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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Actin microfilaments and mitochondria distribution are considered useful markers of cytoplasmic maturation, but no information is available regarding their distribution in cat oocytes and embryos. Thus, the purpose of this study was to (i) assess cytoplasmic characteristics of the oocyte by mitochondria and actin staining in immature and in vitro/in vivo matured cat oocytes and (ii) characterize mitochondria and actin distribution in in vitro produced blastocysts by confocal laser scanning microscopy. Additionally, in vivo matured oocytes were collected to assess mitochondria and actin. Transzonal cumulus cell projections were more abundant in immature oocytes than in matured oocytes. A relocation of mitochondria throughout meiosis was not clearly observed. However, most in vitro produced blastocysts were of good quality, according to their actin cytoskeleton integrity and mitochondria distribution. The functional significance of mitochondria distribution in cat oocytes in relation to their developmental competence requires further research. This study represents the original description of actin and mitochondrial patterns in cat oocytes and embryos.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The spatial relocation of mitochondria throughout oocyte maturation and the actin polymerization into microfilaments are important for meiosis progression and subsequent embryo development. Both parameters are useful markers of cytoplasmic maturation (Ferreira et al. 2009). However, basic information regarding the distribution of mitochondria as well as actin is lacking in regards to cat oocytes and embryos. Therefore, the purpose of this study was to (i) assess cytoplasmic characteristics of the oocyte by mitochondria and actin staining in immature (germinal vesicle stage) and in vitro/in vivo matured (metaphase II) cat oocytes and (ii) characterize mitochondria and actin distribution in in vitro produced blastocysts by confocal laser scanning microscopy. Additionally, in vivo matured oocytes were collected to assess mitochondria and actin cytoskeleton.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Oocyte collection, in vitro maturation, fertilization and embryo culture

Oocytes were recovered by slicing ovaries after ovariohysterectomy in collection medium and incubated for 24 h in maturation medium. Afterwards, fertilization and in vitro culture procedures were performed as explained previously (Gañán et al. 2009). Additionally, in vivo matured oocytes were collected by laparoscopy from two gonadotrophin-treated females as described (Pope et al. 2009).

Confocal microscopy evaluation of actin microfilaments and mitochondria

F-actin microfilaments and actively respiring mitochondria patterns were evaluated using Alexa Fluor-488 Phalloidin and MitoTracker Orange, respectively (Fig. 1). Hoechst 33342 was used for DNA staining. Samples were analysed using a confocal laser scanning microscope (LSM 510; Carl Zeiss AB, Jena, Germany). The zen 2008 light edition Software (Carl Zeiss) was used for image analysis. (http://www.zeiss.com). All images were via a double-blind procedure evaluated twice by the same person.

image

Figure 1. Representative images showing actin microfilaments and actively respiring mitochondria in cat oocytes and embryos. Oocyte showing a peripheral (a); semi-peripheral (b); or diffuse (c) distribution of mitochondria. Actin staining showing cortex microfilaments and transzonal cumulus cell projections in an immature oocyte (d). Representative blastocyst with grade I actin cytoskeleton (e) and grade I mitochondrial distribution (f). Bar = 20 μm (a–d) and 50 μm (e–f)

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In oocytes, cortical microfilaments were classified as complete or absent. The presence of cumulus cell projections through the zona pellucida was classified as absent; incomplete loss; or complete presence (Albarracin et al. 2005; Suzuki et al. 2000). Blastocysts were classified into grades I–III according to the integrity of their actin cytoskeleton (Zijlstra et al. 2008). Grade I was characterized by defined actin staining in the borders of the blastomeres with homogeneous microfilament complement in the cytoplasm. Grade II was typified by gross maintenance of the cell outline, but with large areas where the cell borders were indistinct or stained only patchily. In grade III, the blastocysts had lost the cytoskeleton integrity.

Regarding actively respiring mitochondria distribution, oocytes were included in one of the following mitochondrial patterns, as defined previously (Brevini et al. 2005; Liu et al. 2010): peripheral; semi-peripheral; central; or diffused. In blastocysts, mitochondria distribution was scored from grade I to III, where grade I blastocysts had an even distribution of mitochondria and grade III blastocysts exhibited heterogeneous distribution of active mitochondria among blastomeres, and 50–75% of the embryo did not present distinct points of staining.

Statistical analysis

Data were analysed using Pearson chi-squared test and Cramér's V coefficient, using statistica (Statsoft, Inc. 2001. statistica, version 6.www.statsoft.com). p < 0.05 was considered significant.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

In all types of oocytes, cortical microfilaments showed a similar pattern (p > 0.05). The presence of transzonal cumulus cell projections was identified by actin staining and differed with the source of oocytes (p < 0.001). Immature oocytes (n = 54) were characterized by the presence of a complete network of transzonal projections (78%), whereas they were absent in 96% of the in vitro matured oocytes (n = 76). In contrast, 76% of the in vivo matured oocytes (n = 21) presented remnants of cumulus cell projections through the zona. There was no clear relocation and aggregation of active mitochondria during oocyte maturation. Mitochondria were localized in the periphery of the oocyte in 84% of the immature (n = 54), 87% of in vitro (n = 73) or 71% of in vivo (n = 21) matured oocytes. In vitro matured oocytes (12%) presented a semi-peripheral pattern, while 29% of in vivo matured oocytes had some active mitochondria in the central part of the ooplasm (p < 0.001).

To assess actin cytoskeleton, 71 blastocysts were analyzed. Most blastocysts had good-quality cytoskeleton (grade I, 63%), 28% showed fair quality (grade II), and 9% had lost most of their cellular integrity (grade III).

For mitochondria staining, 50 blastocysts were analyzed. Of them, 52% were classified as having even distribution (grade I), 36% had intermediate (grade II), and 12% had very heterogeneous (grade III) distribution of mitochondria. In blastocysts, actin and mitochondrial patterns were related (p < 0.001).

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The present study represents the original description of actin and mitochondrial patterns in cat oocytes and embryos. Transzonal cumulus cell projections reflected the maturation status of the oocyte, being more abundant in immature oocytes than in matured oocytes. This reflects the disconnection of cumulus-oocyte transzonal projections as a result of cumulus expansion (Suzuki et al. 2000). The translocation of mitochondria from the periphery of the oocyte towards central areas and a homogenous distribution of mitochondria throughout the cytoplasm have been associated with oocyte competence in the pig (Brevini et al. 2005). A relocation of mitochondria throughout meiosis was not clearly observed in the present study. However, most in vitro produced blastocysts were of good quality, according to their actin cytoskeleton integrity and mitochondria distribution. The functional significance of mitochondria distribution in cat oocytes in relation to their developmental competence requires further research.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

The authors are greatly acknowledged to the staff of the University Animal Hospital, especially to Dr Rangvi Hagman and Dr Odd Höglund, Faculty of Veterinary Medicine and Animal Sciences (SLU) and the staff of various local veterinary clinics in Uppsala, for providing us with gonads for this study. This study was funded by Carl Tryggers Stiftelse för Vetenskaplig Forskning, Stockholm, Sweden.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

R.G. contributed to the design of the study, performed data acquisition, data analysis and manuscript preparation. M.C.G and C.E.P contributed to data acquisition and reviewed the manuscript. Y.C.B.B. contributed to the design of the study, data acquisition and reviewed the manuscript.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References
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