Oocyte maturation and fertilization of marine invertebrates (Fertilization process of A. aranciacus, courtesy of L. Santella, Stazione Zoologica Anton Dohrn).

Background of the study

Reproduction is one of the fundamental subjects in the field of developmental biology. In the case of most animals, fertilization process is involved in the reproduction. In our laboratory, we study the mechanism of the fertilization especially how the oocytes obtain the ability for re-initiation of meiosis and further development after fertilization, how the fertilizing sperm enter the oocyte, and how the activation of the oocytes takes place by fertilizing sperm. Marine invertebrates, such as echinoderms, mollusks, ascidians, and hydrozoas are our favorite materials. Around our Marine Biological Station, it is easy to obtain a plenty of these fresh animals. Generally, the oocytes dissected from these gametes are easy to fertilize and this process is easy to observe under a microscope.

Some of our experimental materials

Ciona savignyi (left panel) and several species of mollusks (rignt panel)

Recent studies

Our main interest now is calcium signaling during oocyte maturation and fertilization. We measure the calcium changes using fluorescent calcium indicators with confocal microscopy, high sensitive CCD camera or photomultipilers. Our research subjects in progress are 1. The role of calcium ions and calcium dynamics during oocyte maturation in starfish oocytes, and 2. The purification and characterization of egg activating substance (sperm factor) from ascidian sperm.

Change of calcium ions in ascidian (Ciona savignyi) oocytes after fertilization

Fertilization of Ciona oocyte takes place at metaphase I of meiotic divisions (0 sec). Calcium wave started at the point where the fertilizing sperm entered and it propagated across the oocyte to the opposite side of the sperm entry point (10, 20, 30 sec). Two minutes after fertilization, egg contraction that courses the cytoplasmic segregation was observed (120 sec). Several calcium oscillations followed after the first calcium wave (180, 300, 400 sec) and the first polar body protruded at 12 min after fertilization (720 sec, PB). Free calcium level was low in the oocyte for 5 min after protrusion of the first polar body. Then second set of calcium oscillations started and it stopped when second polar body protruded at 30 min after fertilization.

Recent Publications

Doticon_grn_NEW.pngSoluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.
Nakano T, Kyozuka K.
Zygote. 2014 Oct 16:1-15.

Doticon_grn_NEW.pngIntracellular calcium signaling in the fertilized eggs of Annelida.
Nakano T, Deguchi R, Kyozuka K.
Biochem Biophys Res Commun. 2014 Aug 1;450(3):1188-94. Review.

Guanine nucleotides in the meiotic maturation of starfish oocytes: regulation of the actin cytoskeleton and of Ca(2+) signaling.
Kyozuka K, Chun JT, Puppo A, Gragnaniello G, Garante E, Santella L.
PLoS One. 2009 Jul 20;4(7):e6296. PMID:19617909

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening.
Mohri T, Sokabe M, Kyozuka K.
Dev Biol. 2008 Oct 15;322(2):251-62. Epub 2008 Jul 29. PMID:18694744

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.
Kyozuka K, Chun JT, Puppo A, Gragnaniello G, Garante E, Santella L.
Dev Biol. 2008 Aug 15;320(2):426-35. Epub 2008 Jun 6. PMID:18599031

Novel two-step Ca2+ increase and its mechanisms and functions at fertilization in oocytes of the annelidan worm Pseudopotamilla occelata.
Nakano T, Kyozuka K, Deguchi R.
Dev Growth Differ. 2008 Jun;50(5):365-79. Epub 2008 Apr 28. PMID:18445067

Two phases of calcium requirement during starfish meiotic maturation.
Tosuji H, Seki Y, Kyozuka K.
Comp Biochem Physiol A Mol Integr Physiol. 2007 Jun;147(2):432-7. Epub 2007 Jan 30. PMID:17317251

Serotonin stimulates [Ca2+]i elevation in ciliary ectodermal cells of echinoplutei through a serotonin receptor cell network in the blastocoel.
Katow H, Yaguchi S, Kyozuka K.
J Exp Biol. 2007 Feb;210(Pt 3):403-12. PMID:17234609

Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae.
Takeda N, Kyozuka K, Deguchi R.
Dev Biol. 2006 Oct 1;298(1):248-58. Epub 2006 Jun 27. PMID:16884710

Modulation of calcium signalling by the actin-binding protein cofilin.
Nusco GA, Chun JT, Ercolano E, Lim D, Gragnaniello G, Kyozuka K, Santella L.
Biochem Biophys Res Commun. 2006 Sep 15;348(1):109-14. Epub 2006 Jul 17. PMID:16875665

NAADP and InsP3 play distinct roles at fertilization in starfish oocytes.
Moccia F, Nusco GA, Lim D, Kyozuka K, Santella L.
Dev Biol. 2006 Jun 1;294(1):24-38. Epub 2006 Mar 20. PMID:16545362

NAADP triggers the fertilization potential in starfish oocytes.
Moccia F, Lim D, Kyozuka K, Santella L.
Cell Calcium. 2004 Dec;36(6):515-24. PMID:15488601

The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton.
Lim D, Ercolano E, Kyozuka K, Nusco GA, Moccia F, Lange K, Santella L.
J Biol Chem. 2003 Oct 24;278(43):42505-14. Epub 2003 Jul 16. PMID:12867432

NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes.
Lim D, Kyozuka K, Gragnaniello G, Carafoli E, Santella L.
FASEB J. 2001 Oct;15(12):2257-67.PMID:11641253

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain.
Santella L, Kyozuka K, Hoving S, Munchbach M, Quadroni M, Dainese P, Zamparelli C, James P, Carafoli E.
Exp Cell Res. 2000 Aug 25;259(1):117-26. PMID:10942584

Nicotinic acid adenine dinucleotide phosphate-induced Ca(2+) release. Interactions among distinct Ca(2+) mobilizing mechanisms in starfish oocytes.
Santella L, Kyozuka K, Genazzani AA, De Riso L, Carafoli E.
J Biol Chem. 2000 Mar 24;275(12):8301-6. PMID:10722659

Cortical granule translocation during maturation of starfish oocytes requires cytoskeletal rearrangement triggered by InsP3-mediated Ca2+ release.
Santella L, De Riso L, Gragnaniello G, Kyozuka K.
Exp Cell Res. 1999 May 1;248(2):567-74. PMID:10222148

Injection of sperm extract mimics spatiotemporal dynamics of Ca2+ responses and progression of meiosis at fertilization of ascidian oocytes.
Kyozuka K, Deguchi R, Mohri T, Miyazaki S.
Development. 1998 Oct;125(20):4099-105. PMID:9735370