Ultrastructural localisation of calcium deposits in pig ovarian follicles

https://doi.org/10.1016/j.anireprosci.2005.03.010Get rights and content

Abstract

Calcium intracellular signaling regulates many intracellular events including oocyte maturation. This signaling is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. In this study, intracellular calcium deposits were detected in follicle-enclosed pig oocytes using the combined oxalate–pyroantimonate method. These deposits were observed in the nucleus, the mitochondria, the cytoplasm, and on the surface of lipid droplets. The amount of calcium deposits was expressed as a percentage of the area of the respective cellular compartment, which is covered with calcium deposits on ultrathin sections. The distribution of calcium deposits in oocytes changed during folliculogenesis. The amount of calcium deposits in nuclei (1.11% of the area of oocyte nuclei) and cytoplasm (1.02%) in oocytes from secondary and early antral follicles (0.90% nuclei; 0.99% cytoplasm) is significantly lower (P < 0.05) than the amount of calcium deposits in these compartments in oocytes from primary follicles (2.51% nuclei; 2.34% cytoplasm) or antral follicles with growing oocyte (2.91% nuclei; 2.21% cytoplasm). The amount of calcium deposits in mitochondria of oocytes from primary follicles (1.27%) or antral follicles with growing oocyte (1.14%) is significantly lower (P < 0.05) than in the nucleus (2.51% in oocytes from primary follicles; 2.91% in growing oocytes from antral follicles) or cytoplasm (2.34% in oocytes from primary follicles; 2.21% in growing oocytes from antral follicles). The amount of calcium deposits in the cytoplasm of fully-grown oocytes (1.46%) dropped to levels significantly lower (P < 0.05) than those observed in the oocyte nucleus (2.29%). On the basis of these data, we can conclude that the population of follicles on pig ovaries differs in the distribution and concentration of calcium deposits in oocytes, and these changes may be involved in the regulation of the meiotic competence of oocytes.

Introduction

The meiotic maturation of mammalian oocytes is first observed during the development of the female fetus. It is blocked at the stage of late diplotene. The ability to resume meiosis and to continue in maturation beyond this stage is acquired gradually during the subsequent period of oocyte growth. In fully-grown oocytes, meiosis can continue after the germinal vesicle breakdown through the stages of metaphase I and anaphase I to the stage of metaphase II, when meiosis is again arrested (Motlík and Fulka, 1976, Greenwald and Terranova, 1988, Wassarman, 1988). The ability to undergo all these stages of meiotic maturation is called meiotic competence. Pig oocytes acquired full meiotic competence after the formation of the follicular antrum (Motlík and Fulka, 1986, Motlík, 1989). Oocytes with partially developed meiotic competence formed numerous populations in pig ovary, but these oocytes cannot be routinely used in reproductive biotechnologies.

The events involved in the acquisition of meiotic competence are not fully understood. Oocytes at different stages of meiotic competence display a wide range of distinctive features (e.g., different levels of RNA synthesis, protein synthesis, etc.) (Crozet et al., 1981, Motlík et al., 1984, Wassarman, 1988). There are also differences in the maturity of intracellular signaling within oocytes having a different level of meiotic competence. Oocytes with different levels of meiotic competence also differ in their ability to release calcium ions into their cytoplasm (Carroll et al., 1994, Lefevre et al., 1997, Gomes et al., 1999). Similar changes in the ability to release calcium ions were observed during the meiotic maturation of oocytes (Macháty et al., 1997).

Calcium is an important intracellular messenger, and calcium ions regulate many key intracellular events (Berridge, 1995). Calcium ions also take part in both the regulation of meiotic maturation of oocytes (Homa et al., 1993) and in processes occurring in the somatic compartment of the follicle (Carnegie and Tsang, 1984, Mattioli et al., 1991, Lebedeva et al., 1998, Jayes et al., 2000).

Intracellular signaling via calcium ions is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. Intracellular calcium deposits undergo a typical sequence of dynamic changes during the development of the male germ cells (Ravindranath et al., 1994), in oocyte maturation in vitro (Petr et al., 2001), or during the preimplantation development of human embryos (Sousa et al., 1997). Only limited data are available on the distribution of intracellular calcium deposits in the ovary, especially in follicles and oocytes at different stages of their development. The aim of this study was to monitor the ultrastructural distribution of calcium deposits in primary, secondary, and antral follicles in the pig ovary.

Section snippets

Animals and their anaesthesia

Four gilts of the laboratory mini-pig, hybrids of several breeds and strains (mainly the strain Hormel imported from the USA), were used in this study. The body weight of the gilts was about 25 kg.

The gilts were bound lying on their backs, and atropine (0.15 mg/kg of body weight) and Rohypnol (Roche) (1 mg per 20 kg of body weight) were injected intravenously. The gilts were kept under anaesthesia by inhalation of a mixture of halothane (Narcotan, Spofa, Czech Republic) and oxygen. During the first

Results

There were calcium deposits detectable using the combined oxalate–pyroantimonate method in pig oocytes and granulosa cells from primary, secondary, early antral, and antral follicles. The specificity of the reaction is supported by control experiments, which did not reveal any deposits in the ultrathin sections exposed to EGTA for the chelation of calcium (Fig. 1). Large amounts of calcium deposits were observed in the nuclei, cytoplasm, and mitochondria of oocytes (see Fig. 2). We also

Discussion

In the present study, we demonstrated marked changes in the distribution of intracellular calcium deposits in pig oocytes during folliculogenesis. In our earlier studies (Petr et al., 1997, Petr et al., 1999), we observed differences in the distribution of intracellular calcium deposits between fully-grown and growing pig oocytes during their culture in vitro. The results of this study indicate that these differences observed during in vitro oocyte maturation may be due to different statuses of

Conclusion

On the basis of our data, we can conclude that the population of follicles on the pig ovary differs in the distribution and concentration of calcium deposits in oocytes. The changes in the distribution of calcium deposits probably reflect the different physiological status of the oocyte and may cause a different mode of calcium signaling, which may be involved in the regulation of meiotic competence of oocytes.

Acknowledgements

This study was supported by grants MZe ČR QD 0085, GAČR 523/03/H076, MSM 6046070901 and MZE 0002701401. We thank Mrs. Lucy Wescott and Mrs. Lois Russell for editorial assistance with this manuscript. The authors would also like to thank Drs. V. Horák, J. Klaudy, J. Fortýn, and V. Hruban for the anaesthesia and surgery on the pigs.

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