T-type Ca2+ current activity during oocyte growth and maturation in the ascidian Styela plicata.

Alessandra Gallo,Elisabetta Tosti,Gian Luigi Russo

doi:10.1371/journal.pone.0054604

Alessandra Gallo, Elisabetta Tosti + Show 1 more

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https://doi.org/10.1371/journal.pone.0054604

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Abstract

Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events.

Highlights

Oocyte maturation represents the last phase of oogenesis and consists of nuclear and cytoplasmic modifications [1]
Oocytes Classification Based on the size, oocyte pigmentation and changes in the morphology of accessory cells, we selected three subcategories of germinal vesicle (GV)-containing oocytes as follow: Stage A (GV-A), corresponding to pre-vitellogenic stage; oocytes were less or equal to 70 mm in diameter, with a transparent cytoplasm and a layer of flat follicle cells; Stage B (GV-B), corresponding to vitellogenic stage; oocytes were 70–140 mm in diameter with a yellow cytoplasm and surrounded by a layer of columnar follicle cells; Stage C (GV-C), corresponding to post-vitellogenic stage; oocytes were higher or equal to 140 mm in diameter, with a brown cytoplasm and surrounded by a layer of columnar follicle cells attached to a vitelline coat, with an innermost adherent layer of test cells (Figure 1)
The activation threshold of the recorded inward current in GV-B and GV-C oocytes was 250 mV and the currents were maximally activated by voltage step to 220 mV in the GV-A and GV-B and to 230 mV in GV-C (Figure 3A)

Summary

Introduction

Oocyte maturation represents the last phase of oogenesis and consists of nuclear and cytoplasmic modifications [1]. Nuclear maturation is characterized by the meiotic process. The PI arrest persists up to the time of the hormonal stimulus that resumes meiosis inducing the germinal vesicle breakdown (GVBD). This leads the oocyte to a second block occurring at different stages such as metaphase I (MI) in ascidians, bivalves and gastropods, and metaphase II (MII) in vertebrates and mammals. The cytoplasmic maturation is a less clear process characterized by morphological and functional changes that are necessary to support fertilization and the following developmental events [6]. Cytoplasmic maturation is associated with a considerable increase in the oocyte size depending on the storage of foodstuffs and informational macromolecules, such as transcripts and proteins, modifications of plasma membrane and calcium (Ca2+) signalling [7,8,9,10,11,12]

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