Category: Embryo

DISCUSSION

This is the first examination of the impact of permeating cryoprotectant (PrOH or EG) on the immature cat oocyte and its ability to undergo subsequent maturation, spindle formation, fertilization, and development in vitro. Although high proportions of oocytes resumed meiotic maturation after CPA exposure, progression past the MI stage generally was impaired at high CPA concentrations. All CPA treatments adversely influenced MII spindle integrity except 1.5 M PrOH at 25°C. When CPA treatment affected the MII spindle integrity without impacting the percentage of nuclear maturation, fertilization success (on the basis of pro-nuclear formation) was not impaired. However, the prevalence of MII spindle abnormalities was consistently related to poor embryo cleavage and further development in vitro. Both CPA type and concentration dictated eventual embryo development, with EG and higher concentrations being more detrimental than PrOH and lower concentrations.

In the present study, percentage of oocytes remaining at the germinal vesicle stage, degenerating, or reaching the MI stage after CPA exposure were comparable to results obtained using our standard IVM conditions in the absence of CPA exposure. Regardless of the CPA treatment, these immature cat oocytes were able to resume meiosis until the MI stage as reported previously for the immature mouse oocyte. Even though cytoplasmic structures are known to be extremely sensitive to low temperatures, introduction of immature cat oocytes to low temperature in the absence of CPA had no influence on nuclear maturation. Similar findings have been reported earlier in the bovine oocytes.

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RESULTS

Effect of CPA Exposure on In Vitro Nuclear Maturation ofOocytes

In experiment 1, percentage of oocytes remaining at the germinal vesicle stage (range of means, 8.8%-15.2%) was not affected (P > 0.05) by the CPA type, concentration, or exposure temperature and was not different (P > 0.05) from controls (6.5 ± 7.7% at 25°C; 12.9 ± 6.9% at 0°C).

Likewise, percentage of degenerated oocytes (range of means, 4.8%-15.3%) was not influenced (P > 0.05) by the CPA type, concentration, or exposure temperature and was not different (P > 0.05) from controls (6.1 ± 6.8% at 25°C; 13.8 ± 4.5% at 0°C). Percentage of MII oocytes was lower (P < 0.05) after exposure to 3.0 M PrOH at 0°C and 3.0 M EG at both temperatures compared to the other CPA treatments, including controls (Fig. 1). Temperature of CPA exposure had no effect on the percentage of MII oocytes (P > 0.05) except after 3.0 M PrOH exposure at 0°C versus 25°C (P < 0.05). Percentage of MI oocytes after exposure to 3.0 M PrOH at 0°C (39.1 ± 5.2%) or 3.0 M EG (40.9 ± 2.4% at 25°C; 38.8 ± 4.5% at 0°C) was higher (P < 0.05) than the percentage of MI oocytes after other CPA treatments (range of means, 11.9%-18.7%) and controls (17.4 ± 5.5% at 25°C; 18.0 ± 4.7% at 0°C).

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METHODS

Immature Oocyte Collection, Cryoprotectant Exposure, and In Vitro Maturation

Ovaries from adult domestic cats were collected during the breeding season (December-June) from local veterinary clinics and transported to the laboratory within 6 h of ovariectomy in PBS at 4°C. Immature oocytes were recovered by slicing the ovaries with a scalpel blade in H-MEM (Hepes-buffered Minimum Essential Medium; Gibco Laboratories, Grand Island, NY) supplemented with 1.0 mM pyruvate, 2.0 mM glutamine, 100 IU/ml penicillin, 100 |xg/ml streptomycin, and 4 mg/ml BSA (Sigma Chemical Co., St. Louis, MO). Only grade I immature oocytes (with homogeneous dark cytoplasm, surrounded by several layers of compacted cumulus) were selected and pooled before CPA exposure. Immature oocytes were plunged directly in 0, 0.75, 1.5, or 3 M PrOH (Sigma) or EG (Sigma) in PBS containing 20% fetal calf serum (FCS; Irvine Scientific, Santa Ana, CA) for 30 min at room temperature (25 ± 2°C) or in a programmable alcohol bath (Biocool III, FTS system, New York, NY) at 0°C. After CPA exposure, immature oocytes were transferred through droplets with decreasing concentrations of the same CPA (1.5, 0.75, 0.25, and 0 M in PBS + 20% FCS) for 10 min each at 25°C or 0°C to remove CPA in a stepwise fashion. Immature oocytes were then washed extensively and cultured in in vitro maturation (IVM) medium composed of MEM supplemented with 1.0 mM L-glutamine, 1.0 mM pyruvate, 100 IU/ml penicillin, 100 |xg/ml streptomycin, 4 mg/ml BSA, 1 |xg/ml FSH (1.64 IU/ml; NIDDK-ovine FSH-18; National Hormone and Pituitary Program, Rockville, MD), 1 ^g/ml LH (1.06 IU/ml; NIDDK-oLH-25; National Hormone and Pituitary Program), and 1 |xg/ml estradiol (Sigma) for 30 h in 50-|xl microdrops (10 oocytes/microdrop) under equilibrated mineral oil at 38.5°C in 5% CO2 in air.

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INTRODUCTION

Oocyte cryopreservation could be a viable tool for preserving the female genome to assist in genetically managing rare individuals and small populations. However, survival and quality of frozen-thawed oocytes are poor compared to fresh counterparts for a host of mammalian species, including the hamster, rabbit, pig, mouse, cow, and human. Oocyte survival and quality in the domestic cat also appear to be low and variable after freeze-thawing. However, studies in this species have been quite limited, and, compared to other mammals, there have been no thorough assessments to understand cat oocyte sensitivity to any cryoprotectant agent (CPA).

Exposure to a hyperosmotic medium containing CPA is the first potentially damaging step for the oocyte because of both osmotic stress and the inherent toxicity of the CPA itself to cell organelles. Among the most important subcellular component, microtubules (homologous polymers of a- and (3-tubulin) provide a dynamic framework in the oocyte for both nuclear and cytoplasmic events (including mitochondrial and mRNA redistribution) during maturation, fertilization, pronuclear formation, and first mitotic division. Several studies have demonstrated that microtubule organization is altered by nonphysiological conditions such as CPA exposure.

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