Role of Mitogen Activated Protein Kinase and Maturation Promoting Factor During the Achievement of Meiotic Competency in Mammalian Oocytes.

The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of maturation promoting factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription factors in cumulus cells or cytostatic factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.

Entry into mitosis: a solution to the decades-long enigma of MPF.

Maturation or M phase-promoting factor (MPF) is the universal inducer of M phase common to eukaryotic cells. MPF was originally defined as a transferable activity that can induce the G2/M phase transition in recipient cells. Today, however, MPF is assumed to describe an activity that exhibits its effect in donor cells, and furthermore, MPF is consistently equated with the kinase cyclin B-Cdk1. In some conditions, however, MPF, as originally defined, is undetectable even though cyclin B-Cdk1 is fully active. For over three decades, this inconsistency has remained a long-standing puzzle. The enigma is now resolved through the elucidation that MPF, defined as an activity that exhibits its effect in recipient cells, consists of at least two separate kinases, cyclin B-Cdk1 and Greatwall (Gwl). Involvement of Gwl in MPF can be explained by its contribution to the autoregulatory activation of cyclin B-Cdk1 and by its stabilization of phosphorylations on cyclin B-Cdk1 substrates, both of which are essential when MPF induces the G2/M phase transition in recipient cells. To accomplish these tasks, Gwl helps cyclin B-Cdk1 by suppressing protein phosphatase 2A (PP2A)-B55 that counteracts cyclin B-Cdk1. MPF, as originally defined, is thus not synonymous with cyclin B-Cdk1, but is instead a system consisting of both cyclin B-Cdk1 that directs mitotic entry and Gwl that suppresses the anti-cyclin B-Cdk1 phosphatase. The current view that MPF is a synonym for cyclin B-Cdk1 in donor cells is thus imprecise; instead, MPF is best regarded as the entire pathway involved in the autoregulatory activation of cyclin B-Cdk1, with specifics depending on the experimental system.

Phosphatases driving mitosis: pushing the gas and lifting the brakes.

Entry into and progression through mitosis depends critically on the establishment and maintenance of protein phosphorylation. For this reason, studies on mitotic progression have focused heavily on the activation of MPF (M phase promoting factor), a cyclin-dependent kinase responsible for phosphorylating proteins that execute the dynamic events of mitosis. Recent work, however, has significantly expanded our understanding of mechanisms that allow accumulation of phosphoproteins at M phase, suggesting that mitotic entry relies not only on MPF activation but also on the inhibition of antimitotic phosphatases. It is now clear that there exists a separate, albeit equally important, signaling pathway for the inactivation of protein phosphatases at the G2/M transition. This pathway, which is governed by the kinase Greatwall is essential for both entry into and maintenance of M phase. This chapter will outline the molecular events regulating entry into mitosis, specifically highlighting the role that protein phosphorylation plays in triggering both MPF activation and the inhibition of phosphatase activity that would otherwise prevent accumulation of mitotic phosphoproteins. These intricate regulatory pathways are essential for maintaining normal cell division and preventing inappropriate cell proliferation, a central hallmark of cancer cells.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep.

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  µM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  µM BCB during 60  min allows selection of (BCB+) the largest (123.66  µm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  µm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

Recent advances in meiotic maturation and ovulation: comparing mammals and pisces.

Meiotic maturation is a complex process that involves resumption of meiosis in response to preovulatory luteinizing hormone (LH) surge just before ovulation. High levels of cAMP in oocytes maintain meiotic arrest at diplotene of prophase I in mammals and pisces. In mammals, the process by which LH induces recommencement of meiosis involves breakdown of oocyte-somatic cells communication, which is followed by a drop in intracellular cAMP levels that in turn causes exit from meiotic arrest. Maturation promoting factor (MPF) then accomplishes progression of oocytes to reach first metaphase followed by second metaphase after reinitiating meiosis. Pisces require precise completion of oocyte growth involving vitellogenesis before the entry of meiotic maturation. Then, both mammalian and fish oocytes enters resumption of meiosis involving germinal vesicle breakdown, chromosome condensation, assembly of meiotic spindle, and formation of first polar body. However, this process in pisces is regulated by three major mediators, LH, 17alpha,20beta-dihydroxy progesterone and MPF which are unique. The molecular mechanisms of meiotic maturation and ovulation by comparing mammalian and piscine research have been dealt in this review.

Effects of puberty and gonadotropins on the molecular events controlling meiotic resumption of mouse oocytes.

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.

[Circadian rhythms and systems biology]. / Biologie des systèmes et rythmes cellulaires.

Cellular rhythms represent a field of choice for studies in system biology. The examples of circadian rhythms and of the cell cycle show how the experimental and modeling approaches contribute to clarify the conditions in which periodic behavior spontaneously arises in regulatory networks at the cellular level. Circadian rhythms originate from intertwined positive and negative feedback loops controlling the expression of several clock genes. Models can be used to address the dynamical bases of physiological disorders related to dysfunctions of the mammalian circadian clock. The cell cycle is driven by a network of cyclin-dependent kinases (Cdks). Modeled in the form of four modules coupled through multiple regulatory interactions, the Cdk network operates in an oscillatory manner in the presence of sufficient amounts of growth factor. For circadian rhythms and the cell cycle, as for other recently observed cellular rhythms, periodic behavior represents an emergent property of biological systems related to their regulatory structure.

Regulation of keratinocyte proliferation in rats with deep, partial-thickness scald: modulation of cyclin D1-cyclin-dependent kinase 4 and histone H1 kinase activity of M-phase promoting factor.

BACKGROUND: Keratinocyte proliferation, which is undergone by its cell cycle transition, is considered a major event during re-epithelialization over the wound size. Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors interact to regulate the cell cycle. We investigated proliferative events associated with cell-cycle control in keratinocytes during wound healing in rats with deep, partial scald injuries. MATERIALS AND METHODS: Male Sprague Dawley rats with starting weights of 200 to 220 g were inflicted with standardized deep partial-thickness burns by scalding 10% of the skin surface. The full thickness skin biopsies were harvested for histological evaluation at following time points: 0 d, post-burn day 3, post-burn day 7, and post-burn day 14. Keratinocytes from wound edge were isolated for cell cycle examination. The cell cycle regulators and their activity were detected. RESULTS: Keratinocytes tended to proliferate and had enlarged nuclei and nucleoli from day 3 after injury. Morphological features became evident on day 14, with an increase in keratinocytes. The percentage of S-phase keratinocytes tended to increase on day 14. The percentage G2/M-phase keratinocytes increased from day 3 and significantly increased on days 7 and 14. Cyclin D1 expression markedly increased from day 3, with down-regulation of cyclin-dependent kinase 4, which re-elevated on day 14. Cyclin B1 expression did not dramatically vary. Histone H1 kinase activity of mitosis phase promoting factor markedly increased on day 14. CONCLUSIONS: These findings suggested early, active DNA synthesis and mitosis in keratinocytes, with marked proliferation on day 14, that depended on the modulation of cyclin D1-cyclin-dependent kinase 4 and histone H1 kinase activity of mitosis phase promoting factor. During wound healing, patterns of cell-cycle control expression differed from those previously known.

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.

Different thresholds of MPF inactivation are responsible for controlling different mitotic events in mammalian cell division.

We present evidence for a paradigm that, during cell division, the decreasing activity of MPF acts as a master signal, which utilizes different thresholds to control the initiation of different mitotic events. The key temporal control here is the degradation of cyclin B1. Using single cell analysis, we measured the kinetics of cyclin B1 degradation and determined quantitatively the thresholds of cyclin B1 level for different mitotic events within a HeLa cell. These observed thresholds were: 1.36 +/- 0.49 microM (for chromosome separation), 0.75 +/- 0.08 microM (for cytokinesis) and 0.54 +/- 0.16 microM (for nuclear reassembly). By comparison, the average concentration of endogenous cyclin B1 within a prometaphase cell was found to be 2.92 +/- 1.7 microM. We suggest that the decreasing order of these thresholds plays an important role in triggering the initiation of successive mitotic events in cell division.

Emi2 at the crossroads: where CSF meets MPF.

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2's APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.

Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea.

The hermaphroditic diploid clam Corbicula fluminea reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as two first polar bodies and subsequently second meiosis did not occur. In eggs treated with cytochalasin D (CD) to inhibit the polar body extrusion, the second meiosis was abortive. After the first meiosis, two centrosomes at the spindle poles remained in the cytoplasm because of the effect of CD. The chromosomes divided into two groups at anaphase-I as observed in the control eggs. Two centrosomes divided into four just after the first meiosis but did not separate completely. The microtubules from the centrosomes were rather short. So at the second meiosis, two monoasters or tetrapolar spindles were formed. The fluorescence signal from microtubules of the monoaster or tetrapolar spindle was weak compared with the spindle at the first meiosis. The maternal chromosomes on the monoaster or tetrapolar spindle did not move, and became large female pronuclei. The pronuclei became the metaphase chromosomes on the spindle for the first cleavage. The present study suggests that second meiosis regulating factors may be abortive in androgenetic diploid C. fluminea.

The C. elegans Myt1 ortholog is required for the proper timing of oocyte maturation.

Maturation promoting factor (MPF), a complex of cyclin-dependent kinase 1 and cyclin B, drives oocyte maturation in all animals. Mechanisms to block MPF activation in developing oocytes must exist to prevent precocious cell cycle progression prior to oocyte maturation and fertilization. This study sought to determine the developmental consequences of precociously activating MPF in oocytes prior to fertilization. Whereas depletion of Myt1 in Xenopus oocytes causes nuclear envelope breakdown in vitro, we found that depletion of the Myt1 ortholog WEE-1.3 in C. elegans hermaphrodites causes precocious oocyte maturation in vivo. Although such oocytes are ovulated, they are fertilization incompetent. We have also observed novel phenotypes in these precociously maturing oocytes, such as chromosome coalescence, aberrant meiotic spindle organization, and the expression of a meiosis II post-fertilization marker. Furthermore, co-depletion studies of CDK-1 and WEE-1.3 demonstrate that WEE-1.3 is dispensable in the absence of CDK-1, suggesting that CDK-1 is a major target of WEE-1.3 in C. elegans oocytes.

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents.

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.

Expression and immunohistochemical localization of Cdc2 and P70S6K in different stages of mouse germ cells.

In order to determine the function and possible relationship between Cdc2 and P(70)S6K, Western blot analysis and immunohistochemistry analysis were used to study the expression and kinase activity of Cdc2 and P(70)S6K in male mouse germ cells. With the maturation of germ cells in the testis, the expression of Cdc2 and P(70)S6K was relatively constant. However, the kinase activity of P(70)S6K was increased and the phosphorylation of Tyr15 residue of Cdc2 was enhanced, which suggests that the kinase activity of Cdc2 is decreasing. Immunohistochemistry analysis also showed that there was a P(70)S6K transfer from nucleus to cytoplasm during spermatogenesis. During spermatogenesis, cell division of the germ cell in male mouse is decelerated; nevertheless, cell growth is enhanced. Cdc2 and P(70)S6K are involved in these two processes. It could be an alternative mechanism to prepare for future fertilization that Cdc2 is able to maintain a subtle balance between the production and growth of male germ cells by regulating P(70)S6K.

Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation.

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRalpha protein levels. A correlation between mPRalpha protein levels and the ability of oocytes to undergo GVBD in response to the MIH (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.

Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space.

During meiotic maturation of mammalian oocytes, two successive divisions occur without an intermediate phase of DNA replication, so that haploid gametes are produced. Moreover, these two divisions are asymmetric, to ensure that most of the maternal stores are retained within the oocyte. This leads to the formation of daughter cells with different sizes: the large oocyte and the small polar bodies. All these events are dependent upon the dynamic changes in the organization of the oocyte cytoskeleton (microtubules and microfilaments) and are highly regulated in time and space. We review here the current knowledge of the interplay between the cytoskeleton and the cell cycle machinery in mouse oocytes, with an emphasis on the two major activities that control meiotic maturation in vertebrates, MPF (Maturation promoting factor) and CSF (Cytostatic factor).

Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in carps.

The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, Cdk1. Densitometric analysis of the immunoblot data collected from the melatonin pre-treated MIH incubated oocytes showed that cyclin B level continued to increase even after 4 h of incubation, and reached the peak after 12 h. Moreover, determination of H1 kinase activity as an indicator of MPF activity in oocytes revealed that melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Thus, the present study demonstrates for the first time that prior incubation with melatonin accelerates the action of MIH on carp oocyte maturation.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Protein kinase A regulates cell cycle progression of mouse fertilized eggs by means of MPF.

Cell cycle of one-cell stage mouse fertilized eggs was accompanied by fluctuation in the concentration of adenosine 3'5'-monophosphate (cAMP) and in the activity of free catalytic subunit of cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of free catalytic subunit of PKA decreased at the onset of mitosis and increased at the transition between mitosis and G1 phase. Stimulation of PKA by microinjection of cAMP into one-cell stage mouse embryos at G2 phase induced interphase arrest and prevented the activation of M-phase promoting factor (MPF). Upon blockage of the activation of PKA by microinjecting a thermostable PKA inhibitor (PKI) into one-cell stage mouse embryos at G2 phase, the increase in the MPF activity occurred 30 min earlier than in control group. When a high dose of PKI was microinjected, a transition into interphase was prevented, and the activity of MPF remained high. Western blot analysis showed that Cdc2 remained phosphorylated in cAMP microinjected embryos by the time when control embryos were at metaphase and showed dephosphorylated Cdc2; conversely, Cdc2 dephosphorylation was accelerated in PKI-microinjected embryos. At the same time, Cdc2 was phosphorylated at Tyr15 at G2 phase and even at M phase when cAMP was microinjected but was dephosphorylated when PKI was microinjected. PKI microinjection also prevented cyclin B degradation and sustained MPF activity, thus delaying the transition from metaphase to anaphase. Our results show that PKA, by inhibiting MPF, regulates cell cycle progression of fertilized eggs.