Introduction
In almost all animal species studied, spermatozoa activate eggs by causing transient increases in the intracellular free Ca2+ concentration (Dale, 1990; Miyasaki et al., 1993; Nuccitelli et al., Reference Nuccitelli, Yim and Smarta1993). In most non-mammals, such as Xenopus and echinoderm, only one transient Ca2+ rise occurs in the fertilized eggs. The Ca2+ rise starts from the site of sperm attachment and travels across the entire egg in the form of a wave (Miyasaki et al., 1993; Swann & Lai, Reference Swann and Lai1997). This Ca2+ signal is sufficient to trigger the events associated with egg activation and embryonic development (Whitaker & Swann, Reference Whitaker and Swann1993; Stricker, Reference Stricker1999).
Several models have been proposed to explain how the spermatozoon achieves Ca2+ release. Some authors hypothesized that, after gamete fusion, the sperm may introduce a soluble factor that triggers the activation process in the oocyte (Dale, 1985; Wilding & Dale, Reference Wilding and Dale1998; Machaty et al., Reference Machaty, Bonk, Kühholzer and Prather2000; Kurokawa et al., Reference Kurokawa, Sato and Fissore2004). In mammals, injection of the sperm extract into the unfertilized eggs causes intracellular Ca2+ release and Ca2+ oscillations (Parrington et al., Reference Parrington, Swann, Shevchenko, Sesay and Lai1996). In intact eggs and egg homogenates, mammalian sperm extract triggers Ca2+ release by stimulating IP3 production (Stith et al., Reference Stith, Goalstone, Silva and Jaynes1993; Jones et al., Reference Jones, Cruttwell, Parrington and Swann1998b, Reference Jones, Matsuda, Parrington, Katan and Swann2000; Rice et al., Reference Rice, Parrington, Jones and Swann2000; Wu et al., Reference Wu, Smyth, Luzzi, Fukami, Takenawa, Black, Allbritton and Fissore2001, Saunders et al., Reference Saunders, Larman, Parrington, Cox, Royse, Blayney, Swann and Lai2002), which indicates the involvement of a IP-specific phospholipase C (PLCζ) in the signal transduction mechanism.
Another hypothesis proposes the interaction between sperm and oocyte surface. In the frog Xenopus laevis, unfertilized eggs can be activated by a sperm extract from the newt Cynops pyrrhogaster, this activation is accompanied by propagative Ca2+ release across the entire egg surface (Iwao et al., Reference Iwao, Kobayashi, Miki, Kubota and Yoshimoto1995). In vertebrates, the surface application of a peptide derived from a sperm surface supports the hypothesis of egg activation that involves a signal transducing receptor for sperm in the oocyte's plasma membrane (Shilling et al., Reference Shilling, Craig and Nuccitelli1998). An extract obtained from Xenopus sperm showed hydrolytic activity, which suggests that a tryptic protease from sperm is involved in fertilization, most probably participating in egg activation (Mizote, 1999). In sea urchin and starfish eggs, these early plasma membrane interactions somehow cause the activation of a Src family kinase, which leads to the activation of phospholipase Cγ and the production of IP3, which releases Ca2+ from the endoplasmic reticulum (Jaffe et al., Reference Jaffe, Giusti, Carroll and Foltz2001). The Ca2+ release during the fertilization of Xenopus eggs requires type I IP3-gated Ca2+ channels (Snow et al., Reference Snow, Yim, Leibow, Saini and Nuccitelli1996), but not SH2 domain-mediated activation of PLCγ or Gq-mediated activation of PLCβ (Runft et al., Reference Runft, Watras and Jaffe1999).
In anuran eggs, after sperm contact, cortical granules exocytose their content, which modifies the egg surface and the vitelline envelope (Gómez et al., Reference Gómez, Santolaya and Cabada1984; Oterino et al., Reference Oterino, Sanchez Toranzo, Zelarayan, Valz-Gianinet and Bühler2001, Reference Oterino, Toranzo, Zelarayan, Ajmat, Bonilla and Buhler2006).
In Bufo arenarum oocytes, the mechanism by which spermatoza induce oocyte activation has not yet been elucidated. The purpose of the present work was to investigate the ability of the different fractions, obtained by chromatography from Bufo arenarum sperm extract, to induce oocyte activation either by microinjection or by external application.
Materials and Methods
Animals
Sexually mature Bufo arenarum males and females were collected in the northwestern area of Argentina from May to August (winter animals) and from September to December (summer animals) and kept at 15 °C until use, which generally took place 15 days after collection.
Sperm extract preparation
Sperm suspensions were obtained by gently disrupting the testes in 4 ml amphibian Ringer solution (AR) (NaCl 6.6 g/l, CaCl2 0.15 g/l, KCl 0.15 g/l) and centrifuging at 1085 g for 10 min; then, the pellet was resuspended in AR. To select motile sperms, we used a swim-up procedure. The high quality sperm suspension was centrifuged at 1085 g for 10 min. Sperm were resuspended in calcium-free Tris-buffered saline (NaCl 7.59 g/l, Tris–HCl 2.40 g, pH 7.4) and lysed by three cycles of freezing (−70 °C) and thawing (25 °C). Lysate was observed under a microscope to verify that intact sperm or sperm heads were not present. The lysate was centrifuged for 30 min at 16 000 g and the supernatant was collected as sperm extract.
The sperm extract with a final protein concentration of 2.5 μg/μl was run through size exclusion chromatography using a Bio-Gel P-60 chromatography column. The column was washed with calcium-free Tris-buffered saline, and the fractions were stored at −20 °C until use.
Electrophoresis
SDS-PAGE of sperm fractions was performed according to Laemmli (Reference Laemmli1970) with 7.5% running gels. Proteins were stained with Coomassie brilliant blue R-250.
Oocyte maturation
Denuded oocytes were obtained according to Lin & Schuetz (Reference Lin and Schuetz1985). Follicle cells were removed by incubation of defolliculated oocytes in AR that contained penicillin G-sodium (30 mg/l) and streptomycin sulphate (50 mg/l), pH 7.4, for 5 min with gentle shaking (100 oscillations/min). Denuded oocytes were kept in AR until use.
The experiments were performed with in vitro matured Bufo arenarum oocytes. Meiosis reinitiation was induced by progesterone treatment (2.5 μM) and scored by the presence of a white spot in the animal pole.
Microinjection and incubation
The biological activity of the different fractions was assayed in Bufo arenarum oocytes matured in vitro by microinjection or external exposure.
Different fractions from the sperm extract were microinjected using ICSI (intracytoplasmic sperm injection) micropipettes Humagentm Fertility Diagnostics. The injection volume was 30 nl. The microinjections were carried out in calcium-free Tris-buffered saline at 20 °C. Injection of the buffer alone did not have any measurable effect on oocytes.
In vitro cultures were carried out at room temperature (22–25 °C) using plastic multiwell culture dishes (Costar 3524). Randomized samples of 20 freshly denuded oocytes were distributed into separate wells containing 2 ml AR; the reagents were added (5 μl) directly to the culture medium. Two-well duplicates were routinely run in each experimental group.
We considered as activation parameters the disappearance of the white spot, the elevation of the vitelline envelope and the exocytosis of the cortical granules.
Results
Size exclusion chromatography of sperm extract
Sperm extract obtained according to the Materials and methods was run using a Bio-Gel P-60 chromatography column and the resulting fractions were screened. Results showed four protein peaks and 25 fractions (Fig. 1).
Figure 1 Size exclusion chromatography of sperm extract. The sperm extract with a final protein concentration of 2.5 μg/μl was run through size exclusion chromatography using a Bio-Gel P-60 chromatography column. The column was washed with calcium-free Tris-buffered saline and the 25 fractions obtained were stored at −20 °C until use.
Effect of the microinjection of different sperm extract fractions on the activation of Bufo arenarum oocytes
The ability of the different sperm extract fractions to induce activation was determined by microinjecting each of them into Bufo arenarum oocytes that had been matured in vitro. Groups of 20 oocytes were injected with 30 nl of sperm extract fractions and cultured in AR at 25 °C. Activation signs were checked 30 min after injection. As the control, another group of oocytes was injected with 30 nl calcium-free Tris-buffered saline.
Results showed that the injection of the fractions 17, 18 and 19, which correspond to protein peak III (Fig. 1), induced about 50, 80 and 30% oocyte activation, respectively. The activated oocytes exhibited all the signs of activation (Fig. 2b, d; Fig. 3).
Figure 2 (a) Oocyte matured in vitro exhibiting a white spot on the animal hemisphere. (b) Oocyte exhibiting the characteristic external signs of activation: elevation of the vitelline envelope and disappearance of the white spot. (c) Mature oocyte exhibiting a line of cortical granules beneath the plasma membrane. Alcian-Blue (×400). (d) In-vitro matured oocyte fixed 30 min after activation; the cortical granules have disappeared. Alcian-Blue (×400).
Figure 3 Biological activity of the chromatographic fractions. Oocytes matured in vitro were incubated or microinjected with the different fractions. Mature oocytes were injected with 30 nl or incubated in the different sperm fractions. Activation parameters were scored after 20 min. Values are the mean ± SEM of three experiments (60–80 oocytes per experiment). Each experiment was performed on a different animal.
To investigate if the active factor was a protein, the fractions were incubated at 90 °C for 30 min before injection into the oocytes. Heat treatment totally abolished the ability of all sperm fractions assayed to induce oocyte activation.
Effect of the incubation with different sperm extract fractions on the activation of matured Bufo arenarum oocytes
To study whether the application of the sperm fractions on the outer surface of the oocytes was able to induce activation, incubation experiments were carried out.
Groups of 20 oocytes were cultured in the different sperm fractions and the activation signs were scored after 30 min of incubation at 25 °C. As the control, another group of oocytes was cultured in calcium-free Tris-buffered saline.
Results (Fig. 3) indicated that the ability to induce oocytes activation was restricted to the 6 to 9 fractions, which correspond to protein peak I (Fig. 1). Fraction 8 induced activation in 70% of oocytes, which exhibited all signs of activation. Incubation in heat-treated sperm fractions did not induce activation.
Electrophoretic analysis of the active sperm fractions
Sperm fractions 8 and 18, which showed highest biological activity, were analysed by SDS-PAGE to determine their macromolecular composition. In fraction 8, electrophoretic analysis revealed the presence of a protein of approximately 36 kDa and in fraction 18 another protein of approximately 24 kDa was present (Fig. 4).
Figure 4 Electrophoresis of the biologically active fractions. Fraction 8, which showed activity when it was microinjected and fraction 18, which was able to induce activation when applied on the oocyte surface were analysed by SDS gel electrophoresis. Lane 1: molecular weight markers. Lane 2: fraction 8. Lane 3: fraction 18.
Discussion
Effect of the microinjection of different fractions of sperm extract on the activation of Bufo arenarum oocytes
Although it is a well accepted fact that oocytes are activated by an increase in free Ca2+ level at fertilization, the details of how spermatozoa induce the release of Ca2+ from intracellular stores is still controversial.
The hypothesis that the Ca2+ increase during fertilization is initiated by the introduction of factors into the egg because of sperm–egg fusion is supported by current evidence. Several candidates have been suggested as the egg-activation protein from sperm. The first was a 33 kDa protein purified from hamster sperm that had high sequence homology with the enzyme glucosamine-6-phosphate isomerase (Parrington et al., Reference Parrington, Swann, Shevchenko, Sesay and Lai1996; Swann & Lai, Reference Swann and Lai1997). This protein is present in several species and tissues (Wolny et al., Reference Wolny, Fissore, Wu, Reis, Colombero, Ergün, Rosenwaks and Palermo1999). However, recent studies indicated that immunodepletion of this protein in sperm extract failed to abolish the Ca2+-releasing ability of the extract, so that it is not likely to be the egg-activation protein.
Another protein proposed as the activating factor in sperm extract is a sperm-specific isoform of PLC, PLCζ, of 74 kDa, which showed a higher sensitivity to Ca2+ than other PLC isoforms (Saunders et al., Reference Saunders, Larman, Parrington, Cox, Royse, Blayney, Swann and Lai2002; Kouchi et al, Reference Kouchi, Fukami, Shikano, Oda, Nakamura, Takenawa and Miyazaki2004). The sperm-specific PLCζ would be responsible for Ca2+ oscillations in mammals (Kurokawa et al., Reference Kurokawa, Sato and Fissore2004). An extract of Xenopus sperm was able to stimulate Ca2+ release when injected into mouse eggs, which indicated that the sperm factor appears not to be species specific in vertebrates (Dong et al., Reference Dong, Tang and Sun2000).
Our results indicate that a protein of 24 kDa, present in the sperm extract of Bufo arenarum, was able to induce activation when it was microinjected in homologous oocytes matured in vitro. The molecular weight of the active protein obtained in Bufo arenarum sperm extract is different to that of the 33 kDa protein described in mammal sperm extract. In addition, the 74 kDa PLCζ protein proposed as sperm factor is very different from the 24 kDa protein, which is able to induce activation in Bufo arenarum eggs.
Other experiments have shown that injection of demembranated sperm heads activated mouse eggs, which suggests that the sperm-derived egg-activating protein may be associated with the sperm perinuclear material (Kuretake et al., Reference Kuretake, Kimura, Hoshi and Yanagimachi1996; Kimura et al., Reference Kimura, Yanagimachi, Kuretake, Bortkiewiez, Perry and Yanagimachi1998).
Effect of the incubation with different sperm extract fractions on the activation of Bufo arenarum oocytes
A possibility for signal transduction at fertilization is that spermatozoa might use transmembrane proteins to trigger Ca2+ release (Sato et al., Reference Sato, Tokmakov, Iwasaki and Fukami2000). This hypothesis involves the Ca2+-releasing second messenger IP3. In support of this hypothesis, the protease cathepsin B or a similar protease of 380 kDa present in Xenopus sperm extract caused an increase in intracellular Ca2+ when applied to Cynops eggs (Mizote et al., Reference Mizote, Okamoto and Iwao1999; Iwao, Reference Iwao2000), but it has not been applied to Xenopus eggs.
Other results suggest that Ca2+ release is initiated in frog eggs when disintegrin molecules on the sperm surface contact an integrin in the egg surface (Iwao & Fujimura, Reference Iwao and Fujimura1996; Shilling et al., Reference Shilling, Craig and Nuccitelli1998).
All the molecules proposed as agonists to trigger Ca2+ released at fertilization have molecular weights higher than the protein present in Bufo arenarum sperm extract capable of activating the eggs when it was added to the culture medium. Interestingly, it had no effect when microinjected into the egg cytoplasm.
In this study, we demonstrated the presence in sperm extract from Bufo arenarum of two proteins that were able to induce oocyte activation. The sperm fraction containing a 24 kDa protein induced 90% activation when it was microinjected into the oocytes, while the sperm fraction containing a 36 kDa protein was able to induce about 70% activation only when it was applied on the oocyte surface.
It is possible that mechanisms for oocyte activation involve signals from soluble sperm factors together with signals generated by the interaction of the sperm with receptors on the oocyte plasma membrane.
Acknowledgements
This work was supported by a grant from the Science Council of the National University of Tucumán (CIUNT).
