Modifications of the Ca2+ release mechanisms of mouse oocytes by fertilization and by sperm factor

Ana Carla Gordo 0 1 Manabu Kurokawa 1 Hua Wu 1 Rafael A.Fissore 1 0 Instituto de Ciencias Biome dicas de Abel Salazar, Universidade Do Porto , Porto , Portugal 1 Department of Veterinary and Animal Sciences, University of Massachusetts , Amherst, MA 01003 , USA *Both these authors contributed equally to this work. A cytosolic factor from sperm (SF) is thought to be responsible for the generation of intracellular calcium oscillations ([Ca2 ]i) associated with fertilization in mammalian oocytes. Whether or not mouse oocytes injected with SF exhibit modifications of their Ca2 release mechanisms similar to those observed in fertilized oocytes is not known and this was investigated here by injecting porcine SF (pSF). First, pSF-activated oocytes injected with CaCl2 showed persistent sensitization of the Ca2 -induced Ca2 release mechanism, but this sensitization was absent in SrCl2-activated oocytes. Second, pSF-injected oocytes re-initiated oscillations when fused with untreated oocytes, although the Ca2 responses were short-lived compared to those initiated by fertilization. Likewise, in the presence of colcemid, pSF-initiated oscillations were prolonged but ceased in advance of those in fertilized zygotes. Also, pronuclear envelope breakdown induced by okadaic acid was not associated with Ca2 release in pSFgenerated zygotes, whereas it was observed in fertilized zygotes. Finally, roscovitine, an inhibitor of maturation promoting factor, blocked pSF-induced [Ca2 ]i oscillations. Together, these results show that pSF-induced [Ca2 ]i responses exhibit properties similar to those triggered by the sperm, although the SF's Ca2 active component(s) may be less stable or more susceptible to degradation, resulting in shorter modification of the oocyte's Ca2 release mechanisms. - When ovulated, mammalian oocytes are arrested at the metaphase stage of the second meiotic division (MII). Fertilization, which initiates a series of intracellular calcium ([Ca2 ]i) oscillations, releases oocytes from the MII arrest, induces activation, and promotes progression into the first mitotic cell cycle (Whitaker and Patel, 1990; Kline and Kline, 1992). Events characteristic of oocyte activation include cortical granule exocytosis, meiosis resumption and extrusion of the second polar body, pronuclear formation, and the first mitotic cleavage. How the sperm signals the initiation of [Ca2 ]i oscillations remains to be fully elucidated, although it is likely to involve activation of the phosphoinositide (PI) pathway (Miyazaki et al., 1993; Schultz and Kopf, 1995; Swann and Parrington, 1999). Stimulation of this pathway causes activation of a phospholipase C that leads to the production of inositol 1,4,5-trisphosphate (IP3), a Ca2 releasing second messenger (Berridge, 1993). IP3 triggers Ca2 release by binding to its receptor, the IP3 receptor (IP3R), which is localized in the endoplasmic reticulum (ER), the Ca2 store of the cell. Numerous reports have demonstrated the presence of the molecular components of this signalling pathway in mammalian oocytes (Schultz and Kopf, 1995). Oocyte activation can also be stimulated parthenogenetically in the absence of the sperm by agents that lead to transient increases in [Ca2 ]i (Whittingham, 1980). Parthenogenetic agents such as ethanol or ionomycin cause a monotonic rise in [Ca2 ]i, unlike the repetitive oscillations induced by the sperm, and although this single [Ca2 ]i rise is adequate to activate aged oocytes, it is not sufficient to activate recently ovulated oocytes (Ozil, 1990; Vitullo and Ozil, 1992). Other commonly used parthenogenetic agents, such as SrCl2 and injection of sperm fractions (SF), induce repetitive [Ca2 ]i oscillations and are effective in causing high rates of activation in recently ovulated mouse oocytes (Stice and Robl, 1990; Kline and Kline, 1992; Wu et al., 1998). Despite the fact that several agents induce Ca2 release and activation, fertilization-associated [Ca2 ]i oscillations exhibit several unique features. For example, sperm-induced [Ca2 ]i oscillations are long-lasting and are mediated by the IP3R (Miyazaki et al., 1992). This receptor is sensitized in fertilized oocytes and exhibits an enhanced Ca2 -induced Ca2 release (CICR) mechanism that allows small and localized [Ca2 ]i rises to induce widespread Ca2 release (Igusa and Miyazaki, 1983; Fissore and Robl, 1994). Furthermore, fertilization is accompanied by down-regulation of the IP3R, which appears to be exclusively associated with [Ca2 ]i oscillations induced by production of IP3 (Parrington et al., 1998; He et al., 1999; Brind et al., 2000; Jellerette et al., 2000). Of the above-mentioned activation procedures, only injection of SF is able to closely replicate these changes in the oocytes Ca2 release mechanism(s) induced by fertilization. For instance, injection of SF initiates [Ca2 ]i oscillations similar to those observed during fertilization in oocytes of all mammalian species studied to date (Swann and Lai, 1997; Wu et al., 1997), and these oscillations evoke oocyte activation and full embryo development (Wu et al., 1998; Sakurai et al., 1999). Similar to fertilization, SF-induced [Ca2 ]i oscillations can be blocked either by injection of heparin, a competitive inhibitor of the IP3R, or by injection of a specific antibody against the IP3R (Wu et al., 1997; Oda et al., 1999). Injection of SF also signals down-regulation of the IP3R (Jellerette et al., 2000) and sensitizes CICR (Swann, 1994). Other presumably exclusive characteristics of fertilized oocytes are the ability to activate oocytes long after fertilization has occurred, and the cell-cycle dependence of [Ca2 ]i oscillations. For instance, fusion of fertilized zygotes with MII-arrested oocytes induces activation of the recipient oocytes, but parthenogenetically generated zygotes are unable to do so (Zernicka-Goetz et al., 1995), and it is thought that this property of fertilized zygotes is due to their ability to stimulate oscillations in the arrested oocytes (Zernicka-Goetz et al., 1995). Regarding the cell-cycle dependence, it has been shown that fertilization-associated oscillations last for several hours and cease during entry into interphase, at the time of pronuclear formation (Jones et al., 1995; Day et al., 2000). Whether or not SF-injected oocytes have the capacity to stimulate fertilization-like oscillations in recipient MII oocytes several hours after the initiated oscillations have ceased, and whether they exhibit the modifications in their Ca2 release mechanism(s) commonly associated with fertilization, is not known and will be examined in this report. Materials and methods Oocyte and zygote collection MII oocytes were collected from the oviducts of CD-1 female mice stimulated with 5 IU of equine chorionic gonadotrophin (Sigma, St Louis, MO, USA) followed 48 h later by 5 IU of HCG (Sigma) to induce ovulation. Fertilized embryos were obtained by mating the females after the injection of HCG. (...truncated)