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BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027920
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BIOLOGY OF REPRODUCTION 71, 598–604 (2004)
DOI: 10.1095/biolreprod.104.027920
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Effect of 1,2-Propanediol Versus 1,2-Ethanediol on Subsequent Oocyte Maturation, Spindle Integrity, Fertilization, and Embryo Development In Vitro in the Domestic Cat1

Pierre Comizzoli2, David E. Wildt, and Budhan S. Pukazhenthi

Department of Reproductive Sciences, Smithsonian's National Zoological Park, Washington, D.C. 20008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature cat oocyte as a prerequisite to formulating a successful cryopreservation protocol. In experiment 1, immature oocytes were exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25°C) or 0°C for 30 min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure to 3.0 M PrOH at 0°C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control, except 1.5 M PrOH at 25°C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25°C) or not (1.5 M PrOH at 25°C). After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to CPA except after exposure to 1.5 M PrOH at 25°C, a treatment that still allowed ~60% of the oocytes to reach MII and ~20% to form blastocysts.

assisted reproductive technology, early development, in vitro fertilization, meiosis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte cryopreservation could be a viable tool for preserving the female genome to assist in genetically managing rare individuals and small populations [1]. However, survival and quality of frozen-thawed oocytes are poor compared to fresh counterparts for a host of mammalian species, including the hamster [2], rabbit [3], pig [4], mouse [5–7], cow [8–10], and human [11, 12]. Oocyte survival and quality in the domestic cat also appear to be low and variable after freeze-thawing [13, 14]. 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 [15–19]. Among the most important subcellular component, microtubules (homologous polymers of {alpha}- and ß-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 [20–25]. Several studies have demonstrated that microtubule organization is altered by nonphysiological conditions such as CPA exposure [3, 5, 26, 27]. Furthermore, incidence of irreversible spindle damage and subsequent aneuploidy are higher for oocytes exposed to CPA at the metaphase II (MII) compared to earlier meiotic stages [3, 11, 28–31]. Oocytes at the germinal vesicle stage (GV) are therefore considered to be more resistant to these damages. In the immature oocyte at the GV stage, an envelope protects the nuclear DNA, microtubules are not polymerized, and the presence of cumulus cells may protect against rapid influx or efflux of CPA [5, 11]. Furthermore, immature oocytes are readily available in raw ovarian material, do not require exogenous hormonal stimulation to collect, and therefore offer a convenient means of "gamete rescue" for potential in vitro embryo production from genetically valuable individuals [32]. However, it is known that nuclear maturation and subsequent embryo development in the cat is compromised in immature oocytes exposed to CPA and cryopreserved [13, 14], an effect similar to that reported in the mouse and human [11, 30, 33].

Toxicity of the CPA depends on the type, concentration, and temperature of exposure [34, 35]. A common permeating CPA used for mammalian oocytes is 1,2 propanediol (PrOH), which has low toxicity and good ability to support maturation and fertilization after thawing [5–7, 11, 35, 36]. Immature cat oocytes have been cryopreserved in 1.5 or 3.0 M PrOH but without survival success [13]. However, the effects of exposing cat oocytes to PrOH alone have never been examined. Another common permeating CPA, 1,2-ethanediol (EG) [37], also is suitable for immature oocytes that are less permeable as demonstrated in the goat and cattle [38, 39]. Luvoni and Pellizzari [14] demonstrated that exposing immature domestic cat oocytes to 1.5 M EG at room temperature (~25°C) has no adverse effect on ability to resume nuclear maturation, but there were poor fertilization and developmental competence of the oocytes after thawing that suggested likely disruption of one or more cellular components. Likewise, there have been no studies of the impact of CPA exposure temperature on cell viability in the cat. It is well known in other species that temperature dependence is due to CPA cytotoxicity (reduced by low temperature) but also to osmotic effects (increased by low temperatures) [34, 40].

This study characterized the influence of exposing the immature cat oocyte to various PrOH or EG concentrations at different temperatures. The impact on subsequent nuclear maturation, spindle integrity, fertilization, and developmental competence in vitro was examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immature Oocyte Collection, Cryoprotectant Exposure, and In Vitro Maturation

Ovaries from adult domestic cats were collected during the breeding season (December–June) [41] 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 µg/ml streptomycin, and 4 mg/ml BSA (Sigma Chemical Co., St. Louis, MO) [41, 42]. Only grade I immature oocytes (with homogeneous dark cytoplasm, surrounded by several layers of compacted cumulus) [43] 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 µg/ml streptomycin, 4 mg/ml BSA, 1 µg/ml FSH (1.64 IU/ml; NIDDK-ovine FSH-18; National Hormone and Pituitary Program, Rockville, MD), 1 µg/ml LH (1.06 IU/ml; NIDDK-oLH-25; National Hormone and Pituitary Program), and 1 µg/ml estradiol (Sigma) for 30 h in 50-µl microdrops (10 oocytes/microdrop) under equilibrated mineral oil at 38.5°C in 5% CO2 in air.

Immunostaining of In Vitro-Matured Oocytes

In experiment 1, cumulus cells were removed after IVM by vortexing at maximum setting (Vortex-Genie, Scientific Industries, Bohemia, NY) for 3 min. Denuded oocytes were then fixed in 2.5% paraformaldehyde for 30 min at 38°C. After three washings in PBS, the nonspecific antigenic sites were saturated in a solution of PBS with 0.5% Triton X100 and 20% FCS for 30 min at 38°C. Oocytes were then incubated overnight at 4°C with anti-{alpha}-tubulin monoclonal antibody (Sigma) diluted 1/2000 in PBS containing 0.5% Triton X100 and 2% FCS. After three washings (15 min each) in PBS, oocytes were incubated with a FITC-labeled anti-mouse IgG (Sigma) diluted 1/150 for 1 h at 38°C. Chromatin was then stained with Hoechst 33342 (1 µg/ml; Sigma) in PBS for 5 min at 38°C. Oocytes were mounted on ring Teflon slides with Vectashield medium (Vector Laboratories, Burlingame, CA) under coverslips sealed with nail polish. Oocytes were then observed with a microscope fitted with epifluorescence (Olympus BX 41, Olympus Corporation, Melville, NY) and with a confocal microscope (LSM 510, Carl Zeiss Inc, Drive Thornwood, NY) to analyze meiotic spindles and capture images.

In Vitro Fertilization of Oocytes and In Vitro Development

In experiments 2 and 3, immature oocytes were subjected to in vitro fertilization (IVF) after CPA exposure and IVM. IVF was performed using a standard protocol originally developed in our laboratory [42]. Briefly, frozen-thawed motile spermatozoa from a single sperm donor were selected by swim-up processing [44] in Ham F-10 medium (Irvine Scientific) supplemented with 25 mM Hepes, 1.0 mM pyruvate, 2.0 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5% fetal calf serum (complete Ham with Hepes). Oocytes were inseminated with 5 x 105 motile spermatozoa/ml in 50-µl microdrops (10 oocytes/microdrop) of complete Ham without Hepes under equilibrated mineral oil at 38.5°C in 5% CO2 in air. A portion of immature oocytes (n = 15/CPA exposure) was incubated separately without sperm to assess the incidence of parthenogenetic activation. At 16 h postinsemination, cumulus cells were removed by vortexing at maximum setting for 2 min. In experiment 2, presumptive zygotes and parthenote controls were fixed and stained with Hoechst 33342 [45] to assess fertilization success (presence of two pronuclei) or parthenogenetic activation (presence of one or two pronuclei), respectively. In experiment 3, presumptive zygotes were cultured in vitro for 7 days in 50-µl microdrops (10 embryos/microdrop) of complete Ham (38.5°C; 5% CO2 in air). In vitro development was observed on Day 8 (Day 0 corresponding to the day of in vitro insemination).

Embryo Staining

On Day 8, blastocysts were stained to assess the proportion of inner cell mass relative to the total number of blastomeres according to the method of Thouas et al. [46]. Blastocysts were incubated for 30 sec in PBS containing 1% Triton X100 (Sigma) and 100 µg/ml Propidium Iodide (Sigma) to stain the trophectoderm cells. Embryos were then fixed overnight at 4°C in ethanol with 25 µg/ml Hoechst 33342 (inner cell mass staining) before mounting on slides with Vectashield medium (Vector Laboratories) under coverslips sealed with nail polish. Other embryos were fixed and stained with Hoechst 33342, as previously described [45], to determine the number of blastomeres. Stained embryos were then observed using epifluorescence (Olympus).

Experimental Design and Statistical Analysis

In experiment 1 (control, n = 165 total oocytes; 0.75 M PrOH, n = 157; 1.5 M PrOH, n = 158; 3.0 M PrOH, n = 162; 0.75 M EG, n = 161; 1.5 M EG, n = 161; 3.0 M EG, n = 160), nuclear maturation was expressed as the number of MII oocytes relative to the total number of oocytes placed in culture. The percentage of degenerated oocytes was expressed as the number of degenerated/dead oocytes (fragmented cytoplasm, abnormal chromatin) relative to total number of oocytes. Percentage of abnormal MI was expressed as the number of abnormal MI (based on microtubule and/or chromatin organization) relative to total number of MI oocytes. Percentage of abnormal MII was expressed as the number of abnormal MII (microtubule and/or chromatin organization) relative to the total number of MII oocytes. Experiments 2 and 3 were designed on the basis of experiment 1 results to assess the impact of spindle abnormalities on subsequent embryo development. Oocytes were exposed to CPA conditions yielding optimal nuclear maturation with high (0.75 or 3.0 M PrOH or 1.5 M EG at 25°C) or normal (1.5 M PrOH at 25°C) percentages of abnormal MII spindle. In experiment 2 (control, n = 42 oocytes; 0.75 M PrOH, n = 42; 1.5 M PrOH, n = 39; 1.5 M EG, n = 51; 3.0 M PrOH, n = 54), oocytes were considered as fertilized when two pronuclei were present within the cytoplasm at 16 h postinsemination. In experiment 3 (control, n = 110 oocytes; 0.75 M PrOH, n = 117; 1.5 M PrOH, n = 117; 1.5 M EG, n = 122; 3.0 M PrOH, n = 115), oocyte developmental competence was expressed as number of blastocysts relative to the total number of cleaved embryos. For each replicate of the study, grade I immature oocytes [43] from different ovaries were pooled, then randomly and equally allocated to the different treatments. Experiments were replicated three times (experiments 1 and 2) or four times (experiment 3) on different days (one replicate per day) with different batches of oocytes. Data were expressed as mean ± SD. Percentage data were transformed using arcsine transformation before analysis. Comparisons between treatments and among replicates were analyzed by ANOVA, Tukey multiple comparison testing, and the Bartlett test for the homogeneity of the variances. Data not normally distributed were analyzed by Kruskal-Wallis ANOVA on ranks and the Dunn method for all pairwise comparisons. Differences were considered significant at P < 0.05 (SigmaStat, SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of CPA Exposure on In Vitro Nuclear Maturation of Oocytes

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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  		FIG. 1. Percentage of in vitro-matured grade I oocytes reaching the metaphase II after different cryoprotectant exposures (Propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage (three replicates, values are mean ± SD; numbers above bars represent total number of oocytes per treatment). Bars with asterisks differ (P < 0.05) from control. Solid bars, 25°C; open bars, 0°C

Effect of CPA Exposure on the Oocyte Spindle Organization after In Vitro Maturation

As shown in Figure 2, A and B, a compact set of chromosomes aligned on the metaphase plate and a brightly stained, barrel-shaped meiotic spindle represented the normal configuration. Different abnormal microtubule configurations were observed (short spindle or spindle disorganization; Fig. 2, C–F). Chromatin abnormalities (clumped or dispersed chromosomes) were always associated with spindle abnormalities (Fig. 2, C–F). The incidence of disorganized microtubules associated with MI or MII spindles (~35%; Fig. 2, C–E) was always lower (P < 0.05) than the incidence of short spindle (~65 %; Fig. 2F). Percentage of MI oocytes with an abnormal spindle (range of means, 17.8%–22.2%) did not differ (P > 0.05) from controls (16.7 ± 6.7% at 25°C; 20.6 ± 4.8% at 0°C).



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  		FIG. 2. Spindle organization and chromosome arrangement in grade I oocytes matured in vitro after different cryoprotectant exposures (Propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. A) Normal metaphase I. B) Normal metaphase II. C) Abnormal chromosome arrangement (arrow) and spindle organization in metaphase I. D) Abnormal chromosome arrangement (arrow) and spindle disorganization in metaphase II. E) Abnormal chromosome arrangement and tripolar spindle in metaphase II. F) Short spindle (arrow) in metaphase II. Bar = 10 µm

However, percentage of MII oocytes with an abnormal spindle varied with CPA exposure (Table 1). At 25°C and compared to the control, CPA presence led to higher (P < 0.05) percentages of abnormal MII spindles with the exception of the 1.5 M PrOH treatment (P > 0.05; Table 1). Among the different CPA treatments at 25°C, percentage of MII oocytes with abnormal spindle was higher (P < 0.05) after 1.5 M EG exposure than 1.5 M PrOH and after 3.0 M EG exposure than 3.0 M PrOH. At 0°C, highest percentages of abnormal MII spindle (P < 0.05) were observed after exposure to 3.0 M PrOH or 3.0 M EG compared to the control (Table 1). When compared to counterpart values after the same treatment at 25°C, CPA exposure at 0°C resulted in more MII oocytes with abnormal spindles (P < 0.05) in the absence of CPA (the control) and after 1.5 M PrOH treatment. There was no effect of temperature (P > 0.05) after other CPA exposures.


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  		TABLE 1. Percentage of in vitro-matured grade I oocytes at the meta phase II stage with abnormal MII spindle after different CPA treatment (propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. (Mean ± SD)

Relationship Between Spindle Organization after CPA Exposure and Developmental Competence of In Vitro-Matured Oocytes

In this set of experiments, we assessed the impact of different incidences of spindle abnormalities arising from CPA exposure on subsequent fertilization and embryo development when nuclear maturation was not affected. In experiment 2, success of in vitro fertilization was not affected (P > 0.05) by the different CPA treatments (range of means, 54.3%–56.0%) compared to the control (63.0 ± 6.6%). Polyspermy was not observed, and parthenogenetic activation never occurred regardless of the CPA exposure. However, in experiment 3, percentage of cleaved embryos was lower (P < 0.05) after CPA exposure leading to a high percentage of MII spindle abnormalities (0.75 M PrOH, 1.5 M EG and 3.0 M PrOH) compared to the control and to the 1.5 M PrOH treatment (Fig. 3A). Blastocyst yield was lower (P < 0.05) after exposure to 0.75 M PrOH and lowest (P < 0.05) after exposure to 1.5 M EG and 3.0 M PrOH compared to the control and to 1.5 M PrOH treatment (Fig. 3A). Percentage of embryos with ≤16 cells relative to the total number of cleaved embryos was higher (P < 0.05) for all groups (0.75 M PrOH, 53.8 ± 9.1%; 1.5 M PrOH, 42.7 ± 4.3%; 1.5 M EG, 63.4 ± 3.3%; 3.0 M PrOH, 66.5 ± 7.6%) compared to the control (31.8 ± 1.4%). Percentage of morulae relative to the total number of cleaved embryos was not different (P > 0.05) from the control except after 0.75 M PrOH (Fig. 3B). Percentage of blastocyst relative to the total number of cleaved embryos was lower (P < 0.05) after exposure to 1.5 M EG and 3.0 M PrOH compared to the control and other CPA exposures (Fig. 3B). Total number of blastomeres and cells of the inner cell mass were lower (P < 0.05) after exposure to 1.5 M EG and 3.0 M PrOH compared to the control and the other CPA exposures (Table 2). Furthermore, percentage of inner cell mass was lower (P < 0.05) after 3.0 M PrOH exposure than other CPA treatments, including control (Table 2).



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  		FIG. 3. Developmental competence (7 days of culture in vitro) of in vitro-matured grade I oocytes after different cryoprotectant exposures (Propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. A) Percentage of cleaved embryos (solid bars) and blastocyst yield (number of blastocyst relative to total number of oocytes; open bars). B) Proportion of morula (solid bars) and blastocyst (open bars) stages relative to the total number of cleaved embryos (four replicates, values are mean ± SD; numbers above bars represent total number of oocytes per treatment). Among CPA exposure, bars with different letters differ (P < 0.05)


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  		TABLE 2. Number and characteristic of blastomeres in blastocysts produced in vitro from grade I immature oocytes exposed to various CPA conditions (propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. (Mean ± SD)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 pronuclear 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 [42]. 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 [47]. Even though cytoplasmic structures are known to be extremely sensitive to low temperatures [5, 48], introduction of immature cat oocytes to low temperature in the absence of CPA had no influence on nuclear maturation [42]. Similar findings have been reported earlier in the bovine oocytes [49]. In contrast to observations by Luvoni and Pellizzari [14], we found no detrimental impact of 1.5 M EG at 25°C on nuclear maturation of the immature cat oocyte. However, we did observe a similar percentage of meiotic arrest to that reported by Luvoni and Pellizzari [14] after treating immature cat oocytes with 3.0 M EG at 25°C.

Our detection of altered spindle lengths and spindle organizations after CPA treatment of cat oocytes was similar to what has been reported for cooled bovine oocytes in the absence of CPA [50]. Of the mammalian species studied to date, the formation of MII spindle in the cat oocyte may be most sensitive to CPA exposure since incidence of abnormalities was higher than that reported in frozen-thawed immature mouse [5, 47] or human [51] oocyte. Despite the fact that there was no overall reduction in meiotic progression from germinal vesicle breakdown (GVBD) to MI in CPA-exposed cat oocytes, higher CPA concentrations (both PrOH and EG) were associated with the lowest percentages of nuclear maturation (arrest at MI). The MI block and erratic MII spindle observed here could be due to detrimental influences on mitogen-activated protein (MAP) kinase activities that are initiated after GVBD and remain at a high level throughout the transition from MI to MII [25]. Other detrimental effects could include disruption of microtubule assembling or depolymerization/polymerization dynamics [3, 7], which leads to abnormal MII as asserted by Park et al. [26] for the human oocyte. Furthermore, similar to that observed earlier for the human oocyte, we observed no effect on cat spindle formation after 1.5 M PrOH exposure at 25°C [26]. Nonetheless, there was a concentration gradient with a detrimental influence observed at 3.0 M PrOH that we suspect resulted from the transient extreme dehydration of the oocyte that likely led to a destabilized cytoskeleton and disruption of anchorage of cytoskeletal elements to the oocyte organelles [5]. Overall, EG appeared to be more detrimental to the cat oocyte than PrOH perhaps because the former tends to damage M-phase promoting factor (MPF) and MAP kinase regulations (c-mos, cyclin B) or other factors influencing meiotic progression and cytoskeletal organization during the MI-to-MII transition [23, 50]. The specific disruptive mechanisms remain to be elucidated in the cat and are the focus of our ongoing studies.

The present study revealed an interesting effect of CPA concentration and temperature on nuclear maturation and meiotic spindle integrity. Specifically, exposure to a low temperature did not decrease the toxic impact of CPA exposure on the meiotic resumption and the microtubule organization contrary to results reported in other mammalian species [34, 52]. It is likely that at 25°C, 1.5 M PrOH had no effect on the dynamic assembly and disassembly of microtubules or the MAP kinase activity in the domestic cat. Since exposures of oocytes to 1.5 M PrOH and 0 M PrOH at 0°C both negatively affected spindle integrity, the observed spindle abnormalities might have primarily resulted from low temperature and not from CPA treatment.

Perhaps the most important finding from the present study was that early oocyte exposure to CPA had a negative effect on cytoplasmic maturation and subsequent embryo development in vitro. Microtubules are known to mediate cytoplasmic maturation during early stages of meiosis resumption by facilitating mitochondrial and mRNA distribution [21, 24]. Thus, cytoplasmic maturation of oocytes (based on their developmental competence) was assessed after CPA exposure leading to similar percentage of nuclear maturation but different incidences of abnormal MII spindle. Introduction of CPA at the germinal vesicle stage influenced neither fertilization success (sperm penetration followed by pronuclear formation) nor the incidences of polyspermy or parthenogenetic activation. Exposure to 1.5 M PrOH at 25°C failed to interfere with cell cycle regulatory mechanisms and had no apparent influence on chromosome integrity or cytoplasmic maturation since developmental competence was comparable to controls. However, at a lower concentration (0.75 M PrOH), there was a toxic influence on MII spindle formation that impaired the first cell cycle and the first cleavage. However, developmental competence (after the first cleavage) was normal, similar to what has been reported for immature bovine oocytes [53]. The toxic effect of 3.0 M PrOH was illustrated by a low cleavage rate, low developmental competence, and poor embryo quality (reduced number of blastomeres). This high concentration seemed to be expressed as an inherent toxicant combined with an osmotic effect that was detrimental to the first cell cycle, developmental competence, and embryo quality, again all similar to what has been reported for the immature bovine oocyte [18]. Meanwhile, the control cat embryos were comparable in terms of blastomere number and percentage of ICM to values previously reported in cow [54] or cat [55] embryos. Therefore, we speculate that the high incidence of MII spindle abnormalities induced by CPA treatment resulted in increased incidence of aneuploidy and nondisjunction anomaly in chromosome position leading to a poor developmental competence [3]. Deficient chromatin remodeling during the first cell cycle or impaired embryonic genome activation also might have given rise to the arrest at the morula stage and poor embryo quality [56]. Thus, exposure to 1.5 M EG or 3.0 M PrOH was more toxic than 0.75 M or 1.5 M PrOH at 25°C. In this case, developmental competence and embryo quality were not strictly related to the incidence of MII spindle abnormalities. This toxicity seemed to be species dependent on the cat, at least in part because IVM and subsequent development of immature oocytes are not affected after exposure to 1.5 M EG at 25°C or 0°C in the rhesus monkey [57].

In conclusion, exposing immature cat oocytes to CPA at different concentrations and temperatures resulted in an array of detrimental effects. The use of 0.75 M PrOH at 25°C resulted in normal nuclear maturation with abnormal MII spindle formation and impaired first cleavage. Treatment with 1.5 M EG or 3.0 M PrOH at 25°C resulted in normal nuclear maturation with abnormal MII spindle formation, poor cleavage, and poor embryo development. Exposing cat oocytes to 3.0 M EG at both temperatures or 3.0 M PrOH at 0°C caused poor nuclear maturation with a high incidence of abnormal MII spindle formation. Finally, the use of 1.5 M PrOH at 25°C resulted in nuclear maturation, spindle formation, fertilization, and embryo development in vitro comparable to control (no CPA) oocytes. Thus, we conclude that a good starting point for future oocyte cryopreservation studies is the use of 1.5 M PrOH at 25°C. Finally, these results illustrate 1) the highly complex and interactive sensitivities of the cat oocyte to agents designed to protect during cooling and freezing and 2) the prudence of a multianalysis strategy for such studies that includes assessing spindle organization and advanced embryo development in vitro.


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Cranfield and Brent Whitaker (Maryland Line Animal Rescue) and Dr. Darby Thornburgh (Petworth Animal Hospital) for providing domestic cat ovaries. Authors also thank Wes Garrett (U.S. Department of Agriculture) and Amalia Dutra (National Institutes of Health) for assisting with the confocal microscopy.


   FOOTNOTES
 
1 Supported by a fellowship to P.C. from the Smithsonian Institution Scholarly Studies Program and the National Institutes of Health (KO1 RR00135). Back

2 Correspondence: Pierre Comizzoli, Department of Reproductive Sciences, Smithsonian's National Zoological Park, 3001 Connecticut Ave. NW, Washington, DC 20008-2598. FAX: 202 673 4733; comizzolip{at}si.edu Back

Received: 28 January 2004.

First decision: 23 February 2004.

Accepted: 5 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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