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PubMed1996

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation.

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0-6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components.

Animals↗
PubMed1996

Development-dependent localization of nuclear antigens in growing mouse oocytes.

We have analyzed the distribution of nuclear and nucleolar proteins during the period of oocyte's growth. Oocytes were isolated mechanically or enzymatically from ovaries of juvenile mice of various ages (from 1 to 28 days after birth). Small nuclear ribonucleoproteins (snRNPs), the splicing factor SC-35, and a protein linked to cell proliferation (p-120) were detected by indirect immunofluorescence. snRNP distribution is consistent with the prophase state of oocyte's nuclei, while SC-35 (and p-120) exhibit a "speckled" distribution throughout the entire period of growth. The number of speckles (or foci) appears maximal around 10 days after birth, i.e., in the period of maximal transcriptional activity, and is sensitive to alpha-amanitin treatment. On the other hand, the immunofluorescent distribution of of nucleolin and p-103 (a nucleolar marker of the granular component) is compared to the ultrastructural distribution of the granular component analyzed by electron microscopy on oocytes of the same age.

Amanitins↗
PubMed1996

Germ cell-specific DNA and RNA binding proteins p48/52 are expressed at specific stages of male germ cell development and are present in the chromatoid body.

Proteins homologous to the Xenopus oocyte mRNA binding proteins mRNP3+4 and designated p48/52 have been identified in male mouse germ cells (1993: Dev Biol 158:90-100). Western and North-western blots of extracts from testes and isolated germ cells indicate that p48/52 are present during meiosis but reach their highest levels postmeiotically at a time when many mRNAs are stored. Here we analyze the cellular and subcellular distribution of p48/52 in rat and mouse testes by LM and EM immunocytochemistry using an anti-mRNP3+4 antibody. Immunolabeling was found to be predominantly cytoplasmic and specific to germ cells at certain periods during their development. p48/52 were first detected in early pachytene spermatocytes at stage V of the seminiferous cycle and progressively increased during the remainder of meiotic prophase to a post-meiotic peak in steps 1-8 round spermatids; thereafter, labeling gradually declined as elongated spermatids underwent nuclear condensation and elongation. A proportionally higher concentration of cytoplasmic immunolabeling was found within the lacunae of the anastomotic granulofilamentous network of the chromatoid body. The pattern of synthesis of these mRNA binding proteins together with their association with the chromatoid body suggests a role as germ cell-specific mRNA stabilizing and/or storage proteins.

Animals↗
PubMed1996

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents.

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K(+)-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with 35S-methionine or 32P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K(+)-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K+ or serotonin (12-15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes.

Adenine↗
PubMed1997

Ribosomal S6 kinase p90rsk and mRNA cap-binding protein eIF4E phosphorylations correlate with MAP kinase activation during meiotic reinitiation of mouse oocytes.

During meiotic reinitiation of the mouse oocyte, entry into M-phase is regulated by changes of protein phosphorylation and by the stimulation of selective mRNA translation following the nuclear membrane dissolution. Our results reveal that M-phase kinases (MAP kinase and histone H1 kinase) are being activated together with S6 kinase and with the phosphorylation of eIF4E, the cap-binding subunit of the initiation factor eIF-4F. In order to test which signaling pathway(s) is(are) involved, okadaic acid and cycloheximide have been used as tools for differentially modulating MAP and histone H1 kinase activities. A role for MAP kinases in the phosphorylation of eIF4E and the activation of S6 kinase is suggested. The possible implication of p90rsk and/or of p70s6k in the overall increase in S6 kinase activity has been examined. p70s6k does not appear to be involved since phosphorylated forms are found in prophase and maturing oocytes. In contrast, p90rsk is phosphorylated and activated in maturing oocytes. p90rSk phosphorylation correlates with the activation of S6 kinase. These results suggest that the overall increase of S6 kinase activity is mostly due to p90rsk activation. The roles of eIF4E phosphorylation and S6 kinase activation in the physiological induction of M-phase and in the okadaic acid-induced premature mitotic events are discussed.

Animals↗
PubMed1997

P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation.

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanisms underlying these radiosensitivity differences, p21(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21(Cip1/WAF1) is a cyclin-dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation. P21(Cip1/WAF1) expression was observed in the normal testis, using Western blotting analysis. After a dose of 4 Gy, but not after 0.3 Gy, an increase in p21(Cip1/WAF1) expression could be determined in whole testis lysates. To investigate which germ cells are involved in p21(Cip1/WAF1) protein expression, immunohistochemical analysis was performed on irradiated testis. In the normal testis a weak staining for p21(Cip1/WAF1) was found in pachytene spermatocytes in epithelial stage V up to step 5 spermatids. A dose of 4 Gy of X-irradiation resulted in a transient increase of p21(Cip1/WAF1) staining in these cells with a maximum at 6 h post irradiation, despite the fact that the irradiation did not induce an increase in the number of apoptotic spermatocytes. When a dose of 0.3 Gy was given, no increase in p21(Cip1/WAF1) staining was observed. Using the TUNEL technique, a 10-fold increase in apoptotic spermatogonia was found after a dose of 4 Gy. However, no staining for p21(Cip1/WAF1) was observed in spermatogonia, suggesting that these cells do not undergo a p21(Cip1/WAF1)-induced G1 arrest prior to DNA repair or apoptosis. These data imply that p21(Cip1/WAF1) is a factor which could be important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in spermatogonial cell cycle delay or apoptosis induction.

Animals↗
PubMed1997

Molecular cloning and tissue-specific expression of the mouse homologue of the rat brain 14-3-3 theta protein: characterization of its cellular and developmental pattern of expression in the male germ line.

The highly conserved 14-3-3 family of proteins, originally reported as brain-specific and then found in various somatic cells and oocytes, interacts with several important signal transduction kinases so that actually the 14-3-3 protein are considered as modulators of multiple signal transduction pathways. Here we show that a 14-3-3 protein is also expressed in the male germ cells, thus extending the protein cellular distribution to a cell line never reported to express 14-3-3 proteins. Screening of a mouse spermatogenic cells lambda gt11 cDNA library with affinity-purified polyclonal antibodies to the tyrosine kinase SP42 allowed the isolation of several positive clones. Sequencing of a positive cDNA clone revealed a 735-nucleotide open reading frame encoding a protein of 245 amino acids (27,778 Da). The predicted protein was found to be identical to the most recently discovered 14-3-3 isoform, the theta subtype from a rat brain. Here we demonstrate that 14-3-3 theta mRNA is highly expressed in testis and brain only. Western immunoblot analyses confirm the Northern blot data. Developmental Northern and Western blot analyses are consistent with an expression and translation of the 14-3-3 theta gene throughout spermatogenesis. However, analysis of RNA from purified populations of spermatogenic cells at different developmental stages and immunohistochemistry on adult testis sections reveal that within the testis the 14-3-3 theta gene products are most abundant in meiotic prophase spermatocytes, and, above all, in differentiating spermatids. Both testicular and epididymal spermatozoa are negative. The present study is the first report on the presence and molecular characterization of the 14-3-3 theta gene product in the male germ line. Our observations suggest that this specific member of the 14-3-3 protein family could play distinct modulatory roles in the complex development of the mammalian male germ cell lineage.

14-3-3 Proteins↗
PubMed1997

Calmodulin content, Ca2+-dependent calmodulin binding proteins, and testis growth: identification of Ca2+-dependent calmodulin binding proteins in primary spermatocytes.

In contrast with the transient pre-replicative increase in calmodulin (CaM) level observed in proliferative activated cells, postnatal development of rat testis was paralleled by 3 specific rises in CaM. The first one occurred between 5 and 10 days, coincident with the appearance and proliferation start of spermatogonia and Sertoli cells. Meiosis accomplishment and spermatid differentiation were paralleled by 2 additional rises, at 24 and 32 days, respectively. The plateau phase of testis growth was coincident with the appearance of maturating spermatids and spermatozoa in the germinal epithelium, and with a decrease in CaM content. Testicular DNA:g wet tissue ratio reached the highest level in 15-day-old rats and gradually decreased up to 35 days, when a constant level was reached. A similar level of Ca2+-CaMBPs was observed in 5- and 20-day-old rat testis. Although all subcellular fractions showed the ability to bind CaM in a Ca2+-dependent manner, CaM was mainly recovered in the nuclear and soluble fractions of adult and immature rat testis. Several Ca2+-CaMBPs with an apparent M(r) of 82, 75, 64, 19, and 14 kD were purified by affinity chromatography from pachytene primary spermatocyte nuclear matrix. Ca2+-CaMBPs showing an M(r) of 120, 78, 72, and 66 kD were also purified from the supernatant obtained after DNA and RNA hydrolysis of meiotic nuclei. Major cytosolic Ca2+-CaMBPs of primary spermatocytes showed an M(r) of 120, 84, 44, and 39 kD. The functions that these Ca2+-CaMBPs might have during the first meiotic prophase is discussed.

Animals↗
PubMed1984

Ewing's sarcoma in female siblings. A clinical report and review of the literature.

The third reported case of Ewing's sarcoma in siblings is presented. All reported siblings to date have been female. Because of the most unusual occurrence, clues for environmental and genetic factors in the origin of the tumors were sought. Human leukocyte antigen (HL-A) genotyping, routine chromosomal, and prophase chromosomal studies were obtained. Despite extensive study, the existence of an infectious agent, exposure to a carcinogen, or genetic abnormality could not be defined.

Adult↗
PubMed2000

Meiosis and apoptosis in germ cells of X-autosome translocation carrier boars.

Meiotic features and fate of germ cells were studied using electron microscopy on surface spread spermatocytes and in situ tests for apoptosis on testicular tissues of normal boars and X-autosome translocation (X-AT) carrier boars. Histological sections of the translocation t(Xp+; 14q-) carrier boars showed accumulation of degenerating germ cells including binucleate and multinucleate cells, as well as pyknosis and nuclear fragmentation characteristic of apoptosis. Synaptonemal complex analysis of X-AT carrier boars revealed 19 bivalents including a large complex made up of the altered X (Xp+) and normal chromosome 14, and a smaller element representing the Y chromosome in synapsis with the derived chromosome 14 (14q-) in most (89.3%) of the germ cells. In situ tests for apoptotic DNA fragmentation revealed positive signals exclusively among early spermatocytes and degenerating germ cells. These findings and the absence of stages beyond pachytene suggest that the meiocytes are arrested at pachytene and eliminated through apoptotic process in spite of the complete synapsis displayed by the chromosomes involved in this translocation. Failure of meiotic progress in our X-AT carriers would appear to be the result of the disruption of gene sequence (or function) caused by the involvement of the X chromosome in this rearrangement, rather than the deleterious consequences of abnormal segregation anticipated in reciprocal translocation carriers. We hypothesize that this disruption could have affected the induction of stage-specific gene products in meiosis such as heat shock proteins and caused the excessive release of endonucleases normally produced by early prophase meiocytes, leading to their apoptosis in our X-autosome translocation carrier boars.

Animals↗
PubMed2000

Nuclear transfer and cell transplantation: making more with less.

Pig cloning can be achieved by transfer of nuclei of differentiated somatic cells into enucleated oocytes. Then, developing embryos are placed into surrogate mothers for further development to full term. Although cloned pigs offer the possibility of unlimited organ supply for compatible xenotransplantation in humans, the yield of a predictable number of offspring is still at an experimental phase. Spermatogonial stem cells from a fertile donor can be transplanted to the testes of infertile recipients and generate sperm. At present, results from xenogeneic spermatogenesis by transplantation indicate that porcine, bovine and equine spermatogonia find the interior of the seminiferous tubular environment not favorable for further differentiation into meiotic prophase spermatocytes. Spermatogenic cell transplantation is a promising experimental alternative for understanding the conditions required by both the donor cells and the recipient testis to coexist and even cooperate towards the full development of fertilizing sperm.

Animals↗
PubMed2000

Fission yeast on the brink of meiosis.

The fission yeast Schizosaccharomyces pombe (S. pombe) is now well established as a versatile genetic model organism. It is widely used to analyse the basic eukaryotic cell cycle during vegetative growth and it is also well suited to studies on the elementary processes of sexual reproduction, including intercellular communication and signal transduction in zygote formation, as well as meiosis before sporulation. Systematic mutant screening has contributed much to our current understanding of unicellular differentiation in S. pombe, and structural analysis has revealed a simplified meiotic prophase with abundant crossing-over but no homologue synapsis. This article is a personal account of how this branch of fission yeast genetics has developed.

Denmark↗
PubMed1990

mos proto-oncogene function.

Maturation promoting factor (MPF) is a cytoplasmic activity that causes oocytes arrested in prophase to resume meiosis. An inactive form of MPF termed pre-MPF exists in fully grown oocytes. In Xenopus oocytes, progesterone induces maturation and pre-MPF activation. These early maturation events require protein synthesis. We have shown that p39mos synthesis is rapidly induced in progesterone-treated Xenopus oocytes during the protein synthesis sensitive period and prior to activation of pre-MPF or germinal vesicle breakdown (GVBD). p39mos may qualify, therefore, as an 'initiator' of maturation. Mouse oocytes undergoing meiotic maturation also express p39mos. Microinjection of antisense mos oligodeoxynucleotides into fully grown mouse and Xenopus oocytes results in the block of meiotic maturation. In Xenopus, antisense-injected oocytes not only lack p39mos, but also lack MPF and fail to undergo GVBD. In the mouse, the microinjected oocytes progress through GVBD, but fail to produce the first polar body; cytogenetic analysis shows they are arrested at the bivalent chromosome stage of metaphase I. This and additional studies with Xenopus oocytes indicate that p39mos is also required throughout maturation. We have shown that p39mos is indistinguishable from the protein product constitutively expressed in NIH/3T3 cells transformed with activated c-mos. It is likely that its activity as a transforming gene may be due to activation of pre-MPF activities in interphase in the somatic cell cycle.

Animals↗
PubMed1994

Specification and development of the germline in Caenorhabditis elegans.

Maternal-effect sterile (mes) genes encode maternal components that are required for establishment and development of the germline. Five such genes have been identified in the nematode Caenorhabditis elegans. Mutations in one of the genes result in defects in the asymmetric division and cytoplasmic partitioning that generate the primordial germ cell P4 at the 16-24-cell stage of embryogenesis. As a result of these defects, the P4 cell is transformed into a muscle progenitor and mutant embryos develop into sterile adults with extra body muscles. Mutations in the other four mes genes do not affect formation of the germline during embryogenesis, but result in drastically reduced proliferation of the germline during post-embryonic stages and in an absence of gametes in adults. The failure to form gametes may reflect a defect in germline specification or may be a consequence of reduced germline proliferation. We are currently testing these two possibilities. In addition to the mes gene products, wild-type function of the zygotic gene glp-4 is required for normal post-embryonic proliferation of the germline. Germ cells in glp-4 mutant worms are arrested in prophase of the mitotic cell cycle and are unable to enter meiosis and form gametes. Thus, following establishment of the germ lineage in the early embryo, both maternal and zygotic gene products work in concert to promote the extensive proliferation of the germline and to enable germ cells to generate functional gametes.

Animals↗
PubMed1983

Steroid and peptide control mechanisms in membrane of Xenopus laevis oocytes resuming meiotic division.

Stage 5-6 Xenopus laevis oocytes are arrested in prophase of the first meiotic division, and can be studied in vitro after removal from their follicle cell environment. While they do not mature spontaneously, they demonstrate germinal vesicle (nucleus) breakdown (GVBD) if exposed to approximately 1 microM-progesterone (the hormone released in vivo at the time of ovulation and maturation). The oocytes' then become eggs ready to be fertilized. The progesterone-oocyte interaction, contrary to what is observed in all endocrine steroid target organs so far studied, takes place at the surface membrane level and is not narrowly progesterone-specific, since other hormones such as cortisol or testosterone can also cause resumption of meiosis in vitro. This is the first description of such a paracrine steroid system, which depends however on a receptor mechanism, as indicated by physicochemical experiments, studies with antagonistic (competitive) steroids, and cell-free specific inhibitory effects on membrane-bound adenylate cyclase. It was found that insulin and related growth factors (mitosis-stimulating activity, MSA; insulin-like growth factor, IGF) are also reinitiators of meiosis. Insulin also potentiates the effects of low progesterone concentration (approximately 1 nM) in completely denuded oocytes (free of the vitelline membrane). From these observations it is suggested that there may be a physiological, cooperative involvement of a steroid (progesterone) and an insulin-like peptide factor within the ovaries which promotes oocyte maturation in vivo. The molecular mechanisms of the hormone-dependent changes in cyclic AMP and Ca2+ remain to be elucidated in detail, as well as the respective roles of these two sets of metabolic events.

Adenylyl Cyclases↗
PubMed1984

Structure, development, and cytochemical properties of the nucleolus-associated "round body" in rat spermatocytes and early spermatids.

The "round body," a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 micron in leptotene to 1.6 micron in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8. Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.

Animals↗
PubMed1990

Three structures associated with the nucleolus in male rat germinal cells: round body, coiled body, and "nubecula" and general presence of round body at male meiosis.

In addition to chromosomes and nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called "coils", which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nm-wide filaments and 8 to 10-nm-wide granules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of "spliceosomes", that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560 nm-wide spheroid. It has been observed only in early pachytene nuclei.

Animals↗
PubMed1981

Familial insertional translocation of a portion of 3q into 11q resulting in duplication and deletion of region 3q22.1 leads to q24 in different offspring.

The use of elongated prophase and prometaphase chromosome preparations has allowed detection of an insertion of a small segment of 3q into 11q in a kindred with 4 balanced carriers and 8 unbalanced offspring. Those with partial 3q deletion have a true multiple congenital anomalies/mental retardation (MCA/MR) syndrome with an appearance suggestive of the Schwartz-Jampel syndrome.

Abnormalities, Multiple↗