Early stimulation of protein kinase activity during hormonal meiosis reinitiation in starfish ovocytes]

Total endogenous protein kinase activity has been compared in homogenates prepared from Marthasterias glacialis and Asterias rubens oocytes treated or not by 1-methyladenine, the hormone which triggers meiosis reinitiation. A 5 mn treatment at the threshold 10(-7) M concentration causes a significant increase (+ 20%) of these activities. This stimulation is maintained or even enhanced when homogenates are complemented with exogenous histones.     

Acid release at meiotic maturation of oocytes in the polychaete annelid Sabellaria alveolata.

ABout 1 pmole of acid per egg is released when prophasic oocytes undergo maturation under the action of sperm, proteases or ionophore A 23187. No similar acid release occurs at fertilization of matured oocytes. These findings are compared with data on Urechis and sea urchin.     

Acid release at meiotic maturation of oocytes in the polychaete annelidSabellaria alveolata

Summary About 1 pmole of acid per egg is released when prophasic oocytes undergo maturation under the action of sperm, proteases or ionophore A 23187. No similar acid release occurs at fertilization of matured oocytes. These findings are compared with data onUrechis and sea urchin.     

Inhibition by the enterotoxin of Vibrio cholerase of meiosis reinitiation of the Xenopus laevis oocyte induced in vitro by progesterone]

Progesterone reinitiates in vitro meiosis in Xenopus laevis oocytes. This action of the hormone can be abolished by the exotoxin of vibrio cholerae. The concentration of toxin which inhibits 50% of the progesterone (10 muM) action in about 2.5 pM. Binding experiments using 125I labelled toxin demonstrated the existence of high affinity binding sites (KD approximately 0.2 nM) located probably on the surface of the oocytes.     

Calmodulin content does not change following hormone-induced meiosis reinitiation in starfish oocytes

Summary Activator-deficient phosphodiesterase from bovine heart was stimulated by heat-treated dialyzed extracts from starfish oocytes. The same dose-response curve was obtained with extracts from oocytes which had or had not been released from prophase block by 1-methyladenine. Such extracts were shown to contain calmodulin by affinity chromatography on troponin I covalently bound to sepharose and SDS polyacrylamide gel electrophoresis. The results suggest that no change in calmodulin content occurs in starfish oocyte following meiosis reinitiation.     

Telomeres and meiosis in health and disease

Meiotic dysfunction increasingly afflicts women as they age, resulting in infertility, miscarriage and handicapped offspring. How aging disrupts meiotic function in women remains unclear, but as women increasingly delay childbearing, this issue becomes urgent. Telomeres, which mediate aging in mitotic cells, may also mediate aging during meiosis. Telomeres shorten during DNA replication. In mammals, oocytes remain quiescent, but their precursors replicated during fetal oogenesis. Moreover, eggs ovulated from older women entered meiosis later during fetal oogenesis than eggs ovulated when younger, and therefore underwent more replications. Telomeres also shorten from reactive oxygen, which triggers a DNA repair response, so the prolonged interval between fetal oogenesis and ovulation in some women would further shorten telomeres. Mice normally do not exhibit age-related meiotic dysfunction (interestingly, their telomeres are manyfold longer than telomeres in women), but genetic or pharmacologic shortening of mouse telomeres recapitulates the reproductive aging phenotype of women. This has led to a telomere theory of age-related meiotic dysfunction in women, and underlined the importance to human health of a mechanistic understanding of telomeres and meiosis.     

Cooperative inhibitory effect of follicular fluid and cAMP on hamster oocyte maturation

Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 microM dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro.     

The effects of diverse agents (Ca++ and K++ ionophores, organomercurials, dithiols, colchicine, and cytochalasin B) on the maturation and differentiation without cleavage in Chaetopterus eggs]

Induction of maturation in Chaetopterus oocytes requires the presence of Ca++ ions in the medium, but differentiation without cleavage can proceed in the absence of this cation. The Ca++ ionophore A 23187 induces both maturation and the cortical reaction provided that Ca++ ions are present in the medium differentiation without cleavage may follow. Valinomycin slowly induces germinal vesicle breakdown, which is followed by a sharp segregation between hyaloplasm and yolk. PHMPS, but not DTT, induces maturation. Differentiation without cleavage is more sensitive to colchicin than to cytochalasin B.     

Mechanisms controlling germline cyst breakdown and primordial follicle formation

In fetal females, oogonia proliferate immediately after sex determination. The progress of mitosis in oogonia proceeds so rapidly that the incompletely divided cytoplasm of the sister cells forms cysts. The oogonia will then initiate meiosis and arrest at the diplotene stage of meiosis I, becoming oocytes. Within each germline cyst, oocytes with Balbiani bodies will survive after cyst breakdown (CBD). After CBD, each oocyte is enclosed by pre-granulosa cells to form a primordial follicle (PF). Notably, the PF pool formed perinatally will be the sole lifelong oocyte source of a female. Thus, elucidating the mechanisms of CBD and PF formation is not only meaningful for solving mysteries related to ovarian development but also contributes to the preservation of reproduction. However, the mechanisms that regulate these phenomena are largely unknown. This review summarizes the progress of cellular and molecular research on these processes in mice and humans.     

Hormonal control of mammalian oocyte meiosis at diplotene stage

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin “arrester” until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3′,5′-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3′,5′-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.     

Structure,function and evolution of haspin and haspinrelated proteins,a distinctive group of eukaryotic protein kinases

The haspins constitute a newly defined protein family containing a distinctive C-terminal eukaryotic protein kinase domain and divergent N termini. Haspin homologues are found in animals, plants and fungi, suggesting an origin early in eukaryotic evolution. Most species have a single haspin homologue. However, Saccharomyces cerevisiae has two such genes, while Caenorhabditis elegans has at least three haspin homologues and approximately 16 haspin-related genes. Mammalian haspin genes have features of retrogenes and are strongly expressed in male germ cells and at lower levels in some somatic tissues. They encode nuclear proteins with serine/threonine kinase activity. Murine haspin is reported to inhibit cell cycle progression in cell lines. One of the S. cerevisiae homologues, ALK1, is a member of the CLB2 gene cluster that peaks in expression at M phase and thus may function in mitosis. Therefore, the haspins are an intriguing group of kinases likely to have important roles during or following both meiosis and mitosis.     

Cooperative inhibitory effect of follicular fluid and cAMP on hamster oocyte maturation

Summary Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 M dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro.     

Biological effects of trilostane in vitro on oocyte maturation and fertilization in the hamster

The effects of the inhibition of steroidogenesis by trilostane on oocyte maturation were examined by studying spontaneous maturation and fertilization in vitro. 10(-6)M trilostane had no influence on the meiotic process, whether the oocytes were naked or not. At a concentration of 10(-6)M and 10(-7)M trilostane, low normal pronuclear formation and high polyspermy were found during in vitro fertilization. However, no retarded male pronuclear development could be detected in the trilostane-treated group. Thus, steroid producing activity within ova is apparently necessary to prevent multiple sperm penetration, but it has no effect on meiosis or the action of the so-called male pronucleus growth factor (MPGF).     

Phosphorylation of p27BBP/eIF6 and its association with the cytoskeleton are developmentally regulated in Xenopus oogenesis

p27^BBP/eIF6 is an evolutionarily conserved regulator of ribosomal function. It is necessary for 60S biogenesis and impedes improper joining of 40S and 60S subunits, regulated by protein kinase C or Efl1p. No data on p27^BBP/eIF6 during early development of Metazoa are available. We studied the distribution, post-translational changes and association with the cytoskeleton of p27^BBP/ eIF6 during Xenopus oogenesis and early development. Results indicate that p27^BBP/eIF6 is present throughout oogenesis, partly associated with 60S subunits, partly free and with little cytoskeleton bound. During prophase I, p27^BBP/eIF6 is detected as a single band of 27-kDa. Upon maturation induced by progesterone or protein kinase C, a serine-phosphorylated 29 kDa isoform appears and is kept throughout development to the neurula stage. Confocal microscopy showed that the distribution of p27^BBP/eIF6 and its association with the cytoskeleton varies according to oogenesis stages. Briefly, in stage 6 oocytes, p27^BBP/eIF6 has a limited dot-like distribution, and does not co-localize with cytokeratin, whereas upon maturation it spreads throughout the cytoplasm. After fertilization, a large fraction coalesces around cytomembranes and a cytochalasin B-sensitive co-localization with cytokeratin occurs. RNAse removes p27^BBP/eIF6 from the cytokeratin fibres. Developmental data suggest a role of p27^BBP/eIF6 in controlling ribosomal availability or regulating cross-talk between ribosomes and the cytoskeleton.Received 7 April 2005; received after revision 11 May 2005; accepted 25 May 2005R. Carotenuto and N. De Marco contributed equally to the paper     

Oocyte maturation in vitro: contribution of the oviduct to total maturation in Xenopus laevis.

Contact of progesterone matured oocytes of Xenopus laevis with the oviduct reduces the time necessary to attain cleavage capacity from 24 h to 3 h. Full maturity has been demonstrated by normal development of the matured eggs after fertilization.     

Oocyte maturation in vitro: Contribution of the oviduct to total maturation inXenopus laevis

Summary Contact of progesterone matured oocytes ofXenopus laevis with the oviduct reduces the time necessary to attain cleavage capacity from 24 h to 3 h. Full maturity has been demonstrated by normal development of the matured eggs after fertilization.     

Biological effects of trilostane in vitro on oocyte maturation and fertilization in the hamster

Summary The effects of the inhibition of steroidogenesis by trilostane on oocyte maturation were examined by studying spontaneous maturation and fertilization in vitro. 10^–6 M trilostane had no influence on the meiotic process, whether the oocytes were naked or not. At a concentration of 10^–6 M and 10^–7 M trilostane, low normal pronuclear formation and high polyspermy were found during in vitro fertilization. However, no retarded male pronuclear development could be detected in the trilostane-treated group. Thus, steroid producing activity within ova is apparently necessary to prevent multiple sperm penetration, but it has no effect on meiosis or the action of the so-called male pronucleus growth factor (MPGF).     

Long-term incubation with mifepristone (MLTI) increases the spine density in developing Purkinje cells: new insights into progesterone receptor mechanisms

Cerebellar Purkinje cells (PC) physiologically reveal an age-dependent expression of progesterone with high endogenous concentrations during the neonatal period. Even if progesterone has been previously shown to induce spinogenesis, dendritogenesis and synaptogenesis in immature PC, data about the effects of progesterone on mature PC are missing, even though they could be of significant therapeutic interest. The current study demonstrates for the first time a progesterone effect, depending on the developmental age of PC. Comparable with the physiological course of the progesterone concentration, experimental treatment with progesterone for 24 h achieves the highest effects on the dendritic tree during the early neonate, inducing an highly significant increase in dendritic length, spine number and spine area, while spine density in mature PC could not be further stimulated by progesterone incubation. Observed progesterone effects are certainly mediated by classical progesterone receptors, as spine area and number were comparable to controls when progesterone incubation was combined with mifepristone (incubation for 24 h), an antagonist of progesterone receptors A and B (PR-A/PR-B). In contrast, an increase in the spine number and area of both immature and mature PC was detected when slice cultures were incubated with mifepristone for more than 72 h (mifepristone long-time incubation, MLTI). By including time-lapse microscopy, electron microscopic techniques, PCR, western blot, and MALDI IMS receptor analysis, as well as specific antagonists like trilostane and AG 205, we were able to detect the underlying mechanism of this diverging mifepristone effect. Thus, our results provide new insights into the function and signaling mechanisms of the recently described progesterone receptor membrane component 1 (PGRMC1) in PC. It is highly suitable that progesterone does not just induce effects by the well-known genomic mechanisms of the classical progesterone receptors but also acts through PGRMC1 mediated non-genomic mechanisms. Thus, our results provide first proofs for a previously discussed progesterone-dependent induction of neurosteroidogenesis in PC by interaction with PGRMC1. But while genomic progesterone effects mediated through classical PR-A and PR-B seem to be restricted to the neonatal period of PC, PGRMC1 also transmits signals by non-genomic mechanisms like regulation of the neurosteroidogenesis in mature PC. Thus, PGRMC1 might be an interesting target for future clinical studies and therapeutic interventions.     

Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001     

Differential water permeability and regulation of three aquaporin 4 isoforms

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K⁺ concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.