MOLECULAR REPRODUCTION AND DEVELOPMENT 75:115–125 (2008) Ultrastructure of Canine Oocytes During In Vivo Maturation CHRISTINE VIARIS DE LESEGNO, 1 * KARINE REYNAUD, 1 CHRISTINE PECHOUX, 2 SANDRA THOUMIRE, 1 AND SYLVIE CHASTANT-MAILLARD 1 1 ENVA, UMR 1198, INRA, CNRS, FRE 2857, Biologie du De´veloppement et Reproduction, Maisons-Alfort, France 2 INRA, UR 1196, Ge´nomique et Physiologie de la Lactation, Plateforme de Microscopie E ´ lectronique, Jouy-en-Josas, Cedex, France ABSTRACT The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmis- sion electron microscopy (TEM), either before the LH surge (n ¼ 10), between the LH surge and ovulation (n ¼ 12) or after ovulation (n ¼ 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferen- tially located in the cortical zone, ‘‘mitochondrial cloud’’, scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption). Mol. Reprod. Dev. 75: 115–125, 2008. ß 2007 Wiley-Liss, Inc. Key Words: oocyte maturation; canine; in vivo meiosis; ultrastructure INTRODUCTION Canine reproduction is quite unusual compared to that of other mammals. It was reported almost a century ago (Van der Stricht, 1908) that canine oocytes are ovulated at an immature Germinal Vesicle (GV) stage instead of a metaphase II stage ready to be fertilized. The bitch oocytes reach the metaphase II stage only after 54–72 hr spent within the oviducts (Tsutsui and Shimizu, 1975; Reynaud et al., 2005). The cause of this delay and the factors triggering the resumption of meiosis are still unknown and very few studies have focused on these phenomena. In other mammals, the LH surge triggers meiotic resumption by inducing a rupture in communications between the cumulus and the oocyte and preventing the transfer of inhibitory signals to the oocyte. The oocyte – cumulus communication in the bitch also seems to follow an unusual pattern since the cumulus cells remain strongly attached to the oocyte for several days after the LH peak and ovulation (Reynaud et al., 2006a) and in vitro denudation of oocytes is far more difficult than in other species (Reynaud et al., 2004). The canine LH surge is quite long (36 hr; de Gier et al., 2006) compared to that in the cattle (10 – 12 hr; Lemon et al., 1975). The time between the LH peak in plasma and ovulation is constant in the bitch (48 hr [36–50]; Phemister et al., 1973; Concannon et al., 1989; England and Yeager, 1993; Marseloo et al., 2004; de Gier et al., 2006) but the time between the LH surge and meiotic resumption is particularly lengthy (around 5 days, Reynaud et al., 2005). Altogether these observations raise the unsolved question of the role of the LH surge in canine cumulus–oocyte complex (COC) structure and in meiotic regulation. Moreover, the timing of meiosis resumption is not precisely known. The few studies performed on in vivo maturation of canine oocytes in the peri-ovulatory period are based on light microscopy (Van der Stricht, 1923; Holst and Phemister, 1971; Tsutsui and Shimizu, 1975) or confocal microscopy (Reynaud et al., 2005). These techniques evaluated meiosis resumption through global nuclear or cytoskeleton organization ß 2007 WILEY-LISS, INC. Grant sponsor: French National Academy of Medicine; Grant sponsor: ABIES. *Correspondence to: Christine Viaris de Lesegno, UMR 1198 INRA/ ENVA/CNRSFRE Biologie du De ´veloppement et Reproduction, Ba ˆti- ment Lagneau, Ecole Nationale Ve ´te ´rinaire d’Alfort, 7 avenue du Ge ´ne ´ral de Gaulle, 94704 Maisons-Alfort Cedex, France. E-mail: cviaris@vet-alfort.fr Received 5 January 2007; Accepted 22 March 2007 Published online 2 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20763