鸟氨酸脱羧酶参与孕酮诱导的卵成熟过程吗?

在孕酮诱导的蟾蜍卵母细胞成熟过程中,ODC活性约增加2倍。佛波酯PMA能促进孕酮诱导的成熟速度,而对ODC的活性无明显影响。精胺对ODC活性有显著的作用:当卵母细胞培养在含5mmol/L精胺的碱性任氏生理盐水(pH11.6)中时,ODC活性下降17%,而孕酮诱导的成熟速度却大为增加;当精胺注入卵母细胞后。ODC活性明显下降,而且即使孕酮诱导的ODC活性增加完全被抑制,仍有80%以上的卵母细胞发生GVBD。上述结果充分表明,ODC活性的变化不参与孕酮诱导蟾蜍卵母细胞成熟的调控过程。由于在孕酮处理的卵母细胞中,ODC活性的增加发生在精胺水平下降之后,又外源精胺能大幅度抑制卵内ODC活性,故精胺很可能是卵母细胞ODC活性的调节物。     

Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.     

DFP-sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad,Bufo japonicus

The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD₅₀ on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD₅₀) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process. These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley-Liss, Inc.     

Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.     

Possible involvement of phosphatidylinositol 3-kinase, but not protein kinase B or glycogen synthase kinase 3beta, in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3beta (GSK3beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3beta is a component of the PI3K signal transduction pathway.     

A marked animal-vegetal polarity in the localization of Na(+),K(+) -ATPase activity and its down-regulation following progesterone-induced maturation

The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. In this study, we show the expression and distribution pattern of Na(+),K(+) -ATPase in stage-VI oocytes, and its changes following progesterone-induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [(3) H]-ouabain autoradiography, and immunofluorescence cytochemistry at light microscopic level, we find that Na(+),K(+) -ATPase activity is mainly confined to the animal hemisphere. Electron microscopy histochemical results also suggest that polarized distribution of Na(+),K(+) -ATPase activity persists following progesterone-induced maturation, and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is an initial up-regulation and then gradual down-regulation of Na(+),K(+) -ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na(+),K(+) -ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na(+),K(+) -ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na(+),K(+) -ATPase activity in stage-VI oocytes, and their down-regulation following progesterone-induced maturation, is likely to have a role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD.     

Protein Kinase A(PKA)-Restrictcive and PKA-Permissive Phases of Oocyte Maturation

Substantial evidence has indicated that cAMP-dependent protein kinase (protein kinase A orPKA) plays a critical role in maintaining meiotic prophase arrest in vertebrate oocytes.However, PKA activity dynamic and its physiological substrate profile remain poorly defined.We have recently developed a novel PKA substrate construct which we employ to monitor PKAactivity in live oocytes. In the current study, we have employed biochemical and imaginganalyses of single cells to determine PKA activity dynamics during oocyte maturation and toinvestigate the consequence of re-activation of PKA during oocyte maturation. Wedemonstrated here that progesterone caused a rapid and permanent inhibition of PKA during theentire maturation process. However, artificial reactivation of endogenous PKA had differentialconsequences, depending on the timing of PKA reactivation. Reactivation of endogenous PKAat any time prior to GVBD inhibited progesterone-induced GVBD. PKA reactivation at GVBD,or thereafter, did not interfere with meiosis I to meiosis II transition, nor did it interfere withmetaphase II arrest. These results demonstrate for the first time a PKA-restricted phase and aPKA-permissive phase during oocyte maturation.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.