A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17α, 20β-dihydroxy-4-pregnen-3-one, 17α, 20β-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17α,20β-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17α,20β-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20β-hydroxysteroid dehydrogenase (20β-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17α, 20β-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH2 terminus at lysine 57.
Vertebrate oocytes, which grow within the ovarian follicles, are arrested at the first meiotic prophase. In teleosts, as in other nonmammalian vertebrates, the principal events responsible for the enormous growth of oocytes are due essentially to the accumulation of yolk proteins within their cytoplasm (Devlin & Nagahama 2002). After the oocyte completes its growth, it becomes ready for the next phase of oogenesis, that is, the resumption of meiosis (completion of the first meiotic division followed by progression to metaphase II), which is accompanied by several maturational processes in the nucleus and cytoplasm of the oocyte. This process, called oocyte maturation, occurs prior to ovulation and is a prerequisite for successful fertilization; it consists of breakdown of the germinal vesicle (GVBD), chromosome condensation, assembly of the meiotic spindle, and formation of the first polar body.
Although oocyte maturation has been studied in a variety of vertebrates and invertebrates, including mammals (Mehlmann 2005), amphibians (Masui & Clarke 1979; Maller & Krebs 1980; Hammes 2004), fishes (Nagahama et al. 1994; Thomas 1994; Nagahama 1997) and starfishes (Kishimoto 1999), the endocrine regulation of oocyte maturation has been investigated most extensively in fishes (see above). Studies using well-characterized in vitro systems as well as those of in vivo systems have revealed that oocyte maturation in fish is regulated by three mediators, gonadotropin (GTH; luteinizing hormone, LH), maturation-inducting hormone (MIH), and maturation-promoting factor (MPF) (Fig. 1). This review will discuss the mechanisms whereby hormones act to induce oocyte maturation (meiotic resumption) through the action of MPF in fish.
Pituitary GTH regulates oogenesis in vertebrates through its stimulation of sex steroid synthesis by ovarian follicles surrounding developing oocytes. The fish pituitary produces two types of GTH, follicle-stimulating hormone (FSH) and LH (Kawauchi et al. 1989; Swanson 1991), which are structurally and probably functionally homologous to their counterparts in other vertebrates (Planas et al. 2000). The temporal pattern of secretion in several fishes suggests that FSH has a dominant role in regulating vitellogenic growth of follicles, partly through stimulation of estradiol-17β (E2) biosynthesis by ovarian follicles. LH is involved in the final maturation of gametes, partly through the stimulation of production of MIH (Nagahama 1994; Nagahama et al. 1994; Fig. 1). E2 regulates ovarian development through its control of vitellogenin synthesis in the liver during the oocyte growth period.
A variety of experimental approaches have been used to investigate the hormonal regulation of oocyte maturation in teleosts. Among them, in vitro oocyte GVBD bioassays have proven to be most useful for determining the potencies of various GTH preparations and steroids in inducing oocyte maturation. Although various pituitary GTH preparations including LH are the primary endocrine factor responsible for the induction of oocyte maturation, this hormone does not seem to act directly on the oocyte to induce meiotic maturation. The definitive proof for the indirect action of GTH on oocyte maturation was obtained from incubation studies using various follicular preparations (see below for the details). In these experiments, GTH was shown to be able to induce oocyte maturation by intact follicles (follicle-enclosed oocytes), but not by defolliculated (denuded) oocytes. It was thus concluded that that GTH induces the final phase of oocyte maturation in teleosts indirectly, as it does in amphibians (Masui & Clarke 1979), by stimulating the production of certain substance(s).
Extensive in vitro incubation studies were performed to examine the effects of various compounds on the induction of oocyte maturation in a number of teleost species. C21 steroids such as 20β-dihydroprogesterone, 17α,20β-DP, 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S), and 11-deoxycorticosterone (DOC) have been shown to be potent steroid inducers of GVBD (Jalabert 1976; Sundararaj & Goswami 1977; Young et al. 1982; Goetz 1983; Nagahama et al. 1983). Among them, 17α,20β-DP is the most effective steroid in the induction of GVBD in the majority of teleost species examined to date (Nagahama et al. 1983). Testosterone, as well as other C19 steroids, was found to induce GVBD only at high concentrations. E2 and other C18 steroids are generally not effective inducer of oocyte maturation in fish oocytes.
The data described above regarding the effects of GTH and C21 steroids on fish meiotic maturation suggest that the maturational action of GTH appears to be dependent on the synthesis of a secondary steroidal mediator, MIH (Fig. 1). We then attempted to purify and characterize the MIH of a salmonid, amago salmon (Oncorhynchus rhodurus), from media in which immature but full-grown folliculated oocytes had been incubated with GTH (Nagahama & Adachi 1985). One of the major features of salmonid ovaries is that the follicles they contain are extremely large (about 3–5 mm in diameter) and develop synchronously, an enormous advantage for biochemical studies since a large number of follicles at the same stage of development can be obtained easily. Among 20 fractions separated by reserved phase high performance liquid chromatography, maturation-inducing activity was found in only one fraction which had a retention time coinciding exactly with 17α,20β-DP. The purity and final characterization of this active fraction were further confirmed by thin layer chromatography and mass spectrometry with authentic 17α,20β-DP. In collaboration with Dr A. Kambegawa (Teikyo University), a specific radioimmunoassay for 17α,20β-DP was developed and was applied to measure blood concentrations of this steroid during sexual maturation of amago salmon (Young et al. 1983c). Plasma levels of 17α,20β-DP were very low or nondetectable in vitellogenic females but were strikingly elevated in mature and ovulated females. Importantly, the increase in plasma 17α,20β-DP levels correlated well with a dramatic rise in plasma GTH levels (Young et al. 1983c). 17α,20β-DP was thus identified as the naturally occurring MIH in amago salmon (Nagahama & Adachi 1985; Nagahama 1987). Further studies from our laboratory and others indicate that 17α,20β-DP functions as the MIH common to many other teleost fishes including most species of salmonids (Nagahama et al. 1994). Later, 20β-S was identified as a naturally occurring MIH of a variety of perciform fishes including Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus) (Trant & Thomas 1989; Thomas 1994).
The identification of MIH in amago salmon as 17α,20β-DP has made it possible to investigate in detail the mechanisms of the synthesis of this steroid in the ovarian follicle and action of this MIH in inducing the resumption of meiosis in teleost oocytes. As in other vertebrates, a complex series of enzymes are responsible for the biosynthesis of sex steroids in fish gonads. The biosynthesis of 17α, 20β-DP is illustrated in Figure 2. The pathway initiates with the synthesis of the steroid precursor pregnenolone via side-chain-cleavage of cholesterol by cholesterol side-chain cleavage cytochrome P450 (P450scc). Pregnenolone is converted to progesterone by 3β-HSD (Δ4 pathway) or to 17α-hydroxypregnenolone by 17α-hydroxylase activity of cytochrome P450c17 (P450c17) (Δ5 pathway). Progesterone and 17α-hydroxypregnenolone are then converted to 17α-hydroxyprogesteone which is followed by the production of 17α,20β-DP by 20β-hydroxysteroid dehydrogenase (20β-HSD). Thus, the high activities of two steroidogenic enzymes, 17α-hydroxylyase (P450c17) and 20β-HSD, seem to be critical to initiate maturational events (Senthilkumaran et al. 2004; Fig. 2).
Two-cell type model
In vertebrates, the growth and maturation of the ovarian follicles is dependent on the appropriate dynamics of sex steroid secretion. The identification of two major steroid hormones to mediate the actions of FSH (E2, oocyte growth) and LH (17α,20β-DP, oocyte maturation) in fish ovarian follicles permitted a study of the role of the follicle layer in the production of these two steroid hormones.
Ovarian follicles of teleosts, like those of other vertebrates, are composed of two major cell layers, an outer thecal cell layer and an inner granulosa cell layer, that are separated by a distinct basement membrane (Nagahama 1983). The development of simple dissection techniques to separate the ovarian follicles of salmonid fishes into two cell layers, the thecal and granulosa cell layers, has made it possible to elucidate the relative contributions of each cell layer and GTH in the overall process of 17α,20β-DP production (Nagahama 1994; Nagahama et al. 1994).
17α,20β-DP. Our in vitro studies using various follicular preparations obtained from amago salmon demonstrated that 17α,20β-DP production by intact follicles at different stages of development showed that the capacity of the follicles to respond to GTH by synthesizing this steroid is acquired in association with the ability of the oocyte to mature in response to GTH (immediately prior to the natural maturation periods) (Young et al. 1983b). GTH stimulates 17α,20β-DP production by intact follicles and thecal and granulosa cell co-culture preparations, but not by the isolated thecal and granulosa cells, indicating that both thecal and granulosa cell layers are necessary for GTH-induced 17α,20β-DP production (Young et al. 1986). Further experiments examining the effects of conditional media from incubates of one follicle cell layer on steroidogenesis by the other cell layer revealed that the thecal cell layer produced a steroid precursor which was metabolized to 17α,20β-DP in the granulosa cell layer. We then identified 17α-hydroxyprogesteone as the major precursor produced by thecal cells in response to GTH. GTH greatly stimulated 17α-hydroxyprogesterone by thecal cell layers but only slightly stimulated 17α-hydroxyprogesterone production by the other follicular preparations. Incubation of granulosa cell layer with exogenous GTH resulted in elevated 17α,20β-DP levels, whereas thecal cell layers incubated with 17α-hydroxyprogesterone produced relatively small amounts of 17α,20β-DP which should be attributed to contamination of thecal cell preparations with granulosa cell layers. Considering all of these data, a two-cell type model for 17α,20β-DP has been proposed. In this model, the thecal cell layer produces 17α-hydroxyprogesterone that transverses the basal lamina and is converted to 17α,20β-DP by the granulosa cell layer where GTH acts to enhance the activity of 20β-HSD, the key enzyme involved in the conversion of 17α-hydroxyprogesterone to 17α,20β-DP (Young et al. 1986; Nagahama et al. 1994). We also found that the thecal cell layer from amago salmon and the granulosa cell layer from the rainbow trout, Oncorhynchus mykiss, could produce the same effect as has been reported using a combination of thecal and granulosa cell layers from the same species. The reciprocal use of amago salmon granulosa and rainbow trout thecal cell layers also is effective. These findings imply that there may be little species specificity of each of these cell layers among salmonids. Further studies are required to determine whether this two-cell type model is applicable to the production of MIH in ovarian follicles of other teleost fishes.
E2. Using incubation techniques similar to those used for the studies on follicular 17α, 20β-DP production, we have investigated the role of each follicular cell layer in the production of E2. In this model, the thecal cell layer produces testosterone that is converted to E2 by the granulosa cell layer where GTH acts to enhance the activity of aromatase, the key enzyme involved in the conversion of testosterone to E2 (Kagawa et al. 1982; Nagahama 1987; Nagahama et al. 1994).
In fish ovaries, E2 is produced during oocyte growth, while MIH is produced during oocyte maturation. Thus, a dramatic shift in the steroidogenic pathway from E2 to 17α, 20β-DP or 20β-S occurs in fish ovarian follicles immediately prior to oocyte maturation (Fig. 2). This switch in the type of steroid hormone produced during ovarian development is likely to be primarily regulated by changes in the abundance of individual steroidogenic enzymes through changes in expression of genes encoding steroidogenesis-related proteins in developing ovarian follicles. There are two important stages, the first being the shift in the synthesis of precursor steroids from testosterone to 17α-hydroxyprogesterone, while the other is the shift in the final steroidogenic enzymes from oP450arom (for the production of E2) to 20β-HSD (for the production of 17α,20β-DP or 20β-S) (Fig. 2).
We have used, in addition to salmonid fishes, the medaka Oryzias latipes (which spawns daily) and the Nile tilapia Oreochromis niloticus (which spawns approximately every 2 weeks) to investigate molecular mechanisms of the steroidogenic shift. These two species are excellent models for studies of the hormonal regulation of ovarian cycles and transcriptional regulation of the genes encoding steroidogenic enzymes. The sequence of events associated with the ovarian activity, such as vitellogenesis, oocyte maturation, and ovulation, can be accurately timed in medaka and tilapia (Sakai et al. 1988; Senthilkumaran et al. 2002; Yoshiura et al. 2003).
P450c17. In the biosynthesis of steroid hormones, cytochrome P450c17 is the single enzyme that catalyzes both the 17α-hydroxylation of C21 steroids and the 17,20-lyase activity that cleaves the C17–C20 bond to produce C19 sex steroids. During the maturation of oocytes, fish postvitellogenic follicles have to synthesize a large amount of MIH, 17α,20β-DP or 20β-S from 17α-hydroxyprogesterone, the immediate precursor of 17α,20β-DP. To achieve this, a shift in precursor steroid production from testosterone (for E2 production) to 17α-hydroxyprogesterone (for 17α,20β-DP production) must occur in fish postvitellogenic follicles immediately prior to oocyte maturation. Because 17α-hydroxyprogesterone is produced from progesterone by the hydroxylase activity of P450c17, its 17,20-lyase activity needs to be downregulated in these postvitellogenic follicles. Sufficient 17α-hydroxylase activity must be sustained to supply substrate for 17α,20β-DP synthesis during the oocyte maturation stage. This is complicated because 17α-hydroxylyase and 17,20-lyase activities are encoded by a single cytochrome P450c17, which drives sex steroid production by converting pregnenolone to 17α-hydroxypregnenolone and converting progesterone to 17α-hydroxyprogesterone. These 17α-hydroxylated steroids may then be converted by 17,20-lyase to dehydroepiandrosterone and androstenedione, respectively. Thus, the P450c17 enzyme is a key branch point in fish steroid hormone synthesis, as these two enzymatic activities distinguishes between synthesis of C21 steroids and C18 and C19 steroids. Until recently no studies were reported in fish to explain the differential regulation of the two enzymatic activities of P450c17.
In mammals, a steroidogenic shift also occurs in the ovary during the transition from the follicular phase to the luteal/maturation phase, but in this case from E2 to progesterone. Since the production of progesterone does not need the activity of P450c17, a cessation of the hydroxylase and lyase activities of P450c17 is sufficient to achieve the steroidogenic shift in mammals (Voss & Fortune 1993; Komar et al. 2001). However, the differential regulation of P450c17 appears to be needed for the mammalian adrenal steroidogenesis during aging. The selective production of corticosteroids and sex steroids during the aging process is especially brought about by the variation in 17,20-lyase and also 17α-hydroxylase activities (Chung et al. 1987; Picado-Leonard & Miller 1987). Further studies demonstrated that the selective elevation of 17,20-lyase activity is brought about by a cyclic AMP (cAMP)-dependent protein kinase (also known as protein kinase A) through serine/threonine phosphorylation (Zhang et al. 1995; Pandey et al. 2003), while dephosphorylation leaves alone the 17α-hydroxylase activity for P450c17. As only a single P450c17 enzyme was also found to exist in fish, the same mechanisms found in mammalian adrenals were thought to be responsible for the switching in the action of P450c17 during the steroidogenic shift in the gonads of fish (Sakai et al. 1992; Kobayashi et al. 1996). However, no studies have proved this claim convincingly.
Recently we identified, in addition to the conventional type of P450c17 (P450c17-I), a novel type of P450c17 (P450c17-II) in the genomes of tilapia, medaka, fugu (Fugu rubripes) and tetraodon (Tetraodon nigroviridis) (Zhou et al. 2007a). These two types of P450c17 are encoded by two different genes. Surprisingly, enzymatic assays reveal that P450c17-II possesses unique 17α-hydroxylase activity, without any 17,20-lyase activity, in contrast to P450c17-I, which showed both 17α-hydroxylase and 17,20-lyase activities. Tissue distribution analyzed by reverse transcription–polymerase chain reaction (RT–PCR) clearly revealed that tilapia P450c17-II was expressed not only in the gonads, but also in the head kidney, while P450c17-I was exclusively expressed in the gonads. In situ hybridization showed that P450c17-II, but not P450c17-I, was expressed in the interrenal cells of the head kidney from 5 days after hatching to adult, whereas no expression of P450c17-I was observed in the head kidney. These results suggest that only P450c17-II is responsible for cortisol production in the interrenal cells (Zhou et al. 2007a).
We then used in situ hybridization and RT–PCR to examine the expression patterns of both P450c17-I and -II in ovarian follicles of medaka and tilapia during the spawning cycle. Interestingly, a temporally controlled switching was observable in the expression of these two genes during the steroidogenic shift from E2 to 17α,20β-DP (Fig. 3). The expression of P450c17-I reaches its peak during pre- and midvitellogenesis, followed by its sharp decrease immediately before oocyte maturation. In both medaka and tilapia, there is a peak in P450c17-II expression during late- to postvitellogenesis, coinciding with the start of 17α,20β-DP production (Zhou et al. 2007a,b). In medaka, there was another peak during active vitellogenesis. These results strongly suggest that P450c17-I is needed mainly for oocyte growth (vitellogenesis), while P450c17-II is required for oocyte maturation. The decrease in P450c17-I and the increase in P450c17-II expression in postvitellogenic follicles just before the final oocyte maturation stage may reduce the C17,20-lyase activity in the follicles, leading to the accumulation of 17α-hydroxyprogesterone, favoring the shift from production of E2 to 17α, 20β-DP.
As stated above, the switching in the expression of P450c17-I to P450c17-II is responsible in part for the switching in the action of P450c17 during the steroidogenic shift in the fish ovary. The next important question is how the expression of two P450c17 genes is regulated at the transcriptional level. We then investigated the regulatory mechanisms involved in the transcription of these two genes. Medaka P450c17-II possesses two putative Ad4BP/SF-1 binding sites in its promoter region, while only a single Ad4BP/SF-1 binding site is present in P450c17-I, suggesting that the transcriptional regulation of these two genes is somewhat different. High expression of Ad4BP/SF-1 was observable in the ovarian follicles during vitellogenesis, in agreement with previous data on the role of Ad4BP/SF-1 in the regulation of aromatase transcription (Watanabe et al. 1999). Substantial levels of Foxl2 expression could be detected throughout the spawning cycle of medaka, but a peak was observed immediately prior to oocyte maturation (Zhou et al. 2007b), coinciding with the peak of P450c17-II expression. We then used luciferase assays to investigate the possible involvement of two transcription factors (Ad4BP/SF-1 and Foxl2) in the expression of medaka P450c17-I and -II. Ad4BP/SF-1 alone activated the transcription of P450c17-II, and Foxl2 further enhanced the Ad4BP/SF-1-activated P450c17-II transcription. P450c17-I expression was also regulated by these transcription factors in a manner similar to that of oP450c17-II, but with twice less the efficiency. Similar enhancing effects of Foxl2 on Ad4BP/SF-1-activated aP450arom transcription have also been found in tilapia (Wang et al. 2007). Thus, it is highly possible that these transcription factors play important roles in the differential expression of P450c17-I and -II by ovarian follicles during two different stages of ovarian development, oocyte growth and maturation.
StAR, P450scc, 3β-HSD. Another mechanism necessary for the production of a large amount of 17α-hydroxyprogesterone in ovarian follicles during oocyte maturation is increases in the amount of steroidogenic acute regulatory protein (StAR) and the activity of steroidogenic enzymes involved in the production of 17α-hydroxypregnenolone (Δ5-pathway) or progesterone (Δ4-pathway). In amago salmon, the stimulatory effects of GTH on 17α-hydroxyprogesterone production by thecal cell layers were all mimicked by forskolin, an adenylate cyclase activator, and dibutyryl cAMP (dbcAMP), a membrane-permeable cAMP analogue (Kanamori & Nagahama 1988). Furthermore, GTH and forskolin stimulated cAMP formation in thecal cell layers. These findings are consistent with the view that the first step of the stimulating effect of GTH in thecal cell layers is receptor-mediated activation of adenylate cyclase and formation of cAMP. Incubation experiments utilizing both protein and RNA synthesis inhibitors have suggested that gonadotropin promotes formation of one or more labile proteins required for the delivery of cholesterol to the mitochondrial cytochrome system in thecal cell layers.
StAR, a key rate-limiting mediator in the acute regulation of steroidogenesis by tropic hormone, mediates biosynthesis of steroid hormones by controlling the transfer of cholesterol to mitochondria, where P450scc, the enzyme that catalyzes the first and rate-limiting step in steroidogenesis, is located (Stocco 2000). In medaka, StAR and P450scc transcripts showed marked increases in ovarian follicles during oocyte maturation (Shibata et al., unpubl. data, 2007). Similarly, in rainbow trout, the abundance of StAR mRNA increased during GVBD, peaked during and just following ovulation, and decreased by 2 weeks post ovulation in follicles during late vitellogenic and post-ovulation stages (Kusakabe et al. 2002). In contrast, transcripts for 3β-HSD and P450c17-I were low in follicles throughout the oocyte maturation stage. Taken together, these results suggest that the large increase in transcripts encoding StAR and P450scc in postvitellogenic follicles of medaka and rainbow trout is responsible for the rapid preovulatory increase in 17α-hydroxyprogesterone.
oP450arom. In viviparous vertebrates, the ovarian form of cytochrome P450 aromatase (oP450arom, P450c19a1) plays a crucial role in oocyte growth (vitellogenesis) (Nagahama 1994; Devlin & Nagahama 2002). Previous studies on amago salmon have indicated that aromatase activity, assessed indirectly by the conversion of exogenous testosterone to E2, is highest in late vitellogenic follicles but declines as follicles acquire the ability to respond to gonadotropins and undergo final maturation in vitro (Young et al. 1983a). Similarly, in medaka, a progressive increase in aromatase activity was observed in ovarian follicles during active vitellogenesis (Fukada et al. 1994). In this species, aromatase activity was markedly enhanced by pregnant mare serum gonadotropin (PMSG) after 18 h of incubation of vitellogenic follicles. This action of PMSG was mimicked by forskolin and dbcAMP which are known to raise the cellular level of cAMP. The enhancing effect of PMSG, forskolin, and dbcAMP on aromatization was completely inhibited by actinomycin D and cyclohexamide, suggesting that the action of PMSG is mediated through an adenylate cyclase-cAMP system and is dependent upon both transcriptional and translational processes (Nagahama et al. 1991). In both medaka and tilapia, the pattern of changes in oP450arom mRNA levels was broadly similar to the changes in the ability of isolated vitellogenic follicles to produce E2 in response to GTH (Tanaka et al. 1992a; Fukada et al. 1996; Yoshiura et al. 2003).
The promoter regions of medaka and tilapia oP450arom genes contain Ad4 binding sites, and their Ad4 oligomeric sequences of the oP450arom gene form a complex with in vitro-translated Ad4BP/SF-1, an orphan member of the nuclear hormone receptor family, indicating that Ad4BP/SF-1 specifically binds to its DNA motif (Tanaka et al. 1995; Fukada et al. 1996; Watanabe et al. 1999; Yoshiura et al. 2003). We also demonstrated the natural presence of Ad4BP/SF-1-like protein in midvitellogenic ovarian follicles. In transient transfection studies, overexpression of Ad4BP/SF-1 resulted in an increased basal expression of oP450arom reporter gene constructs, while mutation of these sites resulted in decreased basal expression of oP450arom promoter activities. Thus, it has been thought that Ad4BP/SF-1 is necessary for the expression of the oP450arom gene in fish ovarian follicles (Watanabe et al. 1999; Yoshiura et al. 2003).
In medaka and tilapia, the expression of both oP450arom and Ad4BP/SF-1 increased in a parallel manner in ovarian follicles during vitellogenesis and declined sharply in follicles immediately prior to oocyte maturation (Fukada et al. 1996; Watanabe et al. 1999; Yoshiura et al. 2003). In tilapia, the expression of both correlates became undetectable in postvitellogenic follicles during the stage of meiotic maturation (Yoshiura et al. 2003). More importantly, our studies revealed that in vitro incubation of postvitellogenic follicles with human chorionic gonadotrophin (hCG) purged both oP450arom and Ad4BP/SF-1 mRNA transcripts. Similarly, in mammals the expression of P450arom is induced by FSH in the granulosa cells of preovulatory follicles and subsequently diminished as a consequence of the LH surge. The decrease in P450arom transcripts was associated with the reduction in mRNA of SF-1 (Fitzpatrick et al. 1997). These findings on mammals are in accordance with our observations on medaka and tilapia described above.
It has been reported in mammals that cAMP-responsive element (CRE) binding protein (CREB) and the SF-1 domains of P450arom gene act in an additive manner to mediate cAMP transactivation in granulosa cells (Carlone & Richards 1997; Young & McPhaul 1998). In teleosts, cAMP has also been considered as an important second messenger for E2 biosynthesis. Since the CRE motif is also found in the teleost oP450arom gene, a role for CREB cannot be excluded. Our recent study (Senthilkumaran et al., unpubl. data, 2007) indicated that one of the CREB (CREB-1) shows a synergistic pattern of expression with oP450arom. Since Ad4 deletion mutant constructs of the oP450arom gene did not lose the promoter activity entirely (Watanabe et al., unpubl. data, 1999), it is plausible that CRE or other motifs play a role.
20β-HSD. 20β-HSD, an enzyme first described in the prokaryote Streptomyces hydrogenans (Hubener & Sahrholz 1958), catalyzes the reduction of 20-carbonyl groups to 20β-hydroxylated products during steroidogenesis. In fish, 20β-HSD is the key enzyme involved in the conversion of 17α-hydroxyprogesterone to MIH (17α,20β-DP and 20β-S). In amago salmon, 20β-HSD activity is induced by GTH (LH) in granulosa cells of postvitellogenic follicles immediately prior to oocyte maturation (Nagahama et al. 1985a). It has been shown that this action of GTH can be mimicked by forskolin and dbcAMP but not by dbcGMP or phosphodiestrase inhibitors. In vitro incubation experiments using protein and RNA synthesis inhibitors have revealed that the GTH and cAMP induction of 20β-HSD activity is dependent on the activation of new RNA and protein synthesis. Taken together, these results suggest that GTH causes the de novo synthesis of 20β-HSD in the amago salmon granulosa cell through a mechanism dependent on RNA synthesis (Nagahama et al. 1985b; Nagahama 1997).
Since, unlike most of the ovarian steroidogenic enzymes, 20β-HSD cDNA had not been cloned in any animal species, we, as a first step, isolated a cDNA encoding pig testis 20β-HSD. Using synthetic oligonucleotides deduced from the partially determined amino acid sequences, a cDNA encoding 20β-HSD was isolated and cloned, for the first time in any vertebrate species, from a pig testis cDNA library (Tanaka et al. 1992b). Surprisingly, it had 85% amino acid homology with human carbonyl reductase. Using this pig cDNA clone, we were able to isolate two types of carbonyl reductase-like 20β-HSD cDNA from rainbow trout ovarian cDNA libraries, and one of them was functional in reducing various substrates, including 17α-hydroxyprogesterone (Guan et al. 1999). Interestingly, the sequence data search showed that fish 20β-HSD cDNA clones belong to the short-chain dehydrogenase/reductase family (Guan et al. 1999; Senthilkumaran et al. 2002; Tanaka et al. 2002). Purification of the Escherichia coli-expressed ayu (Plecoglossus altivelis) 20β-HSD cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and that 17α-hydroxyprogesterone was one of the preferred substrates (Tanaka et al. 2002). The significance of the similarity between 20β-HSD and carbonyl reductase is unclear at present.
Using northern blot and RT–PCR analyses, changes in 20β-HSD mRNA expression were examined in ovarian follicles of several fishes during the natural ovarian cycle. These studies revealed that 20β-HSD mRNA levels began increasing in late/postvitellogenic follicles and peaked in postovulatory follicles, consistent with the enhanced capacity of these follicles to produce 17α,20β-DP and the elevated plasma levels seen during oocyte maturation (ayu, Tanaka et al. 2002; tilapia, Senthilkumaran et al. 2002; and rainbow trout, Nakamura et al. 2005). Furthermore, in vitro incubation of postvitellogenic immature follicles of tilapia with hCG induced the expression of 20β-HSD mRNA within 1–2 h, followed by the final meiotic maturation of oocytes. It is important to note that the hCG-induced elevation in the level of 20β-HSD transcripts occurs only in the ovary. Although the expression of 20β-HSD mRNA was also found in other tissues, such as gill, muscle, brain, and pituitary, these tissues did not show any changes in the expression of 20β-HSD after incubation with hCG, ultimately demonstrating its importance in the meiotic maturation of oocytes (Senthilkumaran et al. 2002). In contrast, a lack of change in 20β-HSD transcripts was reported in rainbow trout follicles during the final maturation period (Bobe et al. 2003).
In tilapia, actinomycin D completely blocked hCG-induced 20β-HSD mRNA expression and oocyte maturation, suggesting the involvement of transcriptional factors. Our preliminary promoter analysis revealed that the action of GTH on trout 20β-HSD gene is different from oP450arom. These studies revealed that the promoter activity of 20β-HSD could be maintained in the presence of the CRE but not Ad4 motif with a TATA box. Further combination of forskolin and IBMX treatments induced the promoter activity of the 20β-HSD gene-derived deletion mutant construct with the CRE motif and a TATA box. More recent observations using gel-shift assays demonstrated that the CRE consensus sequence of the 20β-HSD gene binds specifically to follicular nuclear extracts (Senthilkumaran et al., unpubl. data, 2007). These results suggest that CREB may serve as an important role in the regulation of cell- and stage-specific expression of carbonyl reductase-like 20β-HSD gene in postvitellogenic follicles during oocyte maturation in the fish ovary.
Fully grown immature oocytes commence maturation when they are immersed in a medium containing MIH; however, they show no response when MIH is injected directly into the oocyte cytoplasm, since the site of MIH action is on the oocyte surface. The presence of an MIH receptor on the oocyte surface had long been suggested, but its biochemical entity remained to be characterized. In 2003, Zhu et al. succeeded in cloning a distinct family of membrane-bound progestin receptors (mPR) from a spotted seatrout ovarian cDNA library, and subsequently succeeded in isolating related genes from other vertebrates, including mammals (Zhu et al. 2003a,b). On the basis of sequence similarity, mPR are classified into three subtypes, mPRα, mPRβ and mPRγ, all of which have characteristics of a G-protein-coupled receptor and seem to mediate non-genomic actions of steroids. The protein expression level of mPRα increases in fully grown oocytes with a further increase in response to GTH treatment during oocyte maturation (Zhu et al. 2003b), which probably correlates to the oocytes becoming responsive to MIH and able to complete oocyte maturation under the influence of GTH (Yoshikuni et al. 1993; Berg et al. 2005). Injection of mPRα antisense oligonucleotides, but not sense or missense ones, blocks MIH-induced oocyte maturation in zebrafish (Zhu et al. 2003b). These findings strongly suggest the involvement of mPRα in oocyte maturation stimulated by MIH under the influence of GTH, although another type of mPR, mPRβ may also participate in zebrafish oocyte maturation, since MIH-induced oocyte maturation is prevented by injection of antisense oligonucleotides designed for mPRβ (Thomas et al. 2004).
Following the identification of mPR in seatrout and zebrafish, their presence has been confirmed at various levels (at genome, mRNA and protein levels) in other teleost species, including puffer fish (fugu), catfish, goldfish and rainbow trout (Zhu et al. 2003a; Thomas et al. 2004, 2006; Kazeto et al. 2005; Tokumoto et al. 2006; Mourot et al. 2006). Detailed analysis at the protein level of mPR during goldfish oocyte maturation has demonstrated that goldfish mPRα is expressed in oocyte plasma membranes as a 40 kDa protein and that its expression level is upregulated by GTH treatment in accordance with the development of oocyte maturation competence. MIH-induced oocyte maturation was inhibited when mPRα protein level was decreased by microinjecting the oocytes with an antisense morpholino oligonucleotide specific to goldfish mPRα (Tokumoto et al. 2006). These results have revealed that mPRα works as the MIH receptor in goldfish oocyte maturation (Fig. 4).
It remains to be determined how MIH interacts with mPR at the molecular level. Concerning this issue, it is notable that endocrine-disrupting chemicals (EDC) can induce or inhibit oocyte maturation in goldfish and zebrafish, probably through the interaction of EDC with mPR (Tokumoto et al. 2004, 2005, 2007). Molecular details of the interaction will reveal the structural basis for steroid recognition by mPR as well as the mechanisms by which EDC interfere with or mimic the authentic endocrine processes in animals.
As described above, much information is now available about the hormonal regulation of oocyte maturation in fishes. In contrast, studies on fish MPF has been limited to only a few species such as the goldfish (Carassius auratus). In the following section we will include a brief comparison between fish and amphibians on the regulatory mechanisms underlying MPF formation and activation, since amphibian oocytes have been an extremely popular model for studies of oocyte maturation, in particular the molecular structure, formation and activation of MPF.
Signal transduction from MIH receptor to MPF
The MIH signal received on the oocyte surface is transduced into the oocyte cytoplasm for the formation and activation of MPF, the final inducer of oocyte maturation. A pertussis toxin-sensitive inhibitory G-protein (Gi) is involved in the initial step of signal transduction during fish oocyte maturation (Yoshikuni & Nagahama 1994; Oba et al. 1997; Pace & Thomas 2005a). Since Gi is stimulated by MIH, treatment of oocytes with MIH will induce a decrease in intracellular levels of cAMP by downregulation of adenylate cyclase activity. Indeed, a decrease in cAMP levels in the oocytes upon MIH stimulation was reported in several fish species (Jalabert & Finet 1986; Finet et al. 1988; Haider & Chaube 1995; Cerd et al. 1998). Moreover, drugs that elevate oocyte cAMP levels, such as adenylate cyclase activators and phosphodiesterase inhibitors, were shown to prevent MIH-induced oocyte maturation (DeManno & Goetz 1987a,b; Haider & Chaube 1996; Haider 2003). It is therefore concluded that cAMP has a negative role in the induction of fish oocyte maturation (Fig. 4).
Generally, a decrease in cAMP levels is linked to a decrease in protein kinase A (PKA) activity, a link that is known as the cAMP/PKA pathway. It is thus expected that PKA plays an important role in oocyte maturation with its decreased activity. Consistent with this, inhibition of PKA activity is sufficient to induce oocyte maturation in the Indian catfish Clarius batrachus (Haider & Baqri 2002). In the Atlantic croaker Micropogonias undulatus (Pace & Thomas 2005b) and the striped bass Morone saxatilis (Weber & Sullivan 2001), however, the cAMP-independent phosphatidylinositol 3-kinase (PI3)/Akt signal transduction pathway operates during MIH-induced oocyte maturation. A similar discrepancy has been reported in amphibians, in which the cAMP/PKA pathway and the PI3/Akt pathway function in the African clawed frog Xenopus laevis (Wang et al. 2006) and the Korean brown frog Rana dybowskii (Ju et al. 2002), respectively. These findings imply that, according to species, different signal transduction pathways work in response to decreased cAMP levels.
There are some differences in molecules that constitute the MIH signal transduction pathway between fish and amphibians (Figs 4,5). Despite the fact that mPRα is mainly involved in fish, mPRβ appears to act as an MIH receptor in Xenopus (Ben-Yehoshua et al. 2007). Unlike fish, classical nuclear progesterone receptors (nPR) may play a role in the reception of MIH signal in Xenopus, in addition to or instead of mPR (Liu et al. 2005; Martinez et al. 2007). In contrast to the participation of Gi in fish, a stimulatory G-protein (Gs) transmits the MIH signal in Xenopus (Lutz et al. 2000; Sheng et al. 2001; Romo et al. 2002). Nevertheless, intracellular cAMP levels decrease in MIH-treated Xenopus oocytes (Wang et al. 2006), since Gs is repressed by MIH (Fig. 5). Irrespective of the use of different types of G-proteins, therefore, the decrease in cAMP levels in MIH-stimulated oocytes is an event common to fish and amphibians.
As discussed above, the cAMP/PKA pathway and the PI3/Akt pathway respond to decreased cAMP levels, but it remains uncertain what molecules follow them to lead the oocytes to the final step of maturation, the formation and activation of MPF. Undoubtedly, identification of substrate proteins for PKA and Akt is an important goal for future work. An attractive hypothesis has been proposed recently (Han & Conti 2006). The hypothesis assumes that Wee1 and cdc25 are PKA substrates and that the PKA-catalyzed phosphorylation activates Wee1 and inhibits cdc25. Since Wee1 (and its relative, Myt1) has an inhibitory effect on MPF and cdc25 has an activating effect (Fig. 5), MPF is maintained in an inactive state under the conditions of elevated PKA activity, whereas MPF is converted to an active state when PKA activity decreases. This hypothesis assumes the most straightforward pathway from the downregulation of PKA to the activation of MPF, but it should be verified by further studies, since results contradictory to the hypothesis have also been reported (Eyers et al. 2005; Wang et al. 2006).
As the site of its action is on the oocyte surface, MIH is unable to induce maturation when directly injected into immature oocyte cytoplasm. When the cytoplasm of maturing oocytes treated with MIH is injected into immature oocytes, however, the injected oocytes mature in time course much faster than that induced by MIH (Kondo et al. 1997). In many vertebrates, new protein synthesis is required for oocyte maturation induced by MIH but not for that induced by injection of maturing oocyte cytoplasm (Yamashita et al. 1992). These findings indicate that a factor responsible for the final induction of oocyte maturation is formed in the oocyte cytoplasm after the reception of MIH signal on the oocyte surface. This factor has been designated as the maturation-promoting factor and abbreviated as MPF (Masui 1996).
GTH and MIH have species-specific action; they do not induce maturation of oocytes in all species. The action of MPF, however, is universal among species. For example, MPF extracted from meiotic pachytene microsporocytes of a plant, the lily Lilium longiflorum, induces maturation when injected into animal (Xenopus) oocytes (Yamaguchi et al. 1991). Moreover, Xenopus oocyte maturation is induced by MPF derived from mammalian cultured cells and yeasts at mitotic metaphase (Kishimoto et al. 1982; Tachibana et al. 1987). Conversely, MPF obtained from eggs acts on somatic cells and induces them to enter the mitotic M-phase, which is associated with events such as nuclear envelope breakdown and chromosome condensation (Miake-Lye & Kirschner 1985). Thus, MPF is present in all eukaryotic cells and functions as the dominant factor to promote M-phase of the cell cycle, irrespective of meiosis and mitosis. Accordingly, MPF is nowadays known as the M-phase-promoting factor rather than the maturation-promoting factor.
After a long-lasting struggle to recognize the molecular entity of MPF since its discovery in 1971 (Masui & Markert 1971), MPF was first purified in 1988 from mature oocytes of Xenopus as a 200 kDa complex containing 32 kDa and 45 kDa proteins (Lohka et al. 1988). The 32 kDa protein was identified as a Xenopus homologue of cdc2, the serine/threonine protein kinase encoded by the fission yeast Schizosaccharomyces pombe cdc2+ gene, and the 45 kDa protein was identified as a Xenopus homologue of cyclin B, which had been discovered in early embryos of marine invertebrates, including the starfish and the clam. Subsequently, MPF was highly purified from eggs of the starfish Marthasterias glacialis (Labbéet al. 1989) and the carp Cyprinus carpio (Yamashita et al. 1992), and it has been confirmed that MPF exhibits a universal molecular structure as a complex of cdc2 and cyclin B in any species. Following the purification of MPF from carp eggs, fish MPF was also purified from the catfish Clarias batrachus (Balamurugan & Haider 1998) and the perch Anabus testudineus (Basu et al. 2004), the results being consistent with the fact that MPF consists of cdc2 and cyclin B.
We investigated behavior of the components of MPF, cdc2 and cyclin B (probably cyclin B1 according to its sequence similarity) during oocyte maturation of the goldfish Carassius auratus (Hirai et al. 1992; Kajiura et al. 1993; Katsu et al. 1993). Experiments with gel-filtration chromatography followed by immunoblotting analysis have shown that all cdc2 molecules exist as a monomer and cyclin B proteins are absent in immature goldfish oocytes, whereas a part of cdc2 forms a complex with cyclin B in mature oocytes (Fig. 6). Subsequent time-course analyses during oocyte maturation have demonstrated the following points: (i) the protein content of cdc2 is constant during oocyte maturation; (ii) cyclin B protein is newly synthesized from its mRNA stored in the oocyte after MIH treatment; and (iii) the synthesized cyclin B is immediately bound to preexisting cdc2, forming active MPF after phosphorylation of cyclin B-bound cdc2 (Fig. 4).
Even under the condition of inhibited protein synthesis, injection of recombinant cyclin B proteins into immature goldfish oocytes induces GVBD (Katsu et al. 1993). Thus, the synthesis of cyclin B in response to MIH is sufficient to induce oocyte maturation in goldfish. The necessity of cyclin B protein synthesis to induce oocyte maturation has been confirmed by the finding that MIH-induced GVBD is blocked by antisense RNA-mediated inhibition of cyclin B synthesis during oocyte maturation in the Japanese brown frog Rana japonica, in which cyclin B is absent in immature oocytes and is newly synthesized during oocyte maturation, like in goldfish (Ihara et al. 1998). Immature goldfish oocytes contain cyclin B mRNA, and actinomycin D, a transcription inhibitor, has no inhibitory effect on MIH-induced oocyte maturation (Katsu et al. 1999), indicating that the amount of cyclin B mRNA stored in immature oocytes is sufficient to induce oocyte maturation. Taken together, it is concluded that cyclin B protein synthesis via translational activation of the dormant mRNA stored in immature oocytes is both necessary and sufficient for initiation of goldfish oocyte maturation.
MPF activation is regulated by biochemical modifications (phosphorylation and dephosphorylation) of cdc2 in maturing oocytes. We investigated changes in phosphorylation states of cdc2 during goldfish oocyte maturation (Yamashita et al. 1995). In maturing oocytes, activation of cdc2 is associated with its phosphorylation and mobility shift on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) from 35 to 34 kDa (Fig. 6). Phosphoamino acid analysis and peptide mapping of native cdc2 proteins purified from mature oocytes have revealed that the activating phosphorylation site of cdc2 is threonine 161 (T161). Substitution of T161 with a non-phosphorylatable residue results in suppression of both kinase activation and the electrophoretic mobility shift from 35 to 34 kDa, demonstrating that cdc2 T161 phosphorylation causes MPF activation, coincident with the change in apparent molecular mass from 35 to 34 kDa, during goldfish oocyte maturation (Fig. 4).
Phosphorylation of cdc2 T161 is catalyzed by cdk-activating kinase (CAK), which contains cdk7 as a catalytic subunit. CAK does not phosphorylate monomeric cdc2 but phosphorylates T161 of cdc2 bound to cyclin B. CAK is active throughout the processes of oocyte growth and maturation (Kondo et al. 1997). Consequently, the binding of newly synthesized cyclin B to preexisting cdc2 during oocyte maturation enables CAK to phosphorylate T161 of cyclin B-bound cdc2, forming active MPF immediately.
In mitotically dividing cells, cdc2 is not only phosphorylated on T161 by CAK but also phosphorylated on threonine 14/tyrosin 15 (T14/Y15) after its binding to cyclin B. T14/Y15 phosphorylation catalyzed by Wee1/Myt1 kinase makes T161-phosphorylated cdc2 inactive and T14/Y15 dephosphorylation by cdc25 phosphatase is required for the activation of T161-phosphorylated cdc2. Thus, the activity of cdc2 is chiefly regulated by T14/Y15 phosphorylation and dephosphorylation rather than by T161 phosphorylation in dividing somatic cells, as in the case of Xenopus oocyte maturation discussed below (Fig. 5). This situation is obviously different from that in goldfish oocyte maturation, in which cdc2 (MPF) is activated by T161 phosphorylation (Fig. 4). The involvement of Wee1/Myt1 and cdc25 in cdc2 activation during goldfish oocyte maturation was therefore carefully investigated. All of the data obtained have clearly shown that cdc2 T14/Y15 is never phosphorylated even after the binding to cyclin B, and thereby its dephosphorylation by cdc25 is unnecessary for MPF (cdc2) activation (Yamashita et al. 1995). It is hence concluded that cdc2 is activated solely by T161 phosphorylation after the binding of preexisting cdc2 and newly synthesized cyclin B during goldfish oocyte maturation (Fig. 4).
Species specificity of MPF formation and activation
Similar to goldfish, cyclin B proteins are absent in immature oocytes of other fish (carp, catfish, zebrafish and lamprey), and this is also true in amphibians, including the frog (Rana japonica), toad (Bufo japonicus) and newt (Cynops pyrrhogaster) (Tanaka & Yamashita 1995; Kondo et al. 1997; Ihara et al. 1998; Sakamoto et al. 1998). These species are likely to employ mechanisms of MPF formation and activation similar to the mechanisms operating in goldfish. In contrast, immature oocytes of Xenopus contain inactive MPF, called pre-MPF, the amount of which is sufficient for inducing oocyte maturation. In this species, therefore, activation of pre-MPF is required for inducing oocyte maturation (Fig. 5). Pre-MPF consists of T161- and T14/Y15-phosphorylated cdc2 bound to cyclin B (cyclins B2 and B5) (Hochegger et al. 2001). Since T14/Y15 phosphorylation inactivates cdc2, its dephosphorylation is a prerequisite for inducing oocyte maturation in Xenopus. MIH (progesterone) activates cdc25 and inhibits Myt1 to dephosphorylate T14/Y15, yielding active MPF that consists of cyclin B-bound cdc2 phosphorylated only on T161 (Schmitt & Nebreda 2002; Haccard & Jessus 2006a).
According to results of initial studies (Tanaka & Yamashita 1995; Kondo et al. 1997), the absence of pre-MPF in immature oocytes seemed to be common in fish. Later, however, it was reported that cyclin B is present in immature oocytes of the freshwater perch (Basu et al. 2004) and rainbow trout (Qiu et al. 2007), suggesting the existence of pre-MPF in these fish. As in Xenopus, therefore, the activation of pre-MPF might be a key step to induce oocyte maturation in some fishes, although it remains to be confirmed that anti-cyclin B immunoreactive proteins found in perch and rainbow trout are actually cyclin B, since their molecular masses are extraordinarily small compared with those of known cyclin B proteins; perch cyclin B and rainbow trout cyclin B2 are 30 kDa and 33 kDa, respectively, whereas B-type cyclins in other species are 46–55 kDa.
Concerning the species-specificity of MPF formation and activation, a remarkable finding has been reported in an urodele amphibian, the axolotl (Vaur et al. 2004). Although this species contains pre-MPF and activates it during oocyte maturation, immature oocytes have very low levels of pre-MPF and exhibit progressive and slow T14/Y15 dephosphorylation of cdc2. It is therefore possible that cyclin B synthesis is a process indispensable for MPF activation in axolotl oocytes, as in the case in many fish. MPF activation in axolotl oocytes could represent an intermediate category between the two extreme models, the goldfish model (Fig. 4) and the Xenopus model (Fig. 2). Similar to the axolotl, intermediate mechanisms might be employed in the perch and rainbow trout that have pre-MPF in immature oocytes (Basu et al. 2004; Qiu et al. 2007). Taken together, the results suggest that the mechanisms of MPF formation and activation have been modified to various extents from species to species, in spite of the fact that the molecular structure and the function of MPF are common to all eukaryotes (Yamashita et al. 2000).
Initiators of oocyte maturation
For the induction of oocyte maturation by MIH, some proteins must be synthesized de novo in MIH-treated oocytes in fishes, amphibians and mammals except for the mouse. The newly synthesized proteins necessary for inducing oocyte maturation are called initiators (Masui & Clarke 1979). Cyclin B is apparently the initiator in species that have no pre-MPF in immature oocytes. Although the situation is complicated in species containing pre-MPF, cyclin B, Ringo and Mos are the most likely candidates for initiators of Xenopus oocyte maturation (Yamashita 2000; Haccard & Jessus 2006a).
B-type cyclins (mainly cyclins B1 and B4) are newly synthesized in progesterone-treated Xenopus oocytes and able to induce GVBD in the absence of protein synthesis when injected into oocytes (Nebreda et al. 1995; Hochegger et al. 2001). Introduction of dominant negative (kinase-dead) cdc2 that has the ability to bind to cyclin B into Xenopus oocytes blocks progesterone-induced GVBD (Nebreda et al. 1995), probably by sequestering newly synthesized cyclin B. These findings support the idea that cyclin B is an initiator of Xenopus oocyte maturation. On the other hand, the finding that antisense oligonucleotide-mediated inhibition of cyclin B (cyclins B1, B2, B4 and B5) synthesis does not prevent progesterone-induced oocyte maturation seems to favor the notion that cyclin B synthesis is unnecessary for pre-MPF activation (Hochegger et al. 2001). Although it is still a controversial issue whether cyclin B works as an initiator of Xenopus oocyte maturation, a recent study has suggested that two distinct pathways, one depending on cyclin B synthesis and the other depending on Mos synthesis, contribute to the activation of pre-MPF redundantly (Haccard & Jessus 2006b). According to this model, a small amount of newly synthesized cyclin B initiates Xenopus oocyte maturation by forming a starter dose of MPF that triggers the activation of pre-MPF (Fig. 5).
Ringo, a cdc2-binding protein, is synthesized after progesterone stimulation and activates cdc2 after a complex formation with monomeric cdc2 (Ferby et al. 1999; Lenormand et al. 1999). Similar to cyclin B, Ringo may also trigger the activation of pre-MPF by forming a starter MPF in Xenopus; however, its crucial role in initiation of maturation remains to be verified, since antisense-mediated ablation of Ringo mRNA delays GVBD but does not suppress pre-MPF activation (Ferby et al. 1999; Lenormand et al. 1999; Haccard & Jessus 2006b).
Mos, a serine/threonine protein kinase, is also synthesized soon after progesterone stimulation. Introduction of Mos into immature Xenopus oocytes induces maturation and, conversely, inhibition of its synthesis by conventional antisense oligonucleotides suppresses progesterone-induced maturation (Sagata 1996, 1997), although inhibition by morpholino antisense oligonucleotides fails to block progesterone-induced maturation (Dupréet al. 2002; Haccard & Jessus 2006b). These findings indicate that Mos is an initiator of Xenopus oocyte maturation. The function of Mos is mediated by mitogen-activated protein kinase (MAPK), since Mos, as a MAPK kinase kinase, activates MAPK through the activation of MAPK kinase (also called MEK) (Fig. 5).
As in the case of Xenopus, does Mos play a role in initiation of maturation in species that contain no pre-MPF? To address this issue, we examined the role of the Mos/MAPK pathway during goldfish oocyte maturation (Kajiura-Kobayashi et al. 2000). Mos is absent in immature oocytes, appears before the onset of GVBD, and increases to a maximum level in mature oocytes. MAPK is activated in accordance with MPF activation during goldfish oocyte maturation. However, ectopic expression of Mos is unable to initiate goldfish oocyte maturation despite full activation of endogenous MAPK in the oocytes. Even when Mos synthesis and MAPK activation are inhibited, the oocytes mature in response to MIH. According to the results obtained so far, it is concluded that Mos does not serve to initiate oocyte maturation via the MAPK pathway during goldfish oocyte maturation. Essentially the same result was obtained using immature frog (Rana japonica) oocytes (Yoshida et al. 2000), in which pre-MPF is also absent as in goldfish.
Structural changes in cyclin B mRNA
In species that contain no pre-MPF in immature oocytes, dormant cyclin B mRNA present in immature oocytes must be translationally activated to induce oocyte maturation. To obtain an insight into the mechanisms of translational activation of cyclin B mRNA, in situ hybridization analysis was performed during oocyte growth and maturation in zebrafish (Kondo et al. 2001). Cyclin B mRNA is uniformly distributed throughout the cytoplasm in previtellogenic oocytes. As oocytes grow, however, the mRNA moves toward the future animal pole and localizes to the animal cortex in fully grown immature oocytes (Fig. 7A). Cyclin B mRNA in mature oocytes disperses throughout the cytoplasm (Fig. 7B). When oocytes are centrifuged in a density gradient of Ficoll, cyclin B mRNA is distinctly aggregated in the cytoplasmic layer of immature oocytes (Fig. 7C), while it is found as indistinct matter in that of mature oocytes (Fig. 7D).
Cytochalasin B, but not nocodazole or taxol, deforms the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like MIH, cytochalasin B induces both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without MIH. A meshwork of microfilaments and the aggregation of cyclin B mRNA in the oocyte cortex disappear just prior to the initiation of cyclin B protein synthesis and of GVBD (Kondo et al. 2001). These findings strongly suggest that MIH induces microfilament-dependent morphological change in cyclin B mRNA from the aggregated form to the dispersed form, which facilitates the translational activation of the mRNA during zebrafish oocyte maturation (Fig. 8).
Translational control of cyclin B in fish oocytes
When in vitro transcribed, and thereby unmasked, cyclin B mRNA is injected into immature zebrafish oocytes, GVBD occurs without stimulation of MIH (Kondo et al. 1997). The presence of unmasked cyclin B mRNA in the oocytes is thus sufficient to induce oocyte maturation. Translation of cyclin B mRNA stored in immature oocytes is probably prohibited by masking proteins that are physically bound to the mRNA directly or indirectly with the aid of cytoskeletal microfilaments. MIH is likely to trigger the translational activation by inducing the release of mRNA from the masking proteins through the destruction of microfilaments (Fig. 8).
Although there are a number of mechanisms by which translation is controlled, the modulation of poly(A) tail length by cytoplasmic polyadenylation has an important role in the regulation of maternal dormant mRNA stored in oocytes (Vasudevan et al. 2006). The biological significance of polyadenylation (elongation of the poly[A] tail) of cyclin B mRNA during MIH-stimulated oocyte maturation was examined using goldfish (Katsu et al. 1999). The poly(A) tail of cyclin B mRNA in mature oocytes is about 100 nucleotides longer than that in immature oocytes. Elongation of the poly(A) tail of cyclin B mRNA takes place at the same time as that of GVBD during oocyte maturation. Moreover, cordycepin, an inhibitor of poly(A) tail elongation, blocks MIH-induced oocyte maturation. These results suggest that the MIH-induced cyclin B mRNA translation requires elongation of the poly (A) tail, but it is still unknown how MIH stimulates the polyadenylation.
Cytoplasmic polyadenylation element-binding protein (CPEB) is an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPE) of maternal mRNA and promotes their polyadenylation (Richter 2007). Goldfish CPEB is bound to the 3′ untranslated region (3′ UTR) of cyclin B mRNA via recognition of the CPE-motif UUUUAUU and a CPE-like motif, UUUUACU. Truncation of the CPEB-recognition region abolishes poly(A) elongation in vitro. These results strongly suggest the participation of CPEB in translational activation of cyclin B mRNA during goldfish oocyte maturation (Katsu et al., unpubl. data, 1999). Besides CPEB, Y box protein is also bound to cyclin B mRNA in immature goldfish oocytes, suggesting the participation of this protein in translational repression of cyclin B mRNA in goldfish (Katsu et al. 1997). However, details of the molecular mechanisms of masking and unmasking of cyclin B mRNA in fish oocytes should be investigated by future work. To do this, several experimental tools have been prepared. These include cDNA clones and antibodies against goldfish poly(A) polymerase (PAP), which catalyzes elongation of the poly(A) tail, and poly(A) binding protein (PABP), which binds to and stabilizes the elongating poly(A) tail (Nakahata et al. 2001b). Further studies using these tools will disclose the molecular basis of cyclin B mRNA translation during fish oocyte maturation.
Translational control of cyclin B1 in Xenopus oocytes
There has been impressive progress in understanding regulatory mechanisms of cytoplasmic polyadenylation-induced translational activation of cyclin B1 mRNA during oocyte maturation in Xenopus, in which the control is mediated by sequences present in the 3′ UTR of cyclin B1 mRNA (Barnard et al. 2004; Sarkissian et al. 2004; Pascreau et al. 2005; Kim & Richter 2006; for a review, see Richter 2007). According to a current model, the translational control of cyclin B1 mRNA involves a balance of two antagonistic activities of polyadenylation and deadenylation. In immature oocyte cytoplasm, cyclin B1 mRNA is bound, via its 3′ UTR, by the cytoplasmic polyadenylation machinery that consists at least of CPEB, cleavage and polyadenylation specificity factor (CPSF), a scaffold protein (Symplekin), atypical cytoplasmic PAP (germ-line-development factor 2, Gld2) and a poly(A)-specific ribonuclease (PARN) (Fig. 9). In this machinery, the deadenylation activity of PARN surpasses the polyadenylation activity of Gld2, and the poly(A) tail thereby remains short. Progesterone downregulates glycogen synthase kinase 3 (GSK3), and the resulting inactivation of GSK3 in turn activates Aurora A, which phosphorylates CPEB at serine 174 (S174). This event then causes the expulsion of PARN from the cytoplasmic polyadenylation machinery, initiating cytoplasmic polyadenylation catalyzed by preexisting Gld2 (Fig. 9).
CPEB is also associated with an eIF4E-binding protein, Maskin (Stebbins-Boaz et al. 1999). The resulting CPEB-Maskin-eIF4E complex on the CPE-containing mRNA precludes the formation of an active initiation complex including eIF4E, eIF4G, eIF3 and 40S ribosomal subunit, thereby repressing translation of the mRNA (Cao & Richter 2002). When the poly(A) tail is short, Maskin remains associated with both CPEB and eIF4E. When poly(A) elongation is induced after CPEB S174 phosphorylation catalyzed by Aurora A, however, PABP binds to the elongating poly(A) tail, which facilitates interaction between PABP and eIF4G, an event necessary for translational activation (Wakiyama et al. 2000, 2001). Finally, the PABP-bound eIF4G induces replacement of eIF4E from the CPEB-Maskin complex to the initiation complex, and thereby the mRNA becomes translated (Fig. 9). Maskin is phosphorylated at multiple residues by several kinases, including cdc2, PKA and Aurora A. This differential phosphorylation controls Maskin activity and its localization on microtubule-rich structures such as mitotic spindles and centrosomes (Barnard et al. 2005; Pascreau et al. 2005; Cao et al. 2006).
Since its discovery as an essential protein for the formation of abdominal segments during fly (Drosophila) embryogenesis, Pumilio and its relatives have been identified in various organisms, including Xenopus (Nakahata et al. 2001a) and rainbow trout (Kurisaki et al. 2007). Pumilio is specifically bound to the region including the sequence UGUA(A) in the 3′ UTR of cyclin B1 mRNA and regulates its translation during Xenopus oocyte maturation. Since Pumilio also binds to CPEB in immature Xenopus oocytes, this protein should be a constituent of the cytoplasmic polyadenylation machinery (Fig. 9). Furthermore, the interaction between Pumilio and CPEB changes during oocyte maturation; that is, phosphorylated CPEB is released from Pumilio in maturing oocytes (Nakahata et al. 2003). More importantly, it has recently been reported that Pumilio forms a complex with deadenylation enzymes such as PARN in the budding yeast Saccharomyces cerevisiae (Goldstrohm et al. 2006) and the fruit fly Drosophila melanogaster (Kadyrova et al. 2007). Therefore, it is highly likely that the phosphorylation-induced dissociation of the Pumilio-PARN complex from the CPEB-containing cytoplasmic polyadenylation machinery triggers the initiation of cyclin B1 mRNA translation during Xenopus oocyte maturation (Fig. 9).
Mechanisms of action
MPF brings about drastic morphological changes in maturing oocytes in accordance with the progression of meiosis, such as GVBD, chromosome condensation and spindle formation (Kotani & Yamashita 2002). Since MPF is a serine/threonine protein kinase, identification of its in vivo substrates is a prerequisite for understanding how MPF leads oocytes to maturation. In fish oocyte maturation, only a few proteins have been identified as in vivo substrates of MPF to date. They include lamin B3 (Yamaguchi et al. 2001, 2006) and eukaryotic polypeptide chain elongation factor 1γ (EF1γ; Tokumoto et al. 2002). Phosphorylation of lamin B3 and phosphorylation of EF1γ by MPF might be related to organization of the germinal vesicle and protein synthesis during oocyte maturation, respectively, but their biological significance remains to be elucidated.
Comprehensive proteomic approaches based on elaborate gene and protein databases are powerful for identifying in vivo substrates for MPF; however, the action mechanisms of MPF still remain elusive even in the most advanced model organisms, the budding yeast and the fission yeast (Ubersax et al. 2003; Archambault et al. 2004; Tyers 2004). Chemical genetics, which has recently been established, might be useful for understanding the mechanisms of action of MPF. In this approach, a mutant Cdk engineered to accommodate bulky, unnatural ATP analogues is introduced into cells and its activity is specifically inhibited by similar bulky, but nonhydrolyzable, ATP analogues (Bishop et al. 2000; Hochegger et al. 2007; Larochelle et al. 2007). This technique, however, requires complete replacement of endogenous wild-type cdk with mutant cdk to discern the cdk functions in vivo. At present, it is impossible to knock out specific genes in fish by the application of homologous recombination. Establishment of gene-targeting technology that enables us to replace a gene arbitrarily is a prerequisite to apply chemical genetics to fish. The rapid generation of transgenic zebrafish lines by simple fertilization of naturally spawned eggs with transgenic sperm produced in vitro will provide an important cue to establish gene-targeting technology in fish (Sakai 2002; Kurita et al. 2004).
The genus Oryzias includes 20 species of medaka (Takehana et al. 2005), in which gametes of different species can be artificially fertilized to produce interspecific hybrids. However, the resulting hybrids exhibit various abnormalities in reproduction and development, being lethal or sterile according to the combination of parent species (Iwamatsu et al. 1984, 1986, 1994; Hamaguchi & Sakaizumi 1992; Sakaizumi et al. 1993; Shimizu et al. 1997, 2000). A hybrid between O. latipes and O. hubbsi (formerly named O. javanicus) is embryonic lethal, since abnormal mitosis eliminates paternal chromosomes from the cells (Iwamatsu et al. 2003; Sakai et al. 2007). Besides wild-type MPF, the hybrid cells contain an MPF molecule that consists of subunits derived from different species (MPF consisting of O. latipes cdc2 and O. hubbsi cyclin B, and vice versa). Artificial production of hybrid MPF in O. latipes embryos by injection of O. hubbsi cyclin B mRNA induces abnormal mitosis, probably due to anomalous phosphorylation by the hybrid MPF (Iwai et al. 2006). It is thus expected that identification of the proteins that are not phosphorylated at M-phase in wild-type cells but phosphorylated by hybrid MPF will give us a new insight into the mechanisms of action of MPF. In particular, this experimental system will help us understand the molecular mechanisms by which MPF recognizes appropriate substrates, an important issue that remains a mystery.
Mechanisms of inactivation
The meiotic cell cycle of mature oocytes of many vertebrates is stopped at metaphase II (MII arrest) until the eggs are fertilized. The high level of MPF activity in MII-arrested eggs decreases rapidly and the cell cycle proceeds from metaphase to anaphase after fertilization or egg activation. Release from MII arrest (inactivation of MPF) is triggered by a transient increase in cytosolic free Ca2+ occurring universally upon fertilization, which causes cyclin B degradation through a ubiquitin-dependent proteolytic system including 26S proteasome and a ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) (Tunquist & Maller 2003; Schmidt et al. 2006).
To investigate the mechanism of cyclin B degradation upon egg activation, 26S proteasome was purified from goldfish oocytes and its role in the regulation of cyclin B degradation was examined (Tokumoto et al. 1997). The purified 26S proteasome digests a recombinant goldfish cyclin B protein (49 kDa) at lysine 57 (K57) independent of ubiquitination, producing a 42 kDa truncated form. The 42 kDa form is also produced by the digestion of native cyclin B that binds to cdc2. Mutant cyclin B, in which K57 was converted to arginine (K57R), is resistant to digestion by the 26S proteasome. Wild-type cyclin B is completely degraded in Xenopus egg extracts, but the mutant K57R cyclin B is not. These results have revealed the sequence of the biochemical reactions involved in cyclin B degradation upon goldfish egg activation: An initial ubiquitin-independent restricted digestion of cyclin B at K57 by the 26S proteasome allows the truncated 42 kDa cyclin B to be ubiquitinated probably with the aid of APC/C, and then the ubiquitinated cyclin B becomes a target of further complete degradation by ubiquitin-dependent activity of the 26S proteasome, leading to inactivation of MPF in the eggs.
The function of Mos as a CSF was also confirmed in goldfish (Kajiura-Kobayashi et al. 2000). Protein level of Mos reaches a maximum in eggs, but it decreases sharply after fertilization. Injection of goldfish Mos mRNA, as well as of Xenopus Mos mRNA, into one blastomere of 2-cell-stage goldfish or Xenopus embryos inhibits cleavage and induces metaphase arrest in the injected blastomeres. These results indicate that, as in Xenopus, Mos contributes to the MII arrest in goldfish eggs.
Mature oocytes must be released from the surrounding follicle cells to be fertilized, an event called ovulation. The ease of manipulating fish oocytes and ovarian fragments in vitro makes fish an excellent experimental model for investigating regulatory mechanisms of ovulation. Actually, many investigations have carried out to understand the molecular mechanisms involved in ovulation, but there are still many unresolved issues, including the biochemical pathways that link the initial stimulation by MIH to the final acting molecules, probably proteinases, essential for follicular rupture in ovulation, although it is known that arachidonic acid and its metabolites, including prostaglandins, are involved in ovulation in fish (Bradley & Goetz 1994; Goetz & Garczynski 1997; Patiño et al. 2003).
Oocyte maturation is regulated by non-genomic action of MIH, while ovulation is regulated by genomic mechanisms; that is, ovulation requires transcriptional activity accompanied by new mRNA synthesis (Theofan & Goetz 1981; Pinter & Thomas 1999; Liu et al. 2005). Hence, analysis using cDNA microarrays will be a potent approach to disclose new players that function in ovulation, since it enables identification of genes that are differentially expressed before and after ovulation (Bobe et al. 2006). Injection of MPF into immature fish and amphibian oocytes induces oocyte maturation (GVBD) but not ovulation (M. Yamashita, unpubl. data, 1989). Accordingly, the pathway leading to oocyte maturation via MPF and that leading to ovulation are clearly distinct, even though both oocyte maturation and ovulation are promoted by MIH. Nonetheless, these two pathways should be communicated to each other for ensuring the correct timing of maturation and ovulation after MIH stimulation (Liu et al. 2005). Recently, we have shown in medaka that gonadotropin (pregnant mare serum gonadotropin, PMSG), but not MIH (17α,20β-DP), can induce ovulation in postvitellogenic follicles in vitro (Shibata et al., unpubl. data, 2007). In this experiment, gonadotropin also induced rapid yet transient expression of nPR mRNA in granulosa cells. Definitive proof that nPR are essential mediators of ovulation has been provided by analysis of the ovarian phenotype of the nPR knockout mouse. Analysis of this model revealed that nPR are required specifically for LH-dependent follicular rupture leading to ovulation (Lydon et al. 1995). Thus, the in vitro induction of ovulation and nPR mRNA expression by gonadotropin in postvitellogenic follicles of medaka may suggest that 17α,20β-DP is involved in the induction of ovulation through its binding to nPR. This observation leads us to postulate that 17α,20β-DP is the key hormone for the induction of not only oocyte maturation (through its membrane receptors), but also ovulation (through its nuclear receptors).
Since ovulation is completed by follicular rupture that is caused by dissolution of the extracellular matrix of the follicular wall, it is reasonable to assume that proteolytic enzymes are involved in the final process of ovulation; however, proteinases that are critical for follicular rupture had not been fully identified until recently. Recently, hydrolytic enzymes essential for follicular rupture were determined for the first time in any vertebrate, by using an in vitro ovulation system of medaka (Ogiwara et al. 2005). The follicular rupture in medaka is accomplished by the cooperation of at least three matrix metalloproteinases (gelatinase A and membrane-type metalloproteinases 1 and 2), together with the tissue inhibitor of metalloproteinase-2b protein. Identification of the hydrolytic enzymes responsible for follicle rupture will provide new perspectives for future studies on the proteolytic events that impact the ovulation process. Using medaka, it should be possible to determine the specific biochemical and molecular events by which gonadotropin (LH) and MIH (17α,20β-DP) mediate the ovulatory process in fish.
The authors thank the many wonderful colleagues who have contributed to the work described herein. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture, Japan and SORST of Japan Science and Technology to Y. N., and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) from the Bio-oriented Technology Research Advancement Institution (BRAIN) to M. Y.
Conflict of Interest
No conflict of interest has been declared by Y. Nagahama or M. Yamashita.