Cumulus-denuded feline oocytes cultured in vitro in a three-dimensional alginate scaffold

Introduction and aim. Cumulus-denuded oocytes (CDOs) are generally not included in the IVM procedure due to their poor nuclear/cytoplasmic competence caused by the lack of surrounding cumulus cells. In endangered species or prepubertal females, they might represent the only source of genetic material. Even in thawed oocytes, the communication between germinal and somatic compartments is compromised for the detrimental effect of cryoprocedures, and their achievement of full maturational competence is greatly variable (1). To improve the CDOs performances in vitro, enriched culture conditions could be developed. Recently, bioengineering and nanotechnology research have been focused on innovative three-dimensional (3D) culture systems to mimic the in vivo follicular architecture and cellular spatial arrangement. The encapsulation of oocytes in a biocompatible matrix allows the maintenance of their physiological structure and behavior (2), as demonstrated in different species (mouse, 3; pig, 4; human, 5), but not in cat. In the present study viability and maturation rates of feline CDOs cultured in barium alginate capsules (3D) or in the traditional drops (2D), with or without cumulus-oocytes complexes (COCs) as companion cells, were investigated. Materials and methods. Grade I COCs (n=282) were collected from ovaries of domestic queens; 174 COCs were mechanically deprived of cumulus cells with a small-bore pipette. Cumulus-denuded oocytes (CDOs), with or without companion COCs, were encapsulated in barium alginate (Sigma Chemical Co., MO, USA) capsules or placed in drops (50 l) of IVM medium (Kreb’s Ringer bicarbonate buffered salt solution supplemented with antibiotics, 3 mg/mL BSA, 0.5 IU/mL eCG, 1 IU/mL hCG, 10 ng/mL EGF and 0.6 mM cysteine; Sigma) and cultured for 24h in a controlled atmosphere (38.5°C and 5% CO2 in air). Oocyte viability and nuclear status were assessed at the end of culture by staining with fluorescein diacetate–propidium iodide (FDA–PI, Sigma) and bisbenzimide (Hoechst 33342, Sigma), respectively. Data were analyzed by Chi-square test and the level of significance was set at p<0.05. Results. The results showed that overall viability is similar in CDOs cultured in 3D capsules and those cultured in 2D drops (81.5% vs 75%), but in capsules the presence of companion COCs resulted in a higher viability than that obtained without COCs or in 2D culture (91.1% vs 71.2%; p<0.01. 91.1% vs 67.3%; p<0.005, respectively). A trend towards higher, although not significant, rates of resumption of meiosis (RM), including full nuclear maturation (metaphase II: MII), was observed in 3D culture with COCs compared to 3D without COCs (RM 55.4% vs 40.4%; MII 7.1% vs 5.8%). Conclusions. The encapsulation of cumulus-denuded oocytes in a biocompatible scaffold with COCs as companion cells increases their viability and slightly improves nuclear competence in vitro as already shown in 2D system (6,7). Other than the beneficial effect of potential paracrine factors produced by the companion cells, an enrichment of capsules with proliferating granulosa cells, under investigation at our laboratory, could make the 3D scaffold more adequate to fulfill the CDOs requirements. References. 1) Luvoni. Reprod Dom Anim, 2012;47 (Suppl. 6):266-8. 2) Desai et al. Reprod Biol Endocrin, 2010;8:119-31. 3) Pangas et al. Tissue Eng, 2003;9:1013-21. 4) Munari et al. Vet Res Commun, 2007;31(Suppl. 1):181-4. 5) Combelles et al. Hum Reprod, 2005;20:1349-58. 6) Chigioni et al. Proc. 4th Ann Congr EVSSAR, Amsterdam (The Netherlands), 2005, pp. 24-5. 7) Godard et al. Fertil Steril, 2009;91 (Suppl 5.):2051-60.