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Changes in protein synthesis pattern during in vitro maturation of goat oocytes.

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis.

Animals↗

Okadaic acid and p13suc1 modulate the reinitiation of meiosis in mouse oocytes.

Short-term exposure to okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, induced resumption of meiosis, including metaphase spindle formation, in mouse oocytes treated with a phosphodiesterase inhibitor, while long incubations with OA arrested oocyte maturation at a step prior to spindle formation. To explore the basis for this difference, the overall patterns of protein synthesis and phosphorylation and the production of tissue-type plasminogen activator (tPA), the synthesis of which is induced after germinal vesicle breakdown (GVBD), were analyzed under various OA treatments. Short-term exposure to OA led to tPA production and did not greatly affect the maturation-associated changes in protein phosphorylation. By contrast, a long application of OA did not result in tPA production and induced more marked changes in protein phosphorylation. Microinjection into prophase oocytes of the product of the fission yeast gene p13suc1, known to inhibit p34cdc2 kinase activation and/or activity, prevented meiotic reinitiation. This effect was overcome by microinjection of OA, at concentrations higher than those required for induction of maturation in the absence of p13suc1. These observations suggest that inhibition of phosphatase 1 or 2A or both triggers meiotic resumption by acting at the same site or at a site proximal to the p13suc1-sensitive step of cdc2 kinase activation.

Animals↗

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.

Animals↗

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: roles of MPF and protein synthesis.

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase II oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase I and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s).

Animals↗

Inhibition of protein synthesis affects histone H1 kinase, but not chromosome condensation activity, during the first meiotic division of pig oocytes.

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed.

Animals↗

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: studies with phorbol 12-myristate 13-acetate.

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca2+ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP3) and diacyl glycerol (DAG), an activator of the CA2+-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.

Animals↗

Effects of a superovulatory dose of pregnant mare serum gonadotropin on follicular steroid contents and oocyte maturation in rats.

Our previous study has shown that superovulatory treatment with pregnant mare serum gonadotropin (PMSG) in rats caused marked alterations in ovarian and serum steroid responses and coincidental increase in degenerate oocytes (Yun et al., 1987). This study examined the effects of superovulatory treatment (20 IU PMSG) on follicular steroid contents and oocyte maturation. Immature female rats aged 28-30 days were injected with 4 or 20 IU PMSG and sacrificed at 24, 48, 60, and 72 hr. Compared to control regimen, follicular content of progesterone (P) in superovulated rats significantly (P less than .05) increased at 48 hr. Androgen (A) content significantly (P less than .01) decreased below control level at 24 hr but significantly (P less than .05) increased above control level at 48 hr and 60 hr. There was no significant change in 17 beta-estradiol (E) content between the two groups. In control regimen, the ratio of A/E sharply decreased and the ratios of P/E and P/A increased steadily from 24 hr. However, superovulatory regimen showed a consistently steady state in the overall ratios of follicular steroids after 24 hr. Nuclear maturation of the majority of control oocytes recovered from oviducts at 72 hr was synchronized at metaphase II, whereas superovulated oocytes displayed different stages varying from prophase I to metaphase II at 24, 48, and 72 hr. The results provide direct evidence of atypical ovulations in superovulated oocytes with premature or asynchronous nuclear maturation and demonstrate a close relationship between meiotically aberrant oocytes and abnormal follicular steroidogenesis following superovulation with PMSG in rats.

Animals↗

Effects of MAP kinase pathway and other factors on meiosis of Urechis unicinctus eggs.

The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs.

1-Methyl-3-isobutylxanthine↗

XMR, a dual location protein in the XY pair and in its associated nucleolus in mouse spermatocytes.

Xlr and Xmr are sex-specific genes which are expressed during the meiotic prophase I in the mouse. In spermatocytes, XMR concentrates on the asynapsed regions of the XY chromosomes, suggesting that XMR plays a role in sex chromosome condensation and silencing. The present study shows that in the mouse, XMR also concentrates in the nucleolus which is closely associated with the XY chromosome pair. In this species, the formation of a large fibrillo-granular nucleolus signals the activation of the ribosomal genes, but release of pre-ribosomal particles is inhibited. Using laser confocal microscopy we characterized the distribution of XMR in the XY body relative to the XY chromatin and the nucleolus. Immunoelectron microscopy showed that XMR concentrates in the fibrillo-granular component and the granular component (GC) of the nucleolus. In (T[X;16]16H) mouse spermatocytes, the nucleolus displays little or no activity and does not associate with the XY pair. XMR concentrated only on the XY chromosomes in (T[X;16]16H) mouse spermatocytes. These data suggest that XMR could play a role both in the XY pair and the nucleolus associated to the sex chromosomes.

Animals↗

Sex-specific windows for high mRNA expression of DNA methyltransferases 1 and 3A and methyl-CpG-binding domain proteins 2 and 4 in human fetal gonads.

DNA methyltransferases (DNMTs) and 5-methyl-CpG-binding domain proteins (MBDs) are involved in the acquisition of parent-specific epigenetic modifications in human male and female germ cells. Reverse Northern blot analyses demonstrated sex-specific differences in mRNA expression for the maintenance DNMT1 and the de novo DNMT3A in developing testis and ovary. In fetal testis DNMT1 and DNMT3A expression peaked in mitotically arrested spermatogonia around 21 weeks gestation. In fetal ovary transcriptional upregulation of DNMT1 and DNMT3A occurred during a very brief period at 16 weeks gestation, when the oocytes proceeded through meiotic prophase. Fetal gonads showed several fold higher DNMT3A expression levels than fetal brain and adult tissues. The most abundant DNMT3A isoform in fetal testis and ovary was DNMT3A2, whereas in all other analyzed tissues DNMT3A1 predominated. The catalytically inactive DNMT3A3 isoform was also present at relatively high levels in developing gonads and may perform a regulatory function(s). In both male and female fetal gonads expression of genes for MBD2 and MBD4, which may be implicated in chromatin remodeling of methylated genomic DNA sequences, was tightly linked to DNMT expression. We propose that the sex-specific time windows for concomitant upregulation of DNMT1, DNMT3A, MBD2, and MBD4 are associated with prenatal remethylation of the human male and female germ line.

DNA (Cytosine-5-)-Methyltransferase 1↗

Genomic imprinting and epigenetic reprogramming: unearthing the garden of forking paths.

Genomic imprinting, an epigenetic form of gene regulation, determines the parent-dependent gene expression of marked or imprinted genes during gametogenesis and embryonic development. Imprinting involves differential allele DNA methylation in one sex cell lineage but not in the other. Egg and sperm each contributes the same DNA sequences to the zygote but epigenetic imprinting of a subset of genes determines that only one of the parent alleles are expressed relative to the parental origin. Primordial germ cells inherit biallelically imprinted genes from maternal and paternal origin and erase their imprints to start de novo monoallelic imprinting during gametogenesis. Epigenetic paternalization is an ongoing process in the mitotically-dividing spermatogonial stem cell and derived meiotically-dividing spermatocyte progeny to endow sperm with imprinted alleles. Epigenetic maternalization is restricted to the oocyte growth phase of folliculogenesis and is unrelated to DNA replication since it takes place while the oocyte remains in the diplotene stage of meiotic prophase I. Sperm and oocyte genomic methylation patterns depend on the activity of DNA methyltransferases (Dnmt). A variant of Dnmt1, designated Dnmt1o, accumulates in oocyte nuclei during the follicular growth phase. Dnmt3L, an isoform of Dnmt3a and Dnmt3b, but lacking enzymatic activity, interacts with Dnmt2a and Dnmt3b and is required for spermatogenesis. In the mouse early zygote, the male pronucleus is demethylated within 4 h of fertilization. Global demethylation takes place gradually up to the morula stage. In the blastocyst, de novo methylation is reestablished in the inner cell mass but not in the trophectoderm. Both the significance of genomic imprinting and the severe developmental defects caused by disrupted Dnmt activity, point to a need for a better understanding of the causes of low cloning efficiency by somatic nuclear transfer to enucleated ovulated oocyte.

Animals↗

Histone-induced condensation of rat testis chromatin: testis-specific H1t versus somatic H1 variants.

Due the likely role of H1 histone variants in inducing the formation of folded DNA filaments with different stabilities, the condensing capacity of the testis-specific H1t versus the somatic variants was tested. Circular dichroism analyses of rat testis H1-depleted oligonucleosomes (5-2kbp) revealed that H1t, which appears in germ cells during the meiotic prophase of mammalian spermatogenesis, exerts the lowest condensing effect as compared to the other variants. The distribution of H1 subtypes among different chromatin fractions was also investigated and gave evidence that H1t is more abundant in chromatin regions which are more sensitive to DNAase I digestion.

Animals↗

A new meiotic protein factor which enhances activity of meiotic DNA polymerase from Coprinus cinereus.

Meiotic cells of Coprinus contain a protein that can be identified by its ability to enhance activity of the meiotic DNA polymerase reported previously (8). Its activity is found only during the prophase stages in meiotic cells, and is accompanied by the meiotic polymerase. The protein, which was purified to near homogeneity, is a single polypeptide with a molecular mass of 30k, and shows no activities of nuclease, ATPase, or DNA-binding protein. The protein could enhance the meiotic polymerase activity by at least 5 fold. The other Coprinus polymerases were not influenced by the protein. The protein could increase Vmax value of the meiotic polymerase, but not the Km. The significance of this protein to meiotic DNA synthesis is considered in relation to other biochemical properties of meiotic cells.

Chromatography, Gel↗

Forskolin and methylxanthines block the increase in intracellular pH during meiosis in Xenopus laevis oocytes.

Xenopus oocytes, arrested in late prophase of the meiotic cell cycle, undergo nuclear membrane breakdown (Germinal Vesicle Breakdown, GVBD) in response to progesterone stimulation. During this prophase/M-phase transition the oocytes undergo an increase in their intracellular pH (pHi) from 7.3 to 7.7 in response to the steroid. This increase in pHi appears to be due to the activation of Na+/H+ antiporters in the oocyte plasma membrane. Several studies have shown that the pathway leading to GVBD is blocked by forskolin or methylxanthines which elevate cAMP levels within the oocyte. We have found that these same compounds also blocked the up-regulation of the Na+/H+ exchangers during oocyte meiotic maturation.

1-Methyl-3-isobutylxanthine↗

A novel cell-cycle-dependent 350-kDa nuclear protein: C-terminal domain sufficient for nuclear localization.

We have screened human scleroderma patients for immunoreactivity with the components of the nucleus and the mitotic apparatus. We announce the identification of a novel cell-cycle-dependent nuclear protein using serum from a CREST patient AH. AH protein first appears at the nucleus of G2-phase and associates with the centrosome throughout the cell cycle. As chromosomes condense during the prophase, AH protein becomes enriched at the kinetochores. During mitosis, AH protein progressively disperses from the kinetochore and becomes diffusely localized in the cytoplasm and in telophase; it appears to be enriched within the intracellular bridge. Molecular cloning and transfection studies reveal that the 350-kDa AH protein contains a coiled-coil and a globular domain at the C-terminus that is sufficient for nuclear localization.

Amino Acid Sequence↗

The transcript for a novel protein with a zinc finger motif is expressed at specific stages of mouse spermatogenesis.

The cDNA for an RNA that is expressed predominantly in mouse spermatogenic cells was cloned and characterized. It was found to encode novel zinc finger protein. We first generated a cDNA fragment from mouse osteoblastic cells by the differential display method. To our surprise, Northern blot analysis revealed that the corresponding transcript was expressed at high levels in the testis rather than in osteoblastic cells. Therefore, using this fragment as a probe, we isolated the full-length cDNA (3340 bp) from a mouse testis cDNA library. Analysis of the open reading frame of the cDNA indicated that the encoded protein was a polypeptide of 942 amino acids residues that included three distinct domains, namely, a zinc finger domain of the Cys(2)-His(2) type, four basic amino acid-rich domains, and a myosin II-homology domain. In situ hybridization indicated that the transcript was present in seminiferous tubules of adult mice. Elevated expression of the transcript during testicular development in mice was restricted to spermatocytes at the pachytene stage of meiotic prophase and to round and elongated spermatids, as indicated by Northern blot analysis and RT-PCR. Our results suggest that this novel zinc finger protein might act as a transcriptional regulator during spermatogenesis and, in particular, during meiotic division.

Amino Acid Sequence↗

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK1 cells have been treated with 5 micrograms/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G1 promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.

Animals↗

Acetylation of alpha-tubilin in different bovine cell types: implications for microtubule dynamics in interphase and mitosis.

Previous work on five cell types isolated from the bovine corpus luteum showed that the mass of acetylated microtubules (acet-MTs) in interphase differed. Endothelial cells, termed type 3, showed few acet-MTs, whereas the interphase cytoskeleton of granulosal-like cells, termed type 5, was rich in acet-MTs. In the present study, these cultured cells were used to determine whether the degree of alpha-tubulin acetylation in interphase had consequences on mitosis. To this end, the distribution of acet-MTs was determined throughout the cell cycle using a monoclonal antibody, 6-11B-1, directed against acetylated alpha-tubulin. For comparison, tyrosinated MTs were visualized with another monoclonal antibody, YL1/2, detecting tyrosinated alpha-tubulin. Although the amount of acet-MTs in interphase differed significantly between both cell types, major differences in the appearance of acet-MTs during mitosis were only apparent in prophase and during transition from late telophase to interphase. Thus, irrespective of different alpha-tubulin acetylation in interphase, spindle structure is uniform. Since acetylation of alpha-tubulin is believed to indicate the presence of relatively stable MTs, we conclude that MT dynamics is differently controlled in interphase and mitosis. Thereby interphase cells are able to carry out functions which involve stable MTs and the cells progress through mitosis in the presence of more dynamic MTs.

Acetylation↗