Masakane Yamashita

Regulation of oocyte growth and maturation in fish

This chapter has briefly reviewed the current status of investigations on the hormonal regulation of oocyte growth and maturation in fish (see Figs. 4 and 9). Pituitary gonadotropins are of primary importance in triggering these processes in fish oocytes. In both cases, however, the actions of gonadotropins are not direct, but are mediated by the follicular production of steroidal mediators, estradiol-17 beta (oocyte growth) and 17 alpha,20 beta-DP or 20 beta-S (oocyte maturation). Investigators have established that both estradiol-17 beta and 17 alpha,20 beta-DP are biosynthesized by salmonid ovarian follicles via an interaction of two cell layers, the thecal and granulosa cell layers (two-cell-type model). The granulosa cell layers are the site of production of these two steroidal mediators, but their production depends on the provision of precursor steroids by the thecal cell layers. A distinct steroidogenic shift from estradiol-17 beta to 17 alpha,20 beta-DP, occurring in salmonid ovarian follicles immediately prior to oocyte maturation, is a prerequisite for the growing oocytes to enter the maturation stage, and requires a complex and integrated network of gene regulation involving cell specificity, hormonal regulation, and developmental patterning. The cDNAs for most of the steroidogenic enzymes responsible for estradiol-17 beta and 17 alpha,20 beta-DP biosynthesis have been cloned from rainbow trout ovaries. Our next task is to determine how gonadotropin and other factors act on ovarian follicle cells to turn the expression of these specific genes on and off at specific times during oocyte growth and maturation. Increasing evidence now suggests that a variety of neuromodulatory, autocrine, and paracrine factors may also be involved in the regulation of steroidogenesis in fish ovarian follicles. Molecular biological technologies should be applied to identify these substances. Of considerable interest is the finding that MIH, unlike most steroid hormones, acts on its receptors at the surface of oocytes. Further studies of the association of the MIH-MIH receptor complex with a Gi protein, probably resulting in the inactivation of adenylate cyclase, should lead to a discovery of a new mechanism of steroid hormone action. The early steps following MIH action involve the formation of the major cytoplasmic mediator of MIH, MPF. Fish MPF, like that of Xenopus and starfish, consists of two components: cdc2 kinase and cyclin B. Nevertheless, the mechanism of MIH-induced MPF activation in fish oocytes differs from that in Xenopus and starfish because the appearance of cyclin B protein is a crucial step for 17 alpha,20 beta-DP-induced oocyte maturation in fish.(ABSTRACT TRUNCATED AT 400 WORDS)

A Monoclonal Antibody Against the PSTAIR Sequence of p34cdc2, Catalytic Subunit of Maturation‐Promoting Factor and Key Regulator of the Cell Cycle

A homolog of the serine/threonine protein kinase (p34cdc2), encoded by the cdc2+ gene of the fission yeast (Schizosaccharomyces pombe), is a catalytic subunit of maturation‐promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of p34cdc2 This antibody recognizes 31–34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti‐PSTAIR antibody are probably either p34cdc2 itself or proteins highly homologous to p34cdc2 in the given species, since, in all species studies to date, they are all precipitated with p13suc1, the fission yeast suc1+ gene product, which binds to p34cdc2 with high specificity. The anti‐PSTAIR immunoprecipitate had no histone H1 kinase activity and did not contain cyclin B, suggesting that the PSTAIR region is masked when p34cdc2 forms a complex with cyclin B as an active kinase. Immunoblotting with the anti‐PSTAIR antibody demonstrated that the fastest‐migrating form of p34cdc2 homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34cdc2 The observed broad cross‐reactivity of the anti‐PSTAIR antibody against p34cdc2 homologues in various species should permit us to examine the role of p34cdc2 homologues in the regulation of the cell cycle in a variety of organisms.

SUPEROXIDE DISMUTASE AND THIOREDOXIN RESTORE DEFECTIVE P34CDC2 KINASE ACTIVATION IN MOUSE TWO-CELL BLOCK

We recently showed that superoxide dismutase (SOD), a free radical scavenger, and thioredoxin, a potent protein disulfide reductase, release mouse two-cell stage developmental block in vitro. To elucidate the mechanism underlying the two-cell block and the effects of these enzymes, we studied the chronological changes in the kinase activity and the immunoblotting pattern of p34cdc2, a key regulator of the cell cycle, during the first and second cell cycles of the mouse embryonic development. In vivo embryos were freshly collected at fixed times, and in vitro embryos cultured from the pronuclear stage were also sampled with the same time-course. A marked elevation of p34cdc2 kinase activity was observed in vivo at 27-32 and 51 h after an injection of human chorionic gonadotropin. These times coincide with the M-phases of embryo cleavage. In vitro embryos showed high kinase activity during the M-phase of the first cleavage, but this activity was not elevated during the second cell cycle. The addition of recombinant human SOD (200 micrograms/ml) or thioredoxin from Escherichia coli (500 micrograms/ml) to the medium enabled kinase activation with a time course similar to that of in vivo embryos. The immunoblotting patterns suggested the dephosphorylation of p34cdc2 at the M-phase of the first and the second cleavages in vivo. Although p34cdc2 was dephosphorylated at the M-phase of the first cleavage and then rephosphorylated for embryos cultured in vitro, the second dephosphorylation was not observed during the second cell cycle. The addition of SOD or thioredoxin permitted the dephosphorylation at the M-phases of both the first and the second cleavage. These results suggest that one of the chief causes of two-cell block in vitro is the impairment in p34cdc2 dephosphorylation, recently shown to be catalyzed by the cdc25 homologue. This impairment is thought to be due to oxidative stress, because both SOD and thioredoxin are known to play a defensive role against it.

Molecular endocrinology of oocyte growth and maturation in fish.

RésuméLes gonadotropines hypophysaires (GtHs) sont de première 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 lintermédiaire dune production ovarienne de médiateurs stéroïdiens, aussi bien pour la croissance (oestradiol-17β) que pour la maturation ovocytaire (hormone inductrice de la maturation, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP chez les salmonidés 17α,20β,21-trihydroxy-4-pregnen-3-one, 20βS chez les sciénidés). Il est acquis que les productions doestradiol-17β et de 17α,20β-DP par les follicules ovariens de salmonidés se font via linteraction de deux couches cellulaires, la thèque et la granulosa (modèle de coopération cellulaire). Chez les salmonidés, un changement net de la stéroïdogenèse, passant de la production doestradiol-17β à celle de 17α,20β-DP, intervient dans les enveloppes folliculaires juste avant la maturation. Il est possible que ce changement soit une conséquence de modifications profondes dans lexpression des gènes codant pour diverses enzymes de la stéroïdogenèse. Comme étape initiale de létude de cette question, nous avons isolé et caractérisé les ADNc codant pour différentes enzymes de la stéroïdogenèse ovarienne, à savoir les ADNc, de truite arc-en-ciel, du cytochrome P-450 coupant la chaîne latérale du cholestérol, de la 3β-hydrostéroïde déhydrogénase (HSD), du cytochrome P-450 17α-hydroxylase/17,20 lyase, et du cytochrome P-450 aromatase, ainsi que lADNc de la 20β-HSD de porc.Loestradiol-17β stimule la synthèse et la sécrétion hépatique du précurseur du vitellus: la vitellogénine. Celle-ci est ensuite transportée vers lovaire où elle est sélectivement incorporée dans lovocyte par un processus impliquant des récepteurs spécifiques situés à la surface de la cellule. Loestradiol-17β induit également la synthèse, par le foie, de protéines membranaires de loeuf. Linduction de la maturation, par la 17α,20β-DP ou la 20βS, se fait par lintermédiaire de leur liaison à la membrane plasmique ovocytaire. Cette interaction, initiale, surface cellulaire-MIH est suivie par la formation du médiateur majeur du MIH, le facteur promoteur de la maturation (MPF). Nous avons purifié le MPF à partir dovocytes maturés de carpe. Le MPF de carpe est constitué de deux composants: lhomologue du produit du gène cdc2+ de levure (p34cdc2, et la cycline B. La protéine kinase cdc2+ est présenté dans les ovocytes immatures aussi bien que dans les ovocytes dont la maturation a été induite par un traitement à la 17α,20β-DP, tandis que les protéines de type cycline B ne peuvent être détectées que dans les ovocytes maturés. Laddition de cycline B de poisson rouge, exprimée dans des bactéries, à des extraits dovocytes immatures de poisson rouge induit lactivation du MPF. Ces résultats suggèrent que lapparition des protéines de type cycline B est une étape cruciale dans linduction de la maturation des ovocytes de poisson par la 17α,20β-DP.AbstractPituitary 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-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-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-17β to 17α,20β-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, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA.Estradiol-17β 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-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-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 (p34cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-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 17α,20β-DP-induced oocyte maturation in fish.

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: Direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.

Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2.

This paper reports the nucleotide and predicted amino acid sequences of the goldfish cdk2, a cognate variant of the cell cycle regulator cdc2. The predicted protein sequence shows strong homology to the other known cdk2 (88% for Xenopus and 90% for human). A monoclonal antibody against the C-terminal sequence of goldfish cdk2 recognized a 34-kDa protein in extracts from various goldfish tissues. The protein level was high in such tissues as testis and ovary containing actively dividing cells. Protein cdk2 binds to p13sucl, the fission yeast suc1+ gene product, but not to cyclin B, with which cdc2 forms a complex. The kinase activity of cdk2 increased 30-fold when oocytes matured, although its protein level did not remarkably change. Anti-cdk2 immunoprecipitates from 32P-labeled mature oocyte extracts contained a 47-kDa protein, which was not recognized by either anti-cyclin A or anti-cyclin B antibody, indicating complex formation of cdk2 with a protein other than cyclins A or B.

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4,6-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.

Testicular Development Induced by a Recessive Mutation during Gonadal Differentiation of Female Common Carp (Cyprinus carpio, L.)

The phenotypic effects of a new recessive mutation mas−1, which in homozygous condition induces testicular development in XX animals of common carp (Cyprinus carpio L.), are described. Sexual differentiation of XX; mas−+/mas−1 and XX; mas−1/mas−1 animals was compared with the gonad development of XX wild type females and XY males. In XX females gonadal differentiation starts with the formation of an ovarian cavity and entry into meiosis of germ cells at around 80 days post hatching (ph). Male gonads remain quiescent until 120 days ph during which period they develop a network of loose connective tissue. Spermatogenesis starts with tubule formation and the differentiation of germ cells into spermatogonia type B. Heterozygous XX; mas−+/mas−1 animals developed as normal females, but in homozygous XX; mas−1/mas−1 animals two types of gonad development were observed. In the first type, germ cells did not enter meiosis until 100 days ph when they differentiated as spermatogonia. An ovarian cavity was not formed but male specific connective tissue developed instead. These gonad developed as normal testes. In the second type, germ cells differentiated at 80 days ph as either oocytes or spermatocytes, which resulted in the gonads developing as ovotestes. The formation of an ovarian cavity was in most cases incomplete. The phenotypic effects of mas−1 are interpreted as a timing mismatch between mas activation and female sex differentiation.

Mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one, a teleost maturation-inducing substance

This article briefly reviews the current status of investigations, mainly based on the amago salmon,Oncorhynchus rhodurus, on the mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog). Pituitary gonadotropin is of primary importance in triggering meiotic maturation in teleost oocytes. However, the maturational action of gonadotropin is not direct, but is mediated by the follicular production of maturation-inducing substance (MIS). It is now well established that 17α,20β-diOHprog is the major MIS of salmonids. Production of this steroid occursvia the interaction of two distinct cell layers, the thecal and granulosa cell layers (2-cell type model). The first step of the stimulating effect of gonadotropin in both layers is the receptor-mediated activation of adenylate cyclase and formation of cAMP. Our findings suggest that the major stimulating action of gonadotropin on 17α,20β-diOHprog biosynthesis is due to the stimulation of 17α-hydroxyprogesterone production by the thecal layer and the selective induction of thede novo synthesis of 20β-hydroxysteroid dehydrogenase in the granulosa layer. 17α,20β-diOHprog acts at the surface of the oocyte. The early steps following 17α,20β-diOHprog action involve the formation of the major cytoplasmic mediator of this steroid, maturation-promoting factor (MPF). It was shown that goldfish MPF induces meiotic maturation inXenopus oocytes andvice versa. The chemical characterization of fish MPF is important for our understanding of the precise mode of maturational action of 17α,20β-diOHprog.

Maturation-promoting factor and p34cdc2 kinase during oocyte maturation of the Japanese quail☆

Maturation-promoting factor and a homolog of fission yeast cdc2+ gene product (p34cdc2) were investigated during the final 24 hr of maturation of quail oocytes. Kinase activity of p34cdc2 in the oocyte germinal disk (GD) increased 15 times at maturation. Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cdc2 monoclonal antibody. A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation. No p34cdc2 could be detected outside the GD. Microinjection of GD extract from mature oocytes caused maturation of Xenopus oocytes.