Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 3-14 (1993) Kugler Publications, Amsterdam/New York

Molecular endocrinology of oocyte growth and maturation in fish Yoshitaka Nagahama, Michiyasu Yoshikuni, Masakane Yamashita, Noriyoshi Sakai and Minoru Tanaka Laboratory of Reproductive Biology, NationalInstitute for Basic Biology, Okazaki 444, Japan

Keywords: oocyte growth, oocyte maturation, gonadotropin, estradiol-173, vitellogenin, maturationinducing hormone, 17a,20-dihydroxy-4-pregnen-3-one, maturation-promoting factor

Resume Les gonadotropines hypophysaires (GtHs) sont de premiere importance dans la stimulation de la croissance et de la maturation ovocytaires. Toutefois, les actions des GtHs ne sont pas directes, mais elles passent par l'interm6diaire d'une production ovarienne de m6diateurs st6roYdiens, aussi bien pour la croissance (oestradiol-173) que pour la maturation ovocytaire (hormone inductrice de la maturation, MIH; 17a,200dihydroxy-4-pregnen-3-one, 17a,200-DP chez les salmonides 17a,201,21-trihydroxy-4-pregnen-3-one, 203S chez les sci6nid6s). I1 est acquis que les productions d'oestradiol-17, et de 17a,200-DP par les follicules ovariens de salmonid6s se font via l'interaction de deux couches cellulaires, la theque et la granulosa (modle de cooperation cellulaire). Chez les salmonid6s, un changement net de la steroYdogenese, passant de la production d'oestradiol-173 a celle de 17a,200-DP, intervient dans les enveloppes folliculaires juste avant la maturation. II est possible que ce changement soit une consequence de modifications profondes dans l'expression des genes codant pour diverses enzymes de la steroidogenese. Comme tape initiale de l'6tude de cette question, nous avons isol1 et caracterise les ADNc codant pour diff6rentes enzymes de la steroYdogenese savoir les ADNc, de truite arc-en-ciel, du cytochrome P-450 coupant la chain lat6rale du ovarienne, cholesterol, de la 3-hydrost6rode dhydrog6nase (HSD), du cytochrome P-450 17a-hydroxylase/17,20 lyase, et du cytochrome P-450 aromatase, ainsi que I'ADNc de la 203-HSD de porc. L'oestradiol-173 stimule la synthese et la s6cr6tion h6patique du precurseur du vitellus: la vitellog6nine. Celle-ci est ensuite transported vers l'ovaire ofi elle est s6lectivement incorpor6e dans l'ovocyte par un processus impliquant des rcepteurs sp6cifiques situ6s a la surface de la cellule. L'oestradiol-170 induit 6galement la synthese, par le foie, de protines membranaires de l'oeuf. L'induction de la maturation, par la 17a,203DP ou la 20S, se fait par l'interm6diaire de leur liaison Ala membrane plasmique ovocytaire. Cette interaction, initiale, surface cellulaire-MIH est suivie par la formation du m6diateur majeur du MIH, le facteur promoteur de la maturation (MPF). Nous avons purifi6 le MPF Apartir d'ovocytes matures de carpe. Le MPF de carpe est constitu6 de deux composants: l'homologue du produit du gene cdc2 + de levure (p34cdc2, et la cycline B. La prot6ine kinase cdc2+ est pr6sent6 dans les ovocytes immatures aussi bien que dans les ovocytes dont la maturation a et6 induite par un traitement a la 17a,20)3-DP, tandis que les prot6ines de type cycline B ne peuvent etre d6tect6es que dans les ovocytes matures. L'addition de cycline B de poisson rouge, exprim6e dans des bact6ries, Ades extraits d'ovocytes immatures de poisson rouge induit l'activation du MPF. Correspondenceto: Dr. Yoshitaka Nagahama, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, Japan.

4 Ces r6sultats suggbrent que l'apparition des prot6ines de type cycline B est une tape cruciale dans l'induction de la maturation des ovocytes de poisson par la 17oe,20-DP.

Abstract Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-173) and maturation (maturation-inducing hormone, MIH; 17a,201dihydroxy-4-pregnen-3-one, 17a,203-DP in salmonid fishes; 17oa,203,21-trihydroxy-4-pregnen-3-one, 20/-S in sciaenid fishes). It is established that production of estradiol-170 and 17a,201,-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-170 to 17a,20S-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3-hydroxysteroid dehydrogenase (HSD), 17a-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAs as well as the pig 20-HSD cDNA. Estradiol-178 stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-173 was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17a,203-DP and 20,f-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2 + gene product of fission yeast (p34cdc 2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17a,20/-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17a,200-DP-induced oocyte maturation in fish.

Introduction Two major events occurring during oogenesis are the growth and maturation of the oocyte. Gonadotropins (GTHs) secreted from the pituitary gland are responsible for these two processes. It is now apparent that teleost pituitary glands, like those of other higher vertebrates, secrete two kinds of GTHs (GTH I and II) that are chemically and biologically similar to tetrapod follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (Kawauchi et al. 1989; Swanson 1991). In salmonids, GTH I is secreted during the period of oocyte growth, and probably functions to stimulate

ovarian growth and steroidogenesis at this stage. In contrast, during the period of oocyte maturation, the pituitary gland secretes GTH II which alters steroidogenesis to promote oocyte maturation (Swanson 1991). In this review, otherwise indicated, the term GTH is referred to as GTH II preparations, some of which are perhaps contaminated by GTH I. Although GTHs are the primary mediator of oocyte growth and maturation, the actions of this hormone are not direct, but are mediated through the production of steroid hormones by the ovarian follicle cells (Nagahama 1987a). The ovaries of teleost fishes consist of many ovarian follicles, each of

5 which is composed of an oocyte and its surrounding somatic cell layers, an outer thecal cell layer and an inner granulosa cell layer (Nagahama 1983). A simple dissection technique to separate the ovarian follicles of salmonids into two cell layers, the thecal and granulosa layers, has made it possible to elucidate the role of each layer and gonadotropin in the overall process of steroid production (Kagawa et al. 1982). Our in vitro incubation studies indicate that the thecal and granulosa cell layers cooperate in the production of steroidal mediators of oocyte growth and maturation (see below).

1. Oocyte growth (vitellogenesis) a. Hormonal regulation of vitellogenesis Oocytes of nonmammalian vertebrates grow while arrested in the first meiotic prophase. The principle events responsible for the enormous growth of fish oocytes occur predominantly during the phase of development termed vitellogenesis, and involve the sequestration and packaging of a hepatically derived plasma precursor, vitellogenin (VT), into yolk protein. It is well established that vitellogenesis in teleosts is promoted by a two step mechanism in which GTH increases ovarian secretion of estradiol-173, which in turn stimulates the hepatic synthesis and VT secretion (Ng and Idler 1983; Wallace 1985; Tyler 1991). In all of the teleost species studied so far, elevated levels of estradiol-170 have been reported in females during active vitellogenesis (Fostier et al. 1983). The primary site of estradiol-173 production in the teleost ovary is follicle cells which surround vitellogenic oocytes. The capacity of intact follicles to produce estradiol-173 in response to GTH stimulation increases during oocyte growth, but rapidly decreases in association with the ability of the oocyte to mature in response to GTH (Kagawa et al. 1983). A two-cell type model for the production of estradiol-173 has been proposed in the salmonid ovarian follicle. In this model, the thecal cell layer, under the influence of GTH, secretes the androgen substrate (probably testosterone) which diffuses into the granulosa cell layer where the aromatase is

located exclusively (Nagahama 1987a; Adachi et al. 1990). Restricted distribution of testosterone and estradiol-17 was confirmed immunohistochemically in vitellogenic ovarian follicles of rainbow trout (Schulz 1986). However, the two-cell type model described above does not seem to be valid for the Fundulus heteroclitusand medaka, Oryzias latipes (Petrino et al. 1989; Onitake and Iwamatsu 1986) ovarian follicles. In these species, in which steroidogenic thecal cells are not evident in the thecal layer, the follicle cells (granulosa cells) are the major site of steroid synthesis and that the production of estradiol-173 does not require the involvement of two cell types. GTH action on the thecal cell layer to stimulate testosterone production is mediated through a receptor-coupled adenylate cyclase-cAMP system (Kanamori et al. 1988; Kanamori and Nagahama 1988a, b; Nagahama 1987a). Other intracellular signalling molecules including calcium, protein kinase, and arachidonic acid are also associated with GTH-induced testosterone production by intact preovulatory follicles of goldfish, Carassius auratus(Van Der Kraak 1990, 1991; Van Der Kraak and Chang 1990). It is still unknown whether GTH enhances aromatase activity in salmonid granulosa cells. However, we have recently shown that in medaka vitellogenic follicles, aromatase activity is markedly enhanced by pregnant mare serum gonadotropin (PMSG) via an adenylate-cyclasecAMP system. Furthermore, the PMSG-induced aromatase activation is completely blocked by actinomycin D and cycloheximide, suggesting that this action of PMSG is dependent upon both transcriptional and translational processes (Nagahama et al. 1991).

b. Hormonal regulationof vitellogenin uptake and egg membrane formation VT is selectively taken up from the blood stream by developing oocytes (Wallace 1985; Tyler 1991); ultrastructural evidence shows that it is incorporated into the oocyte by micropinocytosis (Selman and Wallace 1982). In vitro culture systems have been developed for studies on VT sequestration

6 into vitellogenic follicles of rainbow trout (Oncorhynchus mykiss) and F. heteroclitus, and have been used to demonstrate receptor-mediated VT incorporation by these oocytes (Tyler et al. 1990; Kanungo et al. 1990; Tyler 1991). The binding for VT was characterized in isolated oocyte membranes of the tilapia, Oreochromis niloticus (Chan et al. 1991). Saturation and Scatchard analyses revealed only a single class of binding site. The number and affinity of these bindings greatly increase from the previtellogenic to the vitellogenic stage, and remained unchanged until the preovulatory stage, at which time the affinity of the bindings was also highest. These findings provide unequivocal evidence that the binding sites in the tilapia oocytes are specific receptors for VT. More recently, proteins with an affinity for VT have been isolated from vitellogenic follicles of coho salmon, Oncorhynchus kisutch, and rainbow trout (Stifani et al. 1991; LeMenn and Nunez Rodriguez 1991; Lancaster and Tyler 1991). Further studies on the expression and regulation of these receptor proteins will provide new insight into the mechanism controlling VT uptake into oocytes. Little is known about the hormonal involvement of VT uptake by oocyte of teleosts. Several hormones have been implicated in regulating oocyte growth; these hormones include GTH, thyroxine, triiodothyronine, insulin and growth hormone, although the role of any of these has yet to be determined fully (Tyler 1991). In vitro studies show that the potency of GTHI in enhancing VT uptake varies with the temporal phase of vitellogenic development; GTH stimulated VT uptake by oocytes only during early to mid-vitellogenic development, and later in vitellogenesis was ineffective in this capacity. We have recently developed an in vitro assay system for VT uptake using follicle-free oocytes of rainbow trout (Shibata et al. 1990, 1991). Using this in vitro system, it was shown that insulin and thyroxine significantly increased VT uptake. In contrast, neither chum salmon GTHI nor GTHII was effective. When using the oocytes that matured in vivo, only 1/15 of VT (compared to immature oocytes) was incorporated. Insulin also had no effect on these mature oocytes. Recently, another possible function of estrogens

during oocyte growth has been demonstrated in the medaka. One of the most striking features observed during oocyte growth in teleosts is the formation of a thick, highly differentiated zone (egg membrane, vitelline membrane, zona radiata, zona pellucida) lying between granulosa layers and oocytes. Although the exact mechanism by which the egg membrane is formed in teleost oocytes is still unknown, recent findings by Hamazaki et al. (1987a, b, 1989) are of great interest. These authors reported that the major glycoprotein constituent of the inner layer of the medaka egg membrane is synthesized in the liver under the influence of estradiol-17 . A similar mechanism also functions in the formation of egg membranes in other teleost species (Hyllner et al. 1991).

2. Oocyte maturation After the oocyte completes vitellogenesis, it becomes ready for the next phase of oogenesis, i.e., the resumption of meiosis 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, and formation of the first polar body (Goetz 1983; Nagahama 1987b, c). It is now established that oocyte maturation in fish is regulated by three major mediators, GTH, maturation-inducing hormone (MIH) and maturation-promoting factor (MPF), that function sequentially at the level of the ovarian follicle cells, the oocyte surface and oocyte cytoplasm (Nagahama 1987b).

a. GTH and MIH Although pituitary maturational GTH is the primary endocrine factor responsible for the induction of oocyte maturation, this hormone does not act directly on the oocyte to induce meiotic maturation. Incubation studies have revealed that the action of GTH is mediated by the production of MIH

7 by the ovarian follicle cells (Young et al. 1982). In a number of teleost species, C21-steroids have been shown to be potent initiators of GVBD in vitro and to be present at high levels in plasma of fish undergoing final oocyte maturation (Jalabert 1976; Fostier et al. 1983; Yamauchi et al. 1984; Scott and Canario 1987; Nagahama 1987b). Among the C21-steroids, however, only two steroids have been identified as the naturally occurring MIH in fish: 17ae,20/-dihydroxy-4-pregnen-3-one (177a,20/-DP) in amago salmon, Oncorhynchusrhodurus, (Nagahama and Adachi 1985) and 17a,20f,21-trihydroxy-4-pregnen-3-one (203-dihydro-11-deoxycortisol, 20/-S) in the Atlantic croaker, Micropogonias undulatus, and spotted seatrout, Cynoscion nebulosus (Trant et al. 1986; Thomas and Trant 1989; Trant and Thomas 1989). Testosterone, as well as other C19-steroids, were found to induce GVBD only at high concentrations. Estradiol-17/3 and other C18-steroids are generally not effective inducer of oocyte maturation in fish oocytes. A two-cell type model involving thecal and granulosa cells was proposed for the production of 17uc,203-DP by salmonid ovarian follicles. GTH acts on the thecal cell layer to enhance 17othydroxyprogesterone production through a receptor-mediated adenylate cyclase-cAMP system. 17a-Hydroxyprogesterone is then converted into 17a,20/-DP by the granulosa cell layer where GTH acts to induce de novo synthesis of 200/-hydroxysteroid dehydrogenase (20/-HSD), the key enzyme involved in the conversion of 17a-hydroxyprogesterone to 17a,20/3-DP, through a mechanism dependent on RNA synthesis (Young et al. 1986; Nagahama 1987a, b). In vitro experiments using both protein and RNA synthesis inhibitors have demonstrated that de novo synthesis of 20/-HSD consists of gene transcriptional events within 6h of exposure to GTH and translational events 6-9h (Nagahama et al. 1985).

b. cDNA cloning of rainbow trout ovarian steroidogenic enzymes In salmonid fishes, two biologically important steroidal mediators of oocyte growth and matura-

tion have been identified (Nagahama 1987a). It is now established that ovarian granulosa cells are the site of production of these two steroidal mediators, but production by the ovarian follicle depends on the provision of precursor steroids by the thecal cell (Nagahama 1987a). A dramatic shift in the salmonid steroidogenesis from estradiol-17/ to 17a,2013-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. To provide a basis for investigation of the molecular mechanisms underlying this distinct steroidogenic shift, we have isolated cDNA clones encoding rainbow trout steroidogenic enzymes responsible for the production of estradiol-17/ and 17a,20/-DP. Mammalian cDNAs, kindly provided by Drs. W.L. Miller, S. Chen and J.I. Mason, U.S.A., have been used as probes. Characterization of these cDNA clones by nucleotide sequencing has led to the complete amino acid sequences of rainbow trout cholesterol sidechain cleavage cytochrome P-450 (P- 4 50scc; M. Takahashi, unpublished), 3-hydroxysteroid dehydrogenase-isomerase (30/-HSD; N. Sakai, unpublished), 17a-hydroxylase/17-20 lyase cytochrome P-450 (P-45017,; Sakai et al. 1992; see also this issue), and aromatase cytochrome P-450 (P- 450 Arom; Tanaka et al. 1992a). The cDNA inserts were confirmed to encode each steroidogenic enzyme by introducing it into non-steroidogenic COS-1 monkey kidney tumour cells. Unlike most of the ovarian steroidogenic enzymes, no cDNA encoding 20/-HSD has been cloned in any animal species. Therefore, we have begun to isolate cDNA encoding pig testis 20,3-HSD, since only pig testis 203HSD has been purified previously (Nakajin et al. 1988). Using synthetic oligonucleotides deduced from the partially determined amino acid sequences, we have, for the first time, isolated and cloned cDNA encoding 20/-HSD from a pig testis cDNA library (Tanaka et al. 1992b). The cDNA contains an open reading frame predicted to encode 289 amino acid residues. Surprisingly it has 85% amino acid homology with human carbonyl reductase. We are now using this cDNA clone to isolate and clone cDNAs encoding rainbow trout 20/-HSD from the rainbow trout ovary cDNA library. Using these cloned cDNAs as probes, we are currently investigating changes in mRNA levels of var-

8 ious steroidogenic enzymes in rainbow trout ovarian follicles during various periods of ovarian development and the gonadotropin- and forskolininduced steroidogenesis. Northern blot analysis of ovarian RNA with a rainbow trout ovarian P-45017, cDNA revealed the presence of a 2.4 Kb mRNA species. The 2.4 Kb transcripts were not found in ovaries during the early vitellogenic stage, barely detected during the mid-vitellogenic stage, and abundant during the postvitellogenic stage and after ovulation (Sakai et al. 1992). Hybridization of rainbow trout RNA to a rainbow trout P-4 05 Arom cDNA clone showed only a single major P-4 50 Arom RNA, 2.6 Kb in length. The 2.6 Kb RNA transcripts were found in the ovary during active vitellogenesis, but could not be found in the ovary in the stage of nuclear migration and nuclear maturation or in the ovary containing postovulatory follicles (Tanaka et al. 1992a). These results are consistent with the rapid decrease in aromatase activity in the granulosa cell layers during the postvitellogenic period (Young et al. 1983). It is thus concluded that the ability of the granulosa cells to produce estradiol-17,3 is regulated by the amount of the 2.6 Kb RNA transcripts present (Tanaka et al. 1992a). Our preliminary results indicate that forskolininduced 17a,20/-DP production is accompanied by a dramatic decrease in P- 4 50 Arom mRNA levels and a marked increase in 20-HSD mRNA levels by granulosa cells isolated from postvitellogenic follicles. A 2- to 3-fold increase in P-450sCC and 3HSD mRNAs and a slight decrease in P-450,1 7 mRNA were also observed during the forskolininduced 17a,203-DP production (Tanaka, unpublished).

c. MIH receptors We demonstrated that 17a,200-DP was ineffective in inducing oocyte maturation when microinjected into the immature oocytes of goldfish, but external application of the steroid was effective (see Nagahama 1987b). These findings suggest that the site of MIH action in inducing meiotic maturation in fish oocytes is at or near the oocyte surface. It is also shown in amphibians that progesterone, a putative

MIH in amphibians, apparently acts via a receptor on the oocyte surface plasma membrane and not through cytoplasmic or nuclear receptors (for review, see Maller 1985). More direct evidence for the existence of MIH receptors in fish oocyte plasma membranes has been obtained by binding studies using labelled MIH. Specific bindings have been demonstrated in salmonid and Japanese flounder, Paralichthys olivaceus, oocytes for 17a,20-DP (Maneckjee et al. 1989; Yoshikuni and Nagahama 1989; Weisbart et al. 1991; Yoshikuni et al. 1992) and in the ovaries of spotted seatrout for 20/-S (Patino and Thomas 1990a). The association of 20/-S to satulable binding sites was extremely fast. Maximum binding was achieved after 5 min of incubation. The binding sites showed high affinity (Kd, 10 - 9 M) and limited capacity (10-13-10 - 12 mol/g ovary) for 203-S to its binding site in seatrout follicles and was several orders of magnitude higher than the affinity of progesterone to its receptor on the amphibian oocyte (Sadler and Maller 1982). In the daily spawners, Kisu, Sillago japonica, and dragonet, Repomucenus beniteguri, there is a phase in which oocytes can mature in vitro in the presence of GTH, but not in the precence of MIH alone. However, these oocytes become sensitive to MIH stimulation if exposed to GTH in vitro (Kobayashi et al. 1988; Zhu et al. 1989). A similar observation was also reported in Atlantic croaker oocytes (Patino and Thomas 1990b). These observations suggest that oocyte maturational competence is regulated by GTH. It is also possible that this action of GTH is mediated by the membrane receptor for MIHs. Recent MIH binding studies on seatrout and flounder appear to support these suggestions (Thomas and Patino 1991; Yoshikuni et al. 1992). The concentration of 17a,203-DP and 203-S binding sites was significantly elevated in these fishes during final oocyte maturation. In vitro treatment of seatrout and flounder ovarian follicles with human chorionic gonadotropin (HCG) also increased MIH binding sites. Moreover, this gonadotropin-induced elevation in MIH binding sites coincides with the development of oocyte maturational competence. These results strongly suggest that a gonadotropin-induced increase in

9 MIH binding sites is essential for the development of oocyte maturational competence in these species (Thomas and Patino 1991; Yoshikuni et al. 1992).

d. Fish MPF The existence of MIH receptors at the surface of oocytes suggests that there is a cytoplasmic factor which mediates the action of MIH. This factor, designated maturation-promoting factor (MPF), was first detected in unfertilized amphibian eggs, from which cytoplasm was withdrawn and microinjected into full-grown amphibian oocytes, causing the oocytes to mature into unfertilized eggs (Masui and Clarke 1979; Nagahama 1987c; Nagahama and Yamashita 1988). It was therefore anticipated that a cytoplasmic factor similar to amphibian MPF is also produced in fish oocytes under the influence of 17tz,203-DP. To determine whether MPF activity is present in the mature fish oocyte, we extracted MPF activity from HCG-injected mature goldfish oocytes and naturally spawned carp oocytes by ultracentrifugation at 100,000 x g for lh. When the supernatant of this extract was microinjected into postvitellogenic, immature goldfish oocytes, the recipient oocytes underwent GVBD, indicating that MPF activity is present in mature goldfish oocytes. MPF activity was also extracted from oocytes matured in vitro by 17ca,203-DP, but not from immature oocytes or activated oocytes. MPF activity extracted from goldfish was also effective when injected into immature goldfish oocytes under the presence of protein synthesis inhibitors. The injected oocytes matured much faster than oocytes induced to mature by incubation with 17a,20f3-DP. GVBD usually occurred at the center of the MPF-injected oocytes, because the movement of the GV to the animal pole did not take place. GV migration always occurred in the oocytes matured naturally, in vivo by HCG, or in vitro by 17ca,200-DP. MPF transfers have been carried out between oocytes of goldfish and a frog, Xenopus laevis. MPF from mature oocytes of either source induced maturation in oocytes of the other species. Furthermore, goldfish MPF can induce maturation when

injected into immature oocytes of the starfish, Asterina pectinifera (see Kishimoto 1988). These results suggest that MPF is similar among vertebrates and invertebrates. It has also been shown that MPF activity is present in a higher plant, lily (Lilium longiflorum) and mitotic somatic cells, and that this factor is not species specific (for review, Nagahama 1987b, c; Kishimoto 1988; Yamaguchi et al. 1991). Thus, MPF is not merely a maturationpromoting factor, but may be a more general factor responsible for the initiation of nuclear membrane breakdown and subsequent cell division of both mitosis and meiosis.

e. MPF and HI kinase purification In an effort to understand the activation and inactivation of fish MPF, we have undertaken purification and characterization of this material from unfertilized eggs of carp, Cyprinus carpio. Carp are an excellent source of MPF, since MPF has been successfully extracted from carp unfertilized eggs and a large number of eggs (300-1000 ml/fish) arrested in a mataphase II can be easily obtained by an in vivo injection of HCG into gravid females. MPF was purified from the 100,000 x g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, pl3sucl-affinity Sepharose, Mono S, and Superose 12. MPF activity was assayed by injecting the sample into full-grown immature Xenopus oocytes in the presence of protein synthesis inhibitor (cycloheximide). GVBD was detected by white spot on the animal pole of the oocyte, and confirmed by manually dissecting oocytes after boiling. H1 kinase activity was also determined using histone H1 and a synthetic peptide (SP-peptide, KKAAKSPKKAKK) as exogenous substrates. MPF activity comigrated with histone H1 kinase activity throughout purification (Yamashita et al. 1992a, b). On Superose 12, MPF and kinase activity coeluted as a single peak with an apparent molecular weight of about 100 Kd. These results suggest the notion that MPF and histone H1 kinase are different manifestations of the same entity. SDSPAGE analysis of the active fractions after Su-

10 perose 12 revealed the presence of four proteins of 33-, 34-, 46-, and 48-Kd. Among the four proteins, the 34-, 46-, and 48-Kd proteins correpond well to MPF and H1 kinase activities, but the peak of 33-Kd protein seems to be different from the peak of MPF and HI kinase activities. The 46- and 48-Kd proteins were labelled when [ 32 P]ATP was applied in the fractions, indicating that these proteins are one of the endogenous substrates for the kinase. The final preparations of MPF was purified over 1,000-fold with a recovery of about 1%. The final preparation was also purified 5,000-fold with a recovery of 5%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP-peptide was used.

f. MPF characterization It has recently been shown from genetic studies in yeast and biochemical studies in frogs and starfish that MPF consists of two components (Lohka et al. 1988; Gautier et al. 1988, 1990; Labbe et al. 1989a, b). A catalytic subunit of MPF is the homolog of the cdc2+ gene product of fission yeast, Schizosaccharomyces pombe, referred to as p3 4 cdc2 (cdc2 kinase). This kinase can use histone HI as an exogenous substrate. A regulatory subunit of MPF is cyclin, which was first discovered, independently of MPF, in the early embryos of marine invertebrates. We have raised a monoclonal antibody against the most conserved amino acid sequence of the p34 cdc2, the PSTAIR sequence (EGVPSTAIREISLLKE) (Yamashita et al. 1991). This antibody recognizes 31-34 Kd proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either p34cdc 2 itself or proteins highly homologous to p34 cdc2 in a given species, since, in all species studied to date, they are all precipitated with pl3SUcl , the fission yeast sucl+ gene product, which binds to p34cdc 2 with high specificity. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes (Hirai et al. 1992a). Sequence comparisons revealed that these two clones are highly homologous (95%) and found to be similar to Xenopus cy-

clin B 1. To obtain sufficient goldfish cyclin B protein with which to produce antibodies, a recombinant cyclin B was produced in Escherichia coli using a cDNA which codes an N-terminal truncated form of goldfish cyclin B. The transformed E. coli produced a 43-Kd protein which was biologically active. It was phosphorylated by MPF purified from mature goldfish oocytes, and the injection of the protein into full-grown immature Xenopus oocytes induced GVBD and chromosome condensation in the presence of cycloheximide. Monoclonal antibodies against goldfish cyclin B were produced by using E. coli-produced goldfish cyclins as antigens. To characterize the four proteins of the purified carp MPF, we carried out an immunoblotting experiment with our anti-PSTAIR, anti-cyclin A and anti-cyclin B monoclonal antibodies. The PSTAIR antibody recognized the 33- and 34-Kd proteins (Yamashita et al. 1992a). More recently, the 33-Kd protein was identified as the goldfish cdk2, a cognate variant of cdc2 (Hirai et al. 1992b). The 46and 48-Kd proteins were recognized by a monoclonal antibody against cyclin B, but not by an anti-cyclin A antibody. These findings indicate that carp MPF is a complex of p3 4 cdc2 (cdc2 kinase) and cyclin B (Yamashita et al. 1992a). The PSTAIR and cyclin B antibodies were also used to examine changes in the levels of p34 cdc2 and cyclin B during goldfish oocyte maturation induced in vitro by 17a,200-DP (Hirai et al. 1992a). Protein p34 cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was absent in immature oocyte extracts and appeared when oocytes underwent GVBD. Cyclin B that appeared during oocyte maturation was labelled with [35 S]methionine, indicating its de novo synthesis (Hirai et al. 1992a). Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 activation, which was associated with threonine phosphorylation of p34 cdc2 and serine phosphorylation of cyclin B, as found in oocytes matured by 17oa,20/DP (Yamashita, unpublished). Cyclin B-induced p3 4 cdc2 activation was not induced by inhibiting threonine phosphorylation of p34 cdc2 by protein kinase inhibitors (6-dimethylaminopurine, H7 and

11 7ca,2013DP 6- DMAP H8

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cyclin B

Fig. 1. Current model of the formation and activation of MPF during fish oocyte maturation. The model incorporates the current findings on MPF formation and activation described in the text, but also contains speculative elements based on our preliminary experimen2 tal data. The catalytic subunit of MPF, p34cdc , is already present in immature oocytes, but its regulatory subunit, cyclin B, is absent. 17a,2003-DP, a MIH of fish, induces oocytes to synthesize cyclin B. The association of the synthesized cyclin B and the preexisting p3 4 cdc2 allow the cdc25 gene product and unidentified threonine kinase to dephosphorylate Tyr 15(Y) and to phosphorylate Thr 161 2 (T) of p34cdc , respectively. The chemically modified p34cdc2 then binds tightly on cyclin B, yielding active MPF, after serine (S) phos2 phorylation of cyclin B catalyzed by the active p34cdc . For further details see the text.

H8). These results suggest that 17ac203-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p3 4 cdc2 through threonine phosphorylation of p34cdc 2 . A hypothesis for the mechanism of the formation and activation of fish MPF, based primarily on the results on studies on the goldfish and carp, is presented in Fig. 1.

Acknowledgements The studies from our laboratory described in this article have been aided in part by Grant-in-Aid for Special Project Research (63640001 and 0120210 to YN) and Scientific Research from the Ministry of Education, Science and Culture, Japan.

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