251 IS THERE A GAP BETWEEN NUCLEAR AND CYTOPLASMIC MATURATION IN IN VITRO-MATURED EQUINE OOCYTES?

Abstract

It is a general belief that as soon as the oocyte is recovered from the follicular environment, the nuclear maturation starts spontaneously in vitro, while specific stimulation for the cytoplasmic maturation is lacking (Gilchrist and Thompson 2007 Theriogenology 67, 6–15; Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). As both nuclear and cytoplasmic maturation are required to prepare the oocyte for successful fertilization and embryonic development, a defective cytoplasmic maturation might be an important cause of low blastocyst rates in vitro (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Nuclear and cytoplasmic maturation can be evaluated using fluorescent dyes. Assessment of nuclear maturation is typically based on the visualisation of chromatin, whereas cytoplasmic maturation is evaluated by the localization of cytoplasmic organelles [i.e. the cortical granules (CG)]. Equine oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation (IVM; Smits et al. 2010 Vlaams Diergen. Tijds. 79, 134–138), oocytes were fixed and permeabilized. Subsequently, CG were labelled by incubation in 10 µg mL–1 FITC-labelled lens culinaris agglutinin during 15 min at RT. Chromatin was counterstained to verify the nuclear status with 20 µg mL–1 Hoechst 33342 during 15 min at RT. Stained oocytes with no or dispersed chromatin were classified as degenerated. Based on the absence or presence of the first polar body (PB), non-degenerated oocytes were either classified as nuclear immature (MI, no PB present) or nuclear mature (MII, PB present). The non-degenerated oocytes were further subdivided in 3 categories based on the migration of the CG: 1) cytoplasmic immature oocytes with (clusters of) CG randomly distributed throughout the ooplasm, 2) oocytes in transition stage with progressing CG migration to the oocyte cortex, and 3) cytoplasmic mature oocytes with a clearly visible CG monolayer just underneath the oolemma. The mean and standard deviation of nuclear and cytoplasmic parameters were calculated using Excel (Excel 2007, Microsoft Corp., Redmond, WA, USA). In 3 replicates, 86.6 ± 2.75% of all oocytes (131/151) demonstrated a corresponding nuclear and cytoplasmic maturation pattern (MI corresponding to CG1 and 2; MII corresponding to CG3). Only 12.0 ± 2.82% of the oocytes (16/133) revealed a cytoplasmic maturation pattern (CG 1 or 2) that lagged behind the nuclear maturation (MII). On the other hand, 22.2 ± 9.8% of the oocytes (4/18) were already cytoplasmic (CG3) but not yet nuclear matured (MI). Since most of the equine in vitro matured oocytes exhibited, surprisingly, a corresponding nuclear and cytoplasmic maturation pattern, it can be concluded that the gap between the nuclear and cytoplasmic maturation in vitro is less important than is generally believed. Consequently, the IVM step is not the main obstacle to increase the efficiency of the in vitro production process in horses.