Keywords

1 Introduction

In starfish, fully grown oocytes in the ripe ovary remain arrested at the prophase of the first maturation division. Resumption of meiosis in these immature oocytes is induced by 1-MeAde, which is produced by ovarian follicle cells on stimulation by GSS released from radial nerves (Kanatani et al. 1969; Kanatani 1985). Recently, GSS was purified from the radial nerves of the starfish Asterina pectinifera and its chemical structure was determined to be a relaxin-like peptide (Mita et al. 2009). In this chapter, 1-MeAde production in follicle cells as a role of GSS is described from the aspect of signal transduction mode, based on incapacity of follicle cells to produce 1-MeAde after washing with CaFSW.

2 Irreversible Incapacity of 1-MeAde Production in CaFSW-Treated Follicle Cells

Starfish (A. pectinifera) were collected from several locations in Japan in the breeding season. Ovarian follicle cells were separated from folliculated oocytes as described previously (Mita 1985). The isolated follicle cells were suspended in modified van’t Hoff’s artificial seawater (ASW) at pH 8.2 (Kanatani and Shirai 1970). In the case of treatment with CaFSW, where NaCl was substituted for CaCl2, follicle cells were washed three times with CaFSW, then suspended in normal ASW (Fig. 11.1). Previous studies have shown that the action of GSS on 1-MeAde production by follicle cells is mediated through the production of cAMP (Mita et al. 1987; Mita and Nagahama 1991). However, after washing the follicle cells with CaFSW, the GSS-dependent production of 1-MeAde and cAMP decreased to a large degree (Fig. 11.2), in agreement with the previous report (Mita 1994). It is considered that the decrease of 1-MeAde production by follicle cells treated with CaFSW is caused by the low level of cAMP. However, it is unclear whether the failure of GSS on 1-MeAde and cAMP production in CaFSW-treated follicle cells is irreversible. Thus, follicle cells were incubated in ASW for 24 h at 4 °C after CaFSW treatment. In spite of incubation, these cells did not produce 1-MeAde and cAMP with 20 nM GSS (Fig. 11.2): the incapacity was irreversible. These results suggest that, once washed with CaFSW, follicle cells lose the ability of 1-MeAde and cAMP production by GSS. On the other hand, the CaFSW-treated follicle cells were capable of producing 1-MeAde in the presence of 1-methyladenosine (1-MeAdo) (Fig. 11.3). Because 1-MeAdo ribohydrolase is present in follicle cells (Shirai and Kanatani 1972), 1-MeAdo is converted to 1-MeAde by 1-MeAdo ribohydrolase independently of the signal transduction pathway for GSS. Presumably, CaFSW treatment damages follicle cells for signal transduction for GSS.

Fig. 11.1
figure 1

Treatment of starfish ovarian follicle cells with Ca2+-free seawater (CaFSW) and preparation of cell extracts: follicle cell suspension in artificial seawater (ASW) (left) and CaFSW (right) by light microscopy. Bars 10 μm

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Fig. 11.2
figure 2

Effect of CaFSW treatment on gonad-stimulating substance (GSS)-induced 1-methyladenine (1-MeAde) (a) and cyclic adenosine monophosphate (cAMP) (b) production in starfish ovarian follicle cells. After treatment with either ASW (blue) or CaFSW (purple), follicle cells were suspended in ASW. With or without for 24 h at 4 °C, these cells were incubated in the absence or presence of GSS (20 nM) for 2 h at 20 °C. Each column and vertical line show the mean for three independent samples and SEM, respectively. ND not detectable. Asterisks indicate significantly lower than value in ASW (*P < 0.05, **P < 0.01)

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Fig. 11.3
figure 3

Effect of CaFSW treatment on GSS- and 1-MeAdo-induced 1-MeAde (a) and cAMP (b) production in starfish ovarian follicle cells. After treatment with either ASW (blue) or CaFSW (purple), follicle cells were suspended in ASW and incubated in the absence or presence of GSS (20 nM) or 1-MeAdo (1 mM) for 2 h at 20 °C. Each column and vertical line show mean for four independent samples and SEM, respectively. ND not detectable. *Significantly lower than value in ASW (P < 0.05); **significantly higher than value in ASW (P < 0.05)

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3 Signal Transduction for GSS in CaFSW-Treated Follicle Cells

Previous studies have shown that the action of GSS on 1-MeAde production in follicle cells is effected through its receptors, G proteins and adenylyl cyclase (Mita and Nagahama 1991). To obtain more information on the failure of GSS to produce 1-MeAde in CaFSW-treated follicle cells, properties of GSS receptors were examined. Membrane preparations from follicle cells after treatment with or without CaFSW were incubated with radioiodinated and radioinert GSS for 2 h at 20 °C. On the basis of the competitive binding experiment, Scatchard plots (Scatchard 1949) were used to estimate the dissociation constant (K d) and the number of binding sites (NBS). Specific binding by GSS was found in CaFSW-treated follicle cells. K d values in CaFSW-treated follicle cells were higher than those in nontreated cells (Table 11.1). NBS also decreased in CaFSW-treated follicle cells, suggesting that the affinity and number of receptors in CaFSW-treated follicle cells were absolutely inferior to those in intact cells.

Table 11.1 Dissociation constant (K d) and number of binding sites (NBS) for gonad-stimulating substance (GSS) in membrane preparations of starfish ovarian follicle cells treated with either ASW or CaFSW
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Previous experiments on immunoblot analysis have shown the presence of two types of G proteins in follicle cell membranes (Mita et al. 2011). To identify the G proteins, immunoblotting using antibodies against the α-subunit of Gs (Gsα) and Gi-3 (Giα) and the β-subunit (Gβ) was performed to determine the presence of G proteins in a membrane preparation of CaFSW-treated follicle cells. In follicle cells after CaFSW treatment, the Gsα and Giα antibodies recognized 45- and 41-kDa proteins, respectively, as well as those in normal cells (Fig. 11.4). The Gβ antibody also recognized a 35-kDa protein in both cells.

Fig. 11.4
figure 4

Immunoblotting after SDS-PAGE of crude membrane preparations obtained from starfish ovarian follicle cells treated with either ASW (A) or CaFSW (C) with anti-Gsα, anti-Giα, and anti-Gβ antibodies. Crude membrane (5 μg protein/well) was separated by SDS-PAGE and used for Western blotting

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Next, an experiment was carried out to examine whether GSS directly influences on adenylyl cyclase activity in follicle cells treated with CaFSW. On the basis of previous observations (Mita and Nagahama 1991), crude membrane preparations of follicle cells were incubated with GSS (20 nM) in the absence or presence of GTP (0.1 mM). Without GTP, GSS hardly stimulated adenylyl cyclase regardless of CaFSW treatment (Fig. 11.5a). The addition of GTP (0.1 mM) markedly enhanced the GSS-stimulated adenylyl cyclase activity in follicle cells without CaFSW treatment. Adenylyl cyclase activity in CaFSW-treated follicle cells was also stimulated by GSS in the presence of GTP, but the activation was a slight. In contrast, nonhydrolyzable GTP analogues, such as GTP-γS and GppNHp, activated adenylyl cyclase in follicle cells regardless of CaFSW treatment (Fig. 11.5b). These findings suggest that signal transduction associated with GSS in CaFSW-treated follicle cells does not transmit well from GSS receptor to Gsα protein. It is considered that the lack of response to GSS in CaFSW-treated follicle cells occurs because of a failure of signal transduction.

Fig. 11.5
figure 5

Effect of GSS (a) and guanosine triphosphate (GTP) analogues (b) on adenylyl cyclase activity in starfish ovarian follicle cells. After treatment of follicle cells with either ASW (blue) or CaFSW (purple), their membrane preparations were used for adenylyl cyclase assay as described previously (Mita and Nagahama 1991). Values shown are means for duplicate determination

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4 Cell Extracts from Follicle Cells Treated with CaFSW

Because the NBS for GSS declined in follicle cells after treatment with CaFSW (Table 11.1), it may be possible that the cell extract contains a receptor or binding protein of GSS. After follicle cells were suspended in CaFSW, supernatant obtained by centrifugation at 500 g for 15 min at 4 °C was dialyzed and lyophilized (Fig. 11.1). The lyophilized sample as a cell extract was dissolved in ASW and used for the experiment. When follicle cells were incubated in ASW containing 10 nM GSS in the presence or absence of cell extract at various concentrations, 1-MeAde production decreased in a dose-dependent manner of cell extract (Fig. 11.6). This result suggests that the cell extract contained some substance that disturbed GSS action on 1-MeAde production. It may be assumed that the cell extract includes a binding protein for GSS such as a component of the GSS receptor. Further studies on GSS receptors in follicle cells should provide useful insights into the hormonal regulation of starfish reproduction.

Fig. 11.6
figure 6

Effect of cell extract on GSS-induced 1-MeAde production by starfish ovarian follicle cells. Isolated follicle cells in normal condition were incubated with 10 nM GSS in the presence of various concentrations of cell extract for 2 h at 20 °C. Symbols and bars represent means for three independent samples and SEM, respectively

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5 Conclusion

  1. 1.

    Brief treatment of follicle cells of the starfish Asterina pectinifera with CaFSW deprived the follicle cells of 1-MeAde and cAMP production upon stimulation by GSS.

  2. 2.

    Loss of capacity in CaFSW-treated follicle cells was irreversible.

  3. 3.

    GSS did not stimulate adenylyl cyclase in CaFSW-treated follicle cells.

  4. 4.

    Affinity and number of receptors in CaFSW-treated follicle cells were relatively inferior to those in intact cells.

  5. 5.

    Both Gsα and Giα were detected immunologically in membranes of CaFSW-treated follicle cells as well as those of nontreated cells.

  6. 6.

    It is possible that signal transduction for GSS in CaFSW-treated follicle cells does not flow well from GSS receptors to G proteins.