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At least 19 recordsLinked to original sources
PubMed1978

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.

Animals↗
PubMed1978

Involvement of the mature domain in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

A precursor of 5S ribosomal RNA from Bacillus subtilis (p5A rRNA, 179 nucleotides in length) is cleaved by RNase M5, a specific maturation endonuclease which releases the mature 5S rRNA (m5, 116 nucleotides) and precursor fragments derived from the 5' (21 nucleotides) and 3' (42 nucleotides) termini of p5A rRNA. Previous results (Meyhack, B., et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3045) led to the conclusion that recognition elements in potential RNase M5 substrates mainly reside in the mature moiety of the precursor. Limited digestion of p5A rRNA with RNase T1 permitted the isolation of a number of test substrates which contained both precursor-specific segments and were unaltered in the immediate vicinity of the cleavage sites, but which differed in that more or less extensive regions of the mature moiety of the p5A rRNA were deleted. Tests of the capacity of these partial molecules to serve as substrates for RNase M5 indicate clearly that the enzyme recognizes the overall conformation of potential substrates, neglecting only the double-helical "prokaryotic loop" (Fox, G.E., & Woese, C.R. (1975) Nature (London) 256, 505).

Bacillus subtilis↗
PubMed2026

Epigenetic safety of in vitro maturation in PCOS: genome-wide DNA methylation profiling of cord blood from a randomized controlled trial.

BACKGROUND: In vitro maturation (IVM) provides a safer alternative to conventional in vitro fertilization (IVF) for women with polycystic ovary syndrome (PCOS) by mitigating the risk of ovarian hyperstimulation. However, concerns persist regarding whether IVM perturbs epigenetic reprogramming in the offspring. Current evidence is constrained by candidate-gene approaches or a lack of parental controls. This study aimed to evaluate the genome-wide DNA methylation safety of IVM compared with conventional IVF using a rigorous trio-based design. METHODS: This secondary epigenetic analysis was nested within a randomized controlled trial (RCT) (ClinicalTrials.gov: NCT03463772). We included 10 nuclear families (trios), comprising five IVM-conceived and five IVF-conceived singleton offspring alongside their biological parents. Both groups utilized a uniform freeze-only single-blastocyst transfer strategy to minimize hormonal confounding. Genomic DNA from umbilical cord blood (UCB) and parental peripheral blood was analyzed using reduced representation bisulfite sequencing (RRBS). Genome-wide methylation patterns and differentially methylated regions (DMRs) were subsequently compared between the groups. RESULTS: Clinical characteristics were comparable between the IVM and IVF groups. Genome-wide analyses demonstrated high concordance in UCB methylation patterns, revealing no significant differences in global CpG methylation levels or distributions across key genomic features (promoters, CpG islands, and gene bodies). Only three rare DMRs were identified in UCB (representing ~ 0.0001% of the genome), none of which mapped to imprinted or developmentally critical loci. Furthermore, methylation variability remained consistent between the groups. CONCLUSIONS: Our findings provide robust mechanistic evidence supporting the epigenetic safety of IVM. The remarkable stability of the neonatal methylome confirms that specific IVM conditions do not compromise early developmental programming, thereby endorsing IVM as a safe and viable alternative for women with PCOS. TRIAL REGISTRATION: ClinicalTrials.gov registry, NCT03463772. Registered on March 13, 2018.

Humans↗
PubMed1977

Involvement of precursor-specific segments in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

In vitro maturation of precursor 5S ribosomal RNA (p5A) from Bacillus subtilis effected by RNase M5 yields mature 5S RNA (m5, 116 nucleotides), and 3' precursor-specific segment (42 nucleotides), and a 5' precursor-specific segment (21 nucleotides) (Sogin, M.L., Pace, B., and Pace, N.R. (1977), J. Biol. Chem. 252, 1350). Limited digestion of p5A with RNase T2 introduces a single scission at position 60 of the molecule; m5 is cleaved at the corresponding nucleotide residue. The complementary "halves" of the molecules could be isolated from denaturing polyacrylamide gels. The isolated fragments of p5A are not substrates for RNase M5, suggesting that some recognition elements can be utilized by RNase M5 only when presented in double-helical form. In exploring the involvement of the precursor-specific segments in the RNase M5-p5A interaction, substrate molecules lacking the 3' or 5' precursor-specific segment were constructed by reannealing complementary "halves" from p5A and m5 RNA. The artificial substrate lacking the 5'-terminal precursor segment was cleaved very much more slowly than the lacking t' segment; the 5' precursor-specific segment therefore contains one or more components recognized by RNase M5 during its interaction with the p5A substrate.

Bacillus subtilis↗
PubMed1975

Failure of gonadotropins to induce in vitro maturation of mouse oocytes treated with dibutyryl cyclic AMP.

In sufficient concentration, dibutyryl cAMP (DBC) prevents the spontaneous in vitro maturation of mouse oocytes. The effects of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on this inhibition were tested in an oil-free chamber-slide culture system. Mouse oocytes devoid of cumulus cells were incubated in the presence of DBC and/or gonadotropins. Oocytes cultured with follicle cells were similarly treated. Whether follicle cells were present or absent, DBC (100 or 500 mug/ml) prevented germinal vesicle breakdown in more than 95% of the oocytes cultured. Neither LH nor FSH in a wide range of concentrations acted directly on the oocytes or indirectly through the follicle cells to initiate maturation in oocytes incubated with 100 mug DBC/ml. The combination of LH (5 mug/ml) and FSH (10 mug/ml) was also ineffective in overcoming the block induced by either 100 or 50 mug DBC/ml. Maturation of oocytes in each of the DBC-free LH or FSH treatments was comparable to that occurring in control medium which did not contain exogenous gonadotropins or DBC. It was concluded that cultured oocytes treated with DBC are not a satisfactory model for studying the steps by which gonadotropins trigger the resumption of meiosis in mammalian oocytes.

Animals↗
PubMed1979

Effects of divalent cations on in vitro maturation of bovine oocytes.

Lowering the external concentrations of both Mg+2 and Ca+2 caused failure of meiotic resumption in vitro of bovine, oocyte-cumulus complexes. Lowering of external Ca+2 levels singly had no effect on either meiotic resumption or completion of the first meiotic division. Lowering of external Mg+2 concentrations alone, although having no effect on meiotic resumption in vitro when Ca+2 was present, did interfere with the completion of the first meiotic division. The result was arrest of oocyte maturation between germinal vesicle breakdown and formation of the first metaphase plate.

Animals↗
PubMed1977

[Infertility of the hamster oocyte having matured in vitro].

The comparative study of fertilization, with the same sperm sample, of in vitro matured oocytes and freshly ovulated ones, shows a new aspect of mammalian oocyte maturation. While 80% of freshly ovulated oocytes are fertilized, in vitro matured eggs are not fertilizable. They present the ability to be penetrated by spermatozoa 4 to 6 hrs. only after HCG injection. This is therefore not on tubal influence but depends on an oocyte specific factor which appears during the end of intrafollicular maturation.

Animals↗
PubMed1978

In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence.

Peanut lectin (PNL) binds to a majority of mouse thymocytes (Thc) in suspension. By using cell affinity chromatography on a column of anti-PNL antibody, Thc populations at least 96 percent pure in PNL + or - cells, as judged by immunofluorescence, were obtained. PNL(+) cells are rich in Thy 1 and poor in H(2) antigens, cortisone sensitive, unresponsive to phytohemagglutinin (PHA), and immunologically incompetent, as judged by mixed lymphocyte reaction, popliteal lymph node graft-versus-host assay, and by testing helper activity in a primary in vitro antibody response to sheep erythrocytes; the converse is true of PNL(-) cells. Thus, PNL(+) and (-) cells appear to correspond to cortical and medullary Thc, respectively, as previously suggested. In culture, PNL(+) Thc show poor viability and a weak proliferative response to concanavalin A (Con A), except when supernate (SUP) of 24 h Con A stimulated lymph node lymphocyte cultures, or irradiated lymph node cells, are added, in which cases a strong proliferative response to the mitogen is observed. A variety of control experiments showed that the proliferating cells did not result from preferential stimulation of a few contaminating PNL(-) Thc present in the PNL(+) Thc cultures. The blasts resulting from PNL(+) Thc proliferation display mitogen-induced cytotoxicity, and give rise to a population of medium-sized lymphocytes, mostly PNL(-), poor in Thy 1 and rich in H(2) antigens, PHA responsive, and immunologically competent in the above-mentioned assays. Fresh PNL(+) Thc responded in mixed lymphocyte reaction in the presence of SUP (lectin depleted) and since incubation in SUP alone did not confer reactivity on PNL(+) Thc, it appears therefore that (a) immature Thc possess alloantigen and mitogen-specific surface receptors but lack the capacity to respond by proliferation to receptor triggering without the help of extracellular factor(s) released by mature lymphoid cells stimulated by mitogens (b) cell division is associated with the acquisition of immunological responsiveness, characteristic of mature T lymphocytes. The implications of these findings for the ontogenesis of thymus-derived lymphocytes, and for the possible traffic of Thc within and from the thymus, are discussed.

Animals↗
PubMed1979

[In vitro maturation of rabbit reticulocytes: oxygen consumption reaction].

With a simple experimental system the changes of endogenous, antimycin A-suppressed, oligomycin-suppressed and antimycin A-resistant oxygen consumption are studied during the maturation of intact cells of the 6th day of bleeding. All functional characteristics of oxygen consumption decrease during maturation. The rate of decrease is strongly increased by high inorganic phosphate concentrations (125 mM). This effect is most obvious for the oligomycin-suppressed and the endogenous respiration. The degree of uncoupling of non-incubated cells is 14%. During 24 h incubation it rises to 75%. Inorganic phosphate accelerates the increase of uncoupling during maturation. Reticulocytes of the 4th day of bleeding are characterized by a higher respiratory capacity and also by a higher rate of maturation of antimycin A-suppressed and endogenous respiration. The degree of uncoupling does not increase during maturation. This may be attributed to the low lipoxygenase activity of these cells. 25% of the endogenous oxygen consumption of unmatured cells are antimycin A-resistant. This type of respiration declines by 50% in 4 h incubation irrespective of inorganic phosphate concentrations and day of bleeding. In nitrogen all functional characteristics of respiration during the maturation decline more rapidly than in oxygen. The antimycin A-resistant respiration, however decreased more slowly and reached 50% after 12 h. A pH dependence of maturation (maximum at pH 8.4) was found for the endogenous and the antimycin A-suppressed respiration. The degree of uncoupling rises most quickly at pH 7.4. This is possibly related to the pH maximum of lipoxygenase.

Animals↗
PubMed1979

In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells.

Long-term survival of mesencephalic and striatal cells from mouse embryos in dissociated primary cultures is described. Catecholaminergic neurons in mesencephalic culutres were identified histochemically and by measuring [3H]dopamine uptake and synthesis from [3H]tyrosine. According to experiments using specific inhibitors of catecholamine uptake, at least two-thirds of the catecholaminergic neurons are dopaminergic. These neurons differentiated whether or not striatal target cells were present, but striatal cells stimulated the development of the dopaminergic neurons. [3H]Dopamine uptake was increased by at least 2-fold regardless of the age of the cocultures (4-15 days). Enhanced [3H]dopamine synthesis was also observed (at least 2-fold) at later times (12-15 days).

Animals↗
PubMed1977

Functional maturation of B cells in vitro.

Maturation of B-cell function was studied in a two-stage tissue culture system. In the first stage, cells were cultured in the absence of antigen and then transferred to microcultures where the frequency of hapten-specific plaque-forming cell (PFC) precursors was determined; Bone-marrow cells and spleen cells from 6--8-day-old mice mice were shown to act as sources of B-cell neogenesis in vitro. Both populations had very low initial frequencies of hapten-specific PFC precursors, but this increased ten- to seventeen-fold during a period of 72 h in the preliminary cultures. This increase could not be accounted for by selective cell death, nor by decay of a suppressor cell subpopulation nor by proliferation of pre-existing Fc-receptor-bearing B cells. The mechanism for the increase in frequency of functional B cells in cultures of bone marrow and neonatal spleen was thus the result of maturation of B-cell precursors to a state of immune competence during the culture interval.

Age Factors↗
PubMed1977

Thymic maturation in vitro by a secretory product from macrophages.

Our results indicate that immature thymocytes can be induced by macrophage culture fluid (MCF) to differentiate in vitro. This maturation occurs during several days of culture but does not require DNA synthesis. It is accompanied by an augmentation of surface H-2 which precedes a loss of susceptibility to cytolysis with anti-TL and complement. Our evidence indicates that there is no physical loss of TL within the thymus. Thymocytes cultured with MCF also acquire the capacity to respond in an MLC. Differentiation was shown not to be due to interferon nor could it be reproduced by 2-ME. The stimulating molecule is distinct from the principal mitogenic protein found in MCF.

Animals↗
PubMed1978

[Anomaly of the nuclear maturation in vitro of golden hamster oocytes].

Nuclear anomalies were observed during maturation in vitro of golden hamster oocytes, as follows: 1) In 33 p. cent of oocytes, the axis of the first meiotic division spindle was oriented incorrectly. This leads either to the formation of "large" polar bodies or to non-expulsion of the polar body if the spindle occupied a central instead of peripherical position. 2) Triploidy was observed in 0.3 p. cent of the oocytes which accomplished their maturation and fertilization in vitro. 3) Two rare anomalies--reconstitution of the nucleus after expulsion of the first polar body and formation of two second division spindles--appeared to be due to degenerescence of the oocytes in culture.

Animals↗
PubMed1979

The effect of LH on the fertilizability and developmental capacity of rat oocytes matured in vitro.

The effect of adding LH (10 microgram NIH-LH-B8/ml) to the medium in which oocytes were undergoing maturation in vitro was studied. The fertilizability of the oocytes was evaluated in the sterile oviduct of a unilaterally ovariectomized, mated recipient. Freshly ovulated oocytes, used as a control of the method, were fertilized at a rate of 72%. Only 14% of oocytes matured in culture (without LH) were penetrated by spermatozoa, and 11% were fertilized normally. Addition of LH to the medium increased these proportions to 43 and 33% respectively. Oocytes matured in the presence of LH were able to develop into apparently normal rats. It is concluded that, although oocytes can mature in vitro spontaneously, and that these matured oocytes can be fertilized, addition of LH increases the numbers 3-fold. LH therefore has a direct maturation-promoting action on the rat oocyte-cumulus complex in vitro.

Animals↗
PubMed1979

Effect of hypophysectomy on mouse oocyte maturation in vitro.

There was no difference in frequency of maturation of oocytes obtained from mice hypophysectomized for 2 weeks compared to those from sham-operated or untreated (control) animals of the same age. By 7 weeks, and also at 12 and 17 weeks, the incidence of polar body formation in vitro was significantly reduced. The number of oocytes which remained meiotically inactive in culture was increased at 7, 12 and 17 weeks after hypophysectomy. This decrease in spontaneous oocyte maturation in vitro could be partly overcome by administering exogenous PMSG, oestradiol-17beta or PMSG + oestradiol-17beta, but not progesteron or hCG, to hypophysectomized mice.

Animals↗
PubMed1979

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro.

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.

Animals↗