously reported in canine oocytes (Jin et al. 2006). Oocytes classified in the type C nuclear stage revealed a DNA condensation similar to that of in GVBD oocytes, suggesting that these oocytes could be classified at GVBD stage. However, in the present study, the nucleolus was still present and the nucleus limits were visible, in contrast, to the nucleolus disappearance and dissolution of the nuclear envelop in GVBD oocytes, explaining why we classified this canine oocytes with DNA condensation at a GV stage. This chromatin pattern is similar to the GV-III stage in the classification of Jin et al. (2006). Type D nuclear stage was rarely observed (3%), and the chromatin arrangement around the nucleolus was comparable to the surrounded-nucleolus (SN) chromatin pattern observed from canine in vivo oocytes (Reynaud et al. 2009) and from in vivo matured mouse and human oocytes (Miyara et al. 2003). Type E nuclear stage was also a rarely observed stage (approximately 2%) but was similar to the nuclear stage reported from in vivo collected canine oocytes (Reynaud et al. 2009).
In canine species, types D and E nuclear stages are probably observed in good quality oocytes, which are originated from the largest follicles present in anoestrous ovaries, and can be used as a marker of good quality oocyte.
Our results indicated that only few oocytes originated from anoestrous ovaries and in vitro matured will present a chromatin configuration close to the ‘in vivo type’. This may be linked to the profound immaturity of anoestrous canine oocytes and/or to the maturation conditions that are not favourable to induce a complete meiotic maturation.
In the mouse, immature GV oocytes are able to reach MII stage without following the ‘classical’ pathway NSN-SN (Debey et al. 1993), which indicates that several pathways of maturation may occur. As a consequence, it is not possible to determine whether canine oocytes classified as types A, B or C could resume meiosis to MII stage.
The authors express their gratitude to Marc Chodkiewicz for careful review of the manuscript.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
K. Reynaud, S. Chastant-Maillard designed the research and wrote the paper. M. Chebrout, C. Tanguy-Dezaux and G. de la Villéon carried out the laboratory experiments and analyzed the data.