In Vitro Fertilization and Cortical Granule Distribution of Bovine Oocytes Having Heterogeneous Ooplasm with Dark Clusters.

Abstract

ABSTRACT. In vitro maturation, fertilization and subsequent development of oocytes with homogeneous (category 1), or heterogeneous ooplasm (category 2) were investigated. No significant differences were observed in the nuclear maturation and total fertilization rates between the two categories. However, category 2 oocytes showed a higher normal fertilization rate due to their lower incidence of polyspermy as compared to category 1 oocytes. Electron microscopic study revealed that all category 2 oocytes had cortical granules lined up next to the plasma membrane, and that some category 1 oocytes still had small clusters of cortical granules after maturation. Although the proportion of cleaved zygotes was higher in category 2, the percentages of cleaved zygotes that developed to the blastocyst stage did not differ between the two categories. These results demonstrate that oocytes with heterogeneous ooplasm have a higher capacity for normal fertilization due to the reduction in polyspermy. This can be attributed to the normal distribution of cortical granules in category 2 oocytes after maturation. -KEY WORDS: bovine, cortical granule, fertilization, morphology.J. Vet. Med. Sci. 61(5): 531-535, 1999 Sigma Chemical Co., St. Louis, U.S.A.), 0.2 mM sodium pyruvate (Sigma) and 50 µg/ml gentamicin sulfate (Sigma). During the washing procedure, oocytes were examined, selected and classified under a stereomicroscope by the following criteria. Category 1 represented oocytes with homogeneous ooplasm and multilayered cumulus investment. Category 2 represented oocytes having heterogeneous ooplasm with dark clusters, and multilayered cumulus investment.In vitro maturation, fertilization and culture: In vitro maturation, fertilization and culture were carried out using the procedure described previously [15]. Briefly, the COCs were washed once in the maturation medium; 25 mM Hepesbuffered TCM 199 supplemented with 10% fetal calf serum (Gibco Laboratories, Grand Island, NY, U.S.A.), 0.02 units/ ml follicle-stimulating hormone (Sigma), 1 µg/ml estradiol-17β (Sigma) and 0.2 mM sodium pyruvate and 50 µg/ml gentamicin sulfate. The oocytes in each category were cultured separately for 22 hr in a 50 µl droplet of maturation medium (10 or 11 oocytes per droplet) covered with paraffin oil at 39°C in a humidified atmosphere of 5% CO 2 in air. Frozen bovine semen collected from a Holstein bull was used for in vitro fertilization. After thawing the semen in a 37°C water bath for 30 sec, the motile sperm were separated using a discontinuous gradient with 45 and 90% Percoll (Pharmacia BioProcess, Uppsala, Sweden) by centrifugation at 700 × g for 20 min. The sperm were subsequently washed with a modified Brackett and Oliphant (mBO) medium [3] by centrifugation at 500 × g for 5 min. The mature oocytes were co-incubated with the sperm at the concentration of 5 × 10 6 sperm/ml in a 100 µl droplet of mBO medium containing 3 mg/ml fatty-acid-free BSA (Sigma) and 2.5 mM theophylline (Sigma) covered with paraffin oil for 20 hr at 39°C in a humidif...

Relationships of Follicle Versus Oocyte Maturity to Ultrasound Morphology, Blood Flow, and Hormone Concentrations of the Preovulatory Follicle in Mares1

“…The changes in percentage of dark clusters in the ooplasm apparently involve the rearrangement of vesicles and lipid droplets from an even distribution to a more polarized appearance in mares [16]. The positive association between the percentage of ooplasm with dark granules and the oocyte maturation rate in the present in vivo study with equine oocytes is consistent with an in vitro study in cattle that indicated that oocytes with dark clusters of granules had a higher potential for maturation [31]. a Within oocyte recovery groups and within oocyte maturity groups, means are significantly different or approach being significantly different as follows: *** P , 0.001; ** P , 0.01; * P , 0.05; # P , 0.1.…”

In vitro maturation of porcine oocytes with retinoids improves embryonic development

“…The better developmental competence achieved by oocytes, and consequently increases in blastocyst formation, after exposure to 5 nm RA during IVM could be related to more complete granular migration in the matured oocyte cytoplasm induced by RA, as suggested to occur in bovine oocytes . This migration provides a block to polyspermy once migrated cortical granules (CGs) have been released (Wang et al 1997b;Nagano et al 1999). It is well known that CGs play an important role in the block to polyspermy in mammalian oocytes (for a review, see Yanagimachi 1994).…”

ATP content and maturational/developmental ability of bovine oocytes with various cytoplasmic morphologies

“…Bovine ovaries obtained at a local abattoir were kept in normal saline at 25–30 °C and delivered to the laboratory within 6 h of their collection. Cumulus–oocyte complexes (COCs) were collected from small antral follicles (2–8 mm in diameter) by aspiration as des-cribed previously (Nagano et al ., 1999). All COCs were washed twice in HEPES-buffered Tyrode's medium (TALP–HEPES) (Bavister et al ., 1983) supplemented with 3 mg/ml of bovine serum albumin (BSA, fraction V, Sigma), 0.2 mM sodium pyruvate and 50 μg/ml of gentamicin sulfate (Sigma).…”