In vitro oocyte maturation in Canids: biological and technical pitfalls

The efficiency of oocyte in vitro maturation in Canids is characterized by a low and greatly variable success. The reason may lie in a very peculiar morphological and functional characteristic of Canid oocyte folliculogenesis and maturation. The selection of oocytes which had acquired the ability to undergo meiotic maturation, through adequate intrafollicular growth, is important for in vitro maturation. Moreover, the identification of parameters indicative of meiotic competence could help in the understanding of the mechanisms which limit the maturation rates in these species. In dogs some of these parameters have already been identified. It has been shown that the size of oocytes is important for their ability to mature in vitro and oocytes with a diameter >100 m have a better chance to reach the metaphase II (Hewitt and England, Theriogenology 1998, 49:957-966). In addition, it has been reported in the fox that oocytes with diameters up to 100 m are meiotically incompetent (Srsen et al., Zygote 1998, 6:299-309). Moreover, advanced preantral and early antral follicles dissected from canine ovaries and cultured in vitro show that their oocytes are competent to resume meiosis to the metaphase II stage (Bolamba et al., Theriogenology 1998, 49: 933-942). Preovulatory maturation is required for the normal development of canine oocytes, although they are ovulated at the germinal vesicle stage. In fact, canine oocytes collected from ovaries before the preovulatory intrafollicular maturation (anoestrous) have lower rates of maturation than oocytes collected from preovulatrory follicles of superovulated bitches (Yamada et al., J. Reprod. Fertil. 1993 Suppl. 47: 227-229). Oocyte and cumulus cell morphology is also important for selecting oocytes. Nickson et al. (J. Reprod. Fertil. 1993 Suppl. 47: 231-240) reported that only oocytes with at least two layers of closely applied cumulus cells are committed to develop in culture. The immature stage of oocytes at ovulation and the persistence of cumulus cells that remain attached in a tight and multilayered mass during the transport and maturation period within the oviduct suggested that the investigation of the communications through gap junctions between the somatic compartment of the follicle and the oocyte, could help to identify competent oocytes. It is well known that such communications are involved in the acquisition of meiotic and developmental competence, and it has been reported in the fox that all junctional contacts between cumulus cells and oocyte are disrupted when metaphase I is reached in vivo (Hyttel et al., Anat Embryol. 1990, 181: 325-331). Moreover, the cumulus, mainly corona radiata cells, controls resumption of meiosis of fox oocytes either in vivo or in vitro conditions (Srsen et al., Zygote 1998, 6:299-309). Recent results (Luvoni et al., J. Reprod. Fertil Suppl., in press) have demonstrated that the functional status of cumulus cells-oocyte communications, through gap junctions, is influenced by the stage of the cycle, and that oocytes collected during late proestrous are capable of completing meiosis at a higher rate than oocytes collected during anoestrous. This suggests that the stage of the cycle at the time of collection influences oocyte meiotic competence. Thus, oocyte diameter, cumulus conformation as well as follicular developmental stage and stage of the cycle are important selection parameters for successful oocyte in vitro maturation in Canids.