Meiotic Maturation Of Xenopus Oocytes Research Articles

Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control.

The meiotic maturation of Xenopus oocytes initiates significant changes in the translation of a number of maternal mRNAs that coincide with alterations in their polyadenylation states. A considerable number of maternal mRNAs are deadenylated in mature oocytes, thereby reducing their translational efficiencies. In this report we demonstrate that deadenylation does not require specific cis-acting sequences. Polyadenylated RNAs derived from either ribosomal protein or beta-globin mRNAs, or that contain non-mRNA-derived sequences, are deadenylated in mature oocytes. Translation of a substrate RNA is not required for its deadenylation. G10 mRNA is representative of a class of mRNAs that is translationally activated at maturation and contains the cytoplasmic polyadenylation element (U)6AU. A deletion mutant G10 transcript that lacks the (U)6AU element is not polyadenylated in mature oocytes but is deadenylated instead. Insertion of the (U)6AU element into the 3'-untranslated region of the ribosomal protein L1 mRNA is sufficient to prevent both its deadenylation and polysomal release in mature oocytes. These results indicate that the deadenylation and translational inactivation of maternal mRNAs during Xenopus oocyte maturation occur by a default pathway in which transcripts lacking a cytoplasmic polyadenylation element undergo poly(A) removal.

P40MO15, a cdc2-related protein kinase involved in negative regulation of meiotic maturation of Xenopus oocytes.

The clone MO15 which codes for a 40 kd protein (p40MO15) with 40% amino acid identity to the human cdc2 protein kinase has been isolated from a Xenopus cDNA library using a synthetic oligonucleotide probe. MO15 mRNA is accumulated during oogenesis, becomes de-adenylated during meiotic maturation, and is degraded after the mid-blastula-transition stage of embryogenesis. Translation of p40MO15 is restricted to non-mature oocytes. Specific inhibition of p40MO15 synthesis in stage VI oocytes by antisense oligonucleotide depletion of MO15 mRNA increases the rate of progesterone induced H1 kinase activation and oocyte maturation. This effect can be reversed by subsequent injection of synthetic MO15 mRNA. These results suggest that p40MO15 is involved in negatively regulating meiosis.

The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes

Protein secretion is blocked in Xenopus oocytes arrested at second meitoic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphase-arrested oocyte's secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitus virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on β- d-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 63% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [ 35S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is 15-fold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the trans-Golgi.

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets.

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.

The Product of the mos Proto-Oncogene as a Candidate "Initiator" for Oocyte Maturation

The endogenous c-mos product, pp39mos, is required for progesterone-induced meiotic maturation in Xenopus oocytes. Treatment of oocytes with progesterone induced a rapid increase in pp39mos that preceded both the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). Microinjection of synthetic mos RNA into oocytes activated MPF and induced GVBD in the absence of progesterone. Thus, the mos proto-oncogene product may qualify as a candidate "initiator" protein of MPF and is at least one of the "triggers" for G2 to M transition.

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.

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.

Induction of meiotic maturation in Xenopus oocytes by 12- O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP 3 − are involved in oocyte maturation was investigated. Microinjection of IP 3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP 3 concentration of 1 μM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12- O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.

Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati.

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.

Amiloride and other blockers of electrolyte flux as inhibitors of progesterone-stimulated meiotic maturation in Xenopus oocytes.

Amiloride and seven of its analogues, as well as seven substances of other structural types, all of which control electrolyte transport, were tested for their effectiveness for inhibiting the progesterone-stimulated meiotic maturation cycle of oocytes from the amphibian Xenopus laevis. Data were also collected on the ability of the oocytes to recover from the drug's inhibitory effect and on the toxicity of each drug. The data revealed that up to a 14-fold difference exists in the inhibitory concentration 50% between substances, that recovery from the drug's inhibitory effect ranged from non-reversible to almost complete reversibility and that toxicity, as measured by failure to exclude trypan blue, ranged from 0 to 57%. In some, but not all cases, the failure to recover from the drug's inhibitory effects could be correlated to the drug's toxic effects. Amiloride which has been shown to be a reversible inhibitor of cell proliferation in rapidly dividing mammalian cell populations has similar properties on the oocyte maturation division cycle of Xenopus.

Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor.

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.

Germinal vesicle breakdown in the Xenopus laevis oocyte: description of a transient microtubular structure.

During progesterone-induced meiotic maturation of Xenopus oocytes in vitro, 7 morphological stages were defined. Using cytological analysis, nuclear breakdown was divided into three stages. Stage 1 corresponded to basal germinal vesicle breakdown. Stage 2 was characterized by the advent and development of a fibrillar network formed by microtubules at the basal part of the nucleus. Below, a lamellar microtubule organizing center (MTOC) was present. Numerous vesicles of smooth endoplasmic reticulum were proximal to both of these structures. During its formation and modification (stages 2 and 3), the fibrillar network migrated towards the animal pole. A prometaphase 1 stage was observed before the formation of the metaphase 1 spindle.

Roles of cyclic amp and calcium in maturation of Xenopus laevis oocytes

Progesterone induces in vitro meiotic maturation in Xenopus oocytes. 1. 1. Early steps of maturation are sensitive to inhibition by phosphodiesterase inhibitors (methyl xanthines) or by cholera toxin, suggesting that elevated levels of intracellular cyclic-AMP block progesterone action. 2. 2. Progesterone causes a significant decrease in the amount of newly synthesized cyclic AMP from microinjected α- 32P-ATP. 3. 3. Ca 2+ ions are involved both in the initiation of progesterone action and in the whole process of the first meiotic division.

Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium.

Progesterone reinitiates meiotic maturation in Xenopus oocytes. Evidence is reported which indicates that the steroid acts at the level of the cell surface and suggests that an induced change of Ca2+ distribution triggers in turn a cascade of cytoplasmic events including protein synthesis and germinal vesicle (nucleus) breakdown. These novel features of steroid hormone action in amphibian oocytes are discussed in relation to presently accepted views of the mechanism of action of steroid hormones in somatic cells.

Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.