Maturation Of Amphibian Oocytes Research Articles

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosis-promoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians ( Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian ( Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0–3 μ M), cAMP (0–3 m M), or dibutyryl cAMP (dbcAMP, 0–2.5 m M) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 m M) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 m M) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0–3 μ M) and IBMX (0-0.09 m M) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 m M) and IBMX (0.09 m M). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.

Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin

AbstractProstaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro‐cultured ovarian follicles. Exposure of follicles to PGF2α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF2α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF2α to FPH‐treated follicles. PGE, in contrast to PGF2α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF2α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF2α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH‐induced ovulation and maturation. PGF2α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH‐induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF2α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.

Intracellular pH and ribosomal protein S6 phosphorylation: role in translational control in Xenopus oocytes

ABSTRACT The induction of amphibian oocyte maturation with progesterone as well as the activation of sea urchin eggs at the time of fertilization result in increased protein synthesis. The increase in both cases involves the recruitment of maternal mRNA onto polysomes. Further, it has been reported that sea urchin eggs, like full-grown Xenopus oocytes, contain no spare translational capacity based on the observation that injected heterologous mRNA is translated only at the expense of endogenous messages. The nature of the limiting component defined by such experiments is not known, but two factors which have been proposed to play a role in regulating protein synthesis are ribosomal protein S6 phosphorylation and intracellular pH. In the current paper, we review the literature and present new evidence on the roles intracellular pH and S6 phosphorylation have in regulating protein synthesis in Xenopus oocytes. We report that pHi does not increase between stage 3 and stage 6, yet the protein synthetic rate increases at least eight fold during the same period. Hence, we conclude that increasing pHi is not a prerequisite for increasing protein synthesis. Moreover, we present three arguments against increased ribosomal protein S6 phosphorylation being sufficient or necessary for increased protein synthesis in Xenopus oocytes. First, the level of S6 phosphorylation does not increase between stages 4 and 6, a period exhibiting a two to three fold increase in protein synthesis. Second, the injection of globin mRNA into stage-4 oocytes increases total protein synthesis two to three fold, but has no effect on S6 phosphorylation. Third, when the injection of globin mRNA into stage-4 oocytes is followed by an injection of MPF, a dramatic increase in S6 phosphorylation is seen, but total protein synthesis is not further stimulated.

Gonadotropins and the timing of progesterone-induced meiotic maturation of Xenopus laevis oocytes

Isolated oocytes from 30 unstimulated Xenopus laevis females required from from 2.50 ± 0.13 to 14.59 ± 0.77 hr after progesterone exposure for the first 50% of each group to complete meiotic maturation. Injecting 8 females with an amount of hCG not causing ovulation (25 μg, 96 IU), lowered oocyte maturation times by 45–83%. An enzyme-linked immunosorbent assay (ELISA) of the blood of 18 unstimulated animals found a constituent which bound to anti-hCG in amounts (equivalent to 0–1.03 μg/ml hCG) that had a direct relationship to the rates of GVBD in oocytes. Preincubation of manually isolated follicles in 0.25–1.25 μg/ml hCG shortens oocyte maturation times by 18–50% in a direct, nonlinear fashion and this priming effect is reversed when hCG is withdrawn. The action of gonadotropins in facilitating germinal vesicle breakdown (GVBD) mimics the previously reported priming effect produced by preincubation of oocytes in subthreshold levels of progesterone. Evidence suggests that individual variation in the time course of progesterone-induced meiotic maturation of amphibian oocytes is the result of priming differences caused by the action on follicle cells of fluctuating blood levels of an LH-like hormone.

Exocytosis of glycogen during maturation of amphibian oocytes

AbstractThe repartition and fate of glycogen β has been followed during progesterone‐induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded.Stacks of annulate lamellae increase during the early phase of in vitro progesterone‐induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.

OOcyte maturation in amphibians.

This chapter presents a complex picture of the regulation of oocyte maturation. The events that have been identified can be expressed temporally on a relative time scale, as shown in Figure 8. A major question that lies ahead concerns which of these events is causal for subsequent events and for GVBD. Clearly, the first event at 0.1 GVBD50, a decrease in cAMP, is both necessary and sufficient to induce GVBD. The increase in protein synthesis at 0.3 GVBD50 is apparently necessary, and the activity of MPF appearing at 0.4 GVBD50 is apparently sufficient, as judged by the microinjection assay. It is important to identify further steps after MPF appearance in order to establish the mechanism of action of MPF. The fact that events highly correlated with cell growth (i.e., pHi increase, S6 phosphorylation, and MPF appearance) all occur during amphibian oocyte maturation provides strong support for continued use of the oocyte in studying the general control of cell proliferation, particularly with the technique of microinjection. Investigation of the process of amphibian oocyte maturation has also led to the discovery of a totally new type of steroid hormone mechanism in which a plasma membrane steroid receptor is linked to the adenylate cyclase system. The oocyte system is the first example of a steroid hormone effect mediated by a direct effect on a target enzyme without a requirement for protein or RNA synthesis. The generality of this type of steroid hormone mechanism remains to be established, but its similarity to the P site on adenylate cyclase is suggestive evidence of a general mechanism, since the P site is present in all eukaryotic plasma membranes. Most steroid hormone systems have been investigated with respect to the mechanism of increased protein synthesis after steroid addition, with little attention to any cell division occurring. In the oocyte, the cell division effect of steroids is the predominant one; it is possible that this type of effect is mediated by a different receptor system than the one associated with changes in gene expression in somatic cells. The information described in this chapter hints at the complexity underlying the activation of development during the process of oocyte maturation. The finding that so many of the specific events and general mechanisms operating in oocyte maturation are also used in differentiated tissues strongly indicates that further study of amphibian oocyte maturation will lead to findings of general significance in terms of how information stored in the oocyte is used during development to result in a new member of the species.

Induction of chromosome replication during maturation of amphibian oocytes.

After fertilization amphibian embryos replicate their chromosomes faster than bacteria replicate their much smaller chromosomes. During oogenesis, materials are accumulated to sustain these rapid cycles of chromosome replication. Thus growth is uncoupled from nuclear division. Most of the machinery for DNA replication and chromatin assembly is present in the oocyte, which lacks only the ability to initiate on a DNA duplex. After maturation and activation a cell cycle clock is triggered which specifies initiation of DNA replication on endogenous chromosomes, injected nuclei or injected plasmid DNA. The ability to reinitiate replication of a replicated molecule is tightly coupled to the cell cycle clock. Each egg can replicate an amount of DNA equivalent to 500 diploid nuclei in only five hours. However, each egg can assemble an amount of purified DNA equivalent to 12 000 diploid nuclei into regularly spaced nucleosomes in only one hour. The molecular basis of these extraordinary rates of DNA replication and chromatin assembly is considered.

In vivo actions of protein phosphatase inhibitor-2 in Xenopus oocytes

Meiotic maturation of amphibian oocytes induced by progesterone is known to be regulated by protein phosphorylation. To investigate a possible role for protein phosphatase-1 in this process, the effect of phosphatase inhibitor-2 was determined on the in vivo rate of dephosphorylation of phosphorylase a and on the rate of oocyte maturation. Dephosphorylation of microinjected phosphorylase a was inhibited up to 40% in the presence of inhibitor-2, with half-maximal inhibition at an intracellular concentration of 0.6 μM. Inhibitor-2 also caused over a 3-fold increase in the half-time for maturation, suggesting a possible role for protein phosphatase-1 in the regulation of meiosis.

Stimulatory and Inhibitory Effects of Human Follicular Fluid on Amphibian Oocyte Maturation and Ovulation hi vitro

Follicular fluid was collected from individual human ovarian follicles and its effects, alone or in combination with frog pituitary homogenate (FPH), on oocyte maturation and ovulation were assessed following incubation with amphibian ovarian follicles in vitro. Oocyte maturation, with little or no concomitant ovulation, was induced by variable amounts of follicular fluid. Some of the individual follicular fluid samples were very active in inducing oocyte maturation, whereas others were inactive. Frog pituitary homogenates exhibited biologic activity (induced oocyte maturation and ovulation) when incubated in the presence of most follicular fluid samples. However, follicular fluid samples from two individuals inhibited ovulation but not maturation in FPH-treated follicles. These results demonstrate that amphibian follicles remain viable and undergo a number of physiologic changes in the presence of unfractionated human follicular fluid. Under appropriate conditions both stimulatory and inhibitory effects of follicular fluid were observed. These data suggest that amphibian ovarian follicles may provide a simple and independent means for detecting and assaying a number of biologic activities present in follicular fluid obtained from single human and other mammalian ovarian follicles. Such results may provide the basis for dissociating endocrine and cellular interactions which occur during normal and abnormal follicular differentiation.

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.

Role of follicle wall in meiosis reinitiation induced by insulin in Rana pipiens oocytes.

The involvement of the ovarian follicle wall in insulin induction of Rana pipiens oocyte maturation in vitro was examined. Complete removal of the follicle wall significantly decreased, but did not obliterate, oocyte maturation (i.e., germinal vesicle breakdown, GVBD) induced by insulin. Dose-response studies of GVBD induction revealed that oocytes within intact follicles were at least 100 times more sensitive to insulin than denuded oocytes. Addition of cyanoketone, a steroid biosynthesis inhibitor, to intact follicles also suppressed insulin-induced GVBD. Inhibitory effects of either follicle wall removal or cyanoketone were not observed when denuded oocytes were treated with progesterone. Addition of either progesterone or pregnenolone to insulin-treated denuded oocytes augmented the oocyte GVBD response compared to either steroid alone and essentially replaced the effect of the follicle wall. In summary, steroidogenesis in the follicle wall appears to be a major factor contributing to the ability of insulin to induce GVBD. However, whether insulin stimulates follicle wall steroidogenesis or simply augments the biological activity of endogenous basal steroid levels is unresolved. The in vitro results show that oocyte maturation can be modulated by the combined actions of several hormones. Such steroid-insulin interactions may also be relevant to understanding the control of oocyte maturation in amphibians and other vertebrates, including mammals, under physiological conditions in vivo.

Changes in the oxidative metabolism during maturation of amphibian oocytes

Some aspects of oxidative metabolism during cytoplasmic maturation of the Bufo arenarum oocyte have been studied. During the autumn-winter period (immature oocyte), carbohydrates are degraded through the glycolytic pathway, followed by the classical tricarboxylic acid cycle. During the spring--summer period (mature oocyte), carbohydrates are mainly used through the pentose phosphate cycle, while the tricarboxylic acid cycle operates as the glutamic-aspartic cycle. The oxidative phosphorylation of ADP does not seem to change during oocyte maturation. Although maturation does not alter the absolute values of ATP and citrate in the oocyte, it determines their different compartmentalization, which, through phosphofructokinase, in turn regulates the glycolytic activity of the oocyte. Oxygen uptake decreases by about 40% during maturation, while simultaneously, a marked increment in respiratory stimulation by 2,4-dinitrophenol is observed.

Xenopus laevis oocyte calmodulin in the process of meiotic maturation.

Calcium ions are postulated to be involved in the process of meiotic maturation of amphibian oocytes. Since several of the calcium effects in eukaryotic cells are mediated by calmodulin, the present study was undertaken to assess the presence and level of calmodulin in Xenopus laevis oocytes before and after progesterone treatment. Calmodulin was shown to be present at a concentration of approximately microM in control oocytes cytosol. This level remained stable for 2 h when the maturation promoting factor appeared, and increased to approximately 44 to 59 microM at the time of the germinal vesicle breakdown. Maturation is therefore associated with calmodulin synthesis. Xenopus calmodulin was isolated from oocyte cytosol after heat treatment, anion exchange chromatography, and gel filtration, with a yield of approximately 23%. When compared to mammalian calmodulins, the amphibian protein exhibited the same ultraviolet absorption spectrum, a similar amino acid composition with 1 residue of trimethyllsine, and the same shape conformers in the absence or presence of divalent metals, as shown by different mobilities upon dodecyl sulfate-polyacrylamide gel electrophoresis. The protein migrated faster in the presence than in the absence of Ca2+ ions, Mn2+ and Mg2+ being less effective. It was able to activate calmodulin-deficient myosin light chain kinase. Its high serine content and the tryptic peptide maps obtained after high performance liquid chromatography point, however, to minor differences in the primary structures of mammalian and amphibian calmodulins.

Calcium dependence of steroid and guanine 3',5'-monophosphate induction of germinal vesicle breakdown in Rana pipiens oocytes.

The action of progesterone in the induction of meiotic maturation in amphibian oocytes is thought to be dependent on the presence of exogenous Ca2+ and/or Mg2+ ions. In the present study we report that exogenous Ca2+ is essential only during the first 5–10 min after exposure of Rana pipiens oocytes to steroid and that there is no significant Mg2+ requirement for germinal vesicle (oocyte nucleus) breakdown (GVBD). The order of effectiveness of divalent cations is Ca > Sr > Ba > Mg. GVBD is inhibited by only about 30% if denuded oocytes are transferred from Ca-containing to Ca-free medium containing inducing levels of steroid. If the diffusible Ca2+ pool (t½ < 15 min) is diminished by preincubation in Ca-free, EDTA-containing medium, GVBD is inhibited by 70-80%. Preincubation in Ca-free medium at 0–4 C results in the loss of 55–60% of the denuded oocyte Ca2+ and complete inhibition of steroid-induced GVBD. Progesterone uptake was independent of [Ca2+]o. Progesterone stimulates net 45Ca2+ uptake by denuded o...

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions

The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 m M Mn 2+ and La 3+ and, to a lesser extent, by 2–4 m M Ca 2+ and Mg 2+. Maturation induced by 1.5 m M Mn 2+ was inhibited by K + concentrations above 0.25 m M. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.

Control of chromosome behavior during progesterone induced maturation of amphibian oocytes

Fully grown oocytes isolated from Rana pipiens ovaries undergo maturation when they are exposed to progesterone. The cytoplasm of these oocytes develops the activity to induce metaphase chromosome condensation in foreign nuclei exposed to it. This chromosome condensation activity (CCA) persists until eggs are fertilized and resume meiosis. However, the present study shows that CCA develops and persists in blastomeres arrested by injection with unfertilized egg cytoplasmic extracts. The stability of metaphase chromosomes induced by CCA in eggs is dependent on substances from germinal vesicles (GV). Chromosomes in enucleated oocytes i.e. oocytes from which GVs had been removed prior to progesterone treatment, disintegrate soon after condensed to the metaphase state, and the ability of the oocytes to sustain metaphase chromosomes can be restored by introduction of GV extracts. Autoradiographic studies reveal that GV proteins as well as cytoplasmic proteins synthesized during the course of maturation are preferentially bound to metaphase chromosomes. It is suggested that cytoplasmic proteins synthesized in maturing oocytes may be responsible for CCA, while proteins discharged from GVs may be required for protection of metaphase chromosomes from cytoplasmic attack on DNA.

Oogenesis and maturation in amphibian oocytes

Study of the processes of oogenesis and maturation is not only embryologically important but reveals much useful biochemical information. This article reviews our understanding of the initial stages of development, which have become as important for the molecular biologist as for the embryologist.

The action of ethidium bromide and chloramphenicol on the steroidogenesis induced by gonadotrophic hormones in the ovaries of amphibia and mammals

Ethidium bromide and chloramphenicol, specific inhibitors of RNA and protein synthesis in mitochondria, suppress the weight increase of the uteri of immature mice, induced by gonadotrophic hormones. They also inhibit in vitro maturation of amphibian oocytes induced by similar stimuli. The action of steroid hormones on the mouse uterus and amphibian oocytes was unaffected by inhibitors of mitochondrial macromolecular syntheses.

Amphibian oocyte maturation and protein synthesis: Related inhibition by cyclic AMP, theophylline, and papaverine

Two inhibitors of cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), theophylline and papaverine, inhibit the maturation of Xenopus laevis oocytes induced by four different stimuli: human chorionic gonadotropin, progesterone, testosterone, and lanthanum ions. Addition of 1 mM cyclic AMP to the medium delays maturation by approximately 2 hr. Papaverine, theophylline, and cyclic AMP inhibit amino acid incorporation into oocyte proteins by 50% or more but do not inhibit amino acid uptake. The capacity of theophylline to block maturation and protein synthesis is reversed in a parallel fashion by addition of 1-5 mM calcium ion to the medium. Addition of papaverine, theophylline, and cycloheximide to oocytes at different times after hormonal treatment shows that the step sensitive to blockage by the three drugs is coincident and precedes germinal vesicle breakdown by about 1.5 hr. Theophylline and papaverine do not increase endogenous cyclic AMP levels in oocytes but do block the decrease of cyclic AMP levels observed 3 hr after progesterone treatment. Both drugs inhibit oocyte cyclic AMP phosphodiesterase measured in vivo and severely inhibit the stimulus of calcium uptake caused by progesterone and human chorionic gonadotropin. These results suggest that cyclic AMP, theophylline, and papaverine may block oocyte maturation by inhibiting protein synthesis, possibly via a cyclic AMP-dependent protein kinase as shown in reticulocytes [Datta, A., De Haro, C., Sierra, J. & Ochoa, S. (1977) Proc. Natl. Acad. Sci., USA 74, 1463-1467].