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J N Caamaño, M Catalá,
R Romaguera,
C Diez,
M Muñoz,
D Martín,
R Morató,
S Carrocera,
T Mogas,
M T Paramio,
E Gomez
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ABSTRACT: The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitro matured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus-oocyte complexes from slaughterhouse sheep ovaries were matured in vitro for 27h. After in vitro maturation, oocytes (n=77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al. 2008 Mol. Reprod. Dev. 75, 191-201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r=0.87; P<0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitro maturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r=1; P<0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitro maturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitro matured sheep and goat oocytes.
Reproduction Fertility and Development 01/2011; 23(1):226-227. · 2.11 Impact Factor
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ABSTRACT: Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (>or=3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.
Theriogenology 10/2010; 74(6):1050-9. · 1.96 Impact Factor
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ABSTRACT: The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (> or =3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of > or =3 mm showed greater mean oocyte diameter (128.27 +/- 7.20 microm vs. 125.35 +/- 7.59 microm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 +/- 3.98 vs. 23.07 +/- 2.44 %) and blastocyst rates (19.77 +/- 3.04 vs. 4.11 +/- 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 +/- 49.62 vs. 104.29 +/- 36.09 for follicles of > or =3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle diameter.
Theriogenology 08/2010; 74(3):364-73. · 1.96 Impact Factor
