Ultrastructural Characteristics of Non-matured and in vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

Objectives - The aim of this experiment is to describe the ultrastructural characteristics of cat oocytes before maturation and after 12 and 24 hour in vitro maturation.

Materials and methods - Oocytes were recovered from pre-pubertal and adult queen ovaries during the non-breeding season (January, February and March) in southeast of Brazil (22°53’ S). Ovaries were recovered after ovariohysterectomy performed at a local veterinary clinic and transported in Dulbecco’s phosphate buffered saline (DPBS; Nutricell®, Brazil) containing 1% antibiotic-antimicotic solution (Sigma®, US) at 4oC using ice packs in a thermal box. Ovaries were processed for gamete collection within 4 hours of gonadectomy. Ovaries were dissected and then sliced into a plastic Petri dish (60 x 15 mm, TPP®, Brazil) containing 5 ml de DPBS (Nutricell®, Brazil) at 38 °C using a scalpel blade to releasecumulus oophorus oocytes (COC). Under stereomicroscope (MZ 125, Leica®, Germany) COC were selected and classified in grade I, II and III. To perform this experiment, only COC grade I, defined by a uniform, dark cytoplasm surrounded by at least four layers of cumulus was selected. Grade I COC were washed three times in Hepes-buffered Minimum Essential Medium (H-MEM; Gibco®, US) supplemented with 3 mg/ml bovine serum albumin (BSA; Sigma®, US), 2.0 mM glutamine (Sigma®, US), 1.0 mM pyruvate (Sigma®, US), 1.2 mM cysteine (Sigma®, US), 100 mg/ml streptomycin and 100 UI/ml penicillin (Gibco®, US). Pre- pubertal and adult non-matured oocytes were stored in glutaraldehyde at 4oC until the performance of transmission electronic microscopy (TEM). Pre-pubertal and adult oocytes from the two in vitro matured groups (12 and 24 hours) were cultured before TEM. Those selected for maturation were incubated (20 to 30/400μl in a four-well dish (Nunc®, Denmark or Ingámed®, Brazil), containing 400 μL DMEM (Dulbecco’s Modified Essential Medium, Gibco®, US) supplemented with3 mg/ml BSA (Sigma®, US), 2.0 mM de glutamine (Sigma®, US), 1.0 mM pyruvate (Sigma®, US), 1.2 mM cysteine (Sigma®, US), 100 mg/ml de streptomycin and 100 UI/ml de penicillin (Gibco®, US), 10μg/mL bovine FSH (Folltropin®- V, Bioniche® Animal Health, Canada), 1μg/ml LH (Lutropin® –V, Bioniche® Animal Health, Canada), 1μg/ml estradiol (Sigma®, US), 20 ng/ml insulin-like growth factor I (IGF-I, Sigma®, US) e 10 ng/ml basic fibroblast growth factor (bFGF, Sigma®, US) for 12 and 24 hours at 38°C in humidified environment of 5% de O2, 5% CO2 e 90% N2. After the maturation time, oocytes were stored in glutaraldehyde at 4oC until TEM performance. Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0); non-matured oocytes from adult queens (A0); 12-hour in vitro matured oocytes from pre-pubertal queens (PP12); 12-hour in vitro matured oocytes from adult queens (A12); 24- hour in vitro matured oocytes from pre-pubertal queens (PP24) and 24-hour in vitro matured oocytes from adult queens (A24).

Results - A gradual increase on thickness could be observed in the perivitelline space (PVS) of PP0 compared to PP12 and to PP24. This same feature was observed in all A group, where the most prominent PVS was seen on A24. Cumulus cell projections penetrating the ZP and forming a junctional complex with the oolemma were observed in all PP group and in A0 and A12. In these same groups the presence of microvilli (MV) was less evident. Only A24 presented an increased amount of MV. In the cytoplasm of PP0, numerous small vesicles were evenly distributed in the ooplasm. On the other hand, PP12 presented very little vesicles and great lipid droplets (LD). PP24 are characterized by a great amount of vesicles and a reduced number of LD. All A group were characterized by a reduced number of lipid droplets. Clusters of mitochondria were observed in PP0 only in the cortical zone. PP12, PP24, A0 and A12 were also characterized by peripheral mitochondrial (M) clusters, but greater clusters could also be seen centrally in the cytoplasm. Differently, A24 were characterized by evenly distributed M within the ooplasm. In PP0 and A0, cortical granules (CG) were seen only in the peripheral area of the cytoplasm, but the number of these organelles appeared to be lower compared to the other groups; and very developed Golgi complexes (GC) were often seen in association with these granules. CG in PP12 and in A12 was higher in the peripheral area, even though, they were also present in central regions of the ooplasm. In all PP group and in A0 and A12, a well developed GC was observed. PP24 and A24 present evenly distributed CG. These observations indicate that in vitro maturation was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes in the three studied periods.